Compositions and methods for the generation of lymphoid tissues

Abstract

The disclosure is directed to synthetic lymphoid organoids, methods of making a synthetic lymphoid organoid, and their uses.

Description

[0001] COMPOSITIONS AND METHODS FOR THE GENERATION OF LYMPHOID TISSUES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims benefit of U.S. Provisional Application No. 63 / 625,544, filed on January 26, 2024, which is incorporated herein by reference in its entirety for any purpose.

[0004] BACKGROUND

[0005] Lymphoid organs such as lymph nodes and tertiary lymphoid structures are specialized tissues which provide the microenvironment where antigen presenting cells can interact with T and B lymphocytes. Thus, these structures play an important role in mounting an adaptive immune response. There remains a need for improved synthetic lymphoid tissues for the treatment of a disorder associated with an immunodeficiency or cancer.

[0006] SUMMARY OF THE INVENTION

[0007] The disclosure is directed towards methods for the generation of synthetic implantable high endothelial venules using human pluripotent stem cells, in combination with lymph node stromal cells. The resulting tissues are termed lymph node high endothelial venule organoids (HEVOs). The disclosure is also directed to such HEVOs and their uses in treating disease.

[0008] In one aspect, the disclosure features a method of making a synthetic lymphoid organoid, the method including culturing a primitive endothelial cell with an isolated lymph node stromal cell in low- attachment tissue culture conditions to form the synthetic lymphoid organoid.

[0009] In some embodiments, the primitive endothelial cell is derived from an induced pluripotent stem cell. In some embodiments, the synthetic lymphoid organoid is a lymph node high endothelial venule organoid. In some embodiments, the lymph node stromal cell is a fibroblastic reticular cell.

[0010] In some embodiments, the primitive endothelial cell and the lymph node stromal cell are autologous human cells. In some embodiments, the primitive endothelial cell and the lymph node stromal cell are allogeneic human cells.

[0011] In some embodiments, the method includes the use of a supporting scaffold. In some embodiments, the supporting scaffold is a Matrigel scaffold.

[0012] In another aspect, the disclosure features a synthetic lymphoid organoid made from a primitive endothelial cell and a lymph node stromal cell.

[0013] In some embodiments, the primitive endothelial cell is derived from an induced pluripotent stem cell. In some embodiments, the synthetic lymphoid organoid is a lymph node high endothelial venule organoid. In some embodiments, the lymph node stromal cell is a fibroblastic reticular cell.

[0014] In some embodiments, the primitive endothelial cell and the lymph node stromal cell are autologous human cells. In some embodiments, the primitive endothelial cell and the lymph node stromal cell are allogeneic human cells. In another aspect, the disclosure features a method of treating a disorder associated with an immunodeficiency, an inflammatory disorder, or a cancer or a condition requiring lymphatic tissue replacement, the method including implanting a lymphoid organoid made from a primitive endothelial cell and a lymph node stromal cell into a patient in need thereof.

[0015] In some embodiments, the method is used in combination with a cellular immunotherapy.

[0016] In some embodiments, the primitive endothelial cell is derived from an induced pluripotent stem cell. In some embodiments, the synthetic lymphoid organoid is a lymph node high endothelial venule organoid. In some embodiments, the lymph node stromal cell is a fibroblastic reticular cell.

[0017] In some embodiments, the primitive endothelial cell and the lymph node stromal cell are autologous human cells. In some embodiments, the primitive endothelial cell and the lymph node stromal cell are allogeneic human cells.

[0018] In some embodiments, the implanting includes injecting the lymphoid organoid into the patient.

[0019] In some embodiments, the cancer is a solid tumor cancer.

[0020] In some embodiments, the method further includes administration of an immunomodulator. In some embodiments, the immunomodulator is an immune checkpoint inhibitor, a cytokine, or an adjuvant. In some embodiments, the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.

[0021] In another aspect, the disclosure features a composition including the synthetic lymphoid organoid of any one of the foregoing aspects and a pharmaceutically acceptable carrier. In some embodiments, the composition further includes a nanocarrier.

[0022] ADVANTAGES OF THE INVENTION

[0023] The methods of the disclosure rapidly generate human lymphatic tissue. In some embodiments, the generated tissue is autologous, precluding rejection after implantation. The method generates the actual anatomical structure where antigen presentation occurs. The process is minimally invasive compared to previous methods which did not employ pluripotent stem cells, and additionally it does not require the use of bone marrow cells.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0026] FIG. 1 shows rapid generation of synthetic high endothelial venule (HEV) organoids in vitro. FIG. 1 a is a schematic representation of the inducible genetic mechanism used for driving the expression of transcription factor Ets variant transcription factor 2 (ETV2) by doxycycline in human induced pluripotent stem cells (hiPSCs). FIG. 1 b is a representation of the protocol used for rapid genetically-driven differentiation of ETI / 2-hiPSCs into inducible endothelial (iEndo) cells in vitro, through a primitive endothelial cell (EC) state that allows tissue-specification. FIG. 1c is representative brightfield images showing E7 2-hiPSCs and Day-6 iEndo derivatives in culture. Scale bar, 25 pm. FIG. 1d is representative immunofluorescence microscopy images showing expression of the endothelial cell marker cluster of differentiation 31 (CD31 ) in ETI / 2-hiPSCs and Day-6 iEndo derivatives. Scale bar, 25 pm. FIG. 1 e is representative immunofluorescence microscopy images of the two endothelial differentiation stages identified by expression of human vascular endothelial growth factor receptor 2 (hVEGFR2), namely primitive iEndo on Day 3, and iEndo on Day 6 of differentiation, distinguished by expression of markers such as von-Willebrand factor (VWF) which is absent in the primitive state. Scale bar, 25 pm. FIG. 1f shows immunodetection of the HEV endothelial cell (HEVEC)-specific marker peripheral lymph node addressin (PNAd) in confocal images taken from Day-6 iEndos cultured alone or co-cultured with prospectively purified lymph node (LN)-derived fibroblastic reticular cells (FRCs), for 3 days. Scale bar, 25 pm. FIG. 1g is a graph showing expression levels of mRNAs encoding HEVEC proteins, measured by means of reverse-transcription polymerase chain reaction in fluorescence- activated cell sorting (FACS)-sorted iEndos that had been cultured alone or with FRCs. Data on the graph represent mean ± SD, n = 7 samples containing 3 culture wells each. *, R<0.05; **, R<0.01 ; P<0.005;

[0027] RcO.001 . Rvalues for individual comparisons were calculated using two-tailed Student’s t-test. FIG. 1 h is representative immunostaining and confocal microscopy images showing the spontaneous interaction between hCD31+iEndos and human platelet-derived growth factor receptor b (hPDGFRb+) FRCs co-cultured under 2-D conditions. A few red splotches can be seen in the iEndo culture Day 6 stack, which do not correspond to specific cell immunostaining of cells. Scale bar, 25 pm. FIG. 1 i is a representative confocal image showing a hCD31 -expressing iEndo with morphological characteristics of tip cell, including the characteristic extended filopodia. Scale bar, 25 pm. FIG. 1j is a schema representing the method used for the aggregation of primitive iEndos and FRCs under low attachment culture conditions for the formation of 3-D lymph node high endothelial venule organoids (HEVOs) shown in FIG.

[0028] 1 k. Scale bar, 50 pm. FIG. 11 shows sequential confocal planes of a HEVO, one-week post-aggregation, depicting hCD31+HEVEC vascular structures (indicated by white arrows) lined by hPDGFRb-expressing FRCs. Scale bar, 50 pm. FIG. 1 m is representative high magnification immunofluorescence images showing HEVECs forming venules that contain a luminal space (indicated by the asterisk), in apposition to FRCs (white arrows). The left image shows a sagittal section while the right image shows a cross section. Scale bar, 12.5 pm. FIG. 1 n is a graph showing representative qRT-PCR expression levels of mRNAs encoding HEV-associated interleukins in iEndos aggregates compared to HEVOs on day 3 after addition of FRCs. Data on the graph represent mean ± SD, n = 7 samples containing 5 organoids each. *, R<0.05; **, P<0.01 ; R<0.005. Rvalues for individual comparisons were calculated using two-tailed

[0029] Student’s t-test.

[0030] FIG. 2 shows formation of tertiary lymphoid structures (TLS)-like structures after subcutaneous implantation of HEVOs in vivo. FIG. 2a is a schematic depiction of the experimental procedures used to investigate HEV properties in vivo. Primitive Day-3 iEndos were aggregated with FRCs at a 3:1 ratio. FIG.

[0031] 2 b is a representative image showing a subcutaneous hiPSC-derived HEVO graft on day 21 postimplantation. Scale bar, 2 mm. FIG. 2 c is representative confocal microscopy images showing HEVO graft sections on day 21 post transplantation, containing hCD31+vasculature associated with hPDGFRb+FRCs. Control grafts were generated by implantation of hCD31+iEndo cells alone (shown) and FRCs alone (not shown, since no grafts or human cells were detected). Scale bar, 25 pm. FIG. 2d is a series of graphs showing quantifications of PNAd-expressing hCD31 + vessels co-immunolabeled with the high endothelial venule marker (MECA79) monoclonal antibody, and FRC content as indicated by human podoplanin (hPDPN) immunolabeling in control iEndo implantation sites and iEndo + FRC (HEVO) graft sections. Values represent mean ± SD, n = 6 grafts. **, P<0.01 . FIG. 2e is representative confocal microscopy images showing expression of mouse lymphatic vessel endothelial hyaluronan receptor 1 (Lyvel ) in cryosections of HEVO grafts sections containing hCD31+HEVs, and in sections of iEndo grafts. Scale bar, 50 pm. FIG. 2f is representative immunofluorescence images showing mCd1 1 c+dendritic cells (DCs) and fibronectin conduits in day 21 HEVO grafts and control iEndo implantation sites. Scale bar, 50 pm. FIG. 2 g is a schematic representation of the experimental procedures followed to test the T cell recruitment capacity of HEVO grafts. FIG. 2h is confocal immunofluorescence images showing adoptively-transferred hCD4+T lymphocytes and HEVs expressing human Vascular Endothelial-Cadherin (hVE-CAD) in HEVO grafts. Scale bars, 1 mm and 100 pm for high magnification. FIG. 2i is a schematic representation of the experimental strategy followed to test addressin-mediated T cell homing mechanisms in engrafted HEVOs. FIG. 2j is representative immunofluorescence images showing hCD4+T cells in HEVO grafts from mice treated with the anti-PNAd antibody and in mice that received vehicle, all of which received peripheral blood mononuclear cells (PBMCs). Scale bar, 50 pm. FIG. 2k is a graph showing quantification of hCD4+T cell aggregates containing ten or more cells in immunofluorescence- labeled cryosections. Values represent mean ± SD, n = 6 mice. **, P<0.01 . FIG. 2I is a representative FACs plot showing adoptively-transferred human CD4+and CD8+lymphocytes within day 28 HEVO grafts.

[0032] FIG. 3 shows engrafted HEVOs provide a lymphoid environment for T cell activation. FIG. 3a is a schematic of the experimental design used to study adoptively-transferred mouse CD3+OT-I and OT-II T cell activation in transplanted HEVOs in response to ovalbumin (OVA). FIG. 3b is representative FACs plots showing expression of the cell proliferation marker Ki67 in CD8+(OT-I) and CD4+(OT-II) mouse cells within HEVOs from mice immune-challenged with OVA plus complete Freund’s adjuvant (OVA + CFA), control mice that remained non-challenged (NC), and control mice that received CFA alone. Numbers within the plots indicate cell population percentage. Quantification values on the bar graphs represent mean ± SD, n = 4 mice with 1 graft each. **, P<0.01 ; ***, P<0.005; ****, P<0.001 . FIG. 3c is a series of graphs showing expression of cytokines I FNy, IL-2 and TNFa in CD8+(OT-I) and CD4+(OT-II) cells in HEVOs from mice that received OVA + CFA, control mice that remained non-challenged (NC), and control mice that received CFA alone. Values on the bar graphs represent mean ± SD, n = 4 mice with 1 graft each. *, P<0.05; **, P<0.01 ; ***, P<0.005; PcO.001 . P values for individual comparisons calculated using Tukey test in one-way ANOVA analysis are indicated.

[0033] FIG. 4 shows that HEVO grafts promote antitumor activity by adoptively-transferred T cells. FIG. 4a is a schematic representation of the experiments performed to investigate anti-tumor activity by adoptively-transferred T cells in HEVO-bearing mice. FIG. 4b is representative photographs of melanomas alone and with HEVO grafts twenty-three days after injection of human B16 melanoma cells and twelve days after adoptive transfer (intravenous injection) of CD3+OT-I and OT-II mouse cells. Scale bar, 2 cm. FIG. 4c is a representative immunofluorescence confocal image of HEVO-melanoma interface showing MelanA-expressing melanoma cells in close apposition with hVE-CAD+hiPSC-derived HEVEC cells and hPDPN+FRCs, prior to T cell injection. The boundary between both tissues is indicated by a dotted white line. Scale bar, 25 pm. FIG. 4d shows quantification of tumor size in the absence or presence of HEVOs in mice receiving adoptively-transferred CD3+cells. The time of cell injection is indicated. Data on the graph represent mean ± SD, n = 5 tumors. **, P<0.01 . FIG. 4e is a representative FACs plot showing human CD4+and CD8+T cell content in HEVO grafts, twelve days after adoptive- transfer injections, and quantification is shown in FIG. 4f. Data on the graph represent mean ± SD, n = 5 tumors. **, P<0.01 . FIG. 4g is representative immunofluorescence microscopy images showing sections of premelanosome protein (PMEL)-expressing melanomas from animals not carrying HEVOs, and of melanomas in apposition to HEVOs. T cells were injected in both control melanoma and HEVO + melanoma groups of mice and were identified on the sections by expression of CD3. Scale bar, 25 pm. FIG. 4h is a graph showing quantification of CD3+T cell clusters containing three or more cells within an area of 50 mm in melanoma sections. Data on the graph represent mean ± SD, n = 18 sections from 6 tumors. PcO.001 . FIG. 4i is representative confocal immunofluorescence images showing clusters of apoptotic caspase 3 (CASP3+) PMEL-expressing melanoma cells in melanoma and in melanoma + HEVO, twelve days after adoptive-transfer of T cells expressing CD3. Scale bars, 50 pm and 25 pm for high magnifications. FIG. 4j is a graph showing quantification of hCASP3+in hPMEL-expressing melanoma cells, in melanoma sections. Data on the graph represent mean ± SD, n = 8 tumors.

[0034] P<0.005. Rvalues for individual comparisons done using Tukey test in one-way ANOVA analysis are indicated.

[0035] FIG. 5 shows a graph and a series of fluorescence microscopy images. FIG. 5a shows the gating strategy used to isolate FRCs from human lymph nodes, by means of fluorescence-activated cell sorting. FIG. 5b shows representative immunofluorescence images of FRCs immunolabeled with antibodies detecting the FRC markers human platelet-derived growth factor receptor b (PDGFRp), podoplanin (PDPN), and chemokine (C-C motif) ligand 19 (CCL19). Scale bar, 25 pm. FIG. 5c and FIG. 5d are immunofluorescence images showing cocultures of PDGFRb+FRCs and iEndos expressing the HEVEC marker PNAd, on Day 6 of differentiation (Day 3 of specification). Scale bars, 50pm. In FIG. 5c the FRCs are not immunolabeled, white arrows indicate prospective FRCs. FIG. 5e shows representative immunofluorescence images showing MADCAM-1 expression in iEndos on Day 6 and Day 12 of differentiation, following three and six days in coculture with FRCs, respectively. Scale bar, 50 pm.

[0036] FIG. 6 shows a series of fluorescence microscopy images. FIG. 6a shows fluorescence microscopy images showing Cd1 1 b+monocytes in close apposition to collagen fibro-reticular structures present within HEVO grafts. Scale bar, 25 pm. FIG. 6b shows representative immunofluorescence images showing adoptively transferred mouse B220+lymphocytes within HEVO grafts, in the vicinity of hCD31+endothelial cells. Scale bar, 25 pm.

[0037] FIG. 7 shows a series of plots, graphs, and images. The gating strategy used to analyze adoptively transferred OT-I CD8+and OT-II CD4+T mouse cells in HEVO grafts and in lymph nodes from mice carrying HEVO grafts treated with OVA + CFA is shown in FIG. 7a and FIG. 7b. Fluorescence minus-one controls were used to determine gate positioning. FIG. 7c shows representative FACS plots showing cytokine content in adoptively-transferred OT-I CD8+T cells and OT-II CD4+T cells within HEVO grafts from non-challenged mice (NC), mice challenged with OVA + CFA and from mice injected with CFA alone. Numbers within the plots indicate cell population percentage. FIG. 7d shows representative immunofluorescence images showing expression of CD25 in adoptively-transferred OT-II CD4+cells within HEVO grafts in the three conditions. Scale bar, 50pm.

[0038] FIG. 8 shows a series of images and a graph. FIG. 8A shows a fluorescence microscopy image of PNAd+hCD31+vessels in peri-tumoral HEVO grafts. Scale bar, 25pm. FIG. 8B is a representative immunofluorescence image showing tumor-infiltrating mouse CD3+T cells expressing Ki67, and an associated bar graph with quantification where values represent mean ± SD, n = 8 grafts. **, P<0.01 . Scale bar, 25pm.

[0039] FIG. 9 shows a series of images showing preservation of HEVO grafts transplanted without a matrix. FIG. 9a shows representative immunofluorescence microscopy images showing HEVO cryosections containing hCD31 -expressing human iPSC-derived vascular structures expressing hCD31 associated with hPDGFRb-expressing human fibroblastic reticular cells. FIG. 9b shows species validation by immunofluorescence imaging showing the detection of human Lamin A / C in hiPSC-derived CD31 cells.

[0040] DEFINITIONS

[0041] As used herein, the term "synthetic lymphoid organoid" refers to a small, synthetically produced engineered cellular structure made from stem cells and made up of lymphoid tissue. A synthetic lymphoid organoid can be used to replicate and recapitulate the structure and functions of a lymphoid organ present within the body of a subject. Synthetic lymphoid organoids can be useful for cancer treatment, or the treatment of a disorder associated with an immunodeficiency.

[0042] As used herein, the term "primitive endothelial cell" refers to the initial or first round of endothelial cells formed during embryonic hematopoiesis from embryonic stem cells. Induced pluripotent stem cells can also be differentiated into primitive endothelial cells.

[0043] As used herein, the term "lymph node stromal cells" refers to a class of connective tissue ceils that help build the architecture and infrastructure of a lymph node. They are non-hematopoietic ceils and help build an environment within the lymph nodes where ceils can interact with one another in a sitespecific manner.

[0044] As used herein, the term "low-attachment tissue culture conditions" refers to tissue culture conditions where ceils or tissues have no or negligible attachment to the plates, dishes, or flasks in which they are being cultured. The ability to remain suspended and unattached supports the growth and development of three-dimensional spheroids and organoids. The formation of spheroids and organoids happens due to the secretion of extracellular matrices which helps cells aggregate with one other. Thus, low-attachment cell culture plates are known as spheroid culture plates.

[0045] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a pluripotent stem cell that can be derived directly from a differentiated somatic cell. Human iPSCs can be generated by introducing specific sets of reprogramming factors into a non-pluripotent cell that can include, for example, Oct3 / 4, Sox family transcription factors (e.g., Sox1 , Sox2, Sox3, Soxl5), Myc family transcription factors (e.g., c-Myc, 1 -Myc, n-Myc), Kruppel-like family (KLF) transcription factors (e.g., KLF1 , KLF2, KLF4, KLF5), and / or related transcription factors, such as NANOG, LIN28, and / or Glisl . Human iPSCs can also be generated, for example, by the use of miRNAs, small molecules that mimic the actions of transcription factors, or lineage specifiers. Human iPSCs are characterized by their ability to differentiate into any cell of the three vertebrate germ layers, e.g., the endoderm, the ectoderm, or the mesoderm. Human iPSCs are also characterized by their ability to propagate indefinitely under suitable in vitro culture conditions. As used herein, the term "lymph node high endothelial venule organoids" or "HEVO" refers to synthetic implantable high endothelial venules that are generated using primitive endothelial cells and lymph node stromal cells. HEVOs possess lymphoid function and upon implantation into animals, HEVOs recruit lymphatic vessels, antigen-presenting immune cells, and T lymphocytes, forming lymphoid structures in which T lymphocytes are activated in response to immune challenge.

[0046] As used herein, the term "fibroblastic reticular cells" or "FRCs" refers to stroma! cells that are found in lymphoid tissue and help organize the microarchitecture of the lymph node. They are heterogeneous in nature and include T ceil zone reticular ceils, B ceil zone reticular ceils, pericytic FRCs, follicular dendritic cells, and marginal reticular cells. They act as immunologically specialized myofibroblasts and pericytic FRCs are known to support the functioning of high endothelial venule (HEV) barriers.

[0047] As used herein, the term "supporting scaffold" refers to any structural material that acts as a support for attachment of cells, tissue development, tissue engineering, three-dimensional tissue formation, cell survival, cell transplantation, and cell differentiation. Supporting scaffolds are generally biocompatible and made of polymers. In some embodiments, the supporting scaffold is a Matrigel scaffold. Matrigel is a gelatinous mixture rich in basement membrane proteins and functions as a matrix supporting different biological processes during cell culture.

[0048] As used herein, the term "immune checkpoint inhibitor" refers to an inhibitor that plays a role at an immune checkpoint. Immune checkpoint inhibitors include compounds and antibodies that block the interaction between programmed death-ligand 1 (PD-L1 ) on a cell (e.g., a tumor cell) and the receptor known as programmed death-1 (PD-1 ) on the T cell, such that the T cell can kill the tumor cell.

[0049] As used herein, the term "immunomodulators" refers to drugs that alter the immune response. Some immunomodulators increase the immune response and are used for the treatment of immunodeficiencies while other immunomodulators attenuate the immune response in patients with inflammatory and autoimmune disorders. The latter group of immunomodulators are known as immunosuppressants. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or excipient such as a compound, material, composition, and / or dosage form, which is suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit / risk ratio.

[0050] DETAILED DESCRIPTION

[0051] Lymph nodes (LNs) and tertiary lymphoid structures (TLS) also known as ectopic lymphoid organs (ELOs), recruit naive lymphocytes for antigen presentation, activation, and differentiation into effector cells (1 -3). While LNs are formed during embryonic development, TLS form postnatally in peripheral tissues, driven by inflammatory processes that may be associated with chronic and autoimmune disease, graft rejection, or cancer (4). In cancer, TLS function as hubs that connect local and systemic anti-tumor responses (4, 5). This notion is supported by the finding that TLS-associated biomarkers have positive prognostic value for primary and metastatic cancers (6-8). Furthermore, increased TLS formation correlates with prolonged overall survival and relapse-free survival in cancer patients, as well as prolonged survival after primary tumor resection (6, 9). LNs are specialized tissues in which antigen presentation occurs, resulting in the activation of T lymphocytes during an adaptive immune response. The process of antigen presentation and T lymphocyte activation occurs in the high endothelial venules (HEVs) of lymph nodes. Increased T lymphocyte activation is beneficial in the context of immunodeficiency or cancer. Within lymph nodes, HEVs recruit antigen presenting cells and T lymphocytes, facilitating their interaction for the activation of T cells, resulting in an adaptive immune response.

[0052] LNs and TLS provide microenvironments anatomically organized to precisely orchestrate the interaction between antigen presenting cells (APCs) and T and B lymphocytes (2, 3). Within LNs and TLS, a specialized type of vasculature known as HEVs forms a network of blood vessels critical for homeostatic lymphocyte trafficking (10). Anatomically, HEVs are post-capillary venules lined by endothelial cells expressing sulfated ligands known as addressins, that mediate the recruitment of naive and memory lymphocytes from the bloodstream (11 , 12). While this mechanism of recruitment is antigenindependent, HEVs determine the type of lymphocyte and the site of entry into lymphoid tissues, where lymphocytes survey dendritic cells (DCs) for cognate antigen (13). Anatomically, HEVs are supported by a thickened basal lamina comprising pericytes and fibroblastic reticular cells (FRCs) (10). FRCs regulate HEV growth through the secretion of vascular endothelial growth factor (VEGF) and organize lymphoid organ remodeling in response to adaptive immune demand (2, 14). The intimate interaction between FRCs and HEVs is critical for selective lymphocyte trafficking in and out of LNs (2, 14, 15). FRCs control HEV permeability and regulate T cell recruitment and homeostasis, as well as the survival of B cells and DCs (2, 14, 15). In addition, the recruitment of fibroblasts with characteristics of FRCs is necessary for ectopic development of TLS and for homing of circulating lymphocytes (16, 17).

[0053] Previous efforts to generate LNs using tissue-bioengineering approaches resulted in the development of lymphatic endothelium-lined micro-structured extracellular matrices (18) and flow- induced in vitro capillary morphogenesis using human lymphatic endothelial cells (18, 19). The combination of a thymus-derived stromal cell line and bone marrow-derived DCs in biocompatible scaffold indicated that the lymphatic stroma instructs formation of lymphoid tissues capable of accumulating lymphocytes and of mounting immune responses in the renal subcapsular space of mice (20, 21 ).

[0054] The disclosure is directed towards a method for the generation of synthetic implantable high endothelial venules using human pluripotent stem cells, in combination with lymph node stromal cells. The resulting tissues are termed lymph node high endothelial venule organoids (HEVOs). When implanted into animals, HEVOs recruit lymphatic vessels, antigen-presenting immune cells, and T lymphocytes, forming lymphoid structures in which T lymphocytes are activated in response to immune challenge. This indicates that HEVOs possess lymphoid function.

[0055] The method may consist of the following steps: (i) Endothelial induction by directed differentiation of patient-derived induced pluripotent stem cells into primitive endothelial cells; (ii) High endothelial venule cell specification by co-culturing primitive endothelial cells generated in the previous step with patient-derived lymph node stromal cells; and (Hi) Organoid formation by aggregation of endothelial cells + stromal cells into three-dimensional structures (organoids) in low-attachment tissue culture conditions. The resulting organoids can be implanted immediately.

[0056] The disclosure exploited orthogonally programmed differentiation of human induced pluripotent stem cells (hiPSCs) (22, 23) for the rapid and scalable generation of synthetic HEV organoids (HEVOs) instructed by lymphoid-derived FRCs, without the need of bone marrow-derived DCs. The results show that transplanted HEVOs attract adoptively transferred human B and T lymphocytes and organize an adaptive immune response. The methodology presented here can be used to elucidate mechanisms of human ectopic lymphoid organ (ELO) development and induction of adaptive immune responses in the HEV niche and could have clinical application in immune deficiency and cancer immunotherapy.

[0057] Bioengineering strategies for the fabrication of implantable lymphoid structures mimicking LNs and TLS could amplify the adaptive cellular response for therapeutic applications such as cancer immunotherapy. No method to date has resulted in the consistent formation of HEVs, which is the specialized vasculature responsible for naive T cell recruitment and education in both LNs and TLS. This disclosure used orthogonal induced differentiation of human pluripotent stem cells (hPSCs) carrying a regulatable ETV2 allele, to rapidly and efficiently induce endothelial differentiation. Assembly of embryoid bodies combining primitive inducible endothelial cells (iEndos) and primary human lymph node FRCs resulted in the formation of HEV-like structures that can aggregate into three dimensional organoids (HEVOs). Upon transplantation into immunodeficient mice, HEVOs successfully engrafted and formed lymphatic structures that recruited both antigen-presenting cells and adoptively-transferred lymphocytes, therefore, displaying basic TLS capabilities. The results further show that functionally, HEVOs can organize an immune response and promote anti-tumor activity by adoptively transferred T lymphocytes. Collectively, these experimental approaches represent an innovative and scalable proof-of-concept strategy for the fabrication of bioengineered tertiary lymphoid structures that can be deployed in vivo to enhance adaptive immune responses.

[0058] Cancers

[0059] The compositions and methods of the disclosure can be used to treat cancer. Cancer refers to a large group of diseases characterized by the uncontrolled growth of abnormal cells and tissues in the body. This unregulated cell division and growth results in the formation of tumors such as malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body using the lymphatic system or bloodstream. Generally, after diagnosis, cancer staging is done to determine the extent of the disease and the stages can be described as follows: Stage 0 - isolated cancer or carcinoma in situ. Stages I, II, and III - higher numbers indicate more extensive disease: larger tumor size and / or spread of the cancer beyond the organ in which it first developed to nearby lymph nodes and / or tissues or organs adjacent to the location of the primary tumor. Stage IV - the cancer has spread to distant tissues or organs using the lymphatic system or bloodstream. Examples of cancers include solid tumors, solid tumors of childhood, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, hematologic malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin’s lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, lymphocytic lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B- lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combinations of said cancers.

[0060] Solid Tumor Cancer

[0061] The compositions and methods of the disclosure can be used to treat solid tumor cancer. A solid tumor is an abnormal mass of tissue or solid cancer cells that usually does not contain cysts, liquids, or liquid areas. They include different types of cells expressing different mutations such as cancer cells, immune cells, connective tissue cells, and cancer stem cells. They can appear anywhere in the body, for example, inside organs such as the breast. Examples of solid tumors include sarcomas, carcinomas, carcinosarcomas, undifferentiated tumors, neuroblastomas, germ cell tumors, bone tumors, kidney tumors, melanomas, solid tumors in different types of cancers (e.g., breast cancers, brain cancers, lung cancers, prostate cancers, thyroid cancers, colon cancers etc.), adrenal tumors, and lymphomas.

[0062] A solid tumor is detectable on the basis of tumor mass, e.g., by procedures such as CAT scan, magnetic resonance imaging, X-ray, ultrasound, or palpation, and / or through histological experiments which can be used to detect the expression of one or more cancer-specific antigens in a tumor sample from a patient. Staging of cancers with solid tumors are dependent on cancer type, tumor size, histological characteristics, tumor markers, and other criteria known to those of skill in the art. Solid tumors can often be treated with surgery.

[0063] Cellular Immunotherapies

[0064] The compositions and methods of the disclosure can be used in combination with adoptively transferred allogeneic or autologous T lymphocytes carrying an engineered T lymphocyte receptor or a chimeric T cell receptor in the context of (T-cell receptor) TCR therapy or chimeric antigen receptor (CAR)-T therapy, respectively.

[0065] Immunodeficiency and Disorders Associated with Immunodeficiencies

[0066] The compositions and methods of the disclosure can be used to treat disorders associated with an immunodeficiency. Immunodeficiency or primary immunodeficiency occurs when the body’s immune system is unable to function properly or lacks different components of the immune system such as phagocytes, lymphocytes etc. This malfunctioning of the immune system leads to infections which last longer, are more severe in nature and / or happen on a recurring basis. Examples of disorders associated with immunodeficiencies include X-linked severe combined immunodeficiency (X-SCID), autoimmune polyglandular syndrome type 1 (APS-1 ) or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), BENTA disease, caspase eight deficiency state (CEDS), common variable immunodeficiency (CVID), CARD9 deficiency, chronic granulomatous disease (CGD), congenital neutropenia syndromes, leukocyte adhesion deficiency (LAD), severe combined immunodeficiency (SCID), B-cell (antibody-deficiency) disorders, adenosine deaminase severe combined immunodeficiency (ADA-SCID), autoimmune lymphoproliferative syndrome (ALPS), Wiskott-Aldrich syndrome (WAS), Bruton disease, and chronic granulomatous disease (CGD), among others. Secondary immunodeficiencies are caused due to the negative impact of environmental factors on the immune system. Secondary causes of immunodeficiency include acquired immune deficiency syndrome (AIDS) caused by a viral infection (e.g., a human immunodeficiency virus (HIV)), immunodeficiencies caused due to other viruses, bacterial infections (e.g., septicemia), parasitic infections, obesity, medications, malnutrition, steroids, chemotherapy, severe burns, and malignancies (e.g., plasma cell tumors, myeloma, plasmacytoma, lymphoma, leukemia, non-Hodgkin’s lymphoma, Hodgkin’s disease etc.).

[0067] Inflammatory Disorders

[0068] The compositions and methods of the disclosure can be used to treat conditions associated with inflammatory disorders. An inflammatory disorder occurs when the body’s immune system is activated in the context of a chronic disease, or when it is triggered by molecules present in the body’s own cells, for example, in autoimmune diseases. Prolonged or chronic inflammation is associated with increased organ damage and cell stress. Examples of inflammatory disorders include gastrointestinal diseases, such as Crohn’s disease, ulcerative colitis, and inflammatory bowel disease; metabolic diseases such as Type 2 diabetes; autoimmune diseases, such as rheumatoid arthritis (RA), lupus, and ankylosing spondylitis (AS).

[0069] Lymphatic Tissue Replacement

[0070] The compositions and methods of the disclosure can be used to treat conditions requiring lymphatic tissue replacement. Lymphatic tissue replacement is necessary in patients who have undergone metastatic lymph node resection or lymphodepletion caused by chemotherapy and may develop lymphedema. Lymphedema is defined as the chronic swelling of an extremity, that occurs when the protein-rich interstitial fluid is not appropriately drained by the lymphatic system. Lymphedema is significantly prevalent in female survivors of ovarian, endometrial, and colorectal cancers.

[0071] Allogeneic and Autologous Cells

[0072] The compositions and methods of the disclosure can be used to generate lymph node high endothelial venule organoids or HEVOs using a combination of primitive endothelial cells and lymph node stromal cells.

[0073] In some embodiments, the primitive endothelial cells and lymph node stromal cells are allogeneic cells. Allogeneic cells refer to cells that are obtained from another individual of the same species, and generally is closely genetically related to the individual being treated and / or closely match the human leukocyte antigens markers of the individual being treated.

[0074] In some embodiments, the primitive endothelial cells and lymph node stromal cells are autologous cells. Autologous cells refer to cells that are obtained from the individual being treated. Immunomodulators

[0075] The compositions and methods of the disclosure can be administered in combination with immunomodulators. Immunomodulators include immune checkpoint inhibitors, cytokines, and adjuvants. Examples of PD-1 / PD-L1 checkpoint inhibitors include pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, atezolizumab, avelumab, and durvalumab. Examples of cytokines include aldesleukin, GM-CSF, interferon alfa-2a, interferon alfa-2b (Intron A), and sylatron. Examples of adjuvants include imiquimod and hiltonol. Immunomodulators with immunosuppressant activity include the biologic drugs infliximab, anakinra, adalimumab, rituximab, and tocilizumab, as well as the antibody abatacept which inhibits cytotoxic T-lymphocyte associated protein 4 (CTLA-4) signaling in T cells.

[0076] Nanocarriers

[0077] The compositions and methods of the disclosure can be administered in combination with nanocarriers. Nanocarriers, also known as nanoparticles, are structures with a diameter within the range of 1-200 nanometers (nm). Nanocarriers may be made of polymer conjugates, lipid-based carriers, dendrimers, carbon nanotubes, or gold nanoparticles, among other nanomaterials. Nanocarriers can be loaded with drugs, allowing targeted drug delivery to specific tissues and cells. Nanoparticles may be conjugated to antibodies or to molecules with specific affinity to surface proteins present in certain cell types. Immunomodulatory drugs can be encapsulated inside nanoparticles conjugated to an antibody that binds to the peripheral node addressin (PNAd), which is expressed on the surface of HEV endothelial cells.

[0078] Pharmaceutical Compositions

[0079] The compositions and methods of the disclosure may be incorporated into a vehicle for administration to a patient, such as a human patient suffering from a disorder associated with an immunodeficiency or cancer, as described herein. Pharmaceutical compositions containing the compositions described herein can be prepared using methods known in the art. For example, such compositions can be prepared using, e.g., physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.

[0080] A pharmaceutical composition containing, for example, the compositions described herein typically includes a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition may include (e.g., consist of), e.g., a sterile saline solution and the compositions described herein. The sterile saline is typically a pharmaceutical grade saline. A pharmaceutical composition may include (e.g., consist of), e.g., sterile water and the compositions described herein. The sterile water is typically a pharmaceutical grade water. A pharmaceutical composition may include (e.g., consist of), e.g., phosphate-buffered saline (PBS) and the compositions described herein. The sterile PBS is typically a pharmaceutical grade PBS. In certain embodiments, pharmaceutical compositions include one or more compositions and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone. In certain embodiments, the compositions described herein may be admixed with pharmaceutically acceptable active and / or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

[0081] EXAMPLES

[0082] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0083] Example 1. Rapid Generation of Human Pluripotent Stem Cell (hPSC)-Derived High Endothelial Venule Organoids with In Vivo Ectopic Lymphoid Tissue Capabilities.

[0084] Objective

[0085] The objective of this study was rapid generation of hPSC-derived high endothelial venule organoids with in vivo ectopic lymphoid tissue capabilities.

[0086] Materials and Methods

[0087] ETV2-PGP1 hiPSC line maintenance and iEndo induction

[0088] The genetic modification of ETV2-PGP1 hiPSCs line was reported previously (22). ETV2-PGP1 hiPSCs were cultured and passaged without antibiotics in mTeSRI medium (STEMCELL Technologies, 05850) on tissue-culture plates coated with growth factor-reduced Matrigel (Corning 354230). The medium was changed daily. The cell line was maintained below passage 32 and mycoplasma contamination was not detected in the cultures. Chromosomal integrity was verified by means of karyotyping. Consistency in cell morphology and sustained expression of pluripotency markers SOX2, OCT3 / 4, and NANOG was verified. Endothelial differentiation was induced by adding 500 ng / mL doxycycline hyclate (Millipore Sigma, D5207). EGM2 (Lonza CC3162) medium was further used to support induced iEndo cell differentiation and expansion. Once reached 90% confluency, iEndo cells were dissociated using TrypLE Express (Life Technologies, 12604013) in 37°C for 10 minutes for further use.

[0089] Isolation and maintenance of human fibroblastic reticular cells (FRCs)

[0090] Human lymph nodes were harvested and digested with 3 ml of enzyme mix containing 0.2 mg / ml collagenase P, 0.1 mg / ml DNase I, and 0.8 mg / ml dispase II, at 37°C for 15 minutes with agitation. Digestion reactions were passed through 40 mm filters and the resulting cell suspensions were centrifuged at 800 rpm for 10min. Supernatants were discarded, and the pellets containing the cells were washed twice and finally resuspended in FACS buffer (DPBS + 2% fetal bovine serum + 1 mM EDTA + 0.1 % sodium azide. Immunostaining with antibodies was performed and incubation was done for 25 minutes at 4°C with antibodies recognizing human PDGFRb and human CD45 (Table 1 ). The gating strategy used for sorting FRCs is shown in FIG. 5a, briefly PDGFRb expression and lack of CD45 expression. The prospectively purified FRCs were cultured in complete DMEM containing 10% FBS, 1 % penicillin / streptomycin, and 1% L-glutamine. Complete DMEM was replaced after 48 hours, and FRCs were allowed to expand for 3 to 4 passages before use. Cells were washed twice with PBS and harvested using 0.25% trypsin-EDTA (Gibco). Expression of FRC markers was verified by means of immunofluorescence after every passage. Table 1 : Antibodies used for immunostaining iEndo and FRCs co-cultures

[0091] To study iEndo and FRCs co-culture in 2D conditions, endothelial differentiation of iEndo hiPSCs line began on day 0 by 500 ng / mL doxycycline treatment. After 6 days of induction, iEndo cells were dissociated using TrypLE Express and cell viability was counted by cell counter (Logos Biosystem, Luna II) with Trypan blue (Gibco, 15250061 ). FRCs were also dissociated as described above. 2.5 x 105iEndo cells and 0.5 x 105FRCs were gently mixed and seeded on the 6-well tissue culture plates at the ratio of 5 to 1 . EGM2 media supplemented with 500 ng / mL doxycycline was used for cell expansion and changed every other day. To generate HEV organoids (HEVOs), iEndo + FRC cocultures at day 6 of differentiation were lifted with Gentle Cell Dissociation Reagent (StemCell Technologies, 100-1077) and transferred on to ultra-low attachment 96 well tissue culture plates (Corning, 7007). The plates were subsequently centrifuged by a horizontal centrifuge (Thermo Scientific, Legend XTR) at 300 g for 3 minutes. 200 pl EGM2 media supplemented with 500 ng / mL doxycycline per well was used and changed every other day. HEVO fabrication began on day 0 (after 1 d of suspension culture). After 3 d in 96 well plates, organoids were transferred into 80 pl cold collagen l / Matrigel gel droplets formed on dimpled parafilm, which was sterilized by UV exposure for 20 min. Final rat tail collagen type I (Corning, 354249) and 25% growth factor-reduced Matrigel were mixed at 1 :1 volume ratio, together with 0.8 pl sterile 1 M NaOH solution to balance pH. Gel droplets containing organoids were solidified at 37°C for 15 mins before being transferred back into ultra-low attachment 96 well plates. 200 pl of EGM2 supplement with 500 ng / mL doxycycline, 100 ng / mL of vascular endothelial growth factor (VEGF) (PeproTech, 100-20) was added per well and changed every other day.

[0092] HEV organoid implantation and melanoma tumor xenograft in NSG mice

[0093] For the study of HEVOs in vivo, organoids or control aggregates containing either iEndos alone or FRCs alone, were embedded in collagen / Matrigel on day 0. After three days of being cultured in 96 well plates, the gel-embedded structures were washed with PBS for 5 minutes and transplanted into the dorsal flank of immunodeficient NOD.Cg PrkdcsadH2rgtm1wi' / SzJ NSG mice (Jackson Laboratory, 005557). All animal experiments were conducted according to the NIH Guidelines for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee of Brigham and Women’s Hospital. After aseptic animal preparation, inhalable isoflurane was used as anesthesia. A subcutaneous pocket was made by a 5 mm incision at the right-side flank. Once HEVO gel was introduced, the incision was closed with 4-0 polypropylene sutures (Ethicon, 8698G). Analgesia was administered in the form of carprofen and buprenorphine during the first 72 h after surgery.

[0094] For the HEVO-melanoma model, B16-F10 murine melanoma cells (ATCC, CRL-6475) were cultured in complete RPMI-1640 medium (Thermo Scientific, 12633012). Cells in exponential growth phase were harvested from culture, washed twice with sterile PBS, counted, and resuspended in PBS. Twelve days post HEVO implantation, 200,000 murine B16-F10 melanoma cells were subcutaneously injected into the edge of HEVO structures grafted in the NSG mice. The growing size of melanoma-HEVO was monitored by a vernier caliper measuring the tumor length and width every 3 days for 24 days.

[0095] Immune cell isolation and adoptive transfer into NSG mice

[0096] For human PBMC isolation, peripheral blood samples obtained from healthy donors after informed and signed consent were collected and processed within 4 hours at the Immunological Core Facility at the Transplant Research Center, Brigham and Women’s Hospital. PBMCs were isolated by density gradient centrifugation at 1 ,000 g for 30 minutes at 20°C using Lymphoprep density gradient medium (Stemcell Technologies, 07820). Cell density and viability was counted by a hemocytometer (Hausser Scientific, 3100) with trypan blue. To reconstitute T cell tracking and immune reaction in NSG mice, CD3+T cells were isolated from spleens of C57BL / 6 mice, OTI mice and OTII mice (Jackson Laboratory, 000664, 003831 , 004194 respectively), using the EasySep Mouse T Cell Isolation Kit (STEMCELL Technologies, 19851 ) according to the manufacture’s instruction. Cell density and viability was counted by hemocytometer with trypan blue. To reconstitute adoptive immunology, 10 million immune cells isolated above in 150 pl normal saline were administrated into NSG mice by retro-orbital intravenous injection respectively. Mice were under anesthesia induction before the injection and monitored 30 minutes post-injection. To simulate adoptive immune reaction, 100 pg of Ovalbumin (OVA, Millipore Sigma, A7641 ) dissolved in 100 pL of complete Freund's adjuvant (CFA, Millipore Sigma, F5881) was subcutaneously administrated into the HEVO grafts in NSG mice that received OTI and OTII CD3+T cells at the same day. Mice were under anesthesia induction before the injection and monitored 30 minutes post-injection. HEVO structures were collected for analysis one week after immune stimulation.

[0097] Cryo-sectioning and immunofluorescent staining

[0098] HEVO and gels were fixed in 4% paraformaldehyde for 30 mins and rinsed with DPBS for 5 minutes 3 times. Fixed organoids were incubated overnight at 4°C in PBS containing 30% w / v sucrose and then transferred into a 1 :1 solution of Optimal Cutting Temperature compound (OCT) (Tissue-Tek, 4583) and PBS containing 30% w / v sucrose for 90 mins. Next, the tissue was placed into a cryostat tissue mold, which was subsequently filled with 100% OCT solution and frozen at -20 °C on a cryostat Peltier cooler. The tissue was sectioned using 8 pm slices and transferred onto a Superfrost Plus glass slide (VWR, 48311 -703). Sections were stored at -20 °C before immunostaining. For immunofluorescent staining of cryo-sections and two-dimensional cell culture, tissues were permeabilized for 30 mins in PBS containing 0.15% Triton-X (MilliporeSigma, T8787), then blocked for 1 h in PBS containing 2% donkey serum. Next, tissue sections were incubated overnight and whole-mount organoids were incubated for 1- 3 d in primary antibodies in PBS containing 2% donkey serum (MilliporeSigma, D9663). Tissues were rinsed 3x in DPBS, then incubated in secondary antibodies in PBS with 2% donkey serum for an equal amount of time as in primary antibodies. Cell nuclei were labelled with 300 nM of 4',6-diamidino-2- phenylindole (DAPI) in PBS for 10 min, followed by 3 rinses in PBS. Tissue sections and whole-mount organoids were imaged on a Zeiss LSM710 confocal microscope. The antibodies used in immunofluorescence experiments are reported in Table 1 .

[0099] Whole tissue mounting and immunofluorescent staining

[0100] Whole HEVO embedded Collagen / Matrigel mixture gel structures were cleared and immunolabelled using an adapted version of the immunolabelling-enabled three-dimensional imaging of solvent cleared organs (iDISCO+) protocol. Briefly, the gel structures were dehydrated using a methanol / water gradient from 1 :3, 1 :1 to 3:1 v / v ratio over the course of 6 h, delipidated using a 3:1 v / v dichloromethane (DCM) / methanol solution for 3 h and bleached with 5% hydrogen peroxide in methanol overnight. Next, they were rehydrated in a reverse manner of methanol / water gradient above and incubated in PBS overnight. Then, the samples were immunolabeled using the procedures described above and dehydrated a second time. The organoids were then rinsed twice in 100% DCM and rehydrated. Finally, they were index-matched using Easylndex (LifeCanvas Technologies EI-Z1001 ) and imaged using a Zeiss LSM710 confocal microscope. A three-dimensional reconstruction of gel-embedded organoids was done using the Zeiss ZEN software. Total RNA isolation and quantitative RT-PCR

[0101] Total RNA from HEVOs or from two-dimension cell cultures was extracted utilizing the Qiagen RNeasy mini kit (Qiagen 74104) according to manufacturer’s instructions. RNA concentration in each sample was validated utilizing Nanodrop 2000 (Thermo Scientific, ND2000). Complementary DNA (cDNA) synthesis was performed using the iScript™ cDNA Synthesis Kit (Bio Rad, 1708891 ) with the input cDNA for each sample normalized to 50 ng / pl for a total of 1 pg per reaction. cDNA was validated with included kit controls by gel electrophoresis. Quantitative PCR was performed with IDT’s PrimeTime qPCR Probe Assays (ZEN / FAM), using SYBR® Green Master Mix (Bio Rad). Curves were obtained on a BioRad CFX96 qPCR machine. Scripts for data processing of quantification cycle (Cq) values were created in Python and graphs were made utilizing Prism 8.4.0. A list of all primers and probes used is provided in Table 2.

[0102] Table 2: List of PrimeTime™ Predesigned qPCR assays used

[0103] Flow cytometry analysis

[0104] Single-cell suspensions were generated from HEVO grafts, lymph nodes, and melanoma tumor grafts and maintained in PBS at 1 x 107cells / ml for FACS immunostaining staining. Live dead blue (Thermo Fisher Scientific) diluted 1 :1000 in DPBS were used to stain the cell viability for 30 minutes at 4°C. The cells were then washed with FACS buffer (DPBS + 2% fetal bovine serum +1 mM EDTA + 0.1% sodium azide) and incubated for 25 minutes at 4°C with antibodies recognizing cell surface markers. Subsequently, the samples were fixed and permeabilized by using an intracellular fixation & permeabilization buffer set (eBioscience™) according to manufacturer’s instructions. The cells were washed in the FACS buffer and subjected to intracellular immunolabeling. The antibodies used in the FACS experiments are reported in Table 1 . Finally, the samples were acquired and were analyzed on a full spectrum flow cytometer, 5-Laser Aurora system (Cytek Biosciences) and data were analyzed using FlowJo V10.0 software.

[0105] Statistical analysis

[0106] Student's t-test and one-way ANOVA with Tukey’s multiple comparisons tests were used. Statistical analysis was performed using GraphPad Prism 9 for Mac OS (GraphPad Software, Inc.). Results

[0107] Induction and specification of hiPSC-derived HEVECs for the generation of HEV organoids.

[0108] In order to generate endothelial cells with an HEV endothelial cell (HEVEC) phenotype, a strategy was devised that combined genetically-induced differentiation of hiPSCs (22) followed by LN stroma- driven specification. A recently developed hiPSC line carrying a Tet-On system that enables overexpression of the transcription factor and hematoendothelial lineage master regulator ETV2 was exploited, resulting in rapid and highly efficient endothelial differentiation (22, 24) (FIG. 1a). To induce endothelial differentiation, ETV2-hiPSCs were incubated with doxycycline for 3 days in endothelial cell growth medium. This initial stage of induction resulted in adoption of a confluent cobblestone cellular morphology, which was concomitant with progressive expression of cluster of differentiation 31 (CD31 ), vascular endothelial vascular endothelial growth factor receptor 2 (VEGFR2), von-Willebrand factor (VWF) over the course of 6 days (FIGS. 1b-1e, FIG. 5a). Because these endothelial cells were generated through a genetic mechanism of induction, they were termed inducible endothelial cells (iEndos) (22).

[0109] Previous studies have highlighted the critical role of stromal cells in secondary lymphoid organ development, maintenance, and remodeling (14, 15, 17), consistent with a well-known general role for tissue-resident stroma in endothelial differentiation and maturation (25-27). This property of stromal cells was taken advantage of to devise a stage of HEVEC specification consisting in co-culturing primitive iEndos (Day 3) with primary human LN-derived FRCs that were isolated by means of FACS sorting (FIG. If, FIGS. 5a-5c).

[0110] Co-culturing iEndos with FRCs accelerated endothelial maturation as indicated by the significantly modified expression of genes encoding the endothelial markers PECAM-1 / CD31 and ICAM1 / CD54, as well as of genes encoding the HEVEC markers VCAM1 and MADCAM1 , recently validated in single cell transcriptomics studies (10, 28-30), by day 3 of differentiation under co-culture conditions (i.e. overall differentiation day 6) (FIG. 1g). Those changes in gene expression were concomitant with accumulation of the HEV signature marker peripheral lymph node addressin (PNAd) (10, 31 ), which was not detected in iEndo cultures alone (FIG. If, FIG. 5d). Co-culturing with FRCs subsequently resulted in reduction of iEndo MADCAM-1 protein levels at Day 12 (FIG. 5e), indicating further maturation of the HEVEC phenotype (32).

[0111] In two-dimensional conditions, co-culturing iEndos and FRCs embedded in a thin layer of basement membrane matrix gel resulted in the assembly of iEndo aggregates, that were lined by FRCs (FIG. 1h). The analysis also revealed the presence of distinctive individual iEndos extending filopodia at the end of those vascular structures, indicative of sprouting angiogenesis (33-35) (FIG. 1i).

[0112] The generation of HEVEC-like cells capable of forming aggregates under two-dimensional culture conditions prompted the designing of a strategy to generate three-dimensional (3-D) HEV organoids (HEVOs). To that end, Day-3 iEndos and LN FRCs co-cultured for three days in 2-D conditions, were gently lifted using a non-enzymatic method of tissue dissociation and embedded in 10-ul hydrogel droplets containing 50% collagen I + 50% low growth factor Matrigel, to form 3-D aggregates on ultra-low attachment surfaces. The resulting HEV / FRC aggregates were grown and maintained in endothelial cell growth medium (FIG. 1j). Histological analyses of the resulting HEVOs one week after aggregation, showed CD31+ endothelial cells forming vascular structures lined by FRCs, indicative of the homotypic endothelial cell-mural cell interactions (FIGS. 1k, 11, 1m). High magnification visualization of the CD31 + vascular structures showed the presence of luminal space (FIG. 1m). In addition, quantitative reversetranscription polymerase chain reaction (qRT-PCR) analysis indicated the upregulation of lymphotoxin beta receptor (LTbR), interleukin 7 (IL7), chemokine ligand 19 (CCL19), and chemokine ligand 21 (CCL21 ) in HEV organoids compared to aggregates containing iEndos alone (FIG. 1n).

[0113] Taken together, these findings indicated that in the presence of FRCs, primitive iEndos derived from hiPSCs through induction of ETV2 expression, can adopt a HEVEC-like phenotype. Under ultra-low attachment conditions, FRC / iEndo aggregates form HEVOs containing vascular structures and specialized endothelial cells with vascular remodeling properties and an HEV chemokine ligand gene expression profile.

[0114] Transplanted HEVOs form TLS that recruit APCs and T cells in vivo.

[0115] To investigate the in vivo properties of synthetic hiPSC-derived HEVs, matrix-embedded HEVOs or either FRCs or iEndos alone were implanted subcutaneously into NOD sc / 'd gamma (NSG) mice (FIG. 2a). Three weeks post-implantation, histological analysis confirmed the preservation of HEV structures in HEVOs, as indicated by expression of human isoforms of CD31 and VE-Cadherin (VE-CAD), concomitant with retention of the HEVEC marker PNAd in grafts containing iEndos + FRCs but not in grafts containing only FRCs or iEndos (FIGS. 2b, 2c, 2d). Critically, transplanted HEVOs contained host-derived lymphatic vessels (LVs) expressing the mouse lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1 ) isoform (FIG. 2e). Further, the grafts also contained collagen I (COL1 )- and fibronectin I (FNI )-expressing human cells assembled into fibers resembling the conduits and fibro-reticular networks characteristic of LNs (FIG. 2f, FIG. 6a) (14, 36). The lymphoid architecture of transplanted HEVOs brought up the question as to whether the grafts contained antigen presenting cells (APCs). The histological analysis identified both Cd11c+and Cd11 b+cells, representing DCs and monocytes, respectively (FIG. 2f, FIG. 6a).

[0116] Given the presence of APCs, the ability of HEVOs to recruit adaptive immune cells in vivo was investigated next. To this end, human peripheral blood mononuclear cells (hPBMCs) were injected intravenously into NSG mice carrying subcutaneous HEVOs (FIG. 2g). Histological analysis of the cells infiltrating the grafts one week after the injection of hPBMCs showed clusters of B220+ and CD4+T cells that had migrated to the constructs, which were not observed in HEVO-bearing mice pre-treated with the anti-PNAd antibody, clone MECA79 (FIGS. 2h-2k, FIG. 6b) (31 ). The presence of CD4+T cells as well as CD8+T cells was further confirmed by means of flow cytometric analysis of HEVO graft cell content (FIG. 21).

[0117] Collectively, the results indicated that transplanted HEVOs can drive lymphoid neogenesis resulting in additional formation of LVs, providing a suitable niche for the migration of DCs and the attraction of T cells via addressin-mediated mechanisms.

[0118] Adaptive immune response in transplanted HEVOs.

[0119] Following confirmation that implanted HEVOs can recruit T cells in vivo, putative mechanisms of immune response taking place within the grafts were investigated. To that end, chicken ovoalbumin (OVA) and OVA-specific OT-I and OT-II mouse CD3+T cells were co-injected into recipient mice carrying HEVOs (FIG. 3a). Chicken OVA elicits T cell-dependent antigen-specific immune responses, in such a manner that pre-trained OT-I CD8+T and OT-II CD4+ T cells rapidly and specifically react with OVA epitopes upon a secondary challenge.

[0120] One week after the injections, the grafts were harvested and dissociated to generate single cell suspensions for flow cytometry studies. The analysis indicated that OVA significantly increased OT-I CD8+T and OT-II CD4+ T cell proliferation within engrafted HEVOs, compared to non-challenged (NC) mice and mice that were injected with complete Freund’s adjuvant (CFA) alone (FIG. 3b, FIG. 7a). In the same mice, scarce numbers of T cells were detected in the underdeveloped lymph nodes (FIG. 7b). It was next asked whether the immune challenge altered cytokine production in the adoptively transferred T cells recruited to the HEVO grafts. It was found that OVA presentation significantly increased production of interferon gamma (IFN-g) in both OT-I and OT-II cells, compared to non-challenged mice and to mice receiving CFA alone (FIG. 3c, FIG. 7c). Similarly, production of interleukin 2 (IL-2) was increased significantly in infiltrating OT-I and OT-II cells in OVA-recipient mice, compared to NC and CFA alone, with a significant increase in OT-II cells also observed for the CFA alone group, compared to NC mice (FIG. 3c, FIG. 7c). These results were consistent with widespread expression of CD25 (interleukin-2 receptor alpha chain) observed in OT-II cells from OVA-treated HEVO grafts compared to NC and CFA counterparts (FIG. 7d). Production of TNFa was significantly increased in both T cell types in OVA recipient mice, compared to the two control groups (FIG. 3c, FIG. 7c), with lesser but significant increases also induced in both T cell populations by CFA alone, compared to NC mice (FIG. 3c, FIG. 7c).

[0121] Taken together these results showed that HEVO grafts recapitulate important functional properties of tertiary lymphoid structure function, providing a niche for T lymphocyte activation and cytokine synthesis in response to antigen challenge.

[0122] HEVOs promote anti-tumor activity by infiltrating T cells.

[0123] Ectopic TLS which form near or adjacent to tumors serve as T cell activation sites (5, 9). The ability of HEVOs was tested to attract and support the activity of T cells against melanoma tumors. To this end, melanoma cells were injected subcutaneously near engrafted HEVOs, and twelve days later 1 x 107isolated mouse CD3+ T cells were injected intravenously (FIG. 4a). Twenty-four days after the injection of melanoma cells, tumors formed in close apposition to HEVO grafts containing mature PNAd+endothelium (FIGS. 4b, 4c; FIG. 8a). The same anatomical features were observed when HEVOs were implanted without a scaffold. Measurement of tumor size every three days for a period of twenty-four days indicated that tumors located near HEVOs were significantly smaller compared to tumors in animals devoid of HEVOs, which also received T cells (FIG. 4d). Reduced tumor size was associated with a significantly higher number of infiltrating CD4+T cells and an increased, albeit not significantly higher, amount of infiltrating CD8+T cells (FIGS. 4e, 4f). Histological analysis confirmed the increased infiltration by CD3+cells in the melanomas associated with HEVOs, compared to control tumors (FIGS. 4g, 4h), and increased CD3+cell proliferation in the interfaces between tumors and HEVOs, compared to the scattered CD3+cells found in areas near the tumors in control mice (FIG. 8b). The analysis further showed the presence of foci containing apoptotic melanoma cells at the interfaces with CD3+cell-containing HEVOs (FIGS. 41, 4j).

[0124] In sum, these results indicated that HEVOs can physically interact with tumors while retaining their TLS architecture and cellular composition. Critically, HEVOs also replicate the anti-tumor activity promoted by TLS, effectively acting as lymphatic hubs supporting T cell interaction with tumors and allowing T cells to infiltrate and kill tumor cells.

[0125] HEVOs without a matrix (Matrigel) persist 4 weeks post-implantation.

[0126] FIGS. 9a and 9b show preservation of HEVO grafts transplanted without a matrix. FIG. 9a includes representative immunofluorescence microscopy images showing HEVO cryosections containing hCD31 -expressing human iPSC-derived vascular structures expressing hCD31 associated with hPDGFRb-expressing human fibroblastic reticular cells. FIG. 9b shows species validation by immunofluorescence imaging showing the detection of human Lamin A / C in hiPSC-derived CD31 cells.

[0127] Summary

[0128] The disclosure reports a tissue-bioengineering approach for the rapid generation of synthetic human HEV-like structures using hiPSCs and lymph node FRCs, that form lymphoid tissue upon implantation in vivo. The strategy exploits the ability of fibroblastic stromal cells to drive the maturation of endothelium in a tissue-specific manner, and to support vasculogenesis (26, 27). Using this approach, HEVECs were derived that when in co-culture with FRCs, formed HEV-like vessels expressing PNAd and other HEV markers. Under low-attachment culture conditions, synthetic HEVs aggregate into three- dimensional HEVOs that are suitable for in vivo applications. As the methods of the disclosure only require nine days, and is both robust and scalable, potentially hundreds of HEVOs could be delivered in a short period of time.

[0129] A previous effort to generate mouse synthetic lymphoid tissues relied on the use of DCs in combination with a thymus-derived stromal cell line, which were mixed into aggregates and implanted in vivo (20, 21 ). The implants formed mouse lymphatic structures with TLS-like anatomy, and adaptive immune function in vivo (21 ). One caveat of that approach is the inconsistent formation of HEVs, which are central to LN and TLS function. Besides, from an applicability standpoint, the method would require invasive procedures for the extraction and purification of DCs from bone marrow and SCs from the thymus (20). By contrast, the methods of the disclosure involve minimally invasive procedures by relying on the use of hiPSCs for the generation of endothelial cells, and of primary autologous or allogeneic FRCs that can be isolated from resected LNs. Because FRCs proliferate rapidly in vitro, a large number of cells can be generated from a small initial sample in just a few days.

[0130] Synthetically generated lymphatic tissue may have therapeutic applications in the context of metastatic LN depletion. Autologous lymphatic tissue generated from patient hiPSCs would be desirable to replace resected metastatic LN, or LN that became atrophied following chemotherapy.

[0131] Critical for this type of application is the finding that after implantation into immunodeficient mice, HEVOs retain their HEV-like constitution for weeks. These results further show that HEVOs drive lymphatic neogenesis by incorporating lymphatic vessels and by forming conduit-like structures similar to those found in LNs and TLS. Consistent with those anatomical features, HEVO grafts host APCs and circulating B lymphocytes, and attract T cells via addressin-mediated mechanisms, further providing a niche for T cell activation in response to antigen challenge. The studies disclosed herein indicate that implanted synthetic HEVs form tertiary lymphoid structures that could be used for lymphatic tissue replacement therapies. Following the advent of T-cell transfer therapies for cancer treatment, approaches to harness and direct the activity of engineered T cells specifically against cancer cells are desirable, in order to increase the efficacy and safety or the treatments (37). This is especially true in the case of solid tumors, against which the efficacy of T cells is lessened by multiple factors, including the ability of T cells to infiltrate the tumor (38, 39). These results with the melanoma model indicated that HEVOs placed in areas adjacent to tumors improve the anti-tumor activity of transferred T cells, partly by increasing their on-site number and their tumor-infiltrating activity. As a result, it was observed that there is significant delay in tumor growth concomitant with increased tumor cell death. Additionally, HEVOs could be used in combination with pharmacologic therapies. For this type of application, HEVOs combined with immune checkpoint inhibitors could be implanted to restore T cell anti-tumor activity. Alternatively, the drugs could be delivered to HEVOs using HEV-targeting nanocarriers (40). Focal delivery of checkpoint inhibitors to HEVOs could have multiple advantages, including the prospective reduction of adverse effects caused by checkpoint inhibitor-associated autoimmune reactions. Alternatively, HEVOs carrying molecules with immunosuppressive or immunomodulating activity could induce immune tolerance and drive the formation of Treg cells. For such applications, HEVOs combined with anti-TNFa, -IL-2, -CTLA4 or -CD40L antibodies, calcineurin inhibitors, IL-10, or CXCL12, could be a potential therapeutic option in the context of organ transplantation, exacerbated inflammatory conditions, and autoimmune disease.

[0132] In summary, the methods of the disclosure for rapid generation of implantable synthetic HEVs make possible the generation of lymphoid structures with TLS functional properties in vivo. Because engrafted HEVOs can effectively harness and focalize T cell activity, this methodology could be a good complement for current therapies aimed at enhancing adaptive immunity, but for which the risk of adverse effects remains considerable.

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[0172] Numbered Embodiments

[0173] 1 . A method of making a synthetic lymphoid organoid, the method comprising culturing a primitive endothelial cell with an isolated lymph node stromal cell in low-attachment tissue culture conditions to form the synthetic lymphoid organoid.

[0174] 2. The method of embodiment 1 , wherein the primitive endothelial cell is derived from an induced pluripotent stem cell.

[0175] 3. The method of embodiment 1 or 2, wherein the synthetic lymphoid organoid is a lymph node high endothelial venule organoid.

[0176] 4. The method of any one of embodiments 1 -3, wherein the lymph node stromal cell is a fibroblastic reticular cell.

[0177] 5. The method of any one of embodiments 1 -4, wherein the primitive endothelial cell and the lymph node stromal cell are autologous human cells. 6. The method of any one of embodiments 1 -4, wherein the primitive endothelial cell and the lymph node stromal cell are allogeneic human cells.

[0178] 7. The method of any one of embodiments 1 -6, wherein the method comprises the use of a supporting scaffold.

[0179] 8. The method of embodiment 7, wherein the supporting scaffold is a Matrigel scaffold.

[0180] 9. A synthetic lymphoid organoid made from a primitive endothelial cell and a lymph node stromal cell.

[0181] 10. The synthetic lymphoid organoid of embodiment 9, wherein the primitive endothelial cell is derived from an induced pluripotent stem cell.

[0182] 11 . The synthetic lymphoid organoid of embodiment 9 or 10, wherein the synthetic lymphoid organoid is a lymph node high endothelial venule organoid.

[0183] 12. The synthetic lymphoid organoid of any one of embodiments 9-11 , wherein the lymph node stromal cell is a fibroblastic reticular cell.

[0184] 13. The synthetic lymphoid organoid of any one of embodiments 9-12, wherein the primitive endothelial cell and the lymph node stromal cell are autologous human cells.

[0185] 14. The synthetic lymphoid organoid of any one of embodiments 9-12, wherein the primitive endothelial cell and the lymph node stromal cell are allogeneic human cells.

[0186] 15. A method of treating a disorder associated with an immunodeficiency, an inflammatory disorder, or a cancer or a condition requiring lymphatic tissue replacement, the method comprising implanting a lymphoid organoid made from a primitive endothelial cell and a lymph node stromal cell into a patient in need thereof.

[0187] 16. The method of embodiment 15, wherein the method is used in combination with a cellular immunotherapy.

[0188] 17. The method of embodiment 15 or 16, wherein the primitive endothelial cell is derived from an induced pluripotent stem cell.

[0189] 18. The method of any one of embodiments 15-17, wherein the synthetic lymphoid organoid is a lymph node high endothelial venule organoid.

[0190] 19. The method of any one of embodiments 15-18, wherein the lymph node stromal cell is a fibroblastic reticular cell.

[0191] 20. The method of any one of embodiments 15-19, wherein the primitive endothelial cell and the lymph node stromal cell are autologous human cells.

[0192] 21 . The method of any one of embodiments 15-19, wherein the primitive endothelial cell and the lymph node stromal cell are allogeneic human cells.

[0193] 22. The method of any one of embodiments 15-21 , wherein the implanting comprises injecting the lymphoid organoid into the patient.

[0194] 23. The method of embodiment 15, wherein the cancer is a solid tumor cancer.

[0195] 24. The method of any one of embodiments 15-23, wherein the method further comprises administration of an immunomodulator.

[0196] 25. The method of embodiment 24, wherein the immunomodulator is an immune checkpoint inhibitor, a cytokine, or an adjuvant. 26. The method of embodiment 25, wherein the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.

[0197] 27. A composition comprising the synthetic lymphoid organoid of any one of embodiments 9-14 and a pharmaceutically acceptable carrier. 28. The composition of embodiment 27, wherein the composition further comprises a nanocarrier.

[0198] Additional embodiments

[0199] All references cited in this specification, including, database-accessioned information (e.g., in GENBANK, UNIPROT, PUBMED), are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

[0200] It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only.

Claims

CLAIMSWhat is claimed is:1 . A method of making a synthetic lymphoid organoid, the method comprising culturing a primitive endothelial cell with an isolated lymph node stromal cell in low-attachment tissue culture conditions to form the synthetic lymphoid organoid.

2. The method of claim 1 , wherein the primitive endothelial cell is derived from an induced pluripotent stem cell.

3. The method of claim 1 or 2, wherein the synthetic lymphoid organoid is a lymph node high endothelial venule organoid.

4. The method of claim 1 , wherein the lymph node stromal cell is a fibroblastic reticular cell.

5. The method of claim 1 , wherein the primitive endothelial cell and the lymph node stromal cell are autologous human cells.

6. The method of claim 1 , wherein the primitive endothelial cell and the lymph node stromal cell are allogeneic human cells.

7. The method of claim 1 , wherein the method comprises the use of a supporting scaffold.

8. The method of claim 7, wherein the supporting scaffold is a Matrigel scaffold.

9. A synthetic lymphoid organoid made from a primitive endothelial cell and a lymph node stromal cell.

10. The synthetic lymphoid organoid of claim 9, wherein the primitive endothelial cell is derived from an induced pluripotent stem cell.11 . The synthetic lymphoid organoid of claim 9 or 10, wherein the synthetic lymphoid organoid is a lymph node high endothelial venule organoid.

12. The synthetic lymphoid organoid of claim 9, wherein the lymph node stromal cell is a fibroblastic reticular cell.

13. The synthetic lymphoid organoid of claim 9, wherein the primitive endothelial cell and the lymph node stromal cell are autologous human cells.

14. The synthetic lymphoid organoid of any one of claim 9, wherein the primitive endothelial cell and the lymph node stromal cell are allogeneic human cells.

15. A method of treating a disorder associated with an immunodeficiency, an inflammatory disorder, or a cancer or a condition requiring lymphatic tissue replacement, the method comprising implanting a lymphoid organoid made from a primitive endothelial cell and a lymph node stromal cell into a patient in need thereof.

16. The method of claim 15, wherein the method is used in combination with a cellular immunotherapy.

17. The method of claim 15 or 16, wherein the primitive endothelial cell is derived from an induced pluripotent stem cell.

18. The method of claim 15, wherein the synthetic lymphoid organoid is a lymph node high endothelial venule organoid.

19. The method of claim 15, wherein the lymph node stromal cell is a fibroblastic reticular cell.

20. The method of claim 15, wherein the primitive endothelial cell and the lymph node stromal cell are autologous human cells.21 . The method of claim 15, wherein the primitive endothelial cell and the lymph node stromal cell are allogeneic human cells.

22. The method of claim 15, wherein the implanting comprises injecting the lymphoid organoid into the patient.

23. The method of claim 15, wherein the cancer is a solid tumor cancer.

24. The method of claim 15, wherein the method further comprises administration of an immunomodulator.

25. The method of claim 24, wherein the immunomodulator is an immune checkpoint inhibitor, a cytokine, or an adjuvant.

26. The method of claim 25, wherein the immune checkpoint inhibitor is a PD-1 or PD-L1 inhibitor.

27. A composition comprising the synthetic lymphoid organoid of claim 9 and a pharmaceutically acceptable carrier.

28. The composition of claim 27, wherein the composition further comprises a nanocarrier.

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