Compositions and methods for the treatment of autoimmune diseases and cancer

Abstract

Aspects of the invention relate to compositions and methods for modulating CD8 Treg recruitment in the treatment of autoimmune disorders, transplanted organ rejection and cancer.

Description

[Technical field]

[0001] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety, and the disclosures of these publications are incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art as of the date of the invention described and claimed herein.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all and any copyright rights whatsoever.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 239291, filed August 31, 2021, the contents of which are incorporated herein by reference.

[0004] Government Interests This invention was made with Government support under Grant No. R01AI037562 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0005] FIELD OF THEINVENTION Aspects of the invention relate to compositions and methods for modulating CD8 Treg recruitment in the treatment of autoimmune disorders (eg, CD8 Treg activation) and cancer (eg, CD8 Treg depletion).

[0006] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy, created above in [], is named [] and is [] bytes in size. [Background technology]

[0007] 2. Background of the Invention Most CD8 + T cells are endowed with the ability to kill cells infected by microbial invaders, but a subset of these cells can regulate immune responses. The regulatory activity of mouse and human CD8 is consigned to a small (<5% CD8 cells) subset that expresses a characteristic triad of surface receptors - CD44, CD122 and Ly49 (mouse) / KIR (human). These cells, referred to herein as CD8+ Treg or CD8 Treg cells, are CD4 + Activated CD4 T cells can be eliminated through targeting of MHC class Ia or class Ib expressed by helper T cells. Summary of the Invention

[0008] Here, we disclose a method to mobilize (activate) or suppress CD8 regulatory T cells (CD8 Treg) to decrease or increase, respectively, CD4 T cell activity and immune response in a mammal. One type of CD8 Treg stimulator includes peptide superagonists for CD8 Treg, which can reduce antibody-mediated rejection (AMR) and allograft tissue damage when administered to mice or used to vaccinate mice. Peptide superagonists can also be used to treat autoimmune diseases. CD8 Treg can also be mobilized or depleted using specific antibodies. Antibodies that can deplete Treg cells can be used to treat cancer in a mammal. Antibodies that bind to CD8 Treg can bind to unique molecules on CD8 Treg cells, including T cell receptors (TCRs).

[0009] Disclosed herein is a method for mobilizing CD8 Treg cells in a mammal, comprising administering a CD8 Treg stimulator to the mammal. In some embodiments, the CD8 Treg cell stimulator can be a peptide / polypeptide agonist or superagonist of CD8 T cells. In some embodiments, the peptide / polypeptide agonist or superagonist binds to a T cell receptor (TCR) on CD8 Treg cells and an MHC class Ib molecule on CD4 T cells. In some embodiments, the CD8 Treg stimulator can be an antibody that binds to CD8 Treg cells. In some embodiments, the antibody can bind to a TCR on CD8 Treg cells. In some embodiments, the antibody can be a bispecific antibody. The method can suppress CD4 cells. The method can be used to treat autoimmune disease and / or reduce allograft rejection.

[0010] Disclosed herein is a method of depleting CD8 Treg cells in a mammal, comprising administering to the mammal a CD8 Treg cell depleting agent. In some embodiments, the CD8 Treg cell depletor can be an antibody that binds to CD8 Treg cells. In some embodiments, the antibody can bind to a TCR or other molecule on CD8 Treg cells. In some embodiments, the antibody can be a bispecific antibody. The method can stimulate CD4 cells. The method can be used to treat cancer.

[0011] CD8 Treg cell stimulator peptide / polypeptide agonists or superagonists are disclosed. Treg cell stimulator antibodies are disclosed. Treg cell depleting antibodies are disclosed. Pharmaceutical compositions of peptide / polypeptide agonists or superagonists, Treg cell stimulator antibodies and Treg cell depleting antibodies are disclosed. Vaccine compositions of peptide / polypeptide agonists or superagonists are disclosed. [Brief description of the drawings]

[0012] [Figure 1A] A schematic diagram showing Ag (Qa-1 peptide) specific recognition of target CD4 cells by CD8 Tregs is provided. Activation of alloreactive CD4 cells upregulates peptides presented on Qa-1. CD8 Tregs express TCRs that recognize the Qa-1 peptide (pQa-1) complex expressed by target CD4 cells and suppress alloreactive CD4 cells. [Figure 1B] We provide schematics showing key molecular interactions between CD8 Tregs and target CD4 cells. I) We show that TCR-pQa-1 interaction between CD8 Tregs and target CD4 cells activates CD8 Tregs. II) We show that binding of pQa-1 by the NKG2A receptor delivers an inhibitory signal resulting in reduced suppressive function. III) We show that stimulatory interactions between NKG2D and NKG2D-L result in CD8 Treg suppressive function. IV) We show that blocking inhibitory Ly49F signaling enhances CD8 Treg function. [Figure 2A] Figures 2A-C provide a representation of the identification of superagonists for FL9 T cells. The peptide library has mutations at each MHC anchoring position (p2, 3, 6, 7 and 9) of the FL9 peptide. FL9 TCR+ hybridoma cells were incubated with EL4 cells and each peptide variant and CD69 expression was measured (Panel A). Panel B shows CD69 expression (%), TCR downregulation and trogocytosis by FL9 TCR+ hybridoma cells + each FL9 peptide variant. Panel C shows CD69 expression by FL9 TCR Tg T cells stimulated with selected FL9 peptide variants. The sequences of selected peptides are shown. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3A]3A-D provide graphical representations showing FL9 Tg CD8 T cell suppression of activated CD4 T cells. In vitro: Con-A stimulated CD4 cells from WT B6, D227K KI and Qa-1 KO mice were co-cultured with 58C hybridomas expressing OT-I or FL9 TCR. After 3 days, stimulation of OT-I TCR+ and FL9 TCR+ hybridomas by these activated CD4 cells was measured based on CD69 expression (panel A). OT-I or FL9 TCR+ hybridomas were co-cultured with Con-A stimulated CD4 cells from WT or ERAAP KO mice. Stimulation of OT-1 or FL9 TCR was measured based on CD69 expression by hybridoma cells (panel B). In vivo: WT or D227K mice were immunized with OT-II peptide in CFA. Seven days later, CD4 (CD4+CD25-) cells were isolated from immunized mice and transferred into WT B6 hosts with or without FL9 TCR Tg T cells, followed by immunization with OTII / CFA (Panels C, D). Detection of OT-II CD4 T cells in the spleen of the host by OT-II tetramer (I-Ab / Ova) (Panel C). Percentage and number of OT-II tetramer+CD4 cells recovered from the adoptive host (Panel D). [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4] FIG. 1 provides a graphical representation showing the Qa-1 peptide yeast library that identified peptides with superagonist activity. FL9 T cells were stimulated with EL4 (Qa-1+) cells for 3 days in the presence of surrogate peptides selected from the peptide Qa-1 yeast library. The activation status of FL9 T cells was measured by CD69 expression. The surrogate peptides (#3, 4, 6, 7, 10, 11) show increased stimulatory activity compared to the FL9 mutant FL9-68. [Figure 5A]Figure 5A-F provides a graphical representation showing the characterization of the Ly49F knockout models. Panel A provides a schematic showing that Ly49F-KO has a disruption specifically in Ly49F, while Pan-KO mice have a disruption in all Ly49-related receptors. Both Ly49F and Pan-KO models lack Ly49F without reducing Helios expression (Panel B), indicating that although Ly49F is deleted, these mice still have a CD8 Treg compartment (Panel C). PD-1 expression was reduced in both knockouts, indicating functional changes within the CD8 Treg compartment (Panel D). High-dimensional cytometry identified 20 distinct clusters of CD44-expressing CD8 T cells in the three genotypes tested (Panels E, F). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] See legend to Figure 5A. [Figure 5F] See legend to Figure 5A. [Figure 6] Provides a schematic diagram showing CD8 Treg expression of inhibitory Ly49F receptor. Blockade of inhibitory Ly49F signaling by Ly49F-deficient CD8 Treg or anti-Ly49F Ab can enhance CD8 Treg function and suppress the proliferation of autoreactive CD4 cells and the production of autoantibodies. [Figure 7] Figure 1 provides a graphical representation showing that blocking Ly49 in vivo can increase activation of CD8 Tregs. WT B6 mice were injected with anti-Ly49F Ab (50ug / mouse) or isotype control Ab on days 0, 2 and 4. Phenotype of CD8 Tregs, as identified by expression of Helios, which is uniquely expressed by CD8 Tregs, was analyzed on day 7. Representative data from two independent experiments are shown. [Figure 8A]8A-E provide a graphical representation of the generation of FL9 TCR Tg mice. Kb- / -Db- / - mice were immunized with FL9 peptide-loaded DCs. FL9-specific CD8 T cells were sorted using PE and APC-conjugated Qa-1 / FL9 tetramers (panel A). TCR repertoire analysis showing predominance of Vα3.2 and Vβ5.1 / 5.2 among FL9-specific CD8 T cells (panel B). Comparison of binding of FL9 tetramers to hybridomas expressing OT-1 (Vα2+Vβ5+) or FL9-specific TCR (Vα2+Vβ5+) (panel C). Confirmation of TCR specificity of FL9-Tg T cells (panel D). Expression of Ly49, NKG2A, and NKG2D by FL9 TCR Tg T cells (panel E). Data are representative of 2-3 experiments. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 9A]9A-B provide a graphical representation showing that depletion of CD8 Tregs enhances antitumor immune responses. WT B6 mice were inoculated with 2×105 EL4 (Qa-1 WT) or EL4-Qa-1 KO cells. Mice receiving EL4 WT or EL4 Qa-1 KO cells were treated with four different conditions: 1) PBS / IFA+isotype control Ab (mIgG1), 2) PBS / IFA+anti-Ly49F Ab, 3) PBS / IFA+anti-Qa-1 Ab, and 4) FL9.68 / IFA+isotype Ab (mIgG1). 100 μg of FL9.68 peptide (days 0 and 10), 50 μg of anti-Ly49 F Ab, and 100 μg of anti-Qa-1 Ab (days 0, 3, and 6) were injected, respectively. A) Status of Ly49+CD8 cells in blood on days 3 and 6 after tumor induction (Panel A). Tumor growth by day 7 in mice inoculated with EL4 WT (left) and EL4 Qa-1 KO (right) tumor cells. Expansion of CD8 Tregs by FL9-68SA vaccination promotes tumor growth, whereas depletion of CD8 Tregs or blocking the interaction of Qa-1 with its receptor results in slower tumor growth. This effect was not observed in mice inoculated with EL4-Qa-1KO tumor cells (Panel B). [Figure 9B] See legend to Figure 9A. [Figure 10A] Figures 10A-C provide graphical representations showing that FL9-68-dependent mobilization of CD8 Tregs attenuates autoantibody responses during MCMV infection. WT B6 mice were infected with MCMV (2x105 pfu) and vaccinated with FL9-68 in IFA or IFA alone (100ug / dose) on days 0, 8, and 12. Mice were bled on days 3, 10, and 18 and the levels of anti-dsDNA Ab (panel A), viral titer (panel B), and frequency of activated CD8 Tregs (panel C, Ly49+NKG2D+CD8) are shown. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 11A]A graphical representation showing the production of FL9 superagonists is provided. Hybridomas were engineered to express a T cell receptor (TCR) that is restricted to FL9-Qa-1, while OT-1 TCR was used as a control. FLP-TCR expressing hybridomas bind to FL9-Qa-1 tetramers, whereas OT-1 TCR expressing hybridomas do not (left panel). A library of modified FL9 peptides was generated and the ability of the peptides to activate (CD69)FL9-TCR hybridomas was tested. FL9-68 was selected as the FL9-superagonist (FL9-SA) (right panel). [Figure 11B] A representative layout of FL9-Qa-1 restricted CD8 Tregs is provided. Hosts immunized with FL9 show an 8-fold expansion of FL9-Qa-1 tetramer-binding CD8 Tregs (CD44+CD122+Ly49+CD8+T). [Figure 11C] Provides a graphical representation showing the immunophenotype of spleen and draining lymph nodes. Tfh: follicular helper T cells (PD-1+CXCR5+CD4+T); GC B: germinal center B cells (GL-7+FAS+B220+); PC: plasma cells (CD138+B220); *: P<0.05; **: P<0.01; ***: P<0.001; ns: not significant. [Figure 11D] FIG. 1 provides a graphical representation showing a donor-specific antibody assay. The X-axis shows the volume to volume ratio of recipient serum to donor splenocytes diluted in PBS at 106 cells / mL. [Figure 12] Figure 1 provides a graphical representation showing the suppression of GC responses after cardiac allograft transplantation by FL9 superagonist peptide vaccine. B6 mice were vaccinated with FL9-68 / IFA or IFA alone on days 0, 10, 13 and 16. On day 27, these B6 mice were transplanted with cardiac allografts from Balb / C mice along with CTLA-Ig injection. One week later, the frequencies of TFH, GC B and plasma cells were analyzed in the dLN. [Figure 13]Provides a graphical representation showing that FL9 superagonist peptide vaccine inhibits AMR and allograft tissue damage. B6 mice were vaccinated with FL9-68 / IFA or IFA alone on days 0, 10, 13, and 16. On day 27, these B6 mice were transplanted with cardiac allografts from Balb / C mice along with CTLA-Ig injection. One week later, donor-specific Ab levels were measured and C4d deposition in the cardiac grafts was evaluated. [Figure 14] The molecules involved in the interaction between CD8+ and CD4+ T cells are presented, as well as strategies (I, II and III) for manipulating this interaction. [Figure 15] 1 provides a schematic showing the CD8 T cell maturation pathway and a graphical representation showing PD1 expression by FL9 thymocytes. Ly49 expression by developing thymocytes rescues the deletion of PD1+FL9-TCR+ thymocytes. [Figure 16] 1 provides a graphical representation showing the LY49F-KO CD8 Treg memory pool replaced by CD8 effector T cells. CD8 T cell subsets are identified by high-dimensional cytometry. [Figure 17] 1 provides a schematic showing the convergent evolution of LY49 and KIR in humans and mice. [Figure 18] Figures 18A-B provide graphical representations showing KIR / Helios phenotypes. Figure 18A shows the KIR+ subset (%) of CD8+ T cells. Figure 18B shows Helios expression by KIR+CD8+ T cells. [Figure 19] 1 provides a graphical representation showing suppression of CD4+ T H cell responses by KIR+ CD8+ T cells. [Figure 20] FIG. 1 provides a schematic showing that chronically activated autoimmune CD4 T cells upregulate the Qa-1 / HLA-E+FL9 peptide. [Figure 21] A diagram showing the docking of TCR to MHC-peptide and the division of roles by CDR1 / CDR2 / CDR3 is provided. [Figure 22]We provide a model for the interaction of peptides with Qa-1 MHC (on CD4+ T cells) and TCR (on CD8+ Treg cells) and a strategy to screen for peptides with altered binding. [Diagram 23] 1 provides a graphical representation showing screening of peptides (FL9.8) with mutations in MHC binding residues. [Figure 24] 1 provides a graphical representation showing screening of peptides with mutations in TCR binding residues. [Diagram 25] 1 provides a graphical representation showing screening of peptides with mutations in TCR binding residues. [Figure 26] FIG. 1 provides a schematic showing the consensus motifs of FL9 superagonist synthetic peptides based on second generation screening of the FL9 mutant library. [Figure 27] Schematic diagram showing that FL9 TCR is type II (conserved CDR1 / CDR2 contacts with Qa-1 / HLA-E). 10 / 10 FL9 TCRs express TRAV 9N3. All Cd8 Treg (FL9) TCRs express common CDR1 and CDR2 sequences. [Figure 28] 1 provides a schematic showing that FL9 TCR is a type II TCR. 10 / 10 FL9 TCRs express TRBV 12-1 / 2. [Figure 29] FIG. 1 provides a schematic showing that Hsp60-TCR is a type II TCR (TCRα). [Diagram 30] FIG. 1 provides a schematic showing that the Hsp60 TCR is a type II TCR (TCRβ). [Diagram 31] FIG. 1 provides a schematic showing 6C5 TCR:Qa-1=proinsulin specific. [Diagram 32] FIG. 1 provides a schematic diagram showing TCR:CMV-induced / peptide-specific (Type I). [Diagram 33]Provides a schematic diagram showing that conserved sequences of CDR1 and CDR2 are expressed by TCR of CD8 Treg. Conserved CDR1 and CDR2 in TCR are reactive to HLA-E self peptides. Non-classical CDR1 and CDR2 are reactive to HLA-E / foreign peptides. TCR8r targeting moieties include toxins, CTLs, and ADT-TCR8r. [Diagram 34] FIG. 1 provides a schematic showing a comparison of regulatory CD4 and CD8 T cells. [Diagram 35] 1 provides a schematic showing the phylogenetic relationships of MHC class Ib molecules. [Diagram 36] 1 provides a schematic diagram showing the contribution of CD8 Tregs to organ transplantation: use of Qa-1-DK mutant mice. 2 shows the analysis of cardiac allograft transplantation in knock-in mice containing a Qa-1 point mutation that disrupts Qa-1 peptide binding in CD8 Tregs. [Figure 37] Provides a schematic showing that CD8 Treg-mediated immunosuppression is important for long-term cardiac graft survival. Cardiac allograft rejection after immunosuppression is primarily dependent on anti-graft allo- or anti-xeno-antibodies. [Figure 38] Figure 1 provides a representation showing an increase in activated CD4+TFH and GC-B cells in B6.DK hosts after heart transplantation. Genetic disruption of the interaction between pQa-1 and TCR enhances TFH and B cell responses upon heart transplantation. (Left) Single dose CTLA4-Ig heart transplantation protocol using BALB / c cardiac allografts and C57BL / 6 (WT) recipients. (Right) Comparison of Qa-1 MFI by activated CD4 (CD62L-CD44+CD4+), TFH (PD-1+CXCR5+CD4+), total CD4 (blue) and TFH (blue), and isotype-switched germinal center B cells (IgM-FAS+B220+) in recipient spleens (WT vs. D227K mice) 28 days after heart transplantation. [Figure 39A]Figure 39A-B provides a graphical representation showing that mutations of Qa-1 fixed residues identify peptides for FL9 TCR+ CD8 Treg activation. Peptide-based mobilization of CD8 Tregs to inhibit antibody-mediated cardiac rejection. (A) A library consisting of 96 FL9 peptide variants (crude peptides) was generated by amino acid mutagenesis at Qa-1 fixed positions (p2, p3, p6, p7 and p9). FL9 TCR+ hybridomas were incubated with EL4 cells (Qa-1+) loaded with each FL9 peptide variant for 12 hours, after which CD69 expression was measured as an indicator of TCR stimulation (B). (C) Activation of FL9.2 T cells after stimulation with FL9 variants selected from the library screening above. Dose-dependent activation of FL9 T cells was measured by culturing FL9.2 T cells with EL4 (Qa-1+) at various concentrations of the indicated peptides (0, 1, 3 and 10 mg / mL). [Figure 39B] See legend to Figure 39A. [Diagram 40] 1 provides a graphical representation showing inhibition of GC responses following heart graft transplantation by FL9 superagonist peptide vaccine. [Diagram 41] Figure 1 provides indications showing that the FL9SA peptide vaccine inhibits donor-specific Ab production and graft tissue damage. [Diagram 42] A schematic diagram showing the search for FL9 superagonists is provided. A Qa-1 yeast library was generated expressing Qa-1 displaying 1×108 peptides by random mutation of amino acids at each position, except p2 and p9 for 9-mers, and p2 and p10 for 10-mers. The library was screened for binding to FL9 TCR. Peptides bound to FL9 TCR were identified by sequencing TCR-binding yeast clones. [Diagram 43] 1 provides a graphical representation showing that the Qa-1 / peptide yeast library identified peptides with superagonist activity. [Diagram 44]FIG. 1 provides a graphical representation showing that amino acid motifs of peptides selected from the library allow for the identification of endogenous self-peptides capable of activating FL9 T cells. [Figure 45-1] FIG. 45A provides a representation showing the identification and isolation of Qa-1-FL9-specific T cells. WT B6 mice were immunized with FL9 peptide-loaded Kb- / -Db- / -DCs on days 0, 8, and 15. On day 22, Qa-1-FL9-specific CD8 T cells were detected by tetramers (Qa-1-FL9-PE and Qa-1-FL9-APC) in CD44+CD122+Ly49+CD8 T cells. FIG. 45B provides a representation showing the TCR repertoire of Qa-1-FL9 Tet+CD8 T cells. Single Qa-1-FL9-PE+Qa-1-FL9-APC+ cells were sorted and subjected to sequencing of TCRα and TCRβ. Thirty-nine TCRα and TCRβ pairs were analyzed based on their TCR V gene segments. The relative usage of TCRα and TCRβV genes by these Tet+ single cells is shown in a donut graph. Figure 45C provides a representation showing the TCR repertoire of Qa-1-Hsp60 Tet+CD8 T cells. Single Qa-1-Hsp60-PE+Qa-1-Hsp60-APC+ cells were sorted and subjected to TCRα and TCRβ sequencing. The relative usage of TCRα and TCRβV genes by these Tet+ single cells is shown in a donut graph. Figure 45D provides a graphical representation showing Qa-1-dependent differentiation of FL9 T cells: tetramer-mediated detection of TCR in 58C hybridomas transduced with FL9.2 and FL9.8 TCRs (upper panel). Responsiveness of FL9.2 TCR and FL9.8 TCR expressing hybridomas upon stimulation with increasing doses of peptide as measured by CD69 expression (lower panel). Figure 45E provides a graphical representation showing the measurement of Qa-1-FL9 binding affinity of FL9.2 and FL9.8 TCR. FL9.2 TCR+ and FL9.8 TCR+ hybridomas were labeled with Qa-1-FL9-PE tetramers and incubated in the presence of anti-Qa-1 Ab for the indicated times. The percentage of PE+ cells was measured at various time points as a measure of the level of tetramer dissociation. [Figure 45-2]Figure 45F provides a graphical representation showing the percentage of Tg TCR+ cells among TCR+ thymocytes and active caspase-3+PD1+ cells among DP (CD4+CD8+) thymocytes in OT-I→WT B6, FL9.2 Tg→WT B6 BM chimeras 8 weeks after BM reconstitution. Figure 45G provides a graphical representation showing Ki67 and CD44 expression by OT-I and FL9.2 TCR Tg CD8+ T cells measured as an index of Ag encounter in the spleen and liver of OT-I→WT B6 and FL9.2 Tg→WT B6 BM chimeras 8 weeks after BM reconstitution. [Figure 46] Figure 46A-B provides a representation showing the TCR repertoire of Qa-1 / FL9 Tet+CD8 T cells. Single Qa-1-FL9-PE+Qa-1-FL9-APC+ cells were sorted and subjected to TCRα and TCRβ sequencing. Thirty-nine TCRα and TCRβ pairs were analyzed based on their TCR V gene segments. Alignment of TCRα (A) and TCRβ (B) sequences obtained from Qa-1-FL9 Tet+ single cells. [Figure 47] Figure 47A-B provides a representation showing the TCR repertoire of Qa-1 / FL9 Tet+CD8 T cells. Single Qa-1-FL9-PE+Qa-1-FL9-APC+ cells were sorted and subjected to TCRα and TCRβ sequencing. Thirty-nine TCRα and TCRβ pairs were analyzed based on their TCR V gene segments. Alignment of TCRα (A) and TCRβ (B) sequences obtained from Qa-1-Hsp60 Tet+ single cells. [Figure 48A]Figures 48A-B and 48C-D provide representations showing the generation of FL9 TCR+ hybridomas and analysis of their responsiveness to FL9 peptides. Figure 48A provides a graphical representation showing the production of Qa-1-FL9 specific hybridomas. 58C α-β-hybridomas were transduced with OT-I TCR or FL9 TCR identified from single cell TCR sequencing from Qa-1-FL9 tet+CD8 T cells. Correct folding and assembly of the transduced TCR and specificity of the TCR were tested by staining with anti-CD3, anti-TCR Vβ and Qa-1-Hsp60 or Qa-1-FL9 tetramer. [Figure 48B] Figures 48A-B and 48C-D provide representations showing the generation of FL9 TCR+ hybridomas and analysis of their responsiveness to FL9 peptides. Figure 48B provides a graphical representation showing that TCRs specific for Qa-1-FL9 can exhibit different binding affinities. Qa-1-FL9 tetramer-based detection of TCRs on 58C hybridomas transduced with individual TCR pairs isolated from Qa-1-FL9-specific single CD8 T cells. [Figure 48C] Figures 48A-B and 48C-D provide representations showing the generation of FL9 TCR+ hybridomas and analysis of their responsiveness to FL9 peptide. Figure 48C provides a graphical representation showing the differential responsiveness of Qa-1-FL9 specific TCR to the cognate peptide FL9. Responsiveness of FL9 TCR+ hybridomas to increasing concentrations of FL9 peptide as indicated by surface CD69 expression. [Figure 48D] Figures 48A-B and 48C-D provide representations showing the generation of FL9 TCR+ hybridomas and analysis of their responsiveness to FL9 peptides. Figure 48D provides a graphical representation showing the frequency of Vα3.2+ or Vβ5+ cells within Ly49+CD8+ cells in spleens and LNs of 8-week-old WT B6, Qa-1.D227K KI and Qa-1 KO mice (n=6 / group). [Figure 49A]Figures 49A-B provide a graphical representation showing NKG2D expression by FL9 Tg T cells. Age-related acquisition of NKG2D expression by FL9.2 (A) and FL9.8 (B) T cells. Percentage of NKG2D+ cells among CD8αβ+FL9 T cells from spleen, LN and liver in 18-day, 9-week and 4-month-old FL9.8 TCR Tg mice (n=5 / group). [Figure 49B] See legend to Figure 49A. [Figure 50A] Figure 50A-C provides a graphical representation showing Qa-1-dependent differentiation of FL9 T cells. (A) Percentage of Tg TCR+ cells (Vα3.2+Vβ5+) among TCR+ thymocytes and active caspase 3+PD1+ cells among DP (CD4+CD8+) thymocytes in OT-I→WT B6, FL9.8 Tg→WT B6 BM chimeras 8 weeks after BM reconstitution. (B) Expression of Ki67 and CD44 by OT-I and FL9.8 TCR Tg CD8+ T cells was measured as an indicator of Ag encounter in the spleen and liver of OT-I→WT B6 and FL9.8 Tg→WT B6 BM chimeras 8 weeks after BM reconstitution. C) TCR and CD8 expression on polyclonal non-Tg CD8 cells, OT-I and FL9.8 Tg T cells. [Figure 50B] See legend to Figure 50A. [Figure 50C] See legend to Figure 50A. [Figure 51-1]Figures 51A-E and 51F provide representations showing Qa-1-dependent differentiation of FL9 T cells. Figure 51A provides a graphical representation showing that Qa-1 deficiency impairs the development of autoreactive FL9 T cells. Frequency of Vα3.2+Vβ5+ T cells in TCRb+ cells from FL9.2 TCR Tg mice on Qa-1 WT and KO background (8 weeks old). Representative FACS plots for detection of Vα3.2+Vβ5+ cells in spleen are shown (left panel) (n=4 / group). Figure 51B provides a graphical representation showing Qa-1 deficiency and autoreactive markers. Expression of CD44 and NKG2D by FL9.2 T cells in spleens of WT.FL9.2 TCR Tg and Qa-1- / -FL9.2 TCR Tg mice. Representative FACS plots of NKG2D+CD44+ cells in spleens of FL9.2 TCR Tg mice are shown on the left. Figure 51C provides a graphical representation showing Qa-1 dependency for maintaining FL9 T cells. CFSE-labeled FL9.2 T cells generated in Qa-1 WT or Qa-1 KO mice were transferred into irradiated (800 rad) Qa-1 WT, Qa-1 KO and D227K KI adoptive hosts. Seven days after transfer, Qa-1 WT or Qa-1 KO FL9.2 T cells were harvested from the spleens of the adoptive hosts. The number of FL9.2 T cells in the spleens of the adoptive hosts is shown. Figure 51D provides a graphical representation showing the expression of Ki67 by FL9.2 T cells in the LNs of WT.FL9.2 TCR Tg and Qa-1- / -FL9.2 TCR Tg mice. Figure 51E provides a graphical representation showing Qa-1-dependent activation and proliferation of FL9 T cells. Percentage of Qa-1 WT FL9.2 T cells undergoing more than three divisions in Qa-1 WT, Qa-1 KO and D227K KI hosts. Figure 51F provides a graphical representation showing Qa-1 restricted CD8 Treg: Vα3.2+Vβ5+ CD8 T cells. Frequency and phenotype of Vα3.2+Vβ5+ cells within Ly49+CD8 cells in spleens and LNs of 8-week-old WT B6, Qa-1.D227K KI and Qa-1 KO mice (n=6 / group). Mean±SEM shown. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 51-2]See description of Figure 51-1. [Figure 52] Figure 52A-C provide a graphical representation showing Qa-1-dependent phenotype acquisition by FL9.8 Tg T cells. Figure 52A provides a graphical representation showing reduced FL9 T cell development in Qa-1 deficiency. Frequency of Vα3.2+Vβ5+ T cells in TCRβ+ cells from FL9.8 TCR Tg mice in Qa-1 WT and KO backgrounds. A representative FACS plot for detection of Vα3.2+Vβ5+ cells in the spleen is shown on the left. Figure 52B provides a graphical representation showing that Qa-1 deficiency affects the expression of markers of autoreactivity. Expression of CD44 and NKG2D by FL9.8 TCR Tg T cells in spleens and LNs of Qa-1 WT and Qa-1 KO mice. A representative FACS plot for NKG2D+CD44+ cells in the spleens of FL9.8 TCR Tg mice is shown on the left. Figure 52C provides a graphic representation showing expression of Ki67 by FL9.8 T cells in LNs of 8-week-old WT.FL9.8 TCR Tg and Qa-1- / -FL9.8 TCR Tg mice. [Figure 53] 1 provides a graphical representation showing detection of Va3.2+Vb5+CD8 cells among Ly49+CD8 cells. Frequency of Vα3.2+Vβ5+CD8 cells among Ly49+ and Ly49-CD8 cells. [Figure 54-1]Figures 54A-B and 54C-D provide graphical representations showing that FL9 Tg CD8 T cells recognize and suppress activated CD4 T cells. Figure 54A provides graphical representations showing that in vitro: activated CD4 cells stimulate FL9 TCR in a Qa-1-dependent manner. In vitro: ConA-stimulated CD4 cells from WT B6, Qa-1.D227K KI, KbDb KO and ERAAP KO mice were co-cultured with FL9.2 T cells isolated from FL9.2 TCR Tg mice. After 20 hours, CD69 expression on FL9 Tg T cells was measured as a readout of TCR stimulation. Figure 54B provides graphical representations showing that in vivo: FL9 Tg T cells suppress activated CD4 T cells (selectivity of response). In vivo: WT or D227K mice were immunized with OT-II peptide in CFA. After 7 days, CD4 (CD4+CD25-) cells were isolated from immunized mice and transferred into WT B6 hosts with or without FL9 TCR Tg T cells, followed by immunization with OT-II / CFA. Detection of I-Ab / Ova323-339 CD4 T cells in the spleen of the host by I-Ab / Ova323-339 tetramer (top left). Percentage and number (center and right) of I-Ab / Ova323 b / Ova-339 tetramer+ (top right) and I-Ab / Ova323-339 tetramer-activated (bottom panel) CD4 cells recovered from the adoptive host (center and right). Figure 54C provides a graphical representation showing Va3.2 T cell depletion and its effect on Ag-specific CD4 cells. B6 WT and B6 D227K mice were immunized with Ova / CFA, then injected with isotype or anti-Vα3.2 Ab on day 0 and boosted with Ova / IFA along with Ab injection on day 8. Expression of Qa-1 on CD4 subsets and frequency of I-Ab / Ova323-339tet+CD4 cells in blood were assessed on day 15. Figure 54D provides a graphical representation showing Qa-1 expression by I-Ab / Ova323-339tet+ and tet-CD4 cells. Mean ± SEM is shown. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 54-2] See description of Figure 54-1. [Figure 55A]Figure 55A-B provides a graphical representation showing that FL9 Tg CD8 T cells recognize activated CD4 T cells. (A) ConA-stimulated CD4 cells from WT B6, Qa-1.D227K KI, KbDb KO and ERAAP KO mice were co-cultured with FL9.8 T cells isolated from spleens and LNs of FL9.8 TCR Tg mice. After 20 hours, CD69 expression on FL9 Tg T cells was measured as a readout of TCR stimulation. (B) ConA-stimulated CD4 cells from WT B6, D227K KI and Qa-1 KO mice were co-cultured with 58C hybridomas expressing OT-I or FL9.8 TCR. After 3 days, stimulation of OT-I TCR+ and FL9 TCR+ hybridomas by these activated CD4 cells was measured according to CD69 expression. (C) OT-I or FL9.8 TCR+ hybridomas were co-cultured with Con-A stimulated CD4 cells from WT or ERAAP KO mice. Stimulation of OT-I or FL9 TCR was measured according to CD69 expression by hybridoma cells. Mean ± SEM is shown. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 55B] See legend to Figure 55A. [Figure 55C] See legend to Figure 55A. [Figure 56] Figure 1 provides a graphical representation showing depletion of Vα3.2+ T cells. WT B6 and D227K mice were immunized with Ova / CFA along with injection of rat IgG2b or anti-Vα3.2 Ab on day 0. The mice were boosted with Ova / IFA on day 8 and also injected with Ab. On day 15, the presence of Vα3.2+Vb5+ cells was assessed in total T cells, CD8 T cells, and Ly49+CD8 T cells. [Figure 57] A graphical representation is provided showing the absence of in vivo stimulation of FL9.2 T cells by FL9 native peptide. FL9.2 T cells were adoptively transferred into CD45.1+B6 hosts and immunized intraperitoneally with FL9 in CFA or no peptide (CFA alone) on day 0. Activation (CD69) proliferation of FL9.2 T cells (Ki67) was measured 3 days later. [Figure 58-1]Figures 58A-C and 58D-E provide diagrams showing the identification of superagonists for FL9 T cells. Figure 58A provides a schematic showing a library composed of 96 FL9 peptide variants (crude peptides) generated by aa mutagenesis at Qa-1 fixed positions (p2, p3, p6, p7 and p9). FL9 TCR+ hybridomas were incubated with EL4 cells (Qa-1+) loaded with each FL9 peptide variant for 12 hours, and CD69 expression and TCR downregulation were measured as indicators of TCR stimulation. Figure 58B provides diagrams showing activation of FL9 TCR+58C hybridomas after stimulation with FL9 peptide variants. CD69 expression by FL9 TCR+58C hybridomas after stimulation with each FL9 peptide variant (left). TCR downregulation is shown as ΔTCR MFI based on the calculation of 100-(test TCR MFI / control TCR MFI) x 100(%) (middle). Expression of Vα3.2 and Vβ5 on EL4 cells (trogocytosis) was measured (right). Figure 58C provides a graphical representation showing activation of FL9.2 T cells after stimulation with FL9 variants selected from the library screening described above. Dose-dependent activation of FL9 T cells was measured by culturing FL9.2 T cells with EL4 (Qa-1+) at various concentrations of the indicated peptides (0, 1, 3, and 10 μg / mL). Figure 58D provides a graphical representation showing that FL9-68 vaccination activates FL9 T cells in vivo. CD45.1+ B6 hosts were adoptively transferred with FL9.2 T cells and immunized intraperitoneally with PBS, FL9, or FL9-68 in CFA on day 0. After 6 days, proliferation of FL9.2 T cells (CD45.2+Vα3.2+Vβ5+) was measured by CFSE dilution (left). CD45.1+B6 mice were immunized with Ova323-339 peptide in CFA on day 6 and the frequency of I-Ab / Ova323-339Tet+CD4 cells among activated (CD44+) CD4 cells was analyzed on day 14. Mean ± SEM is shown. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05.Figure 58E, CD45.1+ B6 mice vaccinated with FL9-68 in CFA or CFA alone on day 0 were immunized with Ova323-339 peptide in CFA on day 6. The frequency of I-Ab / Ova323-339Tet+ CD4 cells among activated (CD44+) CD4 cells in the spleen and dLN was analyzed on day 14. [Figure 58-2] See description of Figure 58-1. [Figure 59] Provides a graphical representation showing the identification of superagonists for FL9 T cells. Activation of FL9.8 T cells after stimulation with FL9 variants selected from the library screening described in Figure 58. Dose-dependent activation of FL9.8 T cells was measured by culturing FL9.8 T cells with EL4 (Qa-1+) at various concentrations of the indicated peptides (0, 1, 3 and 10 μg / mL). [Figure 60A]Figures 60A-F provide graphical representations showing that superagonist peptide vaccination inhibits AMR in heart transplants. Figure 60A provides graphical representations showing the expansion of FL9-specific CD8 Tregs after FL9-SA vaccination. B6 mice were vaccinated with FL9-68 peptide in IFA or IFA alone on day 0 and sensitized with Balb / C skin on day 7. Mice were further vaccinated with FL9-68 in IFA or IFA alone on days 10, 13, and 16. Balb / C hearts were heterotopically transplanted into the peritoneal cavity of B6 recipients. 250 μg of CTLA-4 Ig was administered intravenously after transplantation and recipients were analyzed on day 34. The frequency of Qa-1-FL9-Tet+ cells in the spleens of IFA or FL9-68 / IFA vaccinated B6 hosts is shown. Figure 60B provides graphical representations showing the inhibition of GC Ab responses by FL9-68 immunization. Number of Tfh, GC B and plasma cells in dLN of naive mice or IFA or FL9-68 / IFA vaccinated B6 recipients. Graphical representation showing that superagonist peptide vaccination inhibits AMR in heart transplantation. Figure 60C provides a graphical representation showing Qa-1 expression by Tfh cells after heart transplantation. Qa-1 expression by total, naive CD4 and Tfh cells in spleens of B6 recipients of Balb / C heart grafts. Figure 60D provides a graphical representation showing that FL9-68 immunization inhibits the production of DSA. Donor-specific Ab (IgG1) in naive mice or IFA-, OT-I / IFA-, FL9 / IFA or FL9-68 / IFA-vaccinated B6 recipients of heart grafts was measured in serum collected on day 16. Balb / C donor splenocytes were incubated with serially diluted serum and then detected with fluorescently labeled anti-mouse IgG1 Ab. Statistical analysis was performed using Two Way-Anova with mixed effects analysis. Figure 60E provides a diagram showing that FL9-68 vaccine inhibits graft tissue damage. C4d deposition in cardiac allografts. Tissue sections from cardiac grafts of B6 mice vaccinated with IFA alone or FL9-68 / IFA were stained with anti-C4d Ab. Figure 60F provides a graphical representation showing that FL9-68 vaccine promotes graft survival.Heart graft survival in mice vaccinated as indicated. Mean ± SEM is shown. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 60B] See legend to Figure 60A. [Figure 60C] See legend to Figure 60A. [Figure 60D] See legend to Figure 60A. [Figure 60E] See legend to Figure 60A. [Figure 60F] See legend to Figure 60A. [Figure 61] A graphical representation showing the responsiveness of recipient CD4 cells to allogeneic donor cells is provided. CD4 cells were isolated (day X) from B6 recipients transplanted with Balb / C hearts and vaccinated with PBS or FL9-68 peptide. CFSE-labeled CD4 cells were co-incubated with irradiated donor splenocytes for 7 days and proliferation levels were measured by CFSE dilution. [Figure 62] Primer sequences for amplification of TCR alpha and TCR beta sequences are shown. [Figure 63] Human TCRα-TRAV8.3 is shown as the Vα gene homologue (Vα3.2[TRAV9]) of mouse CD*Treg TCR. [Figure 64] 1 shows the optimized nucleotide sequence of anti-Ly49F. [Figure 65] The amino acid sequence of the anti-Ly49F variable region is shown. [Figure 66] The consensus motif of FL9 superagonist synthetic peptides is shown. [Figure 67] The KIR amino acid sequences are shown. [Figure 68] The KIR and LY49 amino acid sequences are shown. [Figure 69]Cancer and CD8 Treg depletion enabling tumor vaccination therapy. Illustrated is tumor growth in mice inoculated with NT MC38 cells and treated with vaccine, anti-Ly49F Ab or vaccine + anti-Ly49F Ab. Irradiated Cl.EZH2KOMC38 cells were used as cancer cell vaccine (day 10, 1x106 / mouse, subcutaneous). Anti-Ly49F Ab (mIgG2a) was injected intraperitoneally on days 10, 13 and 16. Anti-Ly49F Ab was engineered to express mIgG2a Fc region to enable deletion of Ly49F+ cells. Tumor growth was monitored (left). Tumor volumes for individual mice in each treatment group on day 26 are shown (right). [Figure 70] 1 shows cancer and CD8 Treg depletion.Tumor growth in B6 mice inoculated with MC38 cells and treated with anti-Ly49 or anti-Va3.2 antibodies. [Figure 71] CD8 Treg depletion reveals robust anti-tumor responses. WT B6 mice were inoculated with MC38 tumor cells and vaccinated with CpG-ODN on day 3. Mice were treated with anti-Ly49F Ab on days 8, 11, 14, and 17. Tumor growth curves in groups treated with isotype Ab (mIgG1), CpG, a-Ly49F Ab, or CpG+a-Ly49F Ab are shown. [Figure 72] 1 shows cancer and CD8 Treg depletion enabling tumor vaccination. [Figure 73] Figure 2 shows that CD8 Treg depletion reveals robust anti-tumor responses. [Figure 74] Figure 74A-B shows CD8 T cell profiles in tumors grown in mice treated with isotype or anti-Ly49F Ab. Shown are the percentage of CD8 T cells among CD45+ cells and the expression of GzmB in CD8 T cells (A) and the percentage of CD8 Tregs (CD44+CD122+Ly49+) among CD45+ cells (B). [Figure 75]Figure 75A-B shows NK and DC profiles within tumors grown in mice treated with isotype or anti-Ly49F Ab. (A) NK cell percentage among CD45+ cells and GzmB expression by NK cells within the tumor. (B) Percentage of MDSC among CD11b+ cells and cDC among CD11c+I-Ab+ cells. [Figure 76] Figure 76A-B shows tumor growth in B6 mice inoculated with B16 melanoma and treated with anti-Ly49F Ab (A). Number of cDCs and MDSCs in tumors treated with isotype or anti-Ly49F Ab (B). [Figure 77] In kidney transplantation, we show that mobilization of CD8 Tregs by synthetic peptide agonists prolongs kidney graft survival and that FL90SA (FL(-68) improves AMR and prolongs graft survival in kidney transplantation. The left kidney of a BALB / c mouse (H-2d) was harvested with its full-length ureter and transplanted into a B6 host (H-2b). The ureter of the remaining native kidney was then ligated on days 2–4 after surgery to inhibit native renal function. The success of the operation was determined when the mouse survived for 7 days after surgery (POD). The transplanted B6 host was treated with either FL9-SA (50 μg) or the adjuvant FL9-SA (50 μg) once a week starting from POD2. (Addavax™). Serum DSA levels and capillary C4d deposition were measured 20 days after kidney transplantation (n=5-7 / group). Kidney allograft survival was measured by survival of recipients without native renal function. (Left) Donor-specific Ab (IgG1) in control or FL9-68 / adj vaccinated B6 recipients of kidney transplants. (Middle) Immunohistochemistry for C4d deposition. (Right) Kidney allograft survival measured by survival of recipients without native renal function. [Figure 78] Schematic diagram showing CD8 Treg generation compared to conventional CD8 T cell generation (top), CD8 Treg cells targeting CD4 T cells (bottom left), and superagonist peptide immunization to mobilize / activate CD8 Treg cells. [Figure 79]Figure 79A-H shows an example of the identification of Qa-1-FL9-specific TCR. (A) WT B6 mice were immunized with FL9 peptide-loaded Kb- / -Db- / -DCs on days 0, 8, and 15. On day 22, Qa-1-FL9-specific CD8 T cells were detected by tetramers (Qa-1-FL9-PE and Qa-1-FL9-APC) in CD44+CD122+Ly49+CD8 T cells. (B) TCR repertoire of Qa-1-FL9 Tet+CD8 T cells. Single Qa-1-FL9-PE+Qa-1-FL9-APC+ cells were sorted and subjected to sequencing for TCRa and TCRb. Thirty-nine TCRa and TCRb pairs were analyzed based on their TCR V gene segments. The relative usage of TCRa and TCRb V genes by these Tet+ single cells is shown in a donut chart. (C) TCR repertoire of Qa-1-Hsp60 Tet+CD8 T cells. Single Qa-1-Hsp60-PE+Qa-1-Hsp60-APC+ cells were sorted and sequenced for TCRa and TCRb. Relative usage of TCRa and TCRb V genes by these Tet+ single cells is shown in a donut chart. (D) Frequency and phenotype of Va3.2+Vb5+ cells within Ly49+CD8 cells in spleens and LNs of 8-week-old WT B6, Qa-1.D227K KI and Qa-1 KO mice (n=6 / group). (E) Qa-1-dependent differentiation of FL9 T cells: tetramer-mediated detection of TCR in 58C hybridomas transduced with FL9.2 and FL9.8 TCRs (upper panel). Responsiveness of FL9.2 TCR and FL9.8 TCR expressing hybridomas upon stimulation with increasing doses of peptide as measured by CD69 expression (lower panel). (F) Measurement of Qa-1-FL9 binding affinity of FL9.2 and FL9.8 TCR. FL9.2 TCR+ and FL9.8 TCR+ hybridomas were labeled with Qa-1-FL9-PE tetramers and incubated in the presence of anti-Qa-1 Ab for the indicated times. The percentage of PE+ cells was measured at various time points as a measure of the level of tetramer dissociation. (G) Percentage of Tg TCR+ cells among TCR+ thymocytes and active caspase-3+PD1+ cells among DP (CD4+CD8+) thymocytes in OT-I→WT B6 and FL9.2 Tg→WT B6 BM chimeras 8 weeks after BM reconstitution.(H) Ki67 and CD44 expression by OT-I and FL9.2 TCR Tg CD8+ T cells was measured as an indicator of Ag encounter in the spleen and liver of OT-I→WT B6 and FL9.2 Tg→WT B6 BM chimeras 8 weeks after BM reconstitution. [Figure 80]Figure 80A-H shows exemplary Qa-1-dependent differentiation of FL9 T cells. (A) Frequency of Tg TCR+ cells (Va2+Vb5+ for OT-I and Va3.2+Vb5+ for FL9 T cells) and Helios expression levels in TCR+ thymocytes in OT-I or FL9.2 TCR Tg mice. (B) Frequency of Tg TCR+ cells and Helios and Ly49 expression levels in TCR+ splenocytes in OT-I or FL9 TCR Tg mice. (C) Frequency of FL9 TCR Tg T cells in TCR+ thymocytes in WT or Qa-1- / -FL9 TCR Tg mice. The frequency of FL9 Tg T cells (Va3.2+Vb5+) in total thymocytes is shown in the graph (right). (D) Frequency of Va3.2+Vb5+ T cells in TCRb+ spleen cells from FL9.2 TCR Tg mice on Qa-1 WT and KO background (8 weeks old). Representative FACS plots for detection of Va3.2+Vb5+ cells in spleen are shown (left panel) (n=4 / group). (E) Expression of CD44 and NKG2D by FL9.2 T cells in spleen of WT.FL9.2 TCR Tg and Qa-1- / -FL9.2 TCR Tg mice. Representative FACS plots for NKG2D+CD44+ cells in spleen of FL9.2 TCR Tg mice are shown on the left. (F) Expression of Ki67 by FL9.2 T cells in LN of WT.FL9.2 TCR Tg and Qa-1- / -FL9.2 TCR Tg mice. (G) CFSE-labeled FL9.2 T cells generated in Qa-1 WT or Qa-1 KO mice were transferred into irradiated (800 rad) Qa-1 WT, Qa-1 KO, and D227K KI adoptive hosts. Seven days after transfer, Qa-1 WT or Qa-1 KO FL9.2 T cells were harvested from the spleens of the adoptive hosts. The number of FL9.2 T cells in the spleens of the adoptive hosts is shown. (H) The percentage of Qa-1 WT FL9.2 T cells undergoing more than three divisions in Qa-1 WT, Qa-1 KO, and D227K KI hosts. Mean ± SEM is shown. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 81]Figure 81A-D shows examples of FL9 Tg CD8 T cell recognition and suppression of activated CD4 T cells. (A) In vitro: ConA-stimulated CD4 cells from WT B6, Qa-1.D227K KI, KbDb KO and ERAAP KO mice were co-cultured with FL9.2 T cells isolated from FL9.2 TCR Tg mice. After 20 hours, CD69 expression on FL9 Tg T cells was measured as a readout of TCR stimulation. (B) In vivo: WT or D227K mice were immunized with OT-II peptide in CFA. Seven days later, CD4 (CD4+CD25-) cells were isolated from immunized mice and transferred into WT B6 hosts with or without FL9 TCR Tg T cells followed by immunization with OT-II / CFA. Detection of I-Ab / Ova323-339 CD4 T cells in host spleens by I-Ab / Ova323-339 tetramer (top left). Percentage and number (center and right) of I-Ab / Ova323-339 tetramer+ (top right) and I-Ab / Ova323-339 tetramer-activated (bottom panels) CD4 cells recovered from adoptive hosts (center and right). (C) WT B6 and D227K mice were immunized with Ova / CFA and injected with isotype or anti-Vα3.2 Ab on day 0, followed by a boost with Ova / IFA along with Ab injection on day 8. Expression of Qa-1 on CD4 subsets and frequency of I-Ab / Ova323-339 tet+ CD4 cells in blood were assessed on day 15. (D) Qa-1 expression by I-Ab / Ova323-339tet+ and tet-CD4 cells. Mean ± SEM is shown. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 82A]Figure 82A-F shows an example of the identification of superagonists for FL9 T cells. (A) A library consisting of 96 FL9 peptide variants (crude peptides) was generated by aa mutagenesis at Qa-1 fixed positions (p2, p3, p6, p7 and p9). FL9 TCR+ hybridomas were incubated with EL4 cells (Qa-1+) loaded with each FL9 peptide variant for 12 hours, and CD69 expression and TCR downregulation were measured as indicators of TCR stimulation. (B) Activation of FL9 TCR+58C hybridomas after stimulation with FL9 peptide variants. CD69 expression by FL9 TCR+58C hybridomas after stimulation with each FL9 peptide variant (left). TCR downregulation is shown as ΔTCR MFI based on the calculation of 100-(test TCR MFI / control TCR MFI) x 100 (%) (middle). Expression of Va3.2 and Vb5 on EL4 cells (trogocytosis) was measured (right). (C) Activation of FL9.2 T cells after stimulation with FL9 variants selected from the library screen described above. Dose-dependent activation of FL9 T cells was measured by culturing FL9.2 T cells with EL4 (Qa-1+) at various concentrations of the indicated peptides (0, 1, 3 and 10 mg / mL). (D) FL9.2 T cells were adoptively transferred into CD45.1+ B6 hosts and immunized intraperitoneally with PBS, FL9, or FL9-68 in CFA on day 0. Six days later, proliferation of FL9.2 T cells (CD45.2+Va3.2+Vb5+) was measured by CFSE dilution (left). (E) CD45.1+ B6 mice vaccinated with FL9-68 in CFA or CFA alone on day 0 were immunized with Ova323-339 peptide in CFA on day 6. The frequency of I-Ab / Ova323-339 Tet+CD4 cells among activated (CD44+) CD4 cells was analyzed on day 14. (F) Comparison of high affinity Ab and auto-Ab responses in WT and Qa-1.D227K mice. On day 10, WT B6 and Qa-1.D227K.KI mice were immunized with NP23-KLH / CFA and boosted with NP23-KLH / IFA. High affinity anti-NP responses were measured on day 15. Levels of anti-dsDNA Ab were measured on day 21. Mean ± SEM is shown.****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Fig. 82B] See legend to Figure 82A. [Fig. 82C] See legend to Figure 82A. [Fig. 82D] See legend to Figure 82A. [Fig. 82E] See legend to Figure 82A. [Fig. 82F] See legend to Figure 82A. [Figure 83]Figure 83A-F shows an example of superagonist peptide vaccination inhibiting AMR in heart transplantation. (A) B6 mice were vaccinated with FL9-68 peptide in IFA or IFA alone on days 0 and 7, followed by Balb / C skin sensitization on day 10. Mice were further vaccinated with FL9-68 in IFA or IFA alone on days 10, 13, and 16. On day 27, Balb / C hearts were heterotopically transplanted into the peritoneal cavity of B6 recipients. 250 μg of CTLA-4 Ig was administered intravenously after transplantation and recipients were analyzed on day 34. The frequency of Qa-1-FL9-Tet+ cells in the spleens of IFA- or FL9-68 / IFA-vaccinated B6 hosts is shown. (B) Numbers of Tfh, GC B, and plasma cells in the dLNs of naive B6 mice or IFA- or FL9-68 / IFA-vaccinated B6 recipients. (C) Qa-1 expression by total naive CD4 and Tfh cells in the spleens of B6 recipients of Balb / C heart grafts. (D) Donor-specific Ab (IgG1) in naive mice or IFA-, OT-I / IFA-, FL9 / IFA-, or FL9-68 / IFA-vaccinated B6 recipients of skin grafts was measured in serum collected on day 26, the day before heart transplantation. Balb / C donor splenocytes were incubated with serially diluted serum and then detected with fluorescently labeled anti-mouse IgG1 Ab. Statistical analysis was performed using Two Way-Anova with mixed effects analysis. (E) C4d deposition (blue) in cardiac allografts (upper panel). Tissue sections from cardiac grafts of B6 mice vaccinated with IFA alone or FL9-68 / IFA were stained with anti-C4d Ab. H&E staining of cardiac allografts showing graft-infiltrating lymphocytes (lower panel). (F) Heart graft survival in mice vaccinated with FL9-68 peptide as indicated. Mean ± SEM is shown. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Fig. 84A]Figure 84A-H shows an example of FL9-SA (FL9-68) ameliorating AMR and prolonging graft survival in kidney transplantation. (A) Schematic of the experimental design. (B) Frequency of FL9-Qa-1 specific CD8 (CD44+CD122+Ly49+) T cells in mice with or without FL9-68 immunization. (C) Frequency of Tfh, activated B cells and plasma cells in graft recipients with or without FL9-68 immunization. (D) Donor-specific Ab (IgG1) in control or FL9-68 / adj vaccinated B6 recipients of kidney grafts. (E) Proliferation of activated CD4+ T cells from control or FL9-SA immunized recipients when co-cultured with irradiated donor (BALB / c) splenocytes. (F) Gross anatomy of kidney allografts at day 20. (G) Immunohistochemistry for C4d deposition (blue). (H) Kidney allograft survival as measured by survival of recipients without native renal function. ***P<0.001, **P<0.01, *P<0.05. [Fig. 84B] See legend to Figure 84A. [Fig. 84C] See legend to Figure 84A. [Fig. 84D] See legend to Figure 84A. [Fig. 84E] See legend to Figure 84A. [Fig. 84F] See legend to Figure 84A. [Fig. 84G] See legend to Figure 84A. [Fig. 84H] See legend to Figure 84A. [Figure 85]Figure 85A-B shows an example of TCR repertoire of Qa-1-FL9 Tet+CD8 T cells. Single Qa-1-FL9-PE+Qa-1-FL9-APC+ cells were sorted and subjected to sequencing for TCRa and TCRb. TCRa and TCRb pairs were analyzed based on their TCR V gene segments. Alignment of TCRaA and TCRbB sequences obtained from Qa-1-FL9 Tet+ single cells. TCR affinity for Qa-1-FL9 complex and TCRαV and Vβ gene usage by each Qa-1-FL9 Tet+CD8 T cell are shown on the right. CD8 T cell clones expressing TRAV9N3 (Va3.2) and / or TRBV12.1 / 2 (Vb5.1 / 2) are highlighted. [Figure 86] Figure 86A-B shows an example of the TCR repertoire of Qa-1-Hsp60 Tet+CD8 T cells. Single Qa-1-Hsp60-PE+Qa-1-Hsp60-APC+ cells were sorted and subjected to sequencing for TCRα and TCRβ, and the TCRa and TCRb pairs were analyzed based on their TCR V gene segments. Alignment of TCRaA and TCRbB sequences obtained from Qa-1-Hsp60 Tet+ single cells. TCRαV and Vβ gene usage by each Qa-1-Hsp 60 Tet+CD8 T cell is shown on the right. CD8 T cell clones expressing TRAV9N3 (Va3.2) and / or TRBV12.1 / 2 (Vb5.1 / 2) are highlighted. [Figure 87] Figure 87A-B shows an example of detection of Va3.2+Vb5+CD8 cells in Ly49+CD8 cells. (A) Gating strategy for Vα3.2+Vb5+CD8 cell detection. Frequency of Va3.2+Vb5+CD8 cells in Ly49+ and Ly49-CD8 cells. (B) Frequency of Va3.2+ or Vb5+ cells in Ly49+CD8 cells in spleens and LNs of 8-week-old WT B6, Qa-1.D227K KI and Qa-1 KO mice (n=6 / group). [Figure 88]Figure 88A-C shows an example of Qa-1-dependent phenotype acquisition by FL9.8 Tg T cells. (A) Frequency of Va3.2+Vb5+ T cells among TCRb+ cells from FL9.8 TCR Tg mice in Qa-1 WT and KO background. A representative FACS plot for detection of Va3.2+Vb5+ cells in the spleen is shown on the left. (B) Expression of CD44 and NKG2D by FL9.8 TCR Tg T cells in the spleen and LN of Qa-1 WT and Qa-1 KO mice. A representative FACS plot for NKG2D+CD44+ cells in the spleen of FL9.8 TCR Tg mice is shown on the left. (C) Expression of Ki67 by FL9.8 T cells in the LN of 8-week-old WT.FL9.8 TCR Tg and Qa-1- / -FL9.8 TCR Tg mice. [Figure 89]Figures 89A-D show an example of CD8 Treg depletion enhancing antitumor responses against MC38 cancer. (A) Growth curves of MC38 tumors in groups of B6 mice treated with ether Ig isotype control or α-Ly49F monoclonal Ab (see Methods) alone or in combination with vaccination with irradiated MC38 tumor cells prepared as described in Methods. B6 mice were inoculated subcutaneously with MC38 tumor cells (2x105 / mouse) on day 0 and treated with α-Ly49F or isotype control (30mg / mouse) alone on days 8, 13 and 16 or in combination with vaccination with irradiated MC38 cells on day 6. (B) Immune cell profile in tumors from mice treated with isotype or α-Ly49F Abs on day 29 after tumor cell inoculation. The percentages of Ly49+CD8 cells among CD122+CD44+CD8+ T cells, CD8 T cells among CD45+ cells, and GzmB+CD8 T and NK cells, and the percentage of CD11+Gr1+ (MDSC) cells among CD45+ cells are shown. (C) WT B6 mice were inoculated (subcutaneously) with MC38 cells (2×105 / mouse) and vaccinated with irradiated (2000 rad) MC38 cells on day 6, followed by treatment with isotype control or α-Vα3.2 Ab (80 mg / mouse) on days 10, 13, and 16. (D) MC38 growth in B6 mice after inoculation with MC38 cells (2×105 / mouse) on day 0 and injection of CpG-ODN (50 mg; subcutaneously) on day 3, prior to treatment alone or with α-Ly49F Ab (30 mg / mouse) on days 8, 11, 14, and 17. Each data point represents 5 mice / group, ***P<0.001, **P<0.01, *P<0.05. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 90]An example of CD8 Treg deletion by α-Ly49F Ab is shown. B6 mice were injected intraperitoneally with isotype (mIgG2a) or α-Ly49F Ab (clone HBF-719, 30 μg / mouse). 48 hours after Ab treatment, the frequency of Ly49+CD8 T cells in blood, LN and spleen was measured by staining cells for TCR, CD8, CD44 and Ly49C / I / F / H (Ab clone: ​​14B11) expression. FACS plots are shown after gating on TCR+CD8+ cells. Representative FACS plots and a summary of Ly49F+CD8 T cell frequency within CD8 T cells are shown. [Figure 91] Figure 91A-C illustrates an example of anti-Ly49F Ab inhibiting the growth of B16F10 melanoma cells and promoting the early migration of cDC1 in the TME. B6 mice were subcutaneously inoculated with 2x105 B16F10 tumor cells on day 0 and injected with either α-Ly49F Ab or isotype control (100μg / mouse) on days 6, 9 and 12. After monitoring tumor growth (A), mice were euthanized on day 17 and the percentage of cDC1 (CD11c+, XCR1+, CD103+) and the percentage of KbDb+ expression in cDC (B) and MDSC (CD11b+, Gr1+) cells (C) were determined. 5 mice / group. ***P<0.001, *P<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Detailed Description of the Invention Regulatory T (Treg) cells can function to control immune responses. One type of Treg cell, CD8+ Treg cell, or CD8 Treg cell (CD44+CD122+Ly49+ in mice; CD44+CD122+KIR+ in humans), can suppress (MHC class Ib molecule) CD4+ T cells in a Qa-1 (mouse) or HLA-E (human) restricted manner. This suppression of CD4+ T cells can be antigen specific, as CD4+ cells recognize CD8+ Treg cells through the T cell receptor (TCR) in the context of Qa-1 / HLA-E. Decreased CD8+ Treg activity can contribute to autoimmune and inflammatory diseases. Decreased CD8+ Treg activity can also contribute to antibody-mediated rejection (AMR) of allografts. Antibody-mediated rejection (AMR) can be a barrier to successful solid organ transplantation. Increased CD8+ Treg activity can suppress these conditions. A decrease in CD8+ Treg activity can increase tumor surveillance by the immune system.

[0014] Herein, we have developed an approach to modulate the activity of CD8+ Treg cells. In some embodiments, CD8+ Treg activity can be increased. In some embodiments, we have developed superagonist peptides that can be used to mobilize / activate CD8+ Tregs. In some embodiments, antibodies can also do this. In some embodiments, such mobilization of CD8 Tregs can be used to suppress CD4+ T cells. In some embodiments, this can be used to suppress antibody-mediated rejection (AMR) of transplanted organs and other immune-mediated responses (e.g., autoimmunity).

[0015] Efficient targeting of Qa-1-FL9 (HLA-EFL9) on CD4+ T cells by CD8 Tregs following expansion of Treg cells by peptide agonists is applicable to ameliorate multiple immune responses characterized by pathogenic antibodies in the context of autoimmune diseases, organ transplantation, and infection. Moreover, the recruitment of CD8 Tregs to regulate Ab-dependent immune responses is advantageous over general immunosuppression that may leave the host immunocompromised.

[0016] In some embodiments, CD8+ Treg activity can be reduced. In some embodiments, this can be done using antibodies. In some embodiments, suppression / killing of CD8+ Tregs using antibodies can increase activity or reduce suppression of CD4+ T cells. In some embodiments, this can be used to increase immune responses, including tumor surveillance and anti-tumor activity in mammals.

[0017] Detailed description of one or more embodiments is provided herein. However, it is understood that the present invention can be embodied in various forms. Therefore, the specific details disclosed herein should not be interpreted as limiting, but as a basis for claims and as a representative basis for teaching a person skilled in the art to use the present invention in any suitable manner.

[0018] The singular forms "a," "an," and "the" include plural references unless the context dictates otherwise. The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0019] Whenever any of the phrases "for example," "such as," "including," and the like are used herein, unless expressly stated otherwise, it is understood that the phrase "without limitation" is accompanying. Similarly, "an example," "exemplary," and the like are understood to be non-limiting.

[0020] The term "substantially" permits deviations from the descriptors that do not adversely affect the intended purpose. It is understood that a descriptor is modified by the term "substantially" even if the word "substantially" is not expressly recited.

[0021] Terms such as "comprising," "including," "having," and "involving" (and similarly "comprises," "includes," "has," and "involves") are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the general U.S. patent law definition of "comprising," and therefore is to be construed as an open term meaning "at least the following," and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the terms "a" or "an" are used, they are to be understood as "one or more," unless such an interpretation is insignificant in the context.

[0022] The term "about" as used herein can refer to approximately, roughly, approximately, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. For example, the term "about" is used herein to modify a numerical value by a variance (high or low) of 20 percent above and below the stated value.

[0023] CD8 Treg cells CD8 Tregs regulate immune responses to pathogens and self-antigens by eliminating chronically activated CD4 cells that upregulate Qa-1 / HLA-E on the CD4 cell surface. Recognition of Qa-1 self-peptides on target cells by CD8 Tregs can suppress pathogenic CD4 cells, but the proliferation and recruitment of CD8 Tregs is constrained by molecular mechanisms that constrain excessive or inappropriate CD8 Treg activation. Herein, a new strategy is disclosed that allows both antigen-specific and antigen-nonspecific therapeutic recruitment of CD8 Tregs in the context of transplant rejection, autoimmune diseases and cancer.

[0024] Mouse and human CD8 regulatory activity constitutes a small (<5%) subset of total CD8 T cells that express a characteristic triad of surface receptors: CD44, CD122, and Ly49 (mouse) / KIR (human). Analysis of autoimmune disorders has demonstrated that these CD8 regulatory T cells (CD8 Tregs) are involved in the regulation of CD4 + It has been shown that disease can be suppressed through targeting MHC class Ia or class Ib expressed by T helper cells.

[0025] Generally, CD8 Tregs can express CD8, Ly49F, CD44 and CD122 (i.e. in mice) or CD8, iKIR, CD44 and CD122 (i.e. in humans). Ly49F is a subtype of the Ly49 receptor family. The Ly49 receptor is a type II C-type lectin-like membrane glycoprotein. KIR receptors are expressed by human cells and functional homologs of the Ly49 receptor are expressed in mice.

[0026] Recognition of MHC-E (human HLA-E or mouse Qa-1)-peptide complexes expressed by target CD4 cells is required for regulatory activity, but the identity of the TCR that recognizes class Ib (Qa-1) target ligands and associated self-peptides is unknown. Disclosed herein is such a TCR.

[0027] Analysis of a panel of more than 30 independent TCRs expressed by Qa-1-restricted CD8 T cells specific for two structurally distinct self-peptides (FL9: FYAEATPML, Hsp60p216: GMKFDRGYI) revealed predominant usage of the TRAV9N3 and TRBV12-1 / 2 genes encoding the TCR Va3.2 / Vb5.1. + Va3.2 / Vb5.1 + The development and function of CD8 Tregs was almost completely suppressed in Qa-1-deficient mice, indicating that the Qa-1-restricted subset of CD8 Tregs is restricted to CD8 cells expressing the Va3.2 / Vb5.1 TCR.

[0028] In some embodiments, the TCRs are depicted in Figures 27-28 (eg, specific for a FL9 peptide) and Figures 29-30 (eg, specific for an Hsp60 peptide) herein. In some embodiments, the CD8 Treg TCR alpha CDR1 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to YFGTPYY, the CD8 Treg TCR alpha CDR2 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to YYPGDPVV, and the CD8 Treg TCR alpha CDR3 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to AVSIWATSSGQKLV;AVTRYGSSGNKLI;AVRANYAQGLT;AVRGQGRALI;AVKDSGYNKLT;AVSSNNAGAKLT;AVRANTGKLT;AVKGGNYKPT; or AVKSTGSKLS; The TCRβ CDR1 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to NSQYPW; SGHSN; or SGHLS, the CD8 Treg TCRβ CDR2 sequence can be at least 90% identical to LRSPGDK; HYEKVER; or HYDKMER, or the CD8 Treg TCRβ CDR3 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to TCSARQGSGNTLY; ASSRRPASAETLY; ASSPRLGSAETLY; ASSHRSFSGNTLY; ASSLTGAYEQY; ASSLAGREQY; ASSGPSQNTLY; ASSLLGGPSAETLY; or ASSPRLGSAETLY.

[0029] In some embodiments, the CD8 Treg TCR alpha CDR 1 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to ATSIAYPN or YFGTPL, the CD8 Treg TCR alpha CDR 2 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to KVITAGQ or KYYPGDPV, and the CD8 Treg TCR alpha CDR 3 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to ALGEASSGSWQL, AVSSNYNVL; AVSRANTGKL; AVSKDSGYNKL; or AVSKSTGSKL, and the CD8 Treg The TCRβ CDR1 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to TNNHN, ISGHL; or LSGHS, the CD8 Treg TCRβ CDR2 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SYGAGS; HYDKME; or HYEKVE, or the CD8 Treg TCRβ CDR3 sequence can be at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to CASGTGDERL, CASSLVSGSAEQ; CASSLAGREQ; CASSLGQGNYAEQ; or CASSRANYEQ.

[0030] The above list of TCRs is not meant to be limiting. In some embodiments, the CD8 Treg TCR can bind to a self-peptide. The TCR can bind to a self-peptide in the context of an MHC Ib molecule. The MHC Ib molecule can be Qa-1 or HLA-E. The MHC Ib molecule can be expressed on a CD4 T cell. Generally, the MHC Ib molecule is present on a CD4 T cell.

[0031] In some embodiments, the TCR or CDRs derived therefrom can be engineered to be expressed on or in a variety of cells, including cell lines or primary cells, as described above. In some embodiments, the TCR / CDRs can be expressed on hybridoma cells or chimeric antigen receptor T cells (CAR-T cells). In some embodiments, the TCR / CDRs can be expressed in a variety of transgenic animals. In some embodiments, the TCR / CDRs can be expressed in transgenic mice. The cells and transgenic animals are part of the disclosed invention.

[0032] Other characteristics of CD8 Treg cells disclosed herein can be seen, for example, in FIGS.

[0033] In some embodiments, the TCRs disclosed herein can be made to be expressed on a variety of cells or transgenic animals. In some embodiments, hybridomas can be engineered to express the TCRs. In some embodiments, transgenic animals (e.g., mice) can be engineered to express the TCRs.

[0034] CD8 Treg agonists As used herein, an agonist of CD8 Treg cells or a CD8 Treg stimulator can recruit or activate these cells. In some embodiments, the Treg stimulator can be a peptide or polypeptide. In some embodiments, the Treg stimulator can be an antibody.

[0035] Peptide agonists can be of various types and have various amino acid sequences. In some embodiments, the disclosed peptide agonists are self-peptides or are derived from self-peptides. Self-peptides are generally capable of binding to molecules expressed on CD8 Treg cells.

[0036] In some embodiments, FL9 and Hsp60 peptides that stimulate CD8 Treg cells have been identified. In some embodiments, superagonist (SA) variants of these self-peptides have been engineered to express potent CD8+ Treg cell stimulatory activity in the context of Qa-1b (or HLA-E). Vaccination with superagonist peptides can efficiently mobilize CD8 Tregs and suppress antibody-mediated allograft rejection, autoimmune diseases, and the like.

[0037] Disclosed herein is an approach based on the application of superagonist self-peptides that can efficiently expand CD8 Tregs, reduce germinal center (GC) responses, and suppress antibody responses, which can be used to mobilize CD8 Tregs and reduce Ab-mediated damage to allogeneic organ transplants.

[0038] Based on previous mass spectrometry studies, we selected two SPs (superagonist peptides), FL9 and Hsp60p216, that associate with Qa-1 under immunological stress conditions. We then selected FL9-tetramer-binding CD8 Tregs, sequenced the TCRs of CD8 Tregs, and expressed them on hybridomas. This is our hybridoma system. We also generated a library of modified FL9 sequences and compared the antigenicity of FL9 sequences using TCR-engineered hybridomas. After selecting FL9-SA (FL9-superagonist peptide), we performed skin transplants from BALB / c to B6 with or without Hsp60p216 and FL9-SA, followed by heart transplants.

[0039] We successfully produced FL9-SA using our TCR-modified hybridoma system. Immunization with SP (superagonist peptide) significantly increased SP-Qa-1 tetramer-binding CD8 Tregs. Compared with the control group, hosts treated with SP during sensitization showed a significant reduction in mature B cells, including Tfh (follicular helper T cells) and plasma cells. FL9-SA was more effective than Hsp60p216. Donor-specific antibodies (DSA) were also significantly reduced in the SP-treated group, protecting the cardiac allograft.

[0040] Induction of CD8 Treg responses by Qa-1-related SPs suppressed germinal center reactions and DSA formation. In particular, the superagonist we created showed good biological efficacy in mobilizing CD8 Tregs. Exploiting the mechanisms of CD8 Tregs through the study of Qa-1-related peptides is a new strategy to suppress AMR, which lacks effective therapeutic options.

[0041] In some embodiments, the agonist / superagonist peptide / polypeptide can bind to the TCR on a CD8 Treg cell. Generally, the peptide / polypeptide can bind to the TCR in the context of an MHC Ib molecule, such as Qa-1 and / or HLA-E. The MHC Ib molecule can be on a cell, such as a CD4 T cell. Generally, the peptide is derived from a protein that is a "self" protein (e.g., from a mouse, from a human). Generally, the interaction of CD8 Treg cells with CD4 T cells involves multiple molecular reactions, some of which are shown, for example, in Figures 1A-B, 6, 14, 36 and 78.

[0042] In some embodiments, the agonist / superagonist peptide / polypeptide can comprise the amino acid sequence FSNEATLML;WYADVTPAL; or an amino acid sequence 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0043] In some embodiments, the agonist / superagonist peptide / polypeptide can comprise the amino acid sequence FYAEATLML (FL9-68) or an amino acid sequence that is 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0044] In some embodiments, the agonist / superagonist peptide / polypeptide may comprise the amino acid sequence IMLDTEIRL (BO-1); FMNDALLFL (BO-2); FMEEYMPFL (BO-3); FMEDAGPRL (BO-5); WMSEDHTLL (BO-6); VMQDEKSRL (BO-9); ISSEDGVPL (BO-10); or an amino acid sequence 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0045] In some embodiments, the agonist / superagonist peptide / polypeptide can comprise the amino acid sequence FISDSFFFL (Endo9); FYAEGTTL (MTb); or an amino acid sequence 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0046] In some embodiments, the agonist / superagonist peptide / polypeptide can comprise the amino acid sequence FYAEATPML(FL9) or 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0047] Generally, these amino acid sequences are capable of stimulating CD8 Tregs and / or suppressing CD4 T cells in a mammal.

[0048] In some embodiments, the peptide / polypeptide may be attached / conjugated to, for example, a lipophilic albumin binding tail conjugate.

[0049] In some embodiments, the Treg stimulator can be an antibody. These and other antibodies are described in the following paragraphs.

[0050] Antibodies against CD8 Tregs Disclosed herein are antibodies specific for and binding to CD8 Treg cells and molecules expressed by CD8 Treg cells.

[0051] As used herein, "antibody" can refer to a molecule(s) that binds to an antigen. As used herein, "antibody" can refer to all types of antibodies, fragments, and / or derivatives. Antibodies include polyclonal and monoclonal antibodies of any appropriate isotype or isotype subclass. As used herein, antibodies can refer to, but are not limited to, Fab, F(ab')2, Fab' single chain antibodies, Fv, single chain, monospecific antibodies, bispecific antibodies, trispecific antibodies, multivalent antibodies, chimeric antibodies, dog-human chimeric antibodies, chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, shark antibodies, nanobodies (e.g., antibodies consisting of a single monomeric variable domain), camelid antibodies (e.g., camelid) microbodies, intrabodies (e.g., intracellular antibodies), and / or defucosylated antibodies and / or derivatives thereof. Antibody mimetics are also provided. In embodiments, the antibodies can have a heavy chain constant region, a light chain constant region, an Fc region / site, or a combination thereof. In embodiments, the antibody can be fully human, humanized, or chimeric. The antibody or fragment can be monoclonal. In some embodiments, the antibody can be used in a CAR-T construct.

[0052] In some embodiments, the antibody can bear a therapeutic moiety (e.g., a toxin), an imaging moiety (e.g., a fluorophore, a chromophore, or a combination thereof), a capture moiety (e.g., a GST tag, a His-tag, or a combination thereof), or a combination thereof.

[0053] An antibody may or may not have an Fc site capable of binding to an Fc receptor (FcR). The FcR may be present on an effector cell, including a natural killer (NK) cell or a macrophage. In some embodiments, the Fc site of the antibody may bind to an FcR comprising an Fc-gamma receptor (FcγR), an Fc-alpha receptor (FcαR), or an Fc-epsilon receptor (FcεR). The FcγR may comprise at least FcγRI, FcγRII, or FcγRIII. In some embodiments, the Fc site of the antibody may be modified to better bind to an FcR as compared to an unmodified Fc site.

[0054] The antibodies disclosed herein generally can have the effect of stimulating or recruiting CD8 Tregs (e.g., perhaps in a similar manner to the agonist / superagonist peptides described above). Other antibodies disclosed herein can have the effect of suppressing or depleting CD8 Treg cells. In some embodiments, antibodies that suppress CD8 Treg cells kill or mediate cell killing. In some embodiments, antibodies that suppress / mediate cell killing of CD8 Treg cells can bind to effector cells (e.g., NK cells, macrophages) such that the effector cells mediate cell suppression / death. In some embodiments, antibodies that bind to molecules (e.g., Ly49, iKIR, TCR on CD8 Tregs) can be screened for a functional effect of binding, such as, for example, recruiting CD8 Treg cells or depleting CD8 Treg cells.

[0055] In some embodiments, the antibodies are specific for binding to molecules expressed by CD8 Treg cells that identify CD8 Treg cells. In some embodiments, the antibodies can be specific for Ly49 (mouse) and / or iKIR (human). In some embodiments, the antibodies can be specific for TCRs expressed on specific CD8 Treg cells. In some embodiments, the antibodies can identify a combination of molecules expressed by CD8 Treg cells (e.g., two or all of LY49 / iKIR, CD8, TCR). In some embodiments, these antibodies can be multispecific antibodies, such as bispecific antibodies or trispecific antibodies. In some embodiments, the bispecific antibodies can bind to iKIR (and / or Ly49) and CD8, iKIR (and / or Ly49) and CD8 Treg cell TCR; CD8 and CD8 Treg cell TCR; or iKIR (and / or Ly49), CD8 and CD8 Treg cell TCR.

[0056] Generally, the TCR on a CD8 Treg cell to which the disclosed antibodies can bind is a TCR that can bind a self-peptide. Generally, the peptide is bound by the TCR in the context of an MHC molecule that can bind the self-peptide. In some embodiments, these MHC molecules can be MHC Ib molecules, such as Qa-1 or HLA-E. Generally, the TCR can bind any self-peptide. Some examples of self-peptides can include FL9, amino acid sequence modified FL9, Hsp60p216, amino acid sequence modified Hsp60p216, and the like (discussed in the paragraph on CD8 Treg agonists). In various embodiments, the TCR can bind any of the peptides described in the previous section entitled "CD8 Treg agonists."

[0057] In embodiments, the antibody that binds to the TCR may bind to the alpha or beta chain of the TCR. In embodiments, the antibody may bind to a CDR of the TCR. In embodiments, the antibody may bind to CDR1, CDR2, or CDR3 of the alpha or beta chain of the TCR. In embodiments, the CDR may be any of the CDRs shown in Figures 27, 28, 29, or 30. In embodiments, the CDR may be any of the CDRs described in the section of this application entitled "CD8 Treg Cells."

[0058] With respect to the exemplary antibody properties disclosed herein, "recombinant" with respect to a polypeptide (such as an antibody) or polynucleotide refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together. As used herein, "polypeptide" can encompass a single "polypeptide" as well as multiple "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to a chain of two or more amino acids can refer to "polypeptides" herein, and the term "polypeptide" can be used in place of or interchangeably with any of these terms. "Polypeptide" can also refer to post-expression modified products of a polypeptide, such as, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but does not necessarily have to be translated from a specific nucleic acid sequence. It can be generated in any manner, including chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, delete, or replace a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as "conservatively modified variants." In some embodiments, the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.Such conservatively modified variants of the antibodies disclosed herein can exhibit increased cross-reactivity compared to the unmodified antibody.

[0059] For example, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art as: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is replaced with another amino acid residue from the same side chain family. In another embodiment, an amino acid chain can be replaced with a structurally similar chain that differs in the order and / or composition of the side chain family members.

[0060] Some embodiments also feature antibodies that have a certain percentage of identity or similarity to the amino acid or nucleotide sequences of the antibodies described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing the position of each sequence, which may be aligned for comparison purposes. If a position of the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity when compared to a specific region or the entire length of any one of the antibodies described herein. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid identity when compared to a specific region or the entire length of any one of the antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and / or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Current Protocols in Molecular Biology, edited by Ausubel et al. (2007). Sequence comparison and / or alignment can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology, edited by Ausubel et al. (2007). For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence identity or similarity of the nucleic acids and proteins of the invention.

[0061] An aspect of the invention provides an isolated. When used herein with respect to a cell, a nucleic acid such as DNA or RNA, the term "isolated" refers to a molecule that is separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated" may also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material or medium, if produced by recombinant DNA technology, or chemical precursors or other chemicals, if chemically synthesized. For example, an "isolated nucleic acid" can include a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state. "Isolated" may also refer to a cell or polypeptide that is isolated from other cellular proteins or tissues. An isolated polypeptide can include both purified and recombinant polypeptides.

[0062] As used herein, an "antibody" or "antigen-binding polypeptide" can refer to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be a whole antibody and any antigen-binding fragment, or single chain thereof. For example, an "antibody" can include any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Non-limiting examples include the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, the variable region of a heavy or light chain, the constant region of a heavy or light chain, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" can refer to immunoglobulin molecules and immunoglobulin (Ig) molecules, i.e., immunologically active portions of molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" means that the antibody reacts with one or more antigenic determinants of a desired antigen and not with other polypeptides.

[0063] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to(ab’)2 , F (ab)2 , F ab ', F ab The term "antibody fragment" refers to a portion of an antibody, such as a fragment of an antibody, such as a fusion protein, Fv, scFv, etc. Antibody fragments, regardless of structure, bind to the same antigen recognized by the intact antibody. The term "antibody fragment" can include aptamers (such as spiegelmers), minibodies, and diabodies. The term "antibody fragment" can also include any synthetic or engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, such as Fab, Fab', F(ab') 2 , Fd, Fvs, single-chain Fv (scFv), single-chain antibodies, dAbs (domain antibodies), minibodies, disulfide-linked Fv (sdFv), fragments containing either the VL or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0064] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L Single-chain Fv ("scFv") polypeptide molecules are covalently linked VH:VL heterodimers that can be expressed from gene fusions containing VH- and VL-encoding genes linked by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some embodiments, the domains are linked by a short linker peptide of 10 to about 25 amino acids. The linker may be rich in glycine for flexibility and serine or threonine for solubility, and may be rich in the VH:VL heterodimers. H N-terminus of V LThe C-terminus of the scFv molecule may be linked to the C-terminus of the antibody V region or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. Many methods have been described for identifying chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from the antibody V region into scFv molecules that will fold into a three-dimensional structure substantially similar to the structure of the antigen binding site. See, for example, U.S. Patent Nos. 5,091,513, 5,892,019, 5,132,405, and 4,946,778, each of which is incorporated herein by reference in its entirety.

[0065] Antibody molecules obtained from humans are classified into five classes of immunoglobulins, IgG, IgM, IgA, IgE, and IgD, which differ from each other in the nature of the heavy chains present in the molecule. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within these (e.g., γ1-γ4). Specific classes include, for example, IgG 1 , IgG 2 , IgG 3 and IgG 4 Immunoglobulin subclasses (isotypes), such as IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgG 5etc. are well characterized and known to confer functional specialization. For IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of about 23,000 daltons, and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains can be linked by disulfide bonds in a "Y" configuration, with the light chains surrounding the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. The immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0066] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y-shape to the C-terminus at the bottom of each chain.

[0067] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in a functional sense. The variable domains (VL and VH) of both the light and heavy chain portions determine antigen recognition and specificity. Conversely, the constant domains (CL, and CH1, CH2 or CH3) of the light and heavy chains confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. The term "antigen-binding site" or "binding portion" can refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent sections within the V regions of the heavy and light chains, called "hypervariable regions", are interposed between more conserved adjacent sections known as "framework regions" or "FRs". Thus, the term "FR" can refer to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface that is complementary to the three dimensional surface of a bound antigen, and each of the three hypervariable regions of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs."

[0068] The six CDRs present in each antigen-binding domain are short non-contiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR regions, show less intermolecular variation. The framework regions adopt a predominantly beta-sheet conformation, and the CDRs form loops that connect and in some cases form part of the beta-sheet structure. The framework regions function to form a scaffold for positioning the CDRs in the correct orientation by non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs provides a surface complementary to the epitope on the antigen in the immune response, facilitating non-covalent binding of the antibody to its cognate epitope. The amino acids which comprise the CDRs and framework regions, respectively, have been previously identified (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDepartment of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)) and can be readily identified for heavy or light chain variable regions by one of ordinary skill in the art.

[0069] Where there are more than one definition for a term used and / or accepted in the art, the definition of the term as used herein is intended to encompass all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region is described by Kabat et al., USDept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The definitions of CDR by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is intended that the application of either definition to refer to the CDR of an antibody or variants thereof is within the scope of the term as defined and used herein. The appropriate amino acid residues encompassing the CDRs defined by each of the above cited references are set forth in the following table for comparison. The exact residue numbers which comprise a particular CDR will vary depending on the sequence and size of the CDR, and one of skill in the art can routinely determine which residues constitute a particular CDR given the variable region amino acid sequence of an antibody. TIFF2024534863000002.tif48128

[0070] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without relying on other experimental data of the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0071] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at about the 33rd amino acid residue after the end of CDR-H2; includes 3-25 amino acids, and ends with the sequence WGXG (where X is any amino acid). CDR-L1 begins at about residue 24 (i.e., after the cysteine ​​residue), includes about 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins at about the 16th residue after the end of CDR-L1, and includes about 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., after the cysteine ​​residue), includes about 7-11 residues, and ends with the sequence F or WGXG (where X is any amino acid).

[0072] As used herein, the term "epitope" can include any protein determinant capable of specifically binding to an immunoglobulin, scFv, or T-cell receptor. The variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, the VL and VH domains of an antibody, or a subset of the complementarity determining regions (CDRs), combine to form the variable regions that define a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of a Y. Epitope determinants can consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N-terminal or C-terminal peptides of a polypeptide. More specifically, the antigen-binding site is defined by the three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VL chains.

[0073] As used herein, the terms "immunological binding" and "immunological binding properties" can refer to the type of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K D ), and K D A smaller K represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, both "on-rate constants" (K on ) and "off rate constant" (K off ) can be determined by calculation of the concentrations and actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / K onThe ratio of D (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies of the present invention may have an equilibrium binding constant (K) as measured by a kinetic assay, e.g., a radioligand binding assay or similar assay known to those of skill in the art, e.g., BIAcore or Octet (BLI). D ) is ≦1 μM, ≦10 μm, ≦10 nM, ≦10 pM, or ≦100 pM to about 1 pM. For example, in some embodiments, D is between about 1E-12M and 1E-11M. D In some embodiments, K D is between about 1E-11M and 1E-10M. D In some embodiments, K D is between about 1E-10M and 1E-9M. D In some embodiments, K D is between about 1E-9M and 1E-8M. D In some embodiments, K D is between about 1E-8M and 1E-7M. D In some embodiments, K D is between about 1E-7M and 1E-6M. D For example, in some embodiments, K D is about 1E-12M, and in other embodiments, K D is about 1E-11M. In some embodiments, K D is about 1E-10M, and in other embodiments, K D is about 1E-9M. In some embodiments, K D is about 1E-8M, and in other embodiments, K D is about 1E-7M. In some embodiments, K D is about 1E-6M, and in other embodiments, K D is about 1E-5M. In some embodiments, for example, K D is about 3E-11M, and in other embodiments, KD is about 3E-12M. In some embodiments, K D is about 6E-11M. "Specifically binds" or "having specificity" can refer to an antibody that binds to an epitope through the antigen-binding domain of the antibody, where the binding involves some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope if it binds to that epitope through its antigen-binding domain more readily than it binds to a random, unrelated epitope.

[0074] For example, antibodies can be monovalent or multivalent (e.g., bivalent) and can comprise single or two chains. Functionally, the binding affinity of an antibody can be greater than or equal to 10 -5 M~10 -12 For example, the binding affinity of an antibody is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 - M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10-9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M or 10 -5 M~10 -6 It's M.

[0075] The proteins, or derivatives, fragments, analogs, homologs, or orthologues thereof, can be used as immunogens in the generation of antibodies.Proteoliposome-bound proteins, or derivatives, fragments, analogs, homologs, or orthologues thereof, can be used as immunogens in the generation of antibodies that immunospecifically bind to these protein components.

[0076] One of skill in the art can determine, without undue experimentation, whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by determining whether the human monoclonal antibody interferes with the binding of the human monoclonal antibody of the invention to an immunogen or target. If the human monoclonal antibody being tested competes with the human monoclonal antibody of the invention, e.g., as indicated by reduced binding by the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same or closely related epitopes.

[0077] Another method of determining whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to preincubate the human monoclonal antibody of the invention with an immunogen or target with which the antibody is normally reactive, and then add the human monoclonal antibody being tested to determine whether the ability of the human monoclonal antibody being tested to bind to the target is inhibited. If the human monoclonal antibody being tested is inhibited, then it likely has the same, or a functionally equivalent, epitopic specificity as the monoclonal antibody of the invention. Screening of human monoclonal antibodies of the invention can also be performed by utilizing an immunogen / target and determining whether the test monoclonal antibody is able to bind to or neutralize the target.

[0078] Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies directed against the proteins of the invention or against their derivatives, fragments, analogs, homologs, or orthologs (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0079] Antibodies can be purified by well-known techniques such as affinity chromatography using Protein A or Protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the desired immunoglobulin, or an epitope thereof, can be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0080] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen binding site that immunoreacts with a particular epitope of an antigen characterized by a unique binding affinity for the antigen.

[0081] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal can be immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0082] Multispecific antibodies (bispecific, trispecific) Multispecific antibodies are antibodies that can recognize two or more different antigens. For example, bispecific antibodies (bsAbs) are antibodies that contain two variable domains or scFv units such that the resulting antibody recognizes two different antigens. For example, trispecific antibodies (tsAbs) are antibodies that contain two variable domains or scFv units such that the resulting antibody recognizes three different antigens. The present invention provides multispecific antibodies, such as bispecific and trispecific antibodies, that recognize Ly49 / iKIR, CD8 and / or CD8 Treg TCR. In some embodiments, bispecific and trispecific antibodies can comprise fusion proteins. For example, the fusion protein can comprise an antibody that contains a variable domain or scFv unit as described herein and a ligand or antigen and / or a third ligand or antigen such that the resulting antibody recognizes an antigen and binds to a ligand-specific receptor. In some embodiments, the fusion protein further comprises a constant region and / or a linker as described herein.

[0083] Bispecific or trispecific antibodies in various formats are also provided herein. In some embodiments, each of the first antigen-specific fragment, the second antigen-specific fragment, and / or the third antigen-specific fragment is independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody. In some embodiments, the bispecific or trispecific antibody further comprises an Fc fragment (e.g., as described in PCT / US2015 / 021529 and PCT / US2019 / 023382, each of which is incorporated by reference in its entirety). The bispecific or trispecific antibody of the present invention can comprise a combination of heavy and light chains or scFv antibodies as described herein.

[0084] Multispecific antibodies (e.g., bispecific and trispecific antibodies) of the invention can be constructed using methods known in the art. In some embodiments, bispecific antibodies are single polypeptides in which two scFv fragments are linked by a long linker polypeptide, the linker being long enough to allow intramolecular association between the two scFv units to form the antibody. In other embodiments, bispecific antibodies are two or more polypeptides linked by covalent or non-covalent bonds. In some embodiments, the amino acid linker (GGGGSGGGGS; "(G 4 S) 2 ") has a longer G to improve flexibility. 4 For example, the linker can also be generated using a "(G 4 S)3" (e.g., GGGGSGGGGSGGGGS); "(G 4 S) 4 " (e.g., GGGGSGGGGSGGGGSGGGGS); "(G 4 S) 5 ” (for example, GGGGSGGGGSGGGGGSGGGGSGGGGS); “(G 4 S) 6 ” (for example, GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS); “(G 4 S) 7 " (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS); etc. For example, (G 4 S) 5 The use of linkers can provide greater flexibility to the ligands described herein and can improve expression. In some embodiments, the linker can also be (GS) n , (GGS) n , (GGGS) n , (GGSG) n , (GGSGG) n , or (GGGGS) nwhere n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Non-limiting examples of linkers known to those of skill in the art that can be used to construct the fusions described herein can be found in U.S. Pat. No. 9,708,412, U.S. Patent Application Publication Nos. 20180134789 and 20200148771, and PCT Publication No. 2019051122, each of which is incorporated by reference in its entirety.

[0085] In another embodiment, multispecific antibodies can be constructed using the "knob into hole" method (Ridgway et al., Protein Eng 7:617-621 (1996)). In this method, Ig heavy chains of two different variable domains are reduced to selectively cleave heavy chain pairing while preserving the heavy-light chain pairing. Two heavy-light chain heterodimers recognizing two or three different antigens / ligands are mixed to promote heterologous ligand binding pairing mediated through engineered "knob into hole" of the CH3 domain.

[0086] In another embodiment, multispecific antibodies can be constructed by exchanging heavy-light chain dimers from two or more different antibodies to generate hybrid antibodies, where a first heavy-light chain dimer recognizes a first antigen and a second heavy-light chain dimer recognizes a second antigen and / or a third antigen. The heavy-light chain dimer mechanism is similar to that of human IgG, which also functions as a bispecific molecule. 4 The dimerization of IgG heavy chains is driven by intramolecular forces such as pairing of the CH3 domains of each heavy chain with disulfide bridges. The presence of a specific amino acid in the CH3 domain (R409) is essential for dimer exchange and the formation of IgG 4 Heavy chain pairing is also further stabilized by inter-heavy chain disulfide bridges in the hinge region of the antibody. 4In the hinge region, the amino acid sequence Cys-Pro-Ser-Cys is contained at amino acids 226-230 (compared to the stable IgG1 hinge region, which contains the sequence Cys-Pro-Pro-Cys). This difference in sequence at serine at position 229 results in a IgG 4 is associated with the propensity to form intrachain disulfides in the hinge region (Van der Neut Kolfschoten, M. et al, 2007, Science 317:1554-1557 and Labrijn, AF et al, 2011, Journal of Immunol 187:3238-3246).

[0087] Multispecific antibodies of the invention can be generated through the introduction of the R409 residue in the CH3 domain and a Cys-Pro-Ser-Cys sequence in the hinge region of an antibody recognizing a first or a second and / or third antigen, such that the heavy-light chain dimers are swapped to produce an antibody molecule with one heavy-light chain dimer recognizing the first antigen and a second heavy-light chain dimer recognizing the second and / or third antigen, where the second and / or third antigen (or ligand) is any antigen disclosed herein. 4 The molecules can also be modified such that the heavy and light chains recognize a first or second and / or third antigen, as disclosed herein. Using this method to construct the multispecific antibodies of the invention allows the construction of IgG subtypes whose Fc region differs from other IgG subtypes in that it interacts poorly with effector systems of the immune response, such as complement and Fc receptors expressed by certain white blood cells. 4 This particular property can be beneficial for these IgG 4 Multispecific antibody-based therapeutics will be attractive for therapeutic applications where the antibody needs to bind to a target and functionally modify a signaling pathway associated with the target, but not induce effector activity.

[0088] The multispecific antibodies described herein can be engineered with a non-depleting heavy chain isotype, such as IgG1-LALA or stabilized IgG4 or one of the other non-depleting variants. In some embodiments, mutations are introduced into the constant region of the bsAb such that the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the bsAb is modified. For example, the mutation is a LALA mutation in the CH2 domain. In one aspect, the multispecific antibody contains a mutation on one scFv unit of the heterodimeric multispecific antibody that reduces the ADCC activity. In another aspect, the multispecific antibody contains a mutation on both chains of the heterodimeric multispecific antibody that completely abolishes the ADCC activity. For example, the mutation introduced into one or both scFv units of the multispecific antibody is a LALA mutation in the CH2 domain. These multispecific antibodies with variable ADCC activity can be optimized such that the multispecific antibody shows maximum selective killing towards cells expressing one antigen recognized by the multispecific antibody, but minimum killing towards a second antigen recognized by the multispecific antibody.

[0089] The multispecific antibodies (e.g., bispecific antibodies) described herein can be engineered as modular tetrameric bispecific antibodies (tBsAbs). See, e.g., WO2018 / 071913, which is incorporated herein by reference in its entirety. For example, a tetravalent antibody can be a dimer of bispecific scFv fragments comprising a first binding site for a first antigen and a second binding site for a second antigen. In an embodiment, the first antibody can be a first binding site for a first antigen. In an embodiment, the second antibody can be a second binding site for a second antigen. The two binding sites can be linked together via a linker domain. In an embodiment, the scFv fragment is a tandem scFv, and the linker domain comprises an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region) amino acid sequence. In an embodiment, the immunoglobulin hinge region amino acid sequence can be, for example, the linker amino acid sequence (GGGS) x1-6 , (GGGGS) x1-6, or GSAGSAAGSGEF. In embodiments, the linker domain comprises at least a portion of an immunoglobulin Fc domain, such as an IgG1, IgG2, IgG3, or IgG4 Fc domain. In embodiments, at least a portion of the immunoglobulin Fc domain does not comprise a CH2 domain. In embodiments, at least a portion of the immunoglobulin Fc domain can be a CH2 domain. Exemplary CH2 domain amino acid sequences include APELLGGPDVFLF. The Fc domain can be linked to the C-terminus of an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region) amino acid sequence. The linker domain can be flanked at one or both ends by a flexible linker amino acid sequence (e.g., (GGGS) x1-6 , (GGGGS) x1-6 , or GSAGSAAGSGEF).

[0090] Antibody-mediated rejection, tumor surveillance In some embodiments, mobilizing CD8 Treg cells in mammals using peptide agonists or antibodies that mobilize or activate CD8 Treg cells can be used in organ transplant patients or patients with autoimmune diseases.

[0091] With respect to treating organ transplant patients, in various embodiments, the reagents and methods described herein can be used to treat patients with hyperacute rejection, acute rejection, or chronic rejection. In embodiments, the reagents and methods can be used to treat antibody-mediated rejection (AMR; see Figures 37-41, 60A-F, 77, 83A-F, 84A-H) in patients. In embodiments, the reagents and methods can be used to treat cell-based allograft rejection (e.g., cytotoxic T cell-based rejection) and antibody-mediated allograft rejection. Generally, the treatment reduces humoral and / or cellular rejection of the allograft.

[0092] In various embodiments, allograft rejection that can be treated using the reagents and methods described herein includes any type of transplant, in embodiments, patients with at least the following allograft transplants can be treated: heart, kidney, lung, liver, pancreas, cornea, trachea, skin, vascular tissues, stem cells, bone, etc.

[0093] With regard to autoimmune diseases, the reagents and methods described herein can be used to treat patients with all different kinds of autoimmune diseases or disorders, including, in some embodiments, at least systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes mellitus (DM1; insulin-dependent diabetes mellitus or IDDM), rheumatoid arthritis, psoriasis or psoriatic arthritis, inflammatory bowel disease, Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, celiac disease, and others.

[0094] The reagents and methods described herein can be used to treat patients with inflammation or inflammatory disorders.

[0095] In some embodiments, antibodies that bind to CD8 Treg cells or molecules of CD8 Treg cells can be used to suppress and / or kill CD8 Treg cells in a mammal, thereby increasing anti-tumor activity in the mammal. The antibodies can be used to treat tumors or cancer in a mammal (see Figures 69, 71, 89A-D, 91A-C). Antibodies used in these embodiments are described in the sections of this application entitled "Antibodies to CD8 Treg" and "Multispecific Antibodies".

[0096] In general, depletion of CD8 Tregs can increase CD4 T cell activity and anti-tumor activity in a mammalian organism.

[0097] Generally, the reagents and methods disclosed herein can be used to treat any cancer. A non-limiting list of cancers covered by the reagents and methods disclosed herein includes bladder, breast, colon and rectum, endometrial, kidney, leukemia, liver, lung, lymphoma (e.g., non-Hodgkin's lymphoma), melanoma, pancreatic, prostate, thyroid, etc.

[0098] In some embodiments, the reagents and methods disclosed herein for treating tumors or cancer in a mammal (e.g., a human) can be combined with other types of anti-cancer therapies. In some embodiments, the treatments disclosed herein can be used in combination with a therapeutic cancer vaccine. In some embodiments, the treatments disclosed herein can be used in combination with an immune checkpoint inhibitor or checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor can include a PD-L1 inhibitor.

[0099] Therapeutic preparations Aspects of the present invention relate to therapeutic preparations. As used herein, the term "therapeutic preparation" can refer to any compound or composition (including, for example, cells) that can be used or administered for a therapeutic effect. As used herein, the term "therapeutic effect" can refer to an improvement in symptoms, e.g., an effect sufficient to treat, cure, prevent or ameliorate an associated medical condition, or to increase the rate of treatment, cure, prevention or amelioration of such a condition. In some embodiments, a therapeutic effect may refer to one resulting from the treatment of cancer in a patient subject. As used herein, a therapeutic preparation can include a pharmaceutical composition.

[0100] The pharmaceutical compositions disclosed herein can comprise a therapeutically effective amount of any of the CD8 Treg stimulators disclosed herein (see the paragraph entitled "CD8 Treg Agonists"). In some embodiments, the CD8 Treg stimulator can comprise a peptide / polypeptide agonist / superagonist discussed in that paragraph. In some embodiments, the CD8 Treg stimulator can comprise an antibody that recruits or activates CD8 Treg cells (see the paragraphs entitled "Antibodies to CD8 Tregs" and "Multispecific Antibodies"). In some embodiments, the pharmaceutical compositions can comprise a combination of a peptide agonist and a stimulator antibody. These pharmaceutical compositions can comprise a pharma- ceutically acceptable carrier. In some embodiments, the peptides and / or antibodies of these compositions can be bound to a lipophilic albumin-binding tail conjugate.

[0101] In general, these pharmaceutical compositions that recruit CD8 Treg cells can suppress CD4 T cell activity in the mammal to which the composition is administered. These pharmaceutical compositions can reduce the expression of follicular T cells (Tfh), germline center B cells, antibody production, or a combination thereof. These pharmaceutical compositions can reduce the production of donor-specific antibodies and / or graft tissue damage. In embodiments, these pharmaceutical compositions can be used to treat organ transplant patients to prevent or reduce the likelihood of the transplanted organ being rejected. In embodiments, these pharmaceutical compositions can be used to treat patients with various autoimmune diseases.

[0102] The pharmaceutical compositions disclosed herein can comprise a therapeutically effective amount of any of the molecules disclosed herein that deplete CD8 Treg cells in a mammal. In some embodiments, these CD8 Treg depleting agents can be antibodies (see the sections of this application entitled "Antibodies to CD8 Treg" and "Multispecific Antibodies"). These pharmaceutical compositions can comprise a pharma- ceutically acceptable carrier. In some embodiments, the peptides and / or antibodies of these compositions can be bound to a lipophilic albumin-binding tail conjugate.

[0103] In general, these pharmaceutical compositions that deplete CD8 Treg cells can increase CD4 T cell activity in the mammal to which the composition is administered. These pharmaceutical compositions can increase the expression of follicular T cells (Tfh), T follicular cells (Tfh), germline center B cells, antibody production, or a combination thereof. These pharmaceutical compositions can increase the production of donor-specific antibodies. In embodiments, these pharmaceutical compositions can be used to treat tumors or cancer in patients.

[0104] The embodiments described herein can be administered to a subject in the form of a pharmaceutical composition or therapeutic formulation prepared for the intended route of administration. Such compositions and formulations can include, for example, an active ingredient(s) and a pharma- ceutically acceptable carrier. Such compositions and formulations can be in a form adapted for oral, subcutaneous, parenteral (e.g., intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, dermal, vaginal, buccal, ocular, or pulmonary administration, e.g., in a form adapted for administration by a peripheral route, or in a form suitable for oral administration, or in a form suitable for parenteral administration. Other routes of administration are subcutaneous, intraperitoneal, and intravenous, and such compositions can be prepared in a manner well known to those skilled in the art, for example, as generally described in "Remington's Pharmaceutical Sciences", 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 and more recent editions, and in the monograph "Drugs and the Pharmaceutical Sciences" series, Marcel Dekker. The compositions and preparations may appear in conventional forms, such as solutions and suspensions for injection, capsules and tablets, enteric formulations such as those disclosed in U.S. Pat. No. 5,350,741, and forms for oral administration.

[0105] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0106] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharma- ceutically acceptable polyol, such as glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal.In many cases, it can be useful to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride.Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0107] Sterile injection can be prepared by incorporating the required amount of compound into suitable solvent with one or combination of components listed here as required, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other necessary components listed here.For the preparation of sterile powder for sterile injection, the example of useful preparation method is vacuum drying and freeze-drying, which obtains powder of active ingredient and any additional desired ingredient from its solution that has been previously sterilized and filtered.

[0108] Oral compositions include inert diluents or edible carriers. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using fluid carriers for use as mouthwashes, where the compound in the fluid carrier is orally applied, swirled in the mouth, and expectorated or swallowed. Oral formulations of drugs can be administered, for example, once a day, twice a day, three times a day, or four times a day, depending on the half-life of the drug.

[0109] Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition administered to a subject. Tablets, pills, capsules, lozenges, etc. can contain any of the following: binders such as microcrystalline cellulose, tragacanth or gelatin; excipients such as starch or lactose; disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or stearic acid; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring ingredients such as peppermint, methyl salicylate, or orange flavoring, or compounds of a similar nature.

[0110] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art.

[0111] In embodiments, administering can include placement of a pharmaceutical composition in a subject by a method or route that results in at least partial localization of the composition at a desired site such that a desired effect occurs.

[0112] For example, the pharmaceutical composition can be administered by bolus injection or infusion. Bolus injection can refer to a route of administration in which the syringe is connected to an IV access device and the drug is injected directly into the subject. The term "infusion" can refer to intravascular injection.

[0113] The embodiments described herein can be administered once to a subject (e.g., as a single injection, bolus, or deposition). Alternatively, administration can be once or twice daily to a subject for a period of time, such as from about 2 weeks to about 28 days. Administration can continue for up to a year. In embodiments, administration can continue for the life of the subject. Embodiments can also be administered once or twice daily to a subject for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.

[0114] In embodiments, the compositions described herein can be administered chronically to a subject. "Chronic administration" can refer to administration in a continuous manner, such as to maintain a therapeutic effect (activity) over an extended period of time.

[0115] The pharmaceutical or therapeutic carrier or diluent used can be a conventional solid or liquid carrier.Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ether of cellulose.Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.Similarly, carriers or diluents can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with wax.

[0116] If a solid carrier is used for oral administration, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form, or in the form of a troche or lozenge. The amount of solid carrier varies widely but can be from about 25 mg to about 1 g.

[0117] If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.

[0118] The composition and / or preparation may also be in a form suitable for local or systemic injection or infusion, and may itself be formulated with sterile water or isotonic saline or glucose solution. The composition may be in a form suitable for peripheral administration only, except for forms that can be administered centrally. The composition and / or preparation may be in a form suitable for central administration.

[0119] The composition and / or formulation can be sterilized by conventional sterilization techniques well known in the art. The resulting aqueous solution can be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized formulation is combined with a sterile aqueous solution before administration. The composition and / or formulation can contain pharma- ceutically and / or therapeutically acceptable auxiliary substances required to approximate physiological conditions, such as buffers, isotonicity adjusting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0120] Vaccine Compositions As used herein, a vaccine composition refers to a therapeutically effective amount of a CD8 Treg agonist, including a peptide / polypeptide agonist / superagonist. A vaccine composition can include any of the peptide / polypeptide agonists / superagonists disclosed in the paragraph of this application entitled "CD8 Treg Agonists". In some embodiments, a vaccine composition can include a pharma- ceutically acceptable carrier, diluent, or excipient. In some embodiments, a peptide / polypeptide agonist / superagonist in a vaccine composition is conjugated to one or more carrier proteins. In some embodiments, a carrier protein can include a lipophilic albumin-binding tail conjugate. In some embodiments, a lipophilic albumin-binding tail conjugate can include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG).

[0121] Exemplary data regarding the use of the vaccine composition are shown in Figures 60A-F and 61.

[0122] method The methods disclosed herein involve administering the disclosed peptide / polypeptide agonists / superagonists, antibodies, combinations thereof, and pharmaceutical compositions of the same to a mammal (e.g., human, mouse) to treat or prevent various conditions.

[0123] In some embodiments, the method comprises administering a CD8 Treg stimulator to the mammal. The method of administering a CD8 T cell stimulator can comprise administering a peptide / polypeptide agonist / superagonist to the mammal. The peptide / polypeptide agonist / superagonist, or combination of different agonists / superagonists, are as described herein (see the paragraph entitled "CD8 Treg Agonists").

[0124] The method of administering a CD8 T cell stimulator can include administering to the mammal an antibody that binds to CD8 Treg cells. These antibodies and multispecific antibodies are described herein (see the sections entitled "Antibodies to CD8 Tregs" and "Multispecific Antibodies").

[0125] Administration of these CD8 Treg stimulators can mobilize CD8 Treg cells in a mammal and suppress or reduce CD4 T cells and activity in the mammal. In some embodiments, these CD8 Treg stimulators can be administered to a mammal undergoing an organ transplant. Upon administration, humoral and / or cellular-based rejection of the transplanted organ in the mammal can be reduced. In some embodiments, these CD8 Treg stimulators can be administered to a mammal with an autoimmune disease or disorder.

[0126] Administration of a CD8 Treg cell stimulator can be used to expand effector CD8 Treg cells, treat an autoimmune disease or condition, and / or treat or prevent rejection of a transplanted organ in a mammal (e.g., antibody-mediated rejection).

[0127] In some embodiments, the method comprises administering a CD8 Treg depleting agent to the mammal. The method of administering a CD8 T cell depleting agent can comprise administering to the mammal an antibody that depletes CD8 Tregs (see the sections herein entitled "Antibodies to CD8 Tregs" and "Multispecific Antibodies").

[0128] Administration of CD8 Treg depleting factors (e.g., antibodies and / or multispecific antibodies) can increase CD4 T cell activity in a mammal. In some embodiments, these CD8 Treg cell depleting factors can be used to treat cancer in a mammal. In embodiments, administration of these antibodies can increase anti-tumor activity in a mammal.

[0129] Administration of a CD8 Treg cell depleting factor can be used to deplete effector CD8 Treg cells and / or to treat cancer in a mammal.

[0130] Also disclosed are methods for screening for autoimmune disorders in a mammal by detecting any of the TCRs described in the section of this application entitled "CD8 Treg Cells." These diagnostic methods can be used prior to treating a mammal with an autoimmune disease or prior to treating a mammal with an autoimmune disease with any of the CD8 Treg stimulators disclosed herein.

[0131] Also disclosed are methods of screening antibodies for reactivity against CD8 Treg cells by contacting the antibody with a TCR from CD8 Treg cells or with CD8 Treg cells and detecting binding of the antibody to the TCR and / or CD8 Treg cells. EXAMPLES

[0132] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, and are for illustrative purposes only, since alternative methods can be used to obtain similar results.

[0133] Example 1 Figure 1A-B CD8 Tregs recognize chronically activated CD4 cells that express Qa-1-FL9 peptide on their surface as a result of reduced ERAAP activity. CD8 Treg recognition of target CD4 cells results in cytolysis of these cells, thus inhibiting their proliferation. CD8 Treg recognition of target CD4 cells involves molecular interactions that transmit either stimulatory or inhibitory signals. TCR-Qa-1 / peptide and NKG2D-NKG2DL interactions activate CD8 Tregs, whereas NKG2A / CD94-Qa-1 / Qdm and Ly49F-ligand interactions inhibit CD8 Treg function.

[0134] Figure 2A-C Screening of FL9 peptide variants for their FL9 T cell stimulatory capacity identified FL9 superagonist peptides (eg, FL9-68) that displayed superior ability to activate FL9 T cells.

[0135] Figure 3A-D FL9 T cells can be activated by chronically activated CD4 cells presenting FL9 peptides in the context of Qa-1. Peptides presented on Qa-1 can be produced in the context of ERAAP reduced function or ERAAP deficiency. In vivo recognition of Qa-1-FL9 by FL9 T cells is shown to be related to Qa-1-FL9. + This results in the elimination of activated CD4 cells (in this experiment, Ova-activated OT-II cells).

[0136] Figure 4 Screening the Qa-1 yeast peptide library for superagonist peptides against FL9 T cells identified multiple peptides (peptides 3, 4, 10, 11) that exhibited superior FL9 T cell stimulatory capacity to FL9-68.

[0137] Figure 5A-F We assessed the contribution of the Ly49 receptor to the CD8 Treg phenotype by generating Ly49F and pan Ly49 KO mice. Comparison of key markers between WT and Ly49F or pan Ly49 KO CD8 Tregs demonstrated that deficiency of Ly49 led to the differentiation of CD8 Tregs with an increased activation phenotype.

[0138] Figure 6 CD8 Treg function may be manipulated by blocking inhibitory Ly49 signaling using antibodies, as observed in Ly49-deficient CD8 Tregs.

[0139] Figure 7 Ab-mediated blockade of Ly49 signaling in CD8 Tregs activates them as evidenced by increased proliferation and upregulation of ICAM1 and TNFR2.

[0140] Figures 8A-E FL9 TCR is Ly49 + Qa-1-FL9 tet + The TCR from CD8 T cells was identified by sequencing. FL9 TCR Tg mice were generated to faithfully express TCR specific for the Qa-1-FL9 peptide. FL9 TCR Tg CD8 T cells express significant levels of Ly49, NKG2A and NKG2D, reflecting the acquisition of a CD8 Treg phenotype.

[0141] Example 2 CD8 Tregs regulate immune responses to pathogens and self-antigens by eliminating chronically activated CD4 cells that upregulate Qa-1 / HLA-E on the CD4 cell surface. Recognition of Qa-1 self-peptide on target cells by CD8 Tregs can suppress pathogenic CD4 cells, whereas proliferation and recruitment of CD8+ Tregs is constrained by molecular mechanisms that constrain excessive or inappropriate CD8 Treg activation. In the context of autoimmune diseases and cancer, we have developed strategies that allow both antigen-specific and antigen-nonspecific therapeutic recruitment of CD8 Tregs.

[0142] Mobilization of CD8 Treg responses Peptide superagonists: Activation of CD8+ Tregs in autoimmune diseases CD8+ Tregs recognize peptides presented by target CD4 cells in the context of Qa-1. We have identified a TCR that recognizes Qa-1 and FL9 self-peptides. This Qa-1-FL9 complex is expressed by a significant proportion of activated CD4+ cells. Activation of CD8+ Tregs by target CD4+ cells, particularly by FL9-Qa-1 / HLA-E complexes expressed on Tfh cells, inhibits antibody responses. Mobilization of Qa-1-restricted CD8+ Tregs uses stimulation with agonist peptides for robust activation of these regulatory cells. HLA-E also presents the same FL9 amino acid sequence, and agonist peptides derived from the FL9 sequence can activate and expand human CD8+ Tregs. Here we describe the identification of a superagonist mutant of the self-peptide engineered to express potent CD8+ Treg stimulatory activity in the context of Qa-1b and HLA-E. In vitro studies showed that superagonist FL9 peptide promoted the proliferation of FL9 T cells and enhanced the killing of activated CD4 cells. Furthermore, after immunization with self-antigen (MOG), the formation of Tfh and GC B cells and antibody production were suppressed after adoptive transfer of FL9 T cells. Vaccination with FL9 superagonist peptide efficiently recruited CD8+ Tregs and inhibited antibody-associated immune responses.

[0143] Anti-costimulatory receptor (Ly49F; KIR) mediated regulation of CD8 Treg activity 1. Autoimmune diseases: CD8+ Tregs express the inhibitory Ly49 receptor (Ly49F, an inhibitory KIR in humans), a type II C-type lectin-like membrane glycoprotein that recognizes class I major histocompatibility complex-I (MHC-I) and MHC-I-like proteins on normal as well as modified cells.

[0144] Mouse: Although several Ly49 receptors are expressed by NK cells, we have shown that the mouse inhibitory Ly49F receptor is selectively expressed by CD8 Tregs and not by conventional CD8+ T cells or NK cells. Studies using a genetic model of Ly49F KO mice have shown that Ly49F-deficient CD8 Tregs exhibit an activated and effector phenotype. Preliminary data show that the formation of GC B cells after immunization with antigen (NP-Ova) is reduced in Ly49F KO compared to WT mice, indicating that Ab production may be inhibited in the absence of Ly49F on CD8 Tregs, next to enhanced CD8 Treg-mediated immune suppression. Based on these studies, and without wishing to be bound by the mechanism of action, blocking Ly49 / KIR expression may enhance the recruitment of CD8+ Tregs and increase suppressive function by releasing the brakes on this regulatory lineage.

[0145] Human: Killer cell immunoglobulin-like receptors (KIR) are functional homologs of Ly49F, and human CD8 Tregs express inhibitory KIRs (e.g., KIR3DL1, KIR2DL3) on their surface. In vitro suppression assays showed that KIR+CD8 T cells suppress the proliferation of human Tfh (CXCR5+CD4) cells in vitro. Without wishing to be bound by theory, the suppressive activity of human CD8 Tregs can be enhanced by releasing the brakes on human CD8 Treg responses using anti-iKIR antibodies. This approach can be used to suppress the proliferation of pathogenic CD4 cells during strong inflammation, autoimmunity, and infection.

[0146] 2. Cancer Anti-Ly49F (anti-iKIR Ab in humans) Ab-mediated depletion of CD8 Treg activity We found that Qa-1 mutant mice (B6.D227K mice) lacking CD8 Treg activity developed enhanced antitumor immune responses against B16 melanoma after GVAX immunization. Reduced tumor growth in B6.D227K mice is associated with enhanced proliferation of Tfh cells, GC B cells, and high titers of antitumor autoantibodies. A recent retrospective analysis of human cancers showed that anti-PD1 Ab treatment of mNSCLC patients, which increases the levels of serum autoantibodies, predicts favorable outcome (Giannicola R Mol Cli Onc 2019). These studies indicate that depleting CD8 Tregs can enhance antitumor immune responses by promoting tumor-associated Ab production.

[0147] We developed a strategy to selectively deplete CD8 Tregs using anti-Ly49F Ab: A) Studies can be performed by tagging an anti-Ly49F Ab (IgG1) with the small fluorescein FITC, followed by an anti-FITC IgG2a Ab to activate complement fixation and depletion of Ly49F+CD8 Tregs. B) CD8 Treg depletion can also be achieved using a toxin-conjugated anti-Ly49F Ab. In one example, a CD8 Treg depletion reagent is developed by fusing an anti-Ly49F Ab with proaerolysin (PA), a potent protein toxin secreted by Aeromonas hydrophila. A mutant form of PA (R336A) prevents binding of PA to the universal GPI anchor, while selectivity is guided by the anti-Ly49F Ab conjugate. CD8 Treg depletion can be achieved using depleting anti-Ly49F Ab (clone: ​​HBF719). Treatment of mice inoculated with EL4 tumor cells (Qa-1+) with anti-Ly49F Ab significantly slowed tumor growth compared to mice treated with isotype control Ab (mIgG1) (Figure 9A-B). These data indicate that ablating or reducing CD8 Treg-dependent immune suppression can enhance anti-tumor immunity. C) Expression / upregulation of Qa-1 is an immune evasion mechanism utilized by tumor cells. In an embodiment, blocking Qa-1 interaction with Qa-1 receptor on CD8 Treg can enhance anti-tumor immune response. This analysis reveals that treatment of mice with blocking anti-Qa-1 Ab (clone: ​​4C2.4A7.5H11) slows tumor growth in B6 mice (Figure 9A). D) In ​​an embodiment, vaccination of mice with FL9-68 SA, but not IFA alone, promotes tumor growth, demonstrating that CD8 Treg expansion promotes tumor growth (Figure 9B). E) Depletion of human CD8 Tregs can be achieved by engineering anti-KIR Abs (anti-KIR3DL1 and anti-KIR2DL3) with complement fixing isotypes (IgG2a, IgG2b or IgG3). Depletion of human CD8 Tregs during immunotherapy with ICB can be a viable approach to circumvent CD8 Treg-mediated inhibition of autoantibodies, promoting ICB efficacy.

[0148] 3. Viral infection Viral infections are often accompanied by robust autoimmune responses that result in tissue damage, morbidity, and in some cases, mortality. Inhibition of self-destructive autoantibody production by CD8 Treg-mediated immunosuppression represents an effective approach to attenuate these infection sequelae. In one embodiment, using a murine MCMV infection model, we show that enhancing CD8 Treg function after vaccination with a peptide superagonist (FL9-68 SA at days 0, 8, and 12 post-viral infection) significantly reduced the production of anti-dsDNA Ab without affecting viral clearance (Figures 10A-C). Inhibition of anti-dsDNA Ab production after FL9-68 SA vaccination was associated with an increase in NKG2D+Ly49+CD8 T cells, consistent with the expansion of activated CD8 Tregs. Without wishing to be bound by theory, vaccination with SA peptide represents a viable approach to attenuate the autoimmune sequelae of viral infection.

[0149] Non-limiting exemplary features of the present disclosure: Mobilization of CD8 Tregs to regulate Ab-dependent immune responses has important advantages over general immunosuppression that may leave the host immunocompromised. Because CD8 Tregs specifically recognize cell surface antigens on Tfh cells that signal the activation state of these cells, we developed approaches to identify superagonist peptides that efficiently mobilize CD8 Tregs, reduce GC responses, and suppress autoantibody production. These included mutagenesis of cognate self-peptides, selection from libraries and testing for activation of CD8 Tregs, as well as mobilization of CD8 Tregs to reduce Ab-mediated autoimmune diseases.

[0150] Ly49F is uniquely expressed by CD8 Tregs, and blockade of this inhibitory receptor can enhance CD8 Treg activity without affecting other cells, including NK cells. Mobilization of CD8 Tregs using blocking Ly49F Abs represents a highly specific approach that can be applied when suppression by CD8 Tregs is most efficient, including in conditions of high levels of autologous Ab production.

[0151] CD8 Tregs primarily target Tfh cells, thereby regulating Ab responses during immune responses. Efficient targeting of Qa-1-FL9 (HLA-E-FL9) by CD8 Tregs after expansion with peptide agonists is applicable to ameliorate multiple immune responses characterized by pathogenic antibodies in the context of autoimmune diseases, organ transplantation, and infections.

[0152] Since Qa-1 (HLA-E) is upregulated on Tfh cells during robust immune responses, recruitment of CD8 Tregs can be applied during the time window when upregulation of target molecules (Qa-1 peptide, HLA-E peptide) is maximal and CD8 Treg recruitment is most beneficial.

[0153] For peptide vaccines, a delivery system (DSPE-PEG-peptide conjugate) can be used that maximizes peptide delivery to lymph nodes, where the peptides are taken up by DCs and presented by Qa-1, thereby increasing immunogenicity (Moynihan et al., 2018). In embodiments, the DSPE (lipophilic albumin-binding tail)-PEG conjugates facilitate their binding to albumin (a molecular chaperone) and lymphatic transport, resulting in robust CD8 T cell responses.

[0154] Both peptide agonists and anti-Ly49F / anti-iKIR Abs can expand CD8 Tregs, and these two approaches can be combined in certain embodiments to maximize CD8 Treg recruitment.

[0155] Additional Embodiments We found that FL9 peptides complexed with Qa-1 are expressed by a significant proportion of activated Tfh cells during immune responses. Peptide mutagenesis can be applied to systemically identify synthetic peptides with superagonist activity that induce robust proliferation, activation and suppressive activity of CD8 Tregs in vivo using mouse models of autoimmune diseases including EAE and lupus. These peptide-based regimens can be evaluated in the context of autoimmune responses for suppression of Ab-mediated pathogenesis and tissue damage. Without wishing to be bound by theory, this approach can be clinically applicable to a large population of patients and avoid issues related to MHC class Ia diversity, since HLA-E and Qa-1 are expressed as only one of two alleles.

[0156] References cited in this example: Moynihan, KD, Holden, RL, Mehta, NK, Wang, C., Karver, MR, Dinter, J., Liang, S., Abraham, W., Melo, MB, Zhang, AQ, et al. (2018). Enhancement of Peptide Vaccine Immunogenicity by Increasing Lymphatic Drainage and Boosting Serum Stability. Cancer Immunol Res 6,1025-1038.

[0157] Example 3 Superantigens that recruit CD8+ regulatory T cells inhibit donor-specific antibodies and protect cardiac allografts from antibody-mediated rejection Antibody-mediated rejection (AMR) is a significant barrier to long-term allograft survival. We have shown that Qa-1 (HLA-E in humans)-restricted CD8+ T cells (CD8 Tregs) play a key role in controlling humoral immunity by killing alloreactive CD4+ T cells, particularly follicular helper T cells (Tfh) that upregulate Qa-1 under immunological stress conditions. We previously showed that disruption of CD8+ T cell receptor (TCR)-bound Qa-1 releases Tfh proliferation, resulting in severe AMR in mouse heart transplants. In this study, we identified stress peptides (SPs) presented on Qa-1, modified one of these peptides to engineer a superagonist (SA), and tested the efficacy of SPs to recruit CD8 Tregs. Finally, we investigated whether SPs suppress allosensitization and protect heart grafts from AMR.

[0158] Methods: Based on previous mass spectrometry studies, we selected two SPs, FL9 and Hsp60p216, that associate with Qa-1 under immune stress conditions. We then selected FL9-tetramer-binding CD8 Tregs, and sequenced and expressed the TCRs of CD8 Tregs on hybridomas. We also generated a library of modified FL9 sequences and compared the antigenicity of FL9 sequences using TCR-engineered hybridomas. After selecting FL9-SA, we performed skin transplants from BALB / c to B6 mice, followed by heart transplants, both with and without Hsp60p216 and FL9-SA.

[0159] Results: We successfully produced FL9-SA using our TCR engineered hybridoma system. Immunization with SP significantly expanded SP-Qa-1 tetramer-binding CD8 Tregs. Compared to the control group, hosts treated with SP during sensitization showed a significant reduction in mature B cells, including Tfh and plasma cells. FL9-SA was substantially more effective than Hsp60p216. Donor-specific antibodies (DSA) were significantly reduced in the SP-treated group, protecting the cardiac allograft (Figure 11A-D).

[0160] Conclusion: Induction of CD8 Treg responses by Qa-1-associated SP suppresses germinal center reactions and DSA formation. Notably, the superagonist we generated showed excellent biological efficacy in mobilizing CD8 Treg. Without wishing to be bound by theory, harnessing the mechanisms of CD8 Treg through the study of Qa-1-binding peptides provides a strategy to suppress AMR, which lacks effective therapeutic options.

[0161] Example 4 Mobilization of CD8 Tregs: A therapeutic approach to suppress anti-graft antibody responses in allograft transplantation Antibody-mediated rejection (AMR) remains a major barrier to successful solid organ transplantation. Based on recent advances in understanding CD8 Treg biology, we have developed approaches to attenuate Ab-mediated damage and promote organ allograft survival. The present disclosure of the application of CD8 Treg-based therapy is relevant to at least the clinical problem of organ transplantation. Qa-1 (HLA-E in humans) is a class Ib MHC molecule with limited polymorphism (unlike the highly polymorphic class Ia MHC molecules). Mouse Qa-1 is strongly expressed by activated helper T cells, particularly follicular helper T (Tfh) cells, allowing targeting and lysis by CD8 Tregs. This interaction regulates allo-Ab responses in a fully mismatched heart transplant model. Alloreactive Tfh cells upregulate Qa-1-self-peptide complexes, including the FL9 self-peptide expressed on a significant proportion of Tfh cells, during the alloimmune response, allowing targeting by Qa1-restricted CD8 Tregs. Here we describe the use of a superagonist (SA) mutant of the FL9 self-peptide engineered to express potent CD8 Treg stimulatory activity in the context of Qa-1b. Vaccination with the FL9 superagonist peptide efficiently recruits CD8 Tregs and inhibits antibody-mediated allograft rejection.

[0162] Improved strategies to attenuate AMR are needed. AMR reflects robust germinal center (GC) allo-Ab responses induced by follicular helper T (Tfh) cells. Our previous studies have revealed that Qa-1 (HLA-E)-restricted CD8 Tregs inhibit these Tfh-dependent GC responses (Nakagawa et al., 2018). Here, we outline a method based on the application of a superagonist self-peptide that can efficiently expand CD8 Tregs, reduce GC responses, and suppress antibody responses. Using this method, we mobilize CD8 Tregs and reduce Ab-mediated damage to allogeneic organ transplants. Nakagawa, H., Wang, L., Cantor, H., and Kim, HJ (2018). New Insights Into the Biology of CD8 Regulatory T Cells. Adv Immunol 140, 1-20.

[0163] CD8 Tregs primarily target Tfh cells, thereby regulating Ab responses during immune responses. Efficient targeting of Qa-1-FL9 by CD8 Tregs after expansion with peptide vaccines is at least applicable to organ transplantation.

[0164] CD8 Tregs express the inhibitory Ly49 receptor (Ly49F, inhibitory KIR in humans). The Ly49 receptor is a type II C-type lectin-like membrane glycoprotein that recognizes class I major histocompatibility complex-I (MHC-I) and MHC-I-like proteins on normal as well as modified cells. Without being bound by theory, Ly49 engagement can inhibit the suppressive activity of CD8 Tregs, so blocking Ly49 / KIR expression may enhance CD8 Treg recruitment and increase suppressive function. In addition to peptide vaccines, Ly49F can be blocked using anti-Ly49F Abs.

[0165] Further exemplary embodiments FL9 peptide complexed with Qa-1 may be expressed by a significant proportion of activated Tfh cells during immune responses. In various embodiments, peptide-based regimens are evaluated in the context of allograft responses for suppression of Ab-mediated damage and graft survival. Without wishing to be bound by theory, this approach is clinically applicable to a large population of patients and avoids issues related to MHC class Ia diversity, since HLA-E and Qa-1 are expressed as only one of two alleles. In embodiments, graft-sensitized mice and Ag-specific heart transplant models can be used to analyze these responses.

[0166] References cited in this example Choi JY,Eskandari SK,Cai S,Sulkaj I,Assaker JP,Allos H,AlHaddad J,Muhsin SA,Alhussain E,Mansouri A,Yeung MY,Seelen MAJ,Kim HJ,Cantor H,Azzi JR.Regulatory CD8 T cells that recognize Qa-1 expressed by CD4 T-helper cells inhibit rejection of heart allografts.PNAS USA.2020 Mar 17;117(11):6042-6046.doi:10.1073 / pnas.1918950117.Epub 2020 Feb 28.PMID:32111690;PMCID:PMC7084119.

[0167] Example 5 Figure 12 The effect of peptide immunization on Tfh, GC B and plasma cell production was examined in a heart transplant model. Data show that these cells are significantly reduced in graft recipients immunized with SA peptide FL9-68.

[0168] Figure 13 FL9 SA peptide immunization reduces the production of donor-specific antibodies and maintains tissue integrity leading to cardiac graft survival.

[0169] Figure 14 CD8 Tregs target HLA-E / peptide in humans and Qa-1 / peptide in mice, expressed on activated CD4 cells. Expression of KIR or Ly49 on CD8 Tregs can inhibit the suppressive activity of peptides. CD8 Treg function can be enhanced by Ab-dependent blockade of these inhibitory receptors, immunization with SA peptides, or stimulation with Abs targeting the CD8 Treg classical TCR.

[0170] Figure 15 CD8 Tregs expressing autoreactive TCRs can escape thymic negative selection by also expressing the inhibitory Ly49 (KIR) receptor and PD1.

[0171] Figure 16 Analysis of Ly49F or pan Ly49 KO CD8 Tregs indicates that the lack of Ly49 expression enhances CD8 Treg activation.

[0172] Figure 17 The human genome has evolved to lose the Ly49 locus, and CD8 Tregs instead express the functional homolog KIR.

[0173] Figure 18A-B Human CD8 Tregs preferentially express the KIR2DL2 / 3 and KIR3DL1 subtypes, which express Helios TF, similar to mouse Ly49+CD8 Tregs.

[0174] Figure 19 KIR+CD8 T cells exhibit suppressive activity. Co-culture of isolated T cells with CD8 T cells demonstrated that only KIR+CD8 T cells exhibited inhibition of T cells proliferation.

[0175] Figure 20 Chronically activated CD4 cells downregulate ERAAP, resulting in the production of FL9 peptide that can be loaded onto Qa-1 and presented on the surface of CD4 T cells, leading to the recognition of these cells by CD8 Tregs.

[0176] Figure 21 The TCRs expressed by CD8 Tregs display common V genes (CDR1 and CDR2) independent of the peptide specificity of the TCR that allow them to focus on MHC (Qa-1 or HLA-E) during development, which may allow the development of these autoreactive cells by circumventing thymic negative selection.

[0177] Figure 22 Peptide superagonists for CD8 Tregs can be screened in peptide libraries composed of amino acid variants with amino acid mutations at MHC anchoring positions (2, 3, 6, 7, 9) or TCR binding positions (1, 4, 5, 8).

[0178] Fig. 23 A FL9 peptide library with variants with amino acid mutations at MHC anchoring positions was screened for the ability of the peptides to activate the FL9 TCR by incubating FL9 T cells with EL4 loaded with these peptides. CD69, TCR expression and TCR trogocytosis by APCs (EL4 cells) were measured.

[0179] Fig. 24 A FL9 peptide library with variants with amino acid mutations at the TCR binding positions was screened for the ability of the peptides to activate the FL9 TCR (FL9.2 and FL9.8 TCR) by incubating FL9 T cells with EL4 loaded with these peptides. CD69 expression by FL9 T cells (both FL9.2 and FL9.8 T cells) was measured as a readout.

[0180] Fig. 25 A FL9 peptide library with variants with aa mutations at the TCR binding positions was screened for the ability of the peptides to activate the FL9 TCR (FL9.2 and FL9.8 TCR) by incubating FL9 T cells with EL4 loaded with these peptides. TCR downregulation by FL9 T cells (both FL9.2 and FL9.8 T cells) was measured as readout.

[0181] Fig. 26 The amino acids showing the highest stimulatory potential at MHC anchoring or TCR binding positions are summarized. The AA sequences of the FL9-68 peptides selected for immunization for CD8 Treg activation are shown.

[0182] Fig. 27 Qa-1-FL9 Tet + TCR α chain sequences isolated from CD8 T cells detected by CD8 T cells.

[0183] Fig. 28 TCR β chain sequences isolated from CD8 T cells detected by Qa-1-FL9 Tet+CD8 T cells.

[0184] Fig. 29 TCR α chain sequences isolated from CD8 T cells detected by Qa-1-Hsp60p216 Tet+CD8 T cells.

[0185] Fig. 30 TCR β chain sequences isolated from CD8 T cells detected by Qa-1-Hsp60p216 Tet+CD8 T cells.

[0186] Fig. 31 Comparison of TCR α and β sequences used by Qa-1-FL9 or Qa-1-proinsulin specific CD8 T cells. Both CD8 T cell clones use Vα3.2 and Vβ5 sequences in the TCR of the cell clone.

[0187] Fig. 32 Qa-1 - TCRα and TCRβ sequences used by CD8 T cells specific for non-self peptides. Unlike autoreactive Qa-1-restricted CD8 T cells, these foreign Ag-specific Qa-1-restricted CD8 T cells do not use Vα3.2 and Vβ5 in their TCR.

[0188] Fig. 33 Conserved CDR1 and CDR2 sequences of the TCR α and β chains used by CD8 Tregs.

[0189] Fig. 34 Both CD4 and CD8 lineages contain regulatory T cells that are important in maintaining immune homeostasis. CD8 Tregs can be identified by CD44, CD122 and Ly49 surface markers and express the Helios transcription factor. CD8 Tregs depend on IL-15 for cell survival and activity. Continuous recognition of Qa-1-self peptide may be important for the maintenance of self peptide in the peripheral T cell pool.

[0190] Fig. 35 HLA-E and Qa-1 have evolved to present peptides in MHC class Ib molecules that resemble classical MHC class I molecules. The Ag-presentation capacity allows these MHC molecules to signal the cellular state (activated, stressed, transformed) of cells that can be recognized by CD8 Tregs.

[0191] Fig. 36 B6.Qa-1.D227K KI mice were generated by mutating AA position 227 on Qa-1 from D to K to disrupt CD8 coreceptor binding. The Qa-1.D227K mutant has no CD8 Treg-mediated immune regulation and is an important genetic model for studying the physiological functions of CD8 Tregs.

[0192] Fig. 37 Using a Balb / C→B6 heart transplant model, we demonstrated rapid rejection of the allograft in the absence of CD8 Treg-mediated immunosuppression (Qa-1.D227K mice). Heart graft tissue in D227K recipients showed immune cell infiltration and C4d deposition, indicating an enhanced immune response to the cardiac allograft.

[0193] Fig. 38 D227K mice exhibit enhanced Ab responses to cardiac allografts as evidenced by increased Tfh and GC B cell formation. Tfh cells express high levels of Qa-1 during immune responses to the allograft, rendering them susceptible to CD8 Treg-mediated suppression.

[0194] Figure 39A-B FL9 peptide libraries with variants with aa mutations at MHC anchoring positions were screened for the ability of peptides to activate FL9 TCR by incubating FL9 T cells with EL4 loaded with these peptides. CD69 expression by FL9 T cells was measured. Selected peptides were tested for their ability to stimulate FL9 T cells using titrated doses of peptides. FL9-68 peptide showed superior ability to stimulate FL9 T cells.

[0195] Fig. 40 Immunization of heart graft recipients with FL9-68 peptide reduced Tfh, GC B and plasma cell formation, indicating suppression of anti-alloimmune responses.

[0196] Fig. 41 Immunization of cardiac graft recipients with FL9-68 peptide reduced donor-specific Ab (DSA) production and graft tissue damage, indicating CD8 Treg-mediated suppression of anti-alloimmune responses.

[0197] Fig. 42 1×10 8A Qa-1 peptide yeast library consisting of random 9- and 10-mer peptides was constructed to screen a large pool of peptides allowing the identification of multiple peptide superagonists.

[0198] Fig. 43 Screening of the Qa-1-peptide yeast library identified peptides that showed a strong ability to stimulate FL9 T cells, and these peptides can be tested for their in vivo activity in expanding CD8 Tregs upon immunization.

[0199] Fig. 44 The surrogate peptides selected from the Qa-1-peptide yeast library were subjected to an endogenous peptide search to identify candidate self-peptides. Among these candidate peptides, a peptide derived from the Stag3 protein showed the ability to stimulate FL9 T cells after in vivo challenge with EL4 cells.

[0200] Example 6 Work title: Definition of an MHC class Ib-restricted subset of CD8+ regulatory T cells by TCR gene expression and target cell recognition summary Although most CD8+ T cells are equipped with the capacity to kill cells infected with microbial invaders, a subset of these cells can suppress immune responses (Nakagawa et al., 2018; Saligrama et al., 2019). Mouse and human CD8 regulatory activity is relegated to a small (<5%) subset of CD8 T cells that express a characteristic triad of surface receptors: CD44, CD122, and Ly49 / KIR (triad+). Analysis of autoimmune disorders has revealed that these CD8 T regulatory cells (CD8 Tregs) suppress disease via targeting MHC class Ia or class Ib expressed by CD4+ T helper cells. However, it is not known whether CD8 Tregs targeting class Ia or class Ib represent distinct subsets.

[0201] Analysis of a panel of more than 30 independent TCRs expressed by Qa-1-restricted CD8 T cells specific for two structurally distinct self-peptides revealed predominant usage of the TRAV9N3 and TRBV12-1 / 2 genes encoding the TCR Vα3.2 / Vβ5.1. The development and function of Ly49F+Vα3.2 / Vβ5.1+ was almost completely suppressed in Qa-1-deficient mice, indicating that the Qa-1-restricted subset of CD8 Tregs is restricted to CD8 cells expressing the Vα3.2 / Vβ5.1 TCR. Genetic Ab-based targeting of Qa1-restricted CD8 Tregs after immunization with OVA selectively increased the number and antibody responses of high-affinity tetramer+OVA CD4 T cells.

[0202] This study shows that the TCRs expressed by almost all Qa-1(MHC-E)-restricted CD8 Tregs are encoded by a highly conserved set of Vα+Vβ genes that detect and eliminate target CD4 T cells without systemic immunosuppression. Insights into the TCR-based specificity of CD8 Tregs have led to novel therapeutic approaches using synthetic peptide agonists to recruit CD8 Tregs to inhibit pathogenic or autoimmune Ab responses.

[0203] Prologue The immune system has evolved complex mechanisms that allow efficient destruction of microbial pathogens while sparing the host's own tissues. Maintenance of this delicate balance depends, in part, on regulatory T cells. Although most CD8+ T cells are equipped with the capacity to kill cells infected with microbial invaders, there is growing evidence that a subset of CD8+ T cells is genetically programmed to suppress immune responses (Nakagawa et al., 2018; J. Saligrama et al., 2019). Mouse and human CD8 regulatory activity is relegated to a small (<5%) subset of CD8 T cells that express a characteristic triad of surface receptors: CD44, CD122, and Ly49 / KIR (Kim et al., 2011; Saligrama et al., 2019), which mediates perforin-dependent killing of chronically activated autoreactive CD4 cells (Saligrama et al., 2019; Vivier and Anfossi, 2004). Analysis of autoimmune disorders has revealed that CD8 T regulatory cells (CD8 Tregs) suppress disease through recognition of MHC class Ia (Saligama et al., 2019) or class Ib (Nakagawa et al., 2018) on target CD4+ helper T cells. However, it is not known whether CD8 Tregs that recognize self-peptides associated with class Ia or class Ib represent separate or overlapping subsets. Here, we define the development of class Ib-restricted CD8 Tregs and distinguish them from class Ia-restricted CD8 Tregs by TCR expression and thymus-dependent development.

[0204] Recognition of MHC-E (human HLA-E or mouse Qa-1)-peptide complexes expressed by target CD4 cells is required for regulatory activity, but the identity of the TCR that recognizes class Ib (Qa-1) target ligands and associated self-peptides is unknown. Processing and cell surface expression of Qa-1 pMHC complexes by activated T cells depends on trimming by several enzymes, including endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP), which digests larger peptides into 9 / 10-mers that can efficiently bind to Qa-1. Shastri and coworkers have shown that reduced or absent ERAAP activity associated with chronic activation of CD4 T cells prevents the destruction of Qa-1-associated self-peptides, termed FL9, and the promotion of FL9-specific memory CD8 T cells (Lazaro et al., 2009; Nagarajan et al., 2012).

[0205] In addition to the FL9 self-peptide, chronically activated CD4 T cells also express self-peptides derived from the Hsp60 protein associated with Qa-1 that enable targeting by CD8 Tregs (Worth et al., 2013). To define the TCRs used to recognize these pQa-1 complexes, we cloned and analyzed two large sets of TCRs expressed by CD8 Tregs bound to either Qa-1-FL9 or Qa-1-Hsp60 tetramers. Analysis of a panel of more than 30 independent TCRs expressed by Qa-1-restricted CD8 T cells specific for these two structurally distinct self-peptides revealed enrichment of the same TRAV and TRBV genes encoding Vα3.2 and Vβ5.1, respectively.

[0206] Analysis of mice expressing TCR transgenes and non-transgenic mice demonstrated that mutation or deletion of Qa-1 significantly affected the maintenance and activation of the Vα3.2+ / Vβ5.1+ fraction of triad+ CD8 T cells, but not Vα3.2 - / Vβ5.1 -It was found that the triplet+ fraction had no detectable effect. Further analysis showed that the development of Qa-1-restricted CD8 Treg subsets requires TCR recognition of pQa-1 by the conserved TRAV and TRBV genes for both thymus-dependent differentiation and maintenance in peripheral lymphoid tissues. The MHC-E focus of the conserved TCR CDR1 / 2 sequence may enable these self-peptide-specific T cells to avoid negative selection in the thymus and mediate efficient recognition and elimination of Qa-1+CD4+Th cells in peripheral tissues.

[0207] Using systematic peptide mutagenesis at MHC contact residues of the FL9 self-peptide, we identified synthetic superagonist peptides that promoted efficient recruitment of CD8 Tregs in vivo. Indeed, immunization with these FL9 superagonist peptides promoted robust proliferation of CD8 Tregs and efficient inhibition of Tfh-driven Ab responses to conventional antigens in a preclinical model of solid organ transplantation. Furthermore, selective deletion of Qa-1-restricted Tregs by anti-TCR Abs that recognize conserved TRAV (Vα3.2) after immunization with a conventional Ag (OVA) significantly increased Qa-1hi CD4 T cells with relatively high avidity for the cognate Ag without affecting normal CD4 T cell activation.

[0208] This study demonstrates that the TCRs expressed by almost all Qa-1 (MHC-E)-restricted CD8 Tregs are Vα T cells that contain germline CDR1 / CDR2 sequences that can mediate efficient interactions with MHC-E-self-peptide complexes expressed by target cells. + Vβ + They are shown to be distinguished by a conserved set of genes. Sensitive detection of changes in MHC-E expression by pathogenic target CD4 cells can mediate elimination of pathogenic CD4 T cells without systemic immunosuppression. The TCR-based specificity of CD8 Tregs represents a novel therapeutic approach to attenuate pathogenic or unwanted Ab responses.

[0209] result Identification of TCR specific for Qa-1-FL9 self-peptide Insights into the specialized functions of both class Ia and class Ib restricted CD8 Tregs have mainly relied on the isolation of both subsets of CD8 Tregs using a triad of shared surface markers - CD44, CD122 and Ly49. Here, we distinguish class Ib (Qa-1 restricted) CD8 Tregs from class Ia restricted Tregs according to the expression of TCRs specific for FL9 and Hsp60, two structurally unrelated self-peptides that associate with Qa-1 and allow targeting of pQa-1 bearing CD4 cells (Lesworth et al., 2013; Nagarajan et al., 2012; Nakagawa et al., 2018). Using Qa-1-FL9 and Qa-1-Hsp60 peptide tetramers, we identified tetramer positive (tet + ) cells were detected, sorted and analyzed for TCR expression. Analysis of paired TCRs of 13 independent Qa-1-FL9 tetramer-binding cells revealed that 10 / 13 TCRs specific for Qa-1-FL9 tetramer expressed the TRAV9N3 gene (Vα3.2) and 9 / 13 expressed TRBV 12-1 / 2 (Vβ5.1,2) (Figure 45B, Figure 46A-B). Analysis of 11 independent Qa-1-Hsp60-specific CD8 T cells revealed that 8 / 11 also expressed TRAV9N3 / Vα3.2 and 6 / 11 expressed TRBV 12-1 / 2 / Vβ5.1,2 (Figure 45C, Figure 47A-B). Both TCR sets expressed nearly identical CDR1 and CDR2 sequences that interact with MHC contacts, but expressed different peptide-specific CDR3 regions. This conserved TCR repertoire used to recognize two structurally distinct peptides indicates that Qa-1 restriction depends on highly constrained interactions between Qa-1 and the CDR1 / CDR2 MHC contact regions of the TCR, and that peptide diversity depends on the CDR3 region.

[0210] TCR-dependent acquisition of a CD8 Treg phenotype by Qa-1-FL9-specific T cells To gain further insight into the contribution of TCR usage to the differentiation and function of autoreactive CD8 Tregs, we cloned each of the 16 TCR pairs specific for Qa-1-FL9 into retroviral vectors and expressed them as 58C(α - β - ) hybridoma cells. Expression of each TCR in 58C cells was accompanied by specific binding to Qa-1-FL9, but not Qa-1-Hsp60 tetramer (Figure 48A-B), likely reflecting the peptide-specific CDR 3 sequences described above (Figure 46A-B). The binding activity of each Qa-1-FL9-specific TCR was then determined according to a dose-response analysis of the concentration of FL9 peptide required for CD69 upregulation by each transduced hybridoma (Figure 48C). Qa-1-FL9-specific TCRs with intermediate (FL9.2) and high (FL9.8) binding activity to Qa-1-FL9 (Figure 45D) were further defined in an antigen dissociation assay, which confirmed the higher affinity of the FL9.8 TCR, as judged by the increased retention of the Qa-1-FL9 tetramer, compared to the FL9.2 TCR (Figure 45E).

[0211] To investigate the contribution of these TCRs to the selection and development of CD8 Tregs, BM chimeras were then generated after reconstitution of lethally irradiated B6 hosts with BM transduced with OT-I, FL9.2 or FL9.8 TCRs. Expression of the FL9.2 and 9.8 self-peptide specific TCRs was dependent on the insertion of (pES.42.1c and pKS913.CD18.31) vectors, which were used to generate TCR Tg mice using methods previously used to generate OT-I TCR Tg mice (Hogquist et al., 1994). Tg TCR in peripheral tissues +The proportion of T cells was approximately 90% for the three chimeras reconstituted with each TCR transgene (Fig. 45F, 49A). Analysis of thymocytes revealed that approximately 20% of FL9.2 thymocytes and 40% of FL9.8 thymocytes expressed negative selection markers, including caspase 3 and PD1, whereas OT-I thymocytes did not express these negative selection markers (Fig. 45F, 50A). Analysis of peripheral T cells revealed that the two FL9 TCR transgenes, but not OT-I, showed increased expression of CD44 and Ki67 (Fig. 45G, 50B) and reduced levels of TCR and CD8, a CD8 T cell phenotype associated with chronic activation by self-antigens (Schonrich et al., 1991; Xiao et al., 2007) (Fig. 50C). Chronic exposure of CD8 T cells to self-antigens can also upregulate expression of the NKG2D receptor (Dhanji et al., 2004; Zloza et al., 2011), which correlates with autoreactivity and potential immunoregulatory functions (Dai et al., 2009). FL9.8 T cells showed an age-dependent upregulation of NKG2D expression (>80% at 4 months), whereas FL9.2 T cells showed a more modest increase (20-40%) (Figures 49A, 49B).

[0212] Qa-1-dependent differentiation and maintenance of FL9 T cells We then analyzed the contribution of Qa-1 to the acquisition of the CD8 Treg phenotype in Qa-1 WT and Qa-1 KO FL9.2 and FL9.8 TCR Tg mice. Deletion of the Qa-1 restriction element reduced the number of FL9.2 CD8 T cells by approximately 80% (Figure 51A), while suppressing the expression of the higher affinity FL9.8 TCR + The same was true for T cells (Figure 52A). CD44 and NKG2D expression (Figures 51B, 52B) and the Ki67 proliferation marker were significantly impaired in both FL9.2 (Figure 51D) and FL9.8 T cells (Figure 52C) in peripheral lymphoid tissues.

[0213] Although the number of TCR Tg FL9.2 T cells was reduced by 70-80% in mice lacking or expressing deleted Qa-1, a significant proportion remained. We investigated whether these remaining TCR Tg CD8 cells in the spleen and lymph nodes of Qa-1-deficient mice were functionally impaired. - / - Transfer of residual FL9.2 T cells from mice into irradiated adoptive Qa-1 WT hosts revealed that only a small percentage (~10%) survived, compared to the robust survival of FL9 T cells from Qa-1 WT donors (Figure 51C). We then investigated whether recognition of Qa-1 in peripheral tissues is essential for the continued survival of mature Qa-1-restricted FL9 T cells that were initially differentiated in a Qa-1-sufficient (Qa-1 WT) environment. We found that FL9 CD8 T cells from Qa-1 WT donors transferred into Qa-1 KO or D227K KI hosts expressing the Qa-1 D227K point mutation, which compromises the interaction between Qa-1 and the CD8 coreceptor, showed a poor survival rate similar to that of CD8 TCR tg cells transferred from Qa-1-deficient donors (Figures 51C, 51D, 51E). These data indicate that a) the early intrathymic development of Qa-1-restricted CD8 Tregs requires expression of Qa-1 and b) the survival of Qa-1-restricted T cells in peripheral tissues also requires continued expression of the Qa-1 restriction element.

[0214] Vα3.2 + CD8 T cells represent Qa-1-restricted CD8 T cells Ly49 binding to FL9 or Hsp60 peptides associated with Qa-1 + Analysis of TCR expression by CD8 T cells revealed highly restricted TCR Vα and Vβ gene expression (Figures 45A-B; Figures 46A-B1, 47A-B2). Analysis of CD8 Tregs in non-transgenic mice revealed that CD44 + CD122 + Ly49 +It has been shown that CD8 Tregs expressing triads include both MHC class Ia and class Ib restricted CD8 Tregs (Kim et al., 2011; Saligrama et al., 2019). Therefore, we investigated whether the selective expression of the Vα3.2 and Vβ5 TCR pair described above for Qa-1 restricted CD8 Tregs could distinguish the MHC class Ib restricted Treg subset of CD8 Tregs in non-transgenic mice. + Vα3.2 in CD8 T cells + / Vβ5.1,2 + We found that the Vα3.2 subset was reduced by 60-80% in mice with Qa-1 deletion or Qa-1 D 227 K point mutation (Figure 53). In contrast, the Vα3.2 - / Vβ5.1,2 - Triad + (Ly49 + CD122 + CD44 + ) The number or percentage of CD8 cells was not significantly affected by Qa-1 deletion or mutation (Figure 51F). Finally, Vα3.2 in Qa-1 KO mice + / Vβ5.1,2 + The remaining 20-30% of CD8 T cells showed a 75% decrease in Ki67 expression, a significant increase over the previous study. Collectively, these findings suggest that Qa-1-restricted CD8 Tregs express a conserved set of TCRs as well as Ly49 + CD44 + CD122 + They are distinguished by the expression of a triplicate of markers and are shown to have the ability to recognize structurally unrelated self-peptides.

[0215] Detection and elimination of antigen-specific CD4 cells by Qa-1-restricted CD8 Tregs Although targeting of CD4 cells by CD8 Tregs may reflect TCR-dependent recognition of the pQa-1 complex expressed by activated CD4 cells (Nakagawa et al., 2018), the nature of the targeting complex is not well understood. Here, we investigated whether the expressed FL9-Qa-1 complex represents a major functional target on Ag-specific CD4 T cells. In vitro analysis showed that FL9 TCR Tg T cells were efficiently stimulated by activated CD4 T cells from B6(Qa-1 WT) mice, but not by activated CD4 T cells from B6.Qa-1 KO or B6.Qa-1-D227K KI mice (Figure 54A). Furthermore, Kb - / - Db - / - CD4 cells express FL9 TCR + Qa-1-FL9 elicited increased responses by CD8 T cells, reflecting the absence of a dominant Qdm default peptide from MHC class Ia that binds to Qa-1 (Figures 54A, 55A, 55B), and (2) activated ERAAP-deficient CD4 cells potently stimulated FL9 TCR Tg T cells, consistent with increased Qa-1-FL9 expression by cells lacking the ERAAP protease that normally destroys this peptide (Nagarajan et al., 2012) (Figures 54A, 55C). Together, these findings indicate that activated CD4 cells express an ERAAP-sensitive Qa-1-FL9 ligand that is recognized by Qa-1-FL9-specific CD8 Tregs.

[0216] CD4 cells with high affinity TCR for antigens can express high levels of Qa-1 (Fazilleau et al., 2009; Nakagawa et al., 2018). To investigate whether CD8 Tregs can selectively target activated CD4 T cells with high affinity for immunized or environmental antigens, we characterized the CD4 cells generated after immunization according to their expression of Qa-1-FL9 and their sensitivity to inhibition by CD8 Tregs in vivo. CD4 cells from WT B6 or B6-D227K mice immunized with OT-II peptide were transferred into B6 hosts with or without FL9 TCR Tg CD8 cells, followed by immunization with OT-II / CFA. OT-II tetramers representing CD4 cells with the highest avidity for immunizing Ag + Analysis of CD4 cells revealed that co-transfer of FL9 TCR Tg CD8 T cells resulted in over 90% Ova-specific (tetramer + ) CD4 T cells (Fig. 54B, top panel), whereas almost all activated CD4 cells that were tetramer-negative were spared (Fig. 54B, bottom panel). Suppression of Ag-specific CD4 cells was dependent on Qa-1 targeting, as FL9 CD8 T cells suppressed the response of B6(WT) CD4 T cells, but not B6-D227K CD4 T cells (Fig. 54B, top panel). These data demonstrate that a) the Qa-1-FL9 peptide complex is expressed on a significant proportion of CD4 cells after activation with Ag, and b) the expression of a highly avid TCR (tetramer-negative) for the cognate Ag is essential for the expression of FL9 CD8 T cells. + We show that CD4+ cells expressing Qa-1 (Qa-1hi) strongly express Qa-1 (Qa-1hi) and are specifically suppressed by Qa-1-restricted CD8 Tregs.

[0217] Based on the finding that Qa-1-restricted CD8 Tregs express the Vα3.2 / Vβ5 pair (FIG. 51F), we investigated the Vα3.2 / Vβ5 pair expression after immunization with OVA. +We investigated whether Ab-mediated depletion of T cells could enhance Ag-specific CD4 T cell responses. Near-complete depletion of Vα3.2+ T cells could be achieved based on blood analysis 8 days after anti-Vα3.2 Ab administration (FIG. 56). Furthermore, B6.WT mice, but not B6.Qa-1 D227K mice immunized with OVA / CFA and boosted with OVA / IFA, showed no significant depletion of Vα3.2+ T cells. + After T cell depletion, Ova-specific CD4 cells (I-Ab / Ova 323-339 Tet + ) increased in frequency (Figure 54C). 323-339 Tet + The significant increase in the levels of Qa-1 expressed by CD4 cells indicates that CD8 Tregs can selectively suppress high-affinity Ag-specific CD4 cells (Figure 54D). These data indicate that Qa-1-restricted CD8 cells express the Vα3.2 / Vβ5 pair, which can be targeted to selectively delete Qa-1-restricted CD8 Tregs.

[0218] Definition of FL9-superagonist peptides Immunization of mice with FL9 self-peptide did not induce detectable proliferation of CD8 Tregs (Figure 57), likely reflecting the relatively low Qa-1 binding affinity and the resulting weak TCR activation of the self-peptide (Kambayashi et al., 2004; Nakagawa et al., 2018; van Hall et al., 2010). We reasoned that efficient recruitment of Qa-1-restricted CD8 Tregs specific for self-peptides may require immunization with peptide analogs with increased Qa-1 binding activity. To systematically improve binding stability to Qa-1, we screened a peptide library consisting of approximately 100 aa-swapped variants of the FL9 peptide at MHC anchor positions 2, 3, 6, 7 and 9 (Figure 58A). FL9 TCR + 58C hybridoma was pulsed with FL9 peptide mutants, EL4 (Qa-1 +) cells and monitored for CD69 upregulation and TCR downregulation by 58C cells to define the stimulatory activity of each peptide variant (Figure 58B, left and center panels). The interaction of the Qa-1 peptide complex with the FL9 TCR was also assessed by measuring TCR trogocytosis, which reports the strength of TCR binding to a defined pMHC ligand (Li et al., 2019) (Figure 58B, right). Then, increased FL9 TCR expression was observed compared to the native FL9 peptide. + Mutant FL9 peptides that stimulated hybridoma responses (as judged by CD69 expression, TCR downregulation and increased trogocytosis) were subjected to dose-response analysis.

[0219] This analysis revealed that a FL9 peptide variant containing a P→L substitution at position 7 (termed FL9-68) displayed significantly enhanced dose-dependent stimulatory activity towards FL9.2 and FL9.8 TCRs compared to the cognate FL9 self-peptide (Figures 58C, 59). Immunization with the FL9-68 agonist peptide also enhanced the stimulatory activity of the congenic (CD45.1) TCR compared to the native FL9 peptide. + B6) Host or TCRα - / - After transfer into the host, FL9 TCR + CD8 T cells were activated (Fig. 58D). 323-339 CD45.1 by + Immunization of B6 hosts and subsequent analysis of activated CD4 T cells demonstrated that FL9-68 vaccination inhibited I-Ab / Ova 323-339 tet + The results revealed that FL9-Qa-1 inhibited the CD4 cell response by 50% but did not reduce the number or percentage of nonspecifically activated CD4 cells that did not bind to the FL9-Qa-1 tetramer (Figure 58E). Collectively, these findings suggest that FL9-specific CD8 Tregs can activate Qa-1 tetramers expressing high avidity TCRs against the immunizing antigen without systemic immunosuppression. hi These findings indicate that FL9-68 peptide analogs can be used to specifically recruit CD8 Tregs.

[0220] Peptide-dependent recruitment of CD8 Tregs and inhibition of alloimmunity The finding that CD8 Tregs primarily target high-affinity CD4 cells indicates that recruitment of CD8 Tregs may enable the suppression of destructive autoimmune or alloresponses without the attendant risks of systemic immunosuppression and increased vulnerability to pathogen infection. Antibody-mediated rejection (AMR) remains a major barrier to successful solid organ transplantation. As the pathogenic alloantibodies that mediate AMR are primarily produced by GC B cells after induction by Tfh cells (Kwun et al., 2017), increased expression of the Qa-1-FL9 complex by activated Tfh cells may enable the targeting and suppression of pathogenic CD4 cells by Ag-specific CD8 Tregs. Indeed, our recent analysis of allograft responses in B6.Qa-1 mutant (B6.Qa-1-D227K) mice showed that disrupting the interaction between CD8 Tregs and Qa-1 in recipients of a full allogeneic heart transplant model led to unchecked Tfh cell proliferation and accelerated Ab-mediated allograft injury ( Choi et al., 2020 ).

[0221] To test the ability of FL9-specific CD8 Tregs to inhibit anti-allograft responses, we first determined whether SA peptide-dependent proliferation of FL9-specific Tg T cells could suppress anti-allograft responses. Balb / C skin-bearing B6 mice given FL9 T cells with FL9-68 peptide SA expressed Ly49 + There was a 4-5-fold increase in FL9-specific CD8 cells in the CD8 T cell pool (Figure 60A). Ten days after transplantation with Balb / C cardiac grafts, secondary lymphoid tissues were enriched in Tfh cells (PD-1 + CXCR5 + CD4 + ), activated GC B cells (FAS + GL - 7 + B220 + ), and plasma cells (B220 - CD138 +(Fig. 60B) revealed a significant reduction in GC responses in hosts receiving FL9-68 / IFA but not IFA alone, along with a reduction in the number of CD4 T cells. Suppression of GC responses by expanded FL9 T cells was associated with upregulation of Qa-1 by Tfh cells in graft recipients (Fig. 60C). Furthermore, the reduction in GC responses was accompanied by a reduction in donor-specific antibody (DSA) responses and a marked reduction in graft pathology, as measured by the levels of C4d deposition and immune cell infiltration (Fig. 60D, E). The impact of CD8 Treg mobilization by SA peptide was also shown by a significantly prolonged graft survival after SA peptide vaccination compared to adjuvant alone (Fig. 60F), which was associated with a reduction in alloreactive activated CD4 cells (Fig. 61). These data indicate that peptide-based mobilization of CD8 Tregs that recognize pathogenic CD4 cells via TCR-Qa-1-peptide interactions represents a viable therapeutic strategy for the reduction of Ab-mediated injury in a mouse model of Ab-associated rejection of cardiac allografts.

[0222] Consideration There is increasing evidence that the contribution of CD8 Tregs to the suppression of pathogenic host responses depends on the specific recognition of MHC class Ia and Ib-peptide complexes expressed by activated CD4 effector cells (Kim et al., 2010; Nakagawa et al., 2018; Saligrama et al., 2019). However, the relationship of these CD8 Tregs to other members of this CD8 T cell subset and to the target pQa-1, as well as the relationship of these Qa-1-restricted CD8 Tregs to CD8 Tregs that recognize class Ia pMHC, is not understood. Characterization of the TCRs expressed by class Ib Qa-1-restricted CD8 Tregs revealed preferential usage of TRAV and TRBV genes, independent of their specificity for two structurally distinct self-peptides. Highly conserved expression of the CDR1 / CDR2 region that can interact with Qa-1 class Ib MHC molecules may govern TCR interactions with pMHC, allowing Qa-1-restricted CD8 T cells specific for diverse self-peptides to evade peptide-mediated negative selection. This Qa-1 central focus may also provide the ability to search for CD4 T cells with high avidity TCRs for immunizing Ags in peripheral lymphoid tissues.

[0223] The preferential TCR usage by clonal CD8 Tregs specific for self-peptides was also evident in polyclonal Qa-1-restricted CD8 Tregs. Indeed, almost all Qa-1-restricted CD8 Tregs in the polyclonal CD8 T cell pool expressed Vα3.2 / Vβ5.1, suggesting that this Treg subset is a triad. + Vα3.2 - / Vβ5.1 - In contrast to CD8 cells, they were dramatically reduced in Qa-1-deficient mice. Definition of the classical TCR pairs specific for CD8 Tregs in mice (and potentially for HLA-E-restricted CD8 Tregs in humans) may allow selective activation or deletion of these MHC-E-restricted CD8 Tregs by appropriate antibodies specific for these TCRs.

[0224] TCR TCR Tg mice were analyzed to define the development and function of CD8 Tregs specific for the FL9-Qa-1 complex. Expression of Qa-1 was essential for both early thymic development and later survival in peripheral tissues. Residual FL9-TCR generated in the absence of Qa-1 + CD8 T cells in the adoptive environment expressed the WT Qa-1 phenotype and showed significantly reduced survival and reduced activation (Figures 51C, 51E). + Since the Qa-1 dependency of the subset was also evident from the analysis of polyclonal CD8 Tregs in peripheral lymphoid tissues, these data indicate that TCR recognition bias against Qa-1 imposed by TRAV9 and TRBV12 gene expression and the highly conserved CDR1 / 2 may successfully allow these self-peptide-specific CD8 T cells to escape negative selection, allowing us to survey activated T cells for increased expression of pQa-1.

[0225] antigen Expression of Qa-1-FL9 complexes and a significant proportion of CD4 T cells may allow sensitive monitoring of the increase of pQa-1 by Ag-activated CD4 T cells, but not by non-specifically activated CD4 T cells, to stimulate CD8 Tregs in vitro and to allow targeting of CD8 Tregs in vivo. In vivo analysis revealed that suppressive activity was dependent on the specific recognition and elimination of relatively high avidity CD4 T cells for cognate Ag. High avidity tetramers + More than 90% of CD4 T cells were deleted, while nonspecifically activated tetramer-negative CD4 cells were spared. + This may reflect robust upregulation of Qa-1 by CD4 T cells (which have relatively high avidity for their cognate Ags), allowing efficient targeting of a major source of helper function and associated B cell-dependent Ab responses without systemic immunosuppression ( Fazilleau et al., 2009 ; Blasiak et al., 2013 ).

[0226] occurrence Analysis of T cells from FL9 TCR Tg mice also revealed that CD8 Tregs express low levels of CD8 and TCR, reflecting their self-reactivity (Figure 50C), and are maintained through continuous recognition of Qa-1. Because autoreactive CD8 Tregs may be significantly more anergic than T cells specific for foreign antigens, they may require stronger TCR stimulation for efficient activation and proliferation by agonist peptide analogs (Yu et al., 2015; Yu et al., 2004). To identify agonists with enhanced binding to pMHCI and increased immunogenicity, we screened APC peptides with altered residues at Qa-1 fixed positions. The FL9-68 peptide variant, containing a P→L amino acid exchange at position 7 of the cognate FL9 self-peptide, showed significantly enhanced stimulatory activity in vitro and in vivo. We found that activation of CD8 Tregs by encounter with FL9-68 peptides expanded CD8 Tregs, reduced Tfh cell and GC B cell responses, and reduced anti-graft Ab production in a heart transplant model (Figure 60A-F). Activation and expansion of clonal CD8 Tregs by superagonist peptides in organ transplant hosts can efficiently inhibit anti-graft Ab production and graft tissue damage.

[0227] Multiple autoimmune diseases are associated with autoantibody production secondary to dysregulation of high affinity Tfh proliferation (Kim et al., 2011; Mishra et al., 2021; Serr and Daniel, 2018). CD8 Treg-mediated control of self-Ab production is an essential mechanism for inhibiting autoimmune disease development (Nakagawa et al., 2018). In contrast to CD4 Tregs, CD8 Tregs can be expanded and activated in a pMHC-specific manner and can efficiently target CD4 Th cells with high affinity to cognate antigens, including self-Ags that upregulate pQa-1 complexes on their surface. Although we have demonstrated a peptide-dependent strategy to stimulate CD8 Tregs, the distinctive TCR Vα / Vβ usage by CD8 Tregs defined in this study may also be exploited for activation and expansion of CD8 Tregs in vivo. Activation of CD8 Tregs via anti-TRAV Ab (targeting the conserved CDR1 / CDR2) recruits a broad repertoire of CD8 Tregs and induces pQa-1 hi This may allow for efficient suppression of pathogenic CD4 cells. The efficacy of inhibition of CD8 Treg proliferation and subsequent auto-Ab production and associated pathology can be tested in mouse models of autoimmune diseases including EAE, T1D (NOD) and SLE (BXSB-Yaa). Expression of non-classical MHC gene products (MHC-E) in mouse (Qa-1) models and humans (HLA-E) is different from the highly polymorphic classical MHC genes, both of which are restricted to two alleles (Nakagawa et al., 2018). It is reasonable to speculate that HLA-E-restricted CD8 Tregs may also express limited TRAV and TRBV repertoires. Identification of homologous TCRs expressed by human CD8 Tregs may allow selective recruitment of HLA-E-restricted human CD8 Tregs as an attractive strategy for the treatment of antibody-mediated pathological conditions.

[0228] method mouse C57BL / 6(B6), B6.SJL-Ptprc a Pepc b / BoyJ(B6.CD45.1), Balb / C, C57BL / 6-Tg(TcrATcrb)1100Mjb / J(OT-1), B6.129P2-H2-K1 tm1Bpe H2-D1 tm1Bpe / DcrJ(Kb - / - Db - / - ), C57BL6-Tg(Ins2-TFRC / OVA)296Wehi / WehiJ(Rip-MoVA), and B6.129 S 2-TCRα tm1Mom / J(TCRα - / - ) mice were obtained from the Jackson laboratory (Bar Harbor ME). B6.Qa-1.D227K KI and B6.Qa-1 - / - (B6.129S6-H2-T23 tm1Cant FL9.2, FL9.8 TCR Tg mice were generated in-house as described below and maintained on Qa-1 WT and KO backgrounds. - / - Mice were provided by Dr. Kenneth Rock (UMASS Medical Center, Worcester). All experiments were performed in accordance with institutional guidelines approved by the Animal Care and Use Committee at Dana-Farber Cancer Institute (DFCI).

[0229] Antibodies and flow cytometry TCRβ (clone: ​​H57-597), CD3ε (17A2), CD44 (IM7), CD122 (TM-β1), Ly49C / I / F / H (14B11), Vα3.2 (RR3 -16), Vα2(B20.1), Vβ5.1 / 5.2(MR9-4), CD4(RM4-5), CD8α(53-6.7), CD8β(YTS156.7.7), CD69(H1 Fluorescently labeled antibodies against Qa-1b (6A8.6F10.1A6), PD-1 (29F.1A12), Act.Caspase 3 (5A1E), Ki67 (16A.8), B220 (RA3-6B2), Fas (SA367H8), CXCR5 (SPRCL5), FoxP3 (FJK-16S), NKG2D (A10) and NKG2A (20d5) were purchased from BD Biosciences, eBioscience and Biolegend. For detection of FL9 T cells, Qa-1 / FL9-PE, Qa1b / FL9-APC, Qa-1b / Hsp60p216-PE, Qa-1b / Hsp60p216-APC tetramers were generated by the NIH Tetramer Core Facility and provided for this study. I-Ab / Ova 323-339 Tetramer was purchased from MBL International.

[0230] Identification and isolation of FL9-specific TCR Bone marrow-derived DCs were cultured in the presence of 20 ng / mL GM-CSF to induce Kb - / - Db - / - DCs were generated from mice. Six days later, DCs were stimulated with 50 ng / mL LPS for 12 hours. DCs were irradiated (30 Gy) and pulsed with FL9 peptide by incubating with 10 μg / ml FL9 peptide at 37° C. for 2 hours. FL9-loaded Kb - / - Db - / - DCs were injected into WT B6 mice on days 0, 8, and 15. On day 22, Qa-1b / FL9 Tet+ cells were cloned into FACS-sorted CD44 + CD122 + Ly49 + CD8 subsets and single Tet +TCRa and TCRb chains were detected in the cells. Identification of TCRa and TCRb chains for each sorted cell was performed according to a previously published protocol (Hamana et al., 2016). Briefly, one-step RT-PCR was performed by adding RT-PCR mix to each well. The primers for the RT-PCR mix contain the leader and constant region sequences of TCR with an adapter sequence added to the 5' end of the leader primer (Hamana et al., 2016). The cDNA from this RT-PCR was used to amplify TCRa and TCRb separately using the nested PCR principle. The PCR products were sequenced using mTRAC_1st2R and mTRBC_1st2R primers for TCRα and TCRβ amplicons, respectively, and analyzed with the IMGT / V-Quest algorithm (http:J. / / www.imgt.org).

[0231] TCR + Hybridoma generation and TCR affinity testing The cDNAs encoding the TCR α and TCR β chains were inserted into a pMIG vector containing a GFP cassette, which was then transfected into PLAT-E cells using FuGENE6 (Promega). The culture medium was replaced with fresh medium at 24 hours, and the supernatant was harvested 72 hours after transfection to detect TCR. - / - The 58C hybridoma was used to transduce the TCRα and TCRβ pair expression on the surface of the 58C hybridoma was analyzed by staining with Qa-1b / FL9 tetramer, anti-CD3ε and anti-TCR Vβ Ab.

[0232] The relative affinities of the FL9.8 and FL9.2 TCRs were analyzed by measuring tetramer staining decay kinetics (Savage et al., 1999). FL9.8 TCR + and FL9.2 TCR + Hybridomas were incubated with PE-conjugated Qa-1b / FL9 tetramer in the presence of anti-Qa-1 Ab. Cells were fixed at different time points (0–120 min) after the start of incubation, and PE staining intensity was measured as an index of tetramer binding by flow cytometry.

[0233] Generation of FL9 TCR Tg mice The FL9.2 and FL9.8 TCR transgenes were generated by replacing the TCR V(D)J elements of the pES.42.1c and pKS 913.CD 18.31 vectors previously used to generate OT-I TCR Tg mice (Hogquist et al., 1994) with the TCRα and TCRβ cDNA fragments of the FL9.2 and FL9.8 TCRs, respectively. The vectors were linearized and used to target C57BL / 6 ES cells using standard methods at the Transgenic Core Facility of the Beth Israel Deaconess Medical Center. Founder lines for FL9.2 and FL9.8 TCR Tg mice were established after genotyping with the following primers: A common primer set for both FL9.2 and FL9.8 TCRα, 5'-CTAGAAGACTCAGGGTCTGA-3' and 5'-TCGGCACATTGATTTGGGAGTCA-3', amplifying 1 kbp for the transgene; Primer set 5'-ACACTGTCGCTGATTCTG-3' and 5'-GATGTGAATCTTACCGAGAACAGTCAGTCTGGTTC-3' for FL9.2 TCRb Primer sets for FL9.8 TCRβ 5'-TAACACTGTCGCTCGCTGAC-3' and ATACAGTCGTCTGCACTAG-3' both amplify 500 bp for the transgene

[0234] Peptide mutagenesis and superagonist peptide screening A peptide library was generated by single mutation of each Qa-1 fixed position (p2, 3, 6, 7 and 9) of the FL9 peptide (FYAEATPML) with 20 amino acids, which consisted of 96 FL9 variant peptides. +The 58C hybridoma was incubated with EL4 cells pulsed with each FL9 mutant. After 12 hours, the levels of CD69 expression and TCR expression were measured by flow cytometry. To analyze the binding strength of the FL9 TCR with the Qa-1-FL9 mutant, the FL9 TCR (Vα3.2) on EL4 cells was expressed as 100% CD69 and 100% TCR. + Vβ5 + Trogocytosis was measured directly by detection of FL9.8 TCR. + The 58C hybridoma was co-cultured with EL4 cells pulsed with FL9 mutant peptides from the library. After 2 hours, Vα3.2 + Vβ5 + The percentage of EL4 cells was assessed by flow cytometry as a measure of trogocytosis.

[0235] Adoptive transfer and in vivo suppression assays Qa-1.WT or Qa-1.D227K KI mice were cultured with 100 μg Ova in CFA. 323~339 The mice were immunized intraperitoneally with peptide / mouse. Seven days later, 1 × 10 5 CD25 - CD4 cells were isolated from these mice and transferred into WT B6 mice along with FL9 Tg T cells. WT B6 adoptive hosts were injected with 20 μg Ova in CFA in the footpad. 323-339 Seven days later, mice were immunized with I-Ab / Ova. 323-339 tetramer + The frequency and number of CD4 cells and activated CD4 cells were assessed in inguinal and popliteal LNs of B6 hosts by flow cytometry.

[0236] FL9 peptide immunization CD45.1 + B6 or TCRα - / - Mice were injected with 2 × 10 6 FL9.2 Tg T cells were transferred and subsequently immunized intraperitoneally with 100 μg FL9 or FL9-68 peptide in CFA. The proliferation and activation of FL9.2 Tg T cells in adoptive hosts was analyzed by assessing CFSE dilution, Ki67 and CD69 expression on days 3 and 6 after transfer.

[0237] Heart transplantation and immune response analysis B6 mice were immunized intraperitoneally with 50 μg FL9-68 / IFA or IFA alone on days 0 and 7, followed by BALB / C→B6 skin transplantation on day 10. FL9-68 / IFA or IFA immunization was repeated on days 10, 13, and 16. On day 27, fully vascularized Balb / C hearts were transplanted intraperitoneally into B6 mice using microsurgical techniques as previously described (Cai et al., 2016). Heart graft survival was determined by monitoring palpable heart beating. On day X after skin sensitization, the levels of FL9 T cells, Tfh, GC B, and plasma cells in the dLN were analyzed by flow cytometry. Serum was collected from heart transplant recipient B6 mice immunized with either FL9-68 / IFA or IFA alone on day 16. Serially diluted serum was diluted to 1 × 10 in a total volume of 100 μl PBS. 6 Following incubation with donor splenocytes for 30 min, surface-bound Abs on CD4 cells were detected using anti-CD4 (Biolegend, clone RM 4-5) and anti-mouse IgG1 Ab (BD Biosciences, clone A 85-1). Histological analysis of the heart explants was performed by InvivoEx using anti-C4d Ab (Hycult Biotech) and the Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories).

[0238] statistical analysis Prism v.9.0 (GraphPad Software) was used for statistical analysis. Statistical significance was calculated according to the Wilcoxon-Mann-Whitney rank sum test for comparison of two conditions, and the Kruskal-Wallis test for comparison of more than two conditions. A P value of <0.05 was considered statistically significant ( * =<0.05, ** =<0.01, *** =<0.001, **** =<0.0001).

[0239] References TIFF2024534863000003.tif11150TIFF2024534863000004.tif231149TIFF2024534863000005.tif224150TIFF2024534863000006.tif190150

[0240] Example 7 Fig. 63 We identified a human TCRα sequence that shows significant homology to mouse TCRα3.2, which is preferentially expressed by mouse CD8 Tregs. The human TRAV8.3 V gene shows the highest level of similarity to mouse TRAV9 (Vα3.2). This in silico data supports the KIR + HLA - E / FL9 tet + It is validated using TCR sequences obtained from CD8 T cells.

[0241] Example 8 Fig. 64 The DNA sequences encoding the heavy and light chains of the anti-Ly49F Ab are shown. CDRs 1, 2 and 3 for the heavy and light chains are shown in green (shaded).

[0242] Fig. 65 The amino acid sequences of the heavy and light chains of the anti-Ly49F Ab are shown. CDR1, 2, and 3 for the heavy and light chains are shown in green (shaded).

[0243] Fig. 66 The amino acids at each position of the FL9 peptide variants that yielded the highest FL9 T cell stimulatory capacity are selected and assembled as candidate amino acids that may exhibit superagonist activity.

[0244] Fig. 67 Human CD8 Tregs express a subset of KIR (Killer Cell Immunoglobulin-Like Receptors) (KIR3DL1, KIR2DL2, KIR2DL3) that can be used to identify these cells. KIR receptors are named based on the number of extracellular Ig-like domains (2D or 3D) of the receptor and by the length of the cytoplasmic tail of the receptor (long (L), short (S)). The amino acid sequences of KIR3DL1 and KIR2DL2 are shown.

[0245] Fig. 68 The full-length amino acid sequences of KIR2DL3 and Ly49F (mouse CD8 Treg marker) are shown.

[0246] Example 9 Fig. 69 Freeing the immune system from CD8 Treg-mediated suppression can enhance antitumor immune responses. Vaccination with modified MC38 cells (with increased immunogenicity) enhances the overall immune response, which may be accompanied by upregulation of Qa-1 on tumor-infiltrating immune cells. Anti-Ly49F Ab treatment (to deplete CD8 Tregs) can be combined with tumor cell vaccines, which almost completely inhibit tumor growth. The mIgG1 Fc portion of anti-Ly49F Ab (HBF-719) was used to inhibit Ly49F in vivo. + A chimeric anti-Ly49F Ab was generated by substituting mIgG2a, which efficiently depletes CD8 Tregs.

[0247] Figure 70 shows cancer and CD8 Treg depletion. Tumor growth in B6 mice inoculated with MC38 cells and treated with anti-Ly49 or anti-Va3.2 antibodies.

[0248] Figure 71 shows the effect of CD8 Treg depletion on antitumor immunity under conditions inducing Th1-biased immune responses, tested by injection of CpG-ODN 3 days after inoculation of MC38 cells. Treatment with CpG-ODN showed no significant therapeutic effect, whereas CD8 Treg depletion on days 8-10 strongly inhibited tumor growth, either alone or together with CpG-ODN treatment. These data indicate that strategies targeting CD8 Treg-dependent immune suppression may represent an effective approach to enhance immune responses against cancer, either as a monotherapy or in combination with other immunotherapies.

[0249] FIG. 72 shows cancer and CD8 Treg depletion enabling tumor vaccination.

[0250] FIG. 73 shows that CD8 Treg depletion reveals robust anti-tumor responses.

[0251] Figure 74A-B shows CD8 T cell profiles in tumors grown in mice treated with isotype or anti-Ly49F Ab. Shown are the percentage of CD8 T cells among CD45+ cells and the expression of GzmB in CD8 T cells (A), and the percentage of CD8 Tregs (CD44+CD122+Ly49+) among CD45+ cells (B).

[0252] Figure 75A-B shows NK and DC profiles within tumors grown in mice treated with isotype or anti-Ly49F Ab. (A) CD45 + (B) Percentage of NK cells in the tumor and expression of GzmB by NK cells in the tumor. + Intracellular MDSC and CD11c + I-Ab + Percentage of cDCs in cells.

[0253] Figure 76A-B shows tumor growth in B6 mice inoculated with B16 melanoma and treated with anti-Ly49F Ab (A). Number of cDCs and MDSCs in tumors treated with isotype or anti-Ly49F Ab (B).

[0254] Fig. 77 The effect of peptide superagonist (SA)-mediated expansion of CD8 Tregs on anti-allograft immunity to completely mismatched kidney grafts was also examined. Administration of FL9-68-IFA, but not IFA alone, significantly reduced donor-specific Ab production. Vaccination with FL9-SA significantly protected kidney grafts from antibody-mediated rejection, as judged by reduced C4d deposition in peritubular capillaries of renal allografts and a substantial prolongation of allograft survival compared to controls (mean survival: 20.5 vs. 40 days). These data indicate that peptide-based mobilization of CD8 Tregs that recognize pathogenic CD4 Tfh cells represents a promising therapeutic approach for drug-free reduction of Ab-mediated injury in a mouse model of kidney allografts.

[0255] Fig. 78 Qa-1-restricted CD8 Tregs arise in the thymus primarily through recognition of Qa-1 / peptide complexes by Qa-1 binding to CDR1 and CDR2 of a defined TCR (Va3.2 in mice), allowing the development of these autoreactive T cells without undergoing negative selection. Although Qa-1-restricted CD8 T cells with foreign reactivity can also arise, the TCR repertoire of these CD8 T cells can be diverse and can display a phenotype similar to conventional MHC class Ia-restricted CD8 T cells. In the periphery, CD8 Tregs recognize the Qa-1 / FL9 complex expressed by activated CD4 cells resulting from ERAAP functional deficiency following Ag-specific stimulation of CD4 cells. TCR-dependent targeting of Ag-activated CD4 cells allows the development of Qa-1 without systemic immunosuppression. hi It allows selective suppression of CD4 cells. Identification of peptide superagonists with the ability to stimulate FL9 T cells allows the expansion / activation of CD8 Tregs by peptide immunization. Peptide-dependent mobilization of CD8 Tregs can be applied in pathogenic conditions where Ab production can cause fatal diseases, including autoimmunity and Ab-associated organ rejection. For example, inhibition of donor-specific Abs in the context of heart and kidney transplantation allows the extension of graft survival in mice.

[0256] Example 10 T cell receptor usage determines thymic differentiation and function of MHC class Ib-restricted CD8+ regulatory T cells summary Most CD8 + T cells have the ability to kill cells infected by microbial invaders, but a subset can also regulate immune responses. In mice and humans, CD8 regulatory activity is consigned to a small (<5% CD8 cells) subset that expresses a characteristic triad of surface receptors, CD44, CD122, and Ly49 / KIR. + Activated CD4 T cells are eliminated through targeting of MHC class Ia or class Ib expressed by helper T cells. Here, we characterize CD8 regulatory T cells (Tregs) that target class Ib according to TCR expression, thymus-dependent development and regulatory function. Expression of TRAV9N3 and TRBV12-1 / 2 TCR genes encoding the Va3.2 / Vb5.1 TCR pair allows recognition and elimination of target cells expressing Qa-1 associated with several distinct self-peptides, including FL9 and Hsp60-216. This interaction selectively boosts high-affinity CD4 T cell responses, sparing non-specifically activated CD4 cells and selectively reducing pathogenic antibody responses without systemic immunosuppression.

[0257] Definition of the TCR specific for Qa-1-FL9 enabled systematic mutagenesis of the FL9 self-peptide and identification of synthetic superagonist peptides that promoted robust recruitment and expansion of CD8 Tregs and efficient inhibition of Tfh-driven Ab responses to both conventional and transplantation antigens. Recruitment of CD8 Tregs with agonist FL9 peptides in preclinical models of MHC-mismatched heart or kidney transplantation reduced Tfh-driven alloantibody responses and significantly extended organ graft survival. These insights into the TCR-based specificity of CD8 Tregs and their peptide ligands pave the way for new therapeutic approaches to attenuate pathogenic Ab responses.

[0258] Prologue The immune system has evolved complex mechanisms that allow efficient destruction of microbial pathogens while sparing the host's own tissues. Maintenance of this balance depends, in part, on regulatory T cells. Most CD8 + T cells have the ability to kill cells infected with microbial invaders, but mouse and human CD8 + There is growing evidence that subsets of T cells are genetically programmed to suppress immune responses. 1~3 Mouse and human CD8 regulatory activity is mediated by chronically activated autoreactive CD4 cells. 2,5 A distinct triad of surface receptors capable of mediating perforin-dependent killing of CD44, CD122 and Ly49 / KIR 2~4 Analysis of autoimmune disorders has revealed that CD8 regulatory T cells (CD8 Tregs) mediate the activation of target CD4 + MHC class Ia or class Ib expressed by helper T cells (MHC-E: mouse Qa-1 and human HLA-E) 1、2 We now show that class Ib-restricted CD8 Tregs inhibit pathogenic responses through the recognition of self-peptides associated with Qa-1. Here, we define class Ib-restricted CD8 Tregs according to their TCR expression, thymus-dependent development, and specific recognition mechanism that allows the elimination of activated CD4 T cells expressing the appropriate Qa-1-self-peptide complex.

[0259] Cell surface expression of Qa-1-peptide complexes by activated T cells depends on trimming by several enzymes, including endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP), which digests larger peptides into 9 / 10-mers that efficiently bind to Qa-1. Shastri and coworkers showed that reduced or absent ERAAP activity associated with chronic activation of CD4 T cells is characterized by the expression of a Qa-1-associated self-peptide called FL9 and an increase in FL9-specific memory CD8 T cells. 6,7Chronically activated CD4 T cells also express self-peptides derived from the Hsp60 protein associated with Qa-1 and recruit CD8 Tregs (Leavenworth et al., 2013). To define the TCRs used to recognize these pQa-1 complexes, we investigated the Qa-1 7,8 We cloned and analyzed two large sets of TCRs expressed by CD8 Tregs that recognize two structurally distinct self-peptides, FL9 and Hsp60-216, complexed with Qa-1 in peripheral tissues. This analysis revealed enrichment for TRAV and TRBV genes encoding highly conserved CDR1 and CDR2 regions and highly variable and relatively heterogeneous CDR3 sequences associated with the recognition of either self-peptide. Analysis of TCR transgenic CD8 T cells engaging these receptors demonstrated that CD8 Tregs are able to avoid negative selection by self-peptides and express Qa-1 in peripheral tissues. + CD4 + A strong bias towards MHC (Qa-1) recognition was identified, which may enable efficient recognition and elimination of Th cells. Indeed, mutation or deletion of Qa-1 almost completely prevented intrathymic development, peripheral survival of CD8 Tregs, and suppressed targeting and elimination of activated CD4 T cells. Furthermore, since almost all Qa-1-restricted CD8 Tregs expressed this Vα3.2 / Vβ5.1 pair, CD8 Tregs could also abolish CD8 Treg activity by depletion of CD8 T cells that expressed the TCR Vα3.2 / Vβ5.1 pair.

[0260] Identification and expression of the TCR expressed by the regulatory lineage of Qa-1-restricted CD8 T cells also allowed the definition of synthetic variants of the FL9 self-peptide that efficiently recruited CD8 Tregs during immune responses and suppressed pathogenic CD4 cells. This approach was used to inhibit Tfh-driven alloantibody responses and prolong survival of cardiac and renal allografts in preclinical mouse models of organ transplantation. These insights into the TCR-based specificity of CD8 Tregs point to new therapeutic approaches to attenuate pathogenic or unwanted Ab responses.

[0261] result Identification of TCR specific for Qa-1-self-peptide complex Insights into the specialized functions of both class Ia and class Ib restricted CD8 Tregs have relied primarily on the isolation of both subsets of CD8 Tregs using a triad of shared surface markers - CD44, CD122 and Ly49. Here, class Ib (Qa-1 restricted) CD8 Tregs were distinguished from class Ia restricted Tregs according to their expression of TCRs specific for two structurally unrelated self-peptides, FL9 and Hsp60, which are presented by Qa-1 and allow specific targeting of CD4 cells by CD8 Tregs. 1、7、8 .

[0262] Using Qa-1-FL9 and Qa-1-Hsp60 peptide tetramers, we identified tetramer-positive (tet +) cells were detected, sorted and analyzed for TCR expression. Analysis of paired TCRs from 12 independent Qa-1-FL9 tetramer-binding cells revealed that 9 / 12 TCRs specific for Qa-1-FL9 tetramer expressed the TRAV9N3 gene (Vα3.2) and 9 / 12 expressed TRBV12-1 / 2 (Vβ5.1,2) (Figure 79B, Figure 85A-B). Analysis of 11 independent Qa-1-Hsp60-specific CD8 T cells revealed that 8 / 11 also expressed TRAV9N3 / Vα3.2 and 6 / 11 expressed TRBV 12-1 / 2 / Vβ5.1,2 (Figure 79C, Figure 86 A-B). Both sets of TCRs expressed nearly identical CDR1 and CDR2 sequences (which may represent MHC contact elements) but had different peptide-specific CDR3 regions. Expression of TRAV9N3 and TRBV12-1 / 2 pairs is essential for the development of high-affinity FL9 T cells, as TCRs composed of non-TRAV9N3 or TRBV12-1 / 2 pairs show a marked decrease in binding affinity to the Qa-1-FL9 complex (Figures 85A-B, 48-C). This conserved TCR repertoire used for the recognition of two structurally distinct self-peptides presented by Qa-1 indicates the contribution of interactions between the highly conserved CDR1 / CDR2 TCR regions and Qa-1, together with secondary interactions between CDR3 and FL9 and Hsp60 self-peptides.

[0263] We next investigated whether Qa-1-restricted CD8 Tregs from non-transgenic mice could also express the Vα3.2 and Vβ5 TCR pairs. + (Ly49 + CD122 + CD44 + )Vα3.2 + / Vβ5.1,2 + We found that CD8 Tregs were reduced by 60-80% in mice with Qa-1 deletion or the Qa-1 D227K point mutation (Figure 79D, Figures 87A-B). In contrast, Vα3.2 - / Vβ5.1, 2 - A triad of +The number or percentage of CD8 T cells was not affected by either Qa-1 deletion or mutation (Figure 79D). Furthermore, the remaining triads in Qa-1 KO mice + Vα3.2 + / Vβ5.1,2 + CD8 Tregs showed a 50-75% reduction in the Ki67 proliferation marker (Figure 79D). Collectively, these findings suggest that Ly49 + CD44 + CD122 + We show that Qa-1-restricted CD8 Tregs expressing the marker triad are also distinguished from MHC class Ia-restricted CD8 Tregs by the expression of a conserved set of TCRs with the capacity to recognize pQa-1 complexes containing structurally unrelated self-peptides.

[0264] CD8 Treg phenotype of Qa-1-FL9-specific T cells To gain further insight into the contribution of TCR usage to the differentiation and function of autoreactive CD8 Tregs, we cloned each of the 12 TCR pairs specific for Qa-1-FL9 into retroviral vectors and expressed them as 58C(α - β - ) hybridoma cells. Expression of each TCR in 58C cells was accompanied by specific binding to Qa-1-FL9, but not Qa-1-Hsp60 tetramers (Figures 48A, 48B), likely reflecting the peptide-specific CDR3 sequences described above (Figures 46A-B). The binding activity of each Qa-1-FL9-specific TCR was then determined according to a dose-response analysis of the concentration of FL9 peptide required for CD69 upregulation by each transduced hybridoma (Figure 48C). Qa-1-FL9-specific TCRs with intermediate (FL9.2) and high (FL9.8) binding activity to Qa-1-FL9 (Figure 79E) were further defined in an antigen dissociation assay, which confirmed the higher affinity of the FL9.8 TCR as judged by increased retention of the Qa-1-FL9 tetramer compared to the FL9.2 TCR (Figure 79F).

[0265] We generated Tg mice expressing FL9.2 and 9.8 self-peptide specific TCRs using methods previously used to generate OT-I TCR Tg mice dependent on vector insertion (pES.42.1c and pKS913.CD18.31). 9 To investigate the contribution of these TCRs to the selection and development of CD8 Tregs, BM chimeras were then generated after reconstitution of lethally irradiated B6 hosts with BM transduced with OT-I, FL9.2 or FL9.8 TCRs. + The proportion of T cells was approximately 90% in the three BM chimeras reconstituted with each TCR transgene (Figure 79G, Figure 0A). Analysis of thymocytes revealed that approximately 20% of FL9.2 thymocytes and 40% of FL9.8 thymocytes expressed negative selection markers including caspase 3 and PD1, whereas OT-I thymocytes did not express these negative selection markers (Figure 79G, Figure 50A). Analysis of peripheral T cells revealed that the two FL9 TCR transgenes, but not OT-I, showed increased expression of CD44 and Ki67 (Figure 79H, Figure 50B) and reduced levels of TCR and CD8, i.e., CD8 T cell phenotypes associated with chronic activation by self-antigens. 10、11 (Fig. 50C). Chronic exposure of CD8 T cells to self-antigens can also upregulate the expression of the NKG2D receptor. 12、13 , which correlates with its immunoregulatory function. 14 FL9.8 T cells showed an age-dependent upregulation of NKG2D expression (>80% at 4 months), whereas FL9.2 T cells showed a more modest increase (20-40%) (Figure 4A-B).

[0266] We next investigated whether FL9 T cells acquire and maintain the characteristic Treg phenotype described here and previously. 1 All TCRs in the thymus +The cells expressed Tg TCRα and TCRβ (Vα3.2, Vβ5) similar to the levels of developing OT-I thymocytes (Vα2, Vβ5), whereas only FL9 T cells expressed the classical CD8 Treg TF Helios (Figure 80A). After maturation, peripheral FL9 T cells maintained this characteristic Treg phenotype and expressed both Helios TF and Ly49 (Figure 80B). We then defined the contribution of Qa-1 by the development of CD8 Tregs in Qa-1 WT and Qa-1 KO FL9.2 Tg mice. In the absence of the Qa-1 restriction element, FL9 T cells did not develop: only about 1% of thymocytes in the Qa-1 KO thymus expressed the FL9 TCR, compared to about 60% of thymocytes that expressed the FL9 TCR in WT Tg mice (Figure 80C). This data indicates that Qa-1 is a TCR that has previously been shown to be associated with this TCR. 7、15、16 Deletion of the Qa-1 restriction element also reduced the number of FL9.2 CD8 T cells by approximately 80% (Figure 80D), indicating that the Qa-1 restriction element is a single restricting MHC molecule for FL9 T cells, rather than cross-reactive with other classical and non-classical MHC molecules. + The same was true for T cells (Figure 88A). Furthermore, expression of CD44 and NKG2D receptors (Figures 80E, 88B) and Ki67 proliferation marker was significantly decreased in both FL9.2 (Figure 80F) and FL9.8 T cells (Figure 88C) in peripheral lymphoid tissues.

[0267] Although the number of TCR Tg FL9.2 T cells was reduced by 70-80% in mice that lacked or expressed deleted Qa-1, a significant proportion remained. We investigated whether these remaining TCR Tg CD8 cells in the spleen and lymph nodes of Qa-1-deficient mice were functionally impaired. Transfer of the remaining FL9.2 T cells from Qa-1 KO mice into irradiated adoptive Qa-1 WT hosts revealed that only a small proportion (~10%) survived, compared to the robust survival of FL9 T cells from Qa-1 WT donors (Figure 80G). We then investigated whether recognition of Qa-1 in peripheral tissues is essential for the continued survival of mature Qa-1-restricted FL9 T cells that were initially differentiated in a Qa-1-sufficient (Qa-1 WT) environment. We found that transfer of FL9 CD8 T cells from Qa-1 WT donors into Qa-1 KO or D227K KI hosts (expressing the Qa-1 D227K point mutation that compromises the interaction between Qa-1 and the CD8 coreceptor) showed poor survival rates similar to those of CD8 TCR tg cells transferred from Qa-1-deficient donors (Figure 80G-H). These data indicate that Qa-1 expression is essential for both the early intrathyroidal development and survival of Qa-1-restricted CD8 Tregs.

[0268] Detection and elimination of antigen-specific CD4 cells by Qa-1-restricted CD8 Tregs Although targeting of CD4 cells by CD8 Tregs may reflect TCR-dependent recognition of the pQa-1 complex expressed by activated CD4 cells, 1 However, the nature of the target complex is not well understood. Here, we investigated whether the FL9-Qa-1 complex represents a major functional target on Ag-specific CD4 T cells. In vitro analysis showed that FL9 TCR Tg T cells were efficiently stimulated by activated CD4 T cells from B6(Qa-1 WT) mice, but not by activated CD4 T cells from B6.Qa-1-D227K KI mice (Figure 81A, Figures 60A, 61A). Furthermore, the Kb - / - Db - / - CD4 cells express FL9 TCR +induced an increased response by CD8 T cells, reflecting the absence of a dominant Qdm default peptide derived from MHC class Ia that competitively binds to Qa-1 (Figure 81A, Figure 54A, 54A). Activated ERAAP-deficient CD4 cells potently stimulated FL9 TCR Tg T cells, consistent with increased Qa-1-FL9 expression by cells lacking the ERAAP enzyme that normally destroys this peptide. 7 (FIG. 81A, FIG. 54A, 55A and 55B). Taken together, these findings indicate that activated CD4 cells express the ERAAP-sensitive Qa-1-FL9 ligand that is recognized by Qa-1-FL9-specific CD8 Tregs.

[0269] CD4 cells expressing high affinity TCRs can co-express high levels of Qa-1 1、17 To investigate whether CD8 Tregs can selectively target activated CD4 T cells with high affinity for immunized or environmental antigens, we characterized the CD4 cells generated after immunization according to their expression of Qa-1-FL9 and their sensitivity to inhibition by CD8 Tregs. CD4 cells from WT B6 or B6-D227K mice immunized with OT-II peptide were transferred into B6 hosts with or without FL9 TCR Tg CD8 cells, followed by immunization with OT-II / CFA. OT-II tetramers representing CD4 cells with the highest avidity for immunizing OVA were expressed in 100% immunized mice. + Analysis of CD4 cells revealed that co-transfection of FL9 TCR Tg CD8 T cells resulted in >90% OVA tetramer + Inhibited CD4+ cells (Fig. 81B, upper panel), and almost all activated (CD44 + CD62L -) CD4 T cells were spared (Fig. 81B, lower panel). Suppression of Ag-specific CD4 T cells was dependent on Qa-1 targeting, as FL9 CD8 T cells suppressed the response of B6(WT) CD4 T cells, but not B6-D227K CD4 T cells (Fig. 81B, upper panel). These data demonstrate that a) the Qa-1-FL9 peptide complex is expressed on a significant proportion of CD4 T cells after activation with Ag, and b) the Qa-1-FL9 peptide complex expresses a highly avid TCR (tetramer) for the cognate Ag. + ) expressing Qa-1 (Qa-1 hi ) and are efficiently suppressed by Qa-1-restricted CD8 Tregs.

[0270] Based on the finding that Qa-1-restricted CD8 Tregs predominantly express the Va3.2 / Vb5 pair (Figure 56), we investigated the expression of Va3.2 after immunization with OVA. + We investigated whether Ab-mediated depletion of T cells could enhance Ag-specific CD4 T cell responses. + Near-complete depletion of T cells could be achieved after anti-Va3.2 Ab administration (FIG. 56). B6.WT mice, but not B6.Qa-1 D227K mice immunized with OVA / CFA and boosted with OVA / IFA, were found to be resistant to Vα3.2 + After T cell depletion, Ova-specific CD4 cells (I-Ab / Ova 323-339 Tet + ) increased in frequency (Figure 81C). b / Ova 323-339 Tet + The significantly increased Qa-1 levels expressed by CD4 cells suggest that CD8 Tregs express Qa-1 hi This supports previous findings that Ag-specific CD4 cells can be selectively targeted (Figure 81D). These data also indicate that Qa-1-restricted CD8 cell targeting with anti-TCRα3.2 Ab can be used to regulate CD8 Treg activity.

[0271] Definition of FL9-superagonist peptides Because chronically activated CD4 cells express the Qa-1-FL9 complex, in vivo expansion of Qa-1-FL9-specific CD8 Tregs may facilitate the elimination of these CD4 T cells in a clinical setting. However, immunization of mice with the FL9 self-peptide did not induce detectable proliferation of CD8 Tregs (Figure 57), likely reflecting the relatively low Qa-1 binding affinity and the resulting weak TCR activation by this self-peptide. 1、18、19 We reasoned that efficient recruitment of self-peptide-specific Qa-1-restricted CD8 Tregs may require immunization with peptide analogs with increased Qa-1 binding activity. To systematically improve binding stability to Qa-1, we screened a peptide library consisting of approximately 100 aa-exchange mutants of the FL9 peptide at MHC anchor positions 2, 3, 6, 7, and 9 (Figure 82A). FL9 TCR + 58C hybridoma was pulsed with FL9 peptide mutants, EL4 (Qa-1 + ) cells and monitored for CD69 upregulation and TCR downregulation to define the stimulatory activity of each peptide variant (Figure 82B, left and center panels). The interaction of Qa-1 peptide complexes with the FL9 TCR was also assessed by measuring TCR trogocytosis, which reports the strength of TCR binding to a defined pMHC ligand. 20 (Figure 82B, right panel). Then, increased FL9 TCR compared to the native FL9 peptide. + Mutant FL9 peptides that stimulated hybridoma responses (as judged by CD69 expression, TCR downregulation and increased trogocytosis) were subjected to dose-response analysis.

[0272] This analysis revealed that a FL9 peptide variant containing a P→L substitution at position 7 (termed FL9-68) displayed significantly enhanced dose-dependent stimulatory activity towards FL9.2 and FL9.8 TCRs compared to the cognate FL9 self-peptide (Figure 82C, Figure 58C). Immunization with the FL9-68 agonist peptide significantly enhanced the stimulatory activity of the congenic (CD45.1) TCR compared to the native FL9 peptide. +B6) Host or TCRα - / - After transfer into the host, FL9 TCR + CD8 T cells were activated (Fig. 82D). 323-339 By CD45.1 + Immunization of B6 hosts and subsequent analysis of activated CD4 T cells demonstrated that FL9-68 vaccination inhibited IA b / Ova 323-339 tet + The results revealed that Tet1 inhibited the CD4 cell response by 50%, but did not decrease the number or percentage of non-specifically activated CD4 cells that did not bind to FL9-Qa-1 tetramers (Figure 82E). + Findings that target CD4 cells (Qa-1 hi ) was consistent with the observation that defective CD8 Treg activity in Qa-1.D227K mutant mice resulted in a significant increase in high affinity Ab responses to the immunizing Ag (NP-Ova) as well as to anti-dsDNA Ab (Figure 82F). Collectively, these findings suggest that FL9-specific CD8 Tregs express high avidity TCRs to the immunizing antigen in Qa-1 mice without systemic immunosuppression. hi We show that FL9-68 peptide analogs preferentially suppress CD4 cells, demonstrating that they can be used to purposefully recruit CD8 Tregs in a clinical setting.

[0273] Peptide-dependent recruitment of CD8 Tregs and inhibition of alloimmunity The finding that CD8 Tregs primarily target high-affinity CD4 cells indicates that recruitment of CD8 Tregs may allow the suppression of destructive autoimmune or alloresponses without the attendant risks of systemic immunosuppression and increased vulnerability to pathogen infection. Antibody-mediated rejection (AMR) remains a major barrier to successful solid organ transplantation. Because pathogenic alloantibodies that mediate AMR are primarily produced by GC B cells following induction by Tfh cells, 21Increased expression of Qa-1-FL9 complexes by activated Tfh cells may enable targeting and suppression of pathogenic CD4 cells by Ag-specific CD8 Tregs. Indeed, our recent analysis of allograft responses in B6.Qa-1 mutant (B6.Qa-1-D227K) mice showed that disrupting the interaction between CD8 Tregs and Qa-1 in recipients of a complete allogeneic heart transplant model led to unrestrained Tfh cell proliferation and accelerated Ab-mediated allograft damage. 22 .

[0274] Because transplantation of hearts into non-sensitized hosts induces strong cellular rejection, we transplanted cardiac allografts into hosts previously sensitized to skin allografts, but only a weak humoral response was generated. To test the ability of FL9-specific CD8 Tregs to inhibit humoral allograft rejection, we first examined whether the expansion of FL9-specific T cells by FL9-68 peptide could suppress anti-cardiac graft responses. 23 Vaccination of B6 mice bearing Balb / C skin allografts with FL9-68 peptides enhances the expression of Ly49F + We showed a 4-5-fold increase in FL9-specific CD8 cells (Figure 83A). Alloantigen-sensitized B6 hosts (derived from Balb / C skin allografts) were transplanted with Balb / C cardiac allografts, which are normally rejected following a strong antigraft antibody response. Seven days later, we demonstrated a reduction in GC responses in B6 hosts vaccinated with FL9-68 / IFA but not IFA alone, as well as a reduction in Tfh cells (PD-1 + CXCR5 + CD4 + ), activated GC B cells (FAS + GL-7 + B220 + ), and plasma cells (B220 - CD138 + The suppression of GC responses after FL9-68 administration was also observed in the Qa-1 GC subpopulation, as measured by the levels of C4d deposition and immune cell infiltration (Figure 83B). hiThis was associated with increased numbers of Tfh cells (Figure 83C), reduced donor specific antibody (DSA) responses, and a significant reduction in graft pathology (Figures 83D-E). The impact of FL9-68 peptide-mediated CD8 Treg mobilization was also evident from the significantly extended graft survival time following FL9-68 peptide vaccination compared to adjuvant administration alone (Figure 83F).

[0275] The effect of peptide superagonist-mediated expansion of CD8 Tregs on anti-allograft immunity to completely mismatched kidney grafts was also examined (Figure 84A). Analysis of allograft-draining lymph nodes 20 days after kidney transplantation (n=5-7 / group) revealed a >7-fold increase in FL9-specific Tregs as measured by FL9-Qa-1 tetramers (Figure 84B). Administration of FL9-68-IFA, but not IFA alone, enhanced the proliferation of Tfh cells (PD-1 + CXCR5 + CD4 + ), activated GC B cells (FAS + GL-7 + B220 + ), and plasma cells (B220 - CD138 + (B6) Celltrace Violet™ stained CD4 + Incubation of T cells with (irradiated) donor (Balb / c) lymphocytes also demonstrated that FL9-68 vaccination inhibited the expression of CD44 + Vaccination with FL9-68 was shown to reduce CD4 T cell memory responses (Figure 84E). Vaccination with FL9-68 induced significant protection from antibody-mediated allograft rejection, as judged by reduced C4d deposition in peritubular capillaries of renal allografts (Figures 84F-G) and a substantial prolongation of allograft survival compared to controls (mean survival time: 20.5 days vs. 40 days) (Figure 84H).

[0276] Collectively, these data indicate that peptide-based mobilization of CD8 Tregs that recognize pathogenic CD4 Tfh cells represents a promising therapeutic approach for drug-free reduction of Ab-mediated injury in mouse models of cardiac and renal allografts.

[0277] Consideration There is increasing evidence that suppression of pathogenic host responses by CD8 Tregs depends on precise recognition of MHC class 1b-self-peptide complexes expressed by activated CD4 effector cells. 1、2、24 However, the basis of this recognition and targeting of chronically activated CD4 T cells is unclear. Our characterization of the TCR expressed by Qa-1-restricted CD8 Tregs surprisingly revealed the restricted expression of the CDR1 / CDR2 region expressed by both the TCR α and β chains. This interaction may allow Qa-1-restricted CD8 T cells specific for diverse self-peptides to escape peptide-mediated negative selection in the thymus and equip them with the ability to explore CD4 T cells that express high-affinity TCRs and strongly upregulate Qa-1 for immunizing antigens. This preferential TCR usage by self-peptide-specific CD8 Tregs expressing TCR transgenes was evident in polyclonal Qa-1-restricted CD8 Tregs. Almost all Qa-1-restricted CD8 Tregs in the polyclonal CD8 Treg population expressed Vα3.2 / Vβ5, which was dramatically reduced in mice with Qa-1 deletions or mutations that impair the interaction with pQa-1. In contrast, Ly49F did not express Vα3.2 / Vβ5. + Helios + CD8 T cells were not affected by changes in Qa-1 expression.

[0278] Analysis of the development of immature thymocytes expressing the Vα3.2 / Vβ5 TCR transgene specific for the Qa-1-FL9 self-peptide demonstrated the acquisition of a CD8 Treg phenotype in the thymus and periphery that was dependent on the expression of Qa-1. Indeed, the FL9-TCR persisted in the absence of Qa-1. +The remaining population of CD8 T cells showed significantly reduced survival and decreased activation in the adoptive environment expressing the WT Qa-1 phenotype (Figure 80G-H).

[0279] MHC-E-restricted non-conventional CD8 T cells have been shown to develop into both effector and regulatory lineages. 1、25、26 Our findings indicate that expression of distinct sets of TCRs may be a critical event in directing immature CD8 thymocytes towards regulatory lineage-specific development rather than towards effector CD8 T cells. We show that MHC-E-restricted CD8 T cells expressing TCRs that recognize self-peptides can differentiate into mature CD8 T cells expressing classical features of Tregs, including Helios and Ly49, and a central memory phenotype that reflects their continuous recognition of self-antigens. Defining the classical TCR pairs expressed by CD8 Tregs allows for selective activation or deletion of these MHC-E-restricted CD8 Tregs by antibodies specific for these TCRs, and modulation of their activity in pathological conditions, including autoimmune diseases and cancer.

[0280] The above TCR-based recognition may explain the precise elimination of CD4 T cells expressing high avidity TCRs for cognate Ag. The elevated expression of Qa-1-FL9 complexes by activated CD4 cells may allow for sensitive monitoring of the expansion of Ag-activated CD4 T cells, but not nonspecifically activated CD4 T cells, by CD8 Tregs. The relatively high avidity tetramers + Over 90% of CD4 T cells were deleted, while non-specifically activated tetramer-negative CD4 cells were spared (Figure 81A-D). + Robust upregulation of Qa-1 by CD4 T cells may allow efficient targeting of B cell-dependent Ab responses, a major cellular source of helper function for Tfh cells, without systemic immunosuppression 17、27 .

[0281] CD8 Tregs express relatively low levels of CD8 and TCR, reflecting their self-reactivity (Fig. 50C), and may be significantly more anergic than T cells specific for foreign antigens. These considerations indicate that specific recruitment of CD8 Tregs requires more self-peptide variants with increased agonistic activity. 28、29 To identify agonists with enhanced binding to pMHCI and increased immunogenicity, synthetic FL9 peptides containing altered residues at Qa-1 fixed positions were screened. FL9-68 peptide variants containing a P→L amino acid exchange at position 7 showed significantly enhanced stimulatory activity in vitro and in vivo. Activation of CD8 Tregs with FL9-68 peptides resulted in the inhibition of Tfh and GC B cell responses and the expansion of CD8 Tregs leading to reduced production of anti-graft Abs and prolonged organ survival in heart and kidney transplant models (Figures 83A-83F, Figures 84A-84G).

[0282] Several autoimmune diseases have been linked to autoantibody production secondary to dysregulated high-affinity Tfh proliferation. 4、30、31 CD8 Treg-mediated regulation of self-Ab production is an essential mechanism for inhibiting the development of autoimmune diseases 1 In contrast to CD4 Tregs, we show here that CD8 Tregs can be expanded and activated in a pMHC-specific manner to efficiently target CD4 Th cells with high affinity for cognate antigens, including self-Ags. While we used a peptide-dependent strategy to recruit CD8 Tregs, the distinct expression of TCR Vα / Vβ by CD8 Tregs may also be exploited for their activation and expansion in vivo. Activation of CD8 Tregs with anti-TRAV Ab (targeting the conserved CDR1 / CDR2) recruited a broad repertoire of CD8 Tregs to recruit pQa-1 hiPathogenic CD4 cells can be efficiently inhibited or eliminated. The efficacy of inhibition of CD8 Treg proliferation and subsequent auto-Ab production and associated pathology can be tested in mouse models of autoimmune disease including EAE, T1D (NOD) and SLE (BXSB-Yaa). Expression of non-classical MHC gene products (MHC-E) in mouse (Qa-1) models and in humans (HLA-E) is restricted to two alleles, both unlike the highly polymorphic classical MHC genes. 1 Human CD8 Tregs 3 Identification of homologous TCRs expressed by HLA-E may enable the selective mobilization of HLA-E-restricted human CD8 Tregs for the treatment of antibody-mediated pathological conditions.

[0283] method mouse C57BL / 6(B6), B6.SJL-Ptprc a Pepc b / BoyJ(B6.CD45.1), Balb / C, C57BL / 6-Tg(TcrATcrb)1100Mjb / J(OT-1), B6.129P2-H2-K1 tm1Bpe H2-D1 tm1Bpe / DcrJ(Kb - / - Db - / - ), and B6.129 S 2-TCRα tm1Mom / J(TCRα - / - ) mice were obtained from the Jackson laboratory (Bar Harbor ME). B6.Qa-1.D227K KI and B6.Qa-1 - / - (B6.129S6-H2-T23 tm1Cant / J) mice were generated in our laboratory and described previously 243233 FL9.2, FL9.8 TCR Tg mice were generated in our laboratory as described below and maintained in the Qa-1 WT and KO backgrounds. - / - Mice were provided by Dr. Kenneth Rock (UMASS Medical Center, Worcester). All experiments were performed in accordance with institutional guidelines approved by the Animal Care and Use Committees of Dana-Farber Cancer Institute (DFCI) and Brigham and Women's Hospital.

[0284] Antibodies and flow cytometry TCRb (clone: ​​H57-597), CD3e (17A2), CD44 (IM7), CD122 (TM-b1), Ly49C / I / F / H (14B11), Va3.2 (RR3 -16), Va2(B20.1), Vb5.1 / 5.2(MR9-4), CD4(RM4-5), CD8a(53-6.7), CD8b(YTS156.7.7), CD69(H1 Fluorescently labeled antibodies against Qa-1b (6A8.6F10.1A6), PD-1 (29F.1A12), activated caspase 3 (5A1E), Ki67 (16A.8), B220 (RA3-6B2), Fas (SA367H8), CXCR5 (SPRCL5), FoxP3 (FJK-16S), NKG2D (A10) and NKG2A (20d5) were purchased from BD Biosciences, eBioscience and Biolegend. For detection of FL9 T cells, Qa-1b / FL9-PE, Qa-1b / FL9-APC, Qa-1b / Hsp60p216-PE, Qa-1b / Hsp60p216-APC tetramers were generated by the NIH tetranmer core facility and provided for this study. b / Ova 323-339 Tetramer was purchased from MBL International.

[0285] Identification and isolation of FL9-specific TCR Bone marrow-derived DCs were cultured in the presence of 20 ng / mL GM-CSF to induce Kb - / - Db - / - DCs were generated from mice. Six days later, DCs were stimulated with 50 ng / mL LPS for 12 hours. DCs were irradiated (30 Gy) and pulsed with FL9 peptide by incubating with 10 mg / mL FL9 peptide at 37° C. for 2 hours. FL9-loaded Kb - / - Db - / - DCs were injected into WT B6 mice on days 0, 8, and 15. On day 22, Qa-1b / FL9 Tet + Cells were sorted by FACS and + CD122 + Ly49+ CD8 subsets and single Tet + Identification of the TCRa and TCRb chains for each sorted cell was performed according to a previously published protocol. 34 Briefly, one-step RT-PCR was performed by adding the RT-PCR mix to each well. The primers for the RT-PCR mix contained the leader and constant region sequences of TCR with an adapter sequence added to the 5' end of the leader primer. 34 (Figure 62). The cDNA from this RT-PCR was used to amplify TCRa and TCRb separately using the nested PCR principle. The PCR products were sequenced using mTRAC_1st2R and mTRBC_1st2R primers for the TCRα and TCRβ amplicons, respectively, and analyzed with the IMGT / V-Quest algorithm (http: / / www.imgt.org).

[0286] TCR + Hybridoma generation and TCR affinity testing The cDNAs encoding the TCR α and TCR β chains were inserted into a pMIG vector containing a GFP cassette, which was then transfected into PLAT-E cells using FuGENE6 (Promega). The culture medium was replaced with fresh medium at 24 hours, and the supernatant was harvested 72 hours after transfection to detect TCR. - / - The FL9.8 and FL9.2 TCRs were used to transduce the 58C hybridoma. The expression of TCRα and TCRβ pairs on the surface of the 58C hybridoma was analyzed by staining with Qa-1b-FL9 tetramer, anti-CD3ε and anti-TCRβV Ab. The relative affinity of the FL9.8 and FL9.2 TCRs was analyzed by measuring the tetramer staining decay kinetics. 35 . FL9.8 TCR + and FL9.2 TCR + Hybridomas were incubated with PE-conjugated Qa-1b / FL9 tetramer in the presence of anti-Qa-1 Ab. Cells were fixed at different time points (0–120 min) after the start of incubation, and the intensity of PE staining was measured as an index of tetramer binding by flow cytometry.

[0287] Generation of FL9 TCR Tg mice FL9.2 and FL9.8 TCR transgenes were used in OT-I TCR Tg mice 9 The FL9.2 and FL9.8 TCR Tg mice were generated by replacing the TCR V(D)J elements of the pES.42.1c and pKS913.CD18.31 vectors previously used to generate the FL9.2 and FL9.8 TCRs with the TCRα and TCRβ cDNA fragments of the FL9.2 and FL9.8 TCRs, respectively. The vectors were linearized and used to target C57BL / 6 ES cells using standard methods at the Transgenic Core Facility at Beth Israel Deaconess Medical Center. Founder lines for FL9.2 and FL9.8 TCR Tg mice were established after genotyping with the following primers: A common primer set for both FL9.2 and FL9.8 TCRα, 5'-CTAGAAGACTCAGGGTCTGA-3' and 5'-TCGGCACATTGATTTGGGAGTCA-3', amplifying 1 kbp for the transgene; Primer set 5'-ACACTGTCGCTGATTCTG-3' and 5'-GATGTGAATCTTACCGAGAACAGTCAGTCTGGTTC-3' for FL9.2 TCRb For FL9.8 TCRβ the primer sets 5'-TAACACTGTCGCTCGCTGAC-3' and ATACAGTCGTCTGCACTAG-3' both amplify 500 bp for the transgene.

[0288] Peptide mutagenesis and superagonist peptide screening A peptide library was generated by single mutation of each Qa-1 fixed position (p2, 3, 6, 7 and 9) of the FL9 peptide (FYAEATPML) with 20 amino acids, which consisted of 96 FL9 variant peptides. +The 58C hybridoma was incubated with EL4 cells pulsed with each FL9 mutant. After 12 hours, the levels of CD69 expression and TCR expression were measured by flow cytometry. To analyze the binding strength of the FL9 TCR with the Qa-1-FL9 mutant, the FL9 TCR (Vα3.2) on EL4 cells was expressed as 100% CD69 and 100% TCR. + Vβ5 + Trogocytosis was measured directly by detection of FL9.8 TCR. + The 58C hybridoma was co-cultured with EL4 cells pulsed with FL9 mutant peptides from the library. After 2 hours, Vα3.2 + Vβ5 + EL4 cells were assessed by flow cytometry as a measure of trogocytosis.

[0289] Adoptive transfer and in vivo suppression assays Qa-1.WT or Qa-1.D227K KI mice were cultured with 100 μg Ova in CFA. 323~339 The mice were immunized intraperitoneally with the peptide. Seven days later, 1 × 10 5 CD25 - WT B6 adoptive hosts were injected with 20 μg Ova in CFA in the footpad. 323-339 The mice were immunized with the peptide. Seven days later, IA b / Ova 323-339 tetramer + The frequency and number of CD4 cells and activated CD4 cells were assessed in inguinal and popliteal LNs of B6 hosts by flow cytometry.

[0290] FL9 peptide immunization CD45.1 + B6 or TCRα - / - Mice were injected with 2 × 10 6 FL9.2 Tg T cells were transferred and subsequently immunized intraperitoneally with 100 μg FL9 or FL9-68 peptide in CFA. Proliferation and activation of FL9.2 Tg T cells in adoptive hosts was analyzed by assessing CFSE dilution, Ki67 and CD69 expression on days 3 and 6 after transfer.

[0291] Heart transplantation in skin-sensitized hosts and analysis of immune responses B6 mice were immunized intraperitoneally with 50 μg FL9-68-adjuvant (IFA or AddaVax™) or adjuvant alone on days 0 and 7, followed by BALB / C→B6 skin transplantation on day 10. FL9-68-Adj or Adj immunizations were repeated on days 10, 13, and 16. On day 27, fully vascularized Balb / C hearts were cultured using ELISA kits as previously described. 36 , and transplanted into the abdominal cavity of B6 mice using microsurgical techniques. Heart graft survival was determined by monitoring palpable heart beating. 16 days after skin sensitization, the levels of FL9 T cells, Tfh, GC B and plasma cells in the dLN were analyzed by flow cytometry. Serum was collected from heart transplant recipient B6 mice immunized with either FL9-68 / IFA or IFA alone on day 16. Serially diluted serum was diluted to 1 × 10 in a total volume of 100 μl PBS. 6 Following incubation with donor splenocytes for 30 min, surface-bound Abs on CD4 cells were detected using anti-CD4 (Biolegend, clone RM 4-5) and anti-mouse IgG1 Ab (BD Biosciences, clone A 85-1). Histological analysis of the heart grafts was performed by InvivoEx using anti-C4d Ab (Hycult Biotech) and Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories). For mixed lymphocyte rejection, CD25 - CD4 T cells were isolated from draining lymph nodes of B6 hosts and co-cultured with irradiated Balb / c donor splenocytes. Proliferation was measured by immunofluorescence with Celltrace violet™.

[0292] Kidney transplantation and immune response analysis BALB / c mice (H-2 d The left kidney of the B6 host (H-2 b). The ureter of the remaining native kidney was then ligated 2-4 days after surgery to inhibit native renal function. Success of the surgery was determined if the mouse survived 7 days after surgery (POD). The transplanted B6 hosts were intraperitoneally treated with FL9-SA (50 μg) or PBS emulsified in adjuvant (Addavax™) once a week starting on POD2. On day 20 after kidney transplantation (n=5-7 / group), allograft-draining lymph node tissue was cultured for 10 min at 4 h after transplantation to detect FL9-specific Tregs (Qa-1-FL9 Tet + ), Tfh, GC B and plasma cells, serum DSA levels, capillary C4d deposition and gross anatomy of the kidney allografts were assessed. Kidney allograft survival was measured by survival of recipients without native renal function.

[0293] statistical analysis Prism v.9.0 (GraphPad Software) was used for statistical analysis. Statistical significance was calculated according to the Wilcoxon-Mann-Whitney rank sum test for comparison of two conditions, and the Kruskal-Wallis test for comparison of more than two conditions. A P value of <0.05 was considered statistically significant ( * =<0.05, ** =<0.01, *** =<0.001, **** =<0.0001).

[0294] References TIFF2024534863000007.tif32148TIFF2024534863000008.tif231150TIFF2024534863000009.tif231150TIFF2024534863000010.tif176150

[0295] Example 11 Depletion of CD8 Tregs enhances antitumor immunity Without wishing to be bound by the mechanism, the suppressive function of Qa-1-restricted CD8 Tregs on activated T cells indicates that antitumor immune responses can be enhanced by reducing CD8 Treg levels.4 Expression of the Ly49F surface marker by lymphocytes (but not other lymphocytes, including lymphocytes) allowed the inventors to detect Ly49F in the spleen, lymphocytes and blood. + It was possible to deplete CD8 Tregs by >95% (Fig. 90). We first investigated whether depletion of CD8 Tregs could enhance antitumor immune responses in response to syngeneic MC38 murine colon cancer together with vaccination with irradiated tumor cells. Vaccination alone moderately delayed MC38 growth. Depletion of CD8 Tregs by α-Ly49F administered after tumor growth, alone or in combination with vaccine, was detectable from day 8 and significantly prevented tumor growth (Fig. 89A), indicating that depletion of CD8 Tregs is a promising therapeutic option to suppress antitumor immune responses. This view was supported by analysis of the MC38 TME, where α-Ly49F Ab treatment reduced total CD8 cells as well as zmB cells, together with a reduction in immunosuppressive MDSCs. + It increased the numbers of both CD8 and NK cells (Figure 89B). Furthermore, depletion of a dominant clone of Qa-1-restricted CD8 Treg expressing TCR Vα3.2 (see Figure 56 for depletion efficiency of α-Vα3.2 Ab) resulted in a similar increase in antitumor immunity (Figure 89C).

[0296] Next, 3 days after inoculation of MC38 cells, CpG-ODN 24 The effect of CD8 Treg depletion under conditions that induce a Th1-biased immune response was examined by injection (subcutaneous) of CD8 Treg. Treatment with CpG-ODN showed no significant therapeutic effect, but CD8 Treg depletion on days 8-10 strongly inhibited tumor growth, either alone or with CpG-ODN treatment (Figure 89D).

[0297] Finally, we investigated whether CD8 Treg depletion could enhance responses against a second syngeneic tumor, B16F10 melanoma. In this case, we could also examine early changes in the TME (day 15) to see if there was a skew of DC responses toward cDC1 enrichment during tumor growth early in the response. We noted that B16F10 tumor growth was accompanied by a marked increase in cDC1 at the expense of MDSC (Figure 91A-C). Taken together, these data indicate that strategies targeting CD8 Treg-dependent immunosuppression may represent an effective approach to enhance immune responses against cancer, either as a monotherapy or in combination with other immunotherapies.

[0298] Depletion of CD8 Tregs can enhance antitumor immune responses (Figure 89A-D). Administration of α-Ly49F or α-Vα3.2Ab enhanced antitumor responses leading to inhibition of growth by syngeneic MC38 murine colon carcinoma. Analysis of the B16F10 melanoma model also showed an early increase in type 1 cDCs that may be essential for the initiation of sustained T cell responses. 33 Although the primary cellular target of CD8 Tregs in the TME has not been clearly defined, increased expression of Qa-1 by activated CD4 cells, CD8 T cells, and cDCs in the TME is a target. Without wishing to be bound by mechanism, depletion of human CD8 Tregs using anti-KIR Abs may improve current immunotherapy protocols. To allow selective mobilization or depletion of HLA-E-restricted human CD8 Tregs, 3 The identification of TCR homologues to the murine Vα3.2 / Vβ5.1 set expressed by T cells may form the basis of novel and effective treatments for disorders reflecting insufficient or exaggerated immune responses.

[0299] Tumor inoculation and Ab treatment C57BL / 6 (B6) mice (8–12 weeks old) were inoculated with 2 × 10 5 Mice were subcutaneously inoculated with tumor cell vaccine (10 6MC38 cells were irradiated with 2000 rads [20 Gy] and treated with α-Ly49F Ab alone or in combination with tumor cell vaccine (n=5-6 / group). Irradiated MC38 tumor cells were injected subcutaneously on days 7-10 on the flank opposite the MC38 tumor inoculation site. Anti-Ly49F or isotype control (30 mg / mouse) was administered on days 8, 10, and 13 after MC38 tumor cell inoculation. For vaccination, MC38-Cas9 cells were transduced with lentivirus containing Ezh2 gRNA (5'-AGAGTACATTATGGCACCG-3') at an MOI of 0.5 in the presence of 2.5 mg / mL puromycin for 72 h. 10-20% EZH2 KO Obtained polyclonal EZH2 containing cells KO A portion of the EZH2 gene was subcloned and highly enriched. KO Cells were irradiated (2000 R) and used for vaccination. In some experiments, 10-20% EZH2 cells showed growth curves that were not significantly different from those of MC38 WT cells. KO MC38 cells containing IL-16 cells were used as test tumor inoculum to increase MHC expression and immunogenicity. 39 For CpG-ODN treatment, WT B6 mice were injected with CpG-ODN (50 mg / mouse) on day 3, followed by treatment with α-Ly49F Ab (30 mg / mouse) on days 8, 11, 14, and 17.

[0300] References TIFF2024534863000011.tif80150

[0301] equivalent Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures specifically described herein, which equivalents are considered to be within the scope of the invention.

Claims

1. A recombinant peptide / polypeptide agonist comprising the amino acid sequence FSNEATLML (SEQ ID NO: 48), WYADVTPAL (SEQ ID NO: 49), or an amino acid sequence 88% identical thereto, wherein the peptide / polypeptide is a CD8 Treg agonist.

2. A recombinant peptide / polypeptide agonist as described in claim 1, conjugated to a carrier protein.

3. A recombinant peptide / polypeptide agonist as described in claim 1, comprising a lipophilic albumin-binding tail conjugate.

4. The recombinant peptide / polypeptide agonist described in claim 3, wherein the lipophilic albumin-binding tail conjugate is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG).

5. A recombinant peptide / polypeptide agonist as described in claim 1, wherein the amino acid sequence has one amino acid substitution.

6. A recombinant peptide / polypeptide agonist as described in claim 5, wherein the amino acid substitution is a conservative amino acid substitution.

7. A vaccine composition comprising a therapeutically effective amount of a CD8 Treg agonist and a pharmaceutically acceptable carrier, diluent or excipient, wherein the CD8 Treg agonist comprises at least one recombinant polypeptide agonist according to any one of claims 1 to 6.

8. A pharmaceutical composition comprising a therapeutically effective amount of a recombinant peptide / polypeptide agonist described in any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8 for use in treating an autoimmune disease or condition or allograft rejection in a subject.

10. The pharmaceutical composition of claim 8 for use in mobilizing CD8 Treg cells in a subject.

11. The pharmaceutical composition of claim 8 for use in suppressing CD4 cell activity when administered to a subject.

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