Physiologic humanized mice for studies of MHC class ii function

Abstract

Non-human animals are provided that co-express mutated or humanized Major Histocompatibility Complex Class II protein (MHCII) complexes with invariant chain polypeptide (CD74), CD4 polypeptide, CD223 (LAG-3) polypeptide, COL2 polypeptide, and / or NCF1 polypeptide along with cells, tissues, embryos, and cell-based production systems that co-express the polypeptides. Methods and constructs for manufacture are also provided and methods for utilizing the animals and cell-based production systems for investigation of rheumatoid arthritis and other immunological phenomena in humans.

Description

PHYSIOLOGIC HUMANIZED MICE FOR STUDIES OF MHC CLASS II FUNCTION

[0001] This application claims priority to U.S. Provisional Appln. Ser. No. 63 / 548,866, filed February 2, 2024, and to U.S. Provisional Appln. Ser. No. 63 / 735,244, filed December 17, 2024, and incorporates said applications in their entireties herein by reference.FIELD OF THE INVENTION

[0002] The present invention relates to non-human models for human disease.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 28, 2025, is named / PHYSIOLOGIC HUMANIZED MICE FOR STUDIES OF MHC CLASS II FUNCTION and is 332 KB in size.BACKGROUND OF THE INVENTION

[0004] The major histocompatibility complex class II (MHCII) region codes for the key cell receptors regulating the adaptive immune system. It is essential for maintaining a healthy immune system, for cleaning of stressed cells and defective proteins as well as tumors, and for protection against infections. To fulfill its dynamic function, the MHCII region is highly polymorphic resulting in variation in capacity to protect from infection and susceptibility to autoimmune diseases.

[0005] Autoimmune diseases affect approximately 5% of the population. Major chronic immune mediated inflammatory disorders like rheumatoid arthritis (RA), multiple sclerosis (MS), and insulin-dependent diabetes mellitus are autoimmune diseases strongly linked to certain MHCII genes — known in humans as HLA (Human leukocyte antigen) class 2. The pathogenesis of MHCII-associated autoimmune diseases is complex and governed by a multitude of interacting genetic and environmental factors.

[0006] RA affects approximately 0.5% of the population worldwide and is characterized by inflammation of peripheral joints and bone destruction due to synovial tissue infiltration with immune and inflammatory cells, secretion of proinflammatory cytokines, and matrix metalloproteinases. The most important genetic locus contributing to development of RA is theMHCII region. RA disease susceptibility is associated with certain MHCII alleles located at the DRB1 locus, which accounts for 30-50% of the total genetic risk of RA. Disease-associated loci include DRBl*01:01, DRBl*10:01, DRBl*04:01, DRBl*04:04, DRBl*04:05 and DRBl*04:08. Each of these loci encodes an HLA class 2 0 (DRB1) chain and, together with DRA, form different DR molecules. The DRBl*04:01 is the predominant allele in the Caucasian population whereas the DRBl*04:05 is the predominant allele in the East Asian population. In contrast, DRBl*04:02 is an allele with only weak or no association with RA and may even be associated with protection from RA.

[0007] Type II collagen (COL2) is a self-antigen involved in immune selection and associated with arthritis pathogenesis. Patients diagnosed with RA exhibit detectable antibodies targeting both native and citrullinated forms of COL2 alongside the presence of COL2-specific T cells. RA-associated DRB1 alleles 0401 and 0101 are found to exert restriction on T-cell responses specifically directed at the dominant COL2259-273 peptide. Critically, the major COL2259-273 peptide can undergo hydroxylation, galactosylation, and glycosylation at lysine residues, with RA-associated T cells demonstrating a predominant recognition of the galactosylated variant.

[0008] Another major genetic polymorphism associated with development of autoimmune diseases is located within the NCF1 (neutrophil cytosolic factor 1) locus, originally cloned using animal models and confirmed as both copy number variation and a disease-causative SNP in humans. The human SNP with amino acid (aa) replacement R90H is the major locus controlling genetic susceptibility to systemic lupus erythematosus and plays a major role in many other autoimmune diseases, including RA. Importantly, the disease causative effect of NCF1 is mediated by a lower reactive oxygen response (ROS), which attenuates activation of MHCII restricted autoreactive T cells. Thus, there has been a suggestion that the NCF1 gene may at least indirectly impact immunological functions resulting from MHCII mechanisms.

[0009] Antibodies to citrullinated proteins (ACPAs) are also understood to play a role in the development of RA but the extent and mechanism have till now not been understood. Individuals who carry variant DRB1 *0401 are at increased risk of developing RA and the presence of the variant is associated with the production of ACPAs, which recognize a citrulline instead of an arginine side chain on proteins. This modification is catalyzed by a family of enzymes calledpeptidylarginine deiminases (PADs). PAD4 is a PAD isoform expressed in neutrophils and macrophages. PAD4 plays a role in RA and has been identified as a therapeutic target. ACPAs are present in up to 70% of patients with RA and their presence is used as a classification marker for the disease.

[0010] Experimental animal models for diseases like RA have been successfully employed to screen new therapeutics that might have the potential for the treatment of human patients. Collagen-induced arthritis (CIA) is considered a gold standard model for RA and is induced by immunization with COL2. This immunization elicits autoreactive T cell responses and the production of arthritogenic autoantibodies leading to synovitis, pannus formation, and finally culminating in cartilage and bone destruction.

[0011] Transgenic and minimally humanized mice expressing human HLA-DR1 and HLA- DR4 have been used as a tool for further understanding disease mechanisms and testing novel therapies in several autoimmune diseases but have proven sub-optimal because of artefacts of expression, including, for example, a critical lack of physiological DR4 expression in antigen- presenting cells (APCs), such as in thymic epithelial cells (TECs) (Romero-Castillo et al., Adv. Sci. 2024, 11, 2401513, incorporated herein by reference), and alterations in B-cell function (Labrecque, Immunity, Vol. 11, 515-516, 1999), among other non-physiologic phenotypical expression.

[0012] One way to overcome artefacts of transgenic expression is to replace mouse genes with human genes using targeted knockin. This can be done by either changing specific nucleotides, specific genes, or larger fragments of the chromosome. A limitation with this technology is that the introduced human DNA sequences, and the human expressed genes, do not interact physiologically with the already existing mouse genome and proteome. It leads to inappropriate or deficient interactions resulting in less than useful physiological models for these complex phenomena.

[0013] U.S. Pat. No. 9,043,996 illustrates genetically modified mice expressing a chimeric HLA-DR4 / H-2E gene. Published US20220322648 illustrates genetically modified mice expressing a humanized T cell co-receptor (e.g., humanized CD4 and / or CD8 such as CD8a and / or CD8p), a human or humanized T cell receptor (TCR) comprising a variable domain encoded by at least one human TCR variable region gene segment, and a human or humanizedmajor histocompatibility complex that binds the humanized T cell co-receptor. U.S. Patent No. 11,102,961 illustrates genetically modified mice expressing a humanized Lymphocyte activation gene 3 (LAG3), also referred to as CD223.

[0014] A chimeric humanized non-human model (like a mouse) that considers all the abovedisclosed physiologic influences in RA has never before been conceived or considered as possible and could not be predicted. Complexities and uncertainties present in humanized chimera for each and every functional polypeptide discussed above and the multiples of complexity and uncertainty presented by a combination of humanized chimera in all the moieties discussed result in myriad implications (each unpredictable and unknown) for the physiologic / immunologic systems of a non-human model.

[0015] A chimeric humanized non-human model that achieves physiologic expression of humanized MHCII systems and nevertheless avoids the many technical difficulties in the current art is much needed. In response to these continuing needs and despite extensive efforts in the art to solve these technical problems, the applicant has now surprisingly provided a humanized model where the immune system functions endogenously while expressing humanized factors functioning seamlessly within the otherwise non-human immune system. These new models provide an immense potential for expanding understanding of human immunology, including RA pathogenesis and robustly facilitate evaluation of novel therapeutic approaches, dynamically extending the study of MHCII-associated diseases.SUMMARY OF THE INVENTION

[0016] The present disclosure provides non-human animals that endogenously co-express mutated or chimeric MHCII (including DR, DQ, DP, DO, and DM), CD74 (invariant chain or il), CD4, CD223 (LAG-3), COL2, and / or NCF1, including human polymorphisms. The present disclosure likewise provides cells, tissues, embryos, and cell-based production systems coexpressing one or more of these proteins. Methods and constructs for manufacture are also provided along with methods for utilizing the animals and cell-based production systems for investigation of RA, MS, insulin-dependent diabetes mellitus and other immunologic phenomena in humans including assessing the therapeutic efficacy of a candidate compound for treating a disease associated with autoimmune disease. The present disclosure further provides a non-human animal model capable of producing antibodies to citrullinated proteins (ACPAs) associated with human rheumatoid arthritis (RA).

[0017] A first non-limiting aspect of the present disclosure provides a non-human animal genome, a non-human animal cell, and a non-human animal comprising: a nucleotide sequence of a human DRA genetic fragment that has replaced a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous MHCII a gene locus of the non-human animal; a nucleotide sequence of a human DRB1 allele genetic fragment that has replaced a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous MHCII 13 gene locus of the non- human animal; a nucleotide sequence of a human CD74 genetic fragment that has replaced a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous CD74 gene locus of the non-human animal; a nucleotide sequence of a human CD4 genetic fragment that has replaced a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous CD4 gene locus of the non-human animal; and, a nucleotide sequence of a human CD223 genetic fragment that has replaced a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous CD223 gene locus of the non-human animal, where the following polypeptides are capable of co-expression from the non-human animal genome and / or are co-expressed in the non-human animal cell or at least one cell of the non- human animal: a humanized MHCII a polypeptide expressed from the at least one endogenous MHCII a gene locus of the non-human animal comprising at least an amino acid sequence expressed from the nucleotide sequence of the human DRA genetic fragment; a humanized MHCII 13 polypeptide expressed from the at least one endogenous MHCII 13 gene locus of the non-human animal comprising at least an amino acid sequence expressed from the nucleotide sequence of the human DRB1 allele genetic fragment; a humanized CD74 polypeptide expressed from the at least one endogenous CD74 locus of the non-human animal comprising at least an amino acid sequence expressed from the nucleotide sequence of the human CD74 genetic fragment; a humanized CD4 polypeptide expressed from the at least one endogenous CD4 locus of the non-human animal comprising at least an amino acid sequence of the nucleotide sequenceexpressed from the nucleotide sequence of the human CD4 genetic fragment; and, a humanized CD223 polypeptide expressed from the at least one endogenous CD223 locus of the non-human animal containing at least an amino acid sequence expressed from the nucleotide sequence of the human CD223 genetic fragment.

[0018] In a non-limiting embodiment, a nucleotide sequence of a human DRA genetic fragment is inserted into a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous MHCII a gene locus of the non-human animal and / or a nucleotide sequence of a human DRB1 allele genetic fragment is inserted into a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous MHCII 13 gene locus of the non-human animal and / or a nucleotide sequence of a human CD74 genetic fragment is inserted into a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous CD74 gene locus of the non-human animal and / or a nucleotide sequence of a human CD4 genetic fragment is inserted into a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous CD4 gene locus of the non- human animal and / or a nucleotide sequence of a human CD223 genetic fragment is inserted into a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous CD223 gene locus of the non-human animal.

[0019] In a non-limiting embodiment, the human DRB 1 allele is selected from the group consisting of DRB1 *04:01, DRBl*04:02, and DRBl*04:05 alleles. In other embodiments, the human DRB1 allele is selected from the group consisting of DRB 1*01:01, DRB1* 10:01, and DRB 1*04: 04 alleles.

[0020] In a non-limiting embodiment, the non-human genome, cell, or animal further comprises a COL2 gene sequence that is humanized. In a non-limiting example, the COL2 gene sequence that is humanized comprises a mutation leading to amino acid replacement D266E or X266E or homologous substitution or retention when expressed from at least one endogenous COL2 gene locus of the non-human animal where a COL2 polypeptide with the amino acid replacement D266E or X266E or homologous substitution or retention is capable of coexpression from the non-human animal genome and / or is co-expressed in the non-human animal cell or in said at least one cell of the non-human animal along with said following polypeptides.In a non-limiting embodiment, the genome, cell, or animal further comprises a COL2 gene sequence, with a mutation leading to amino acid replacement R360Q or X360Q or homologous substitution or retention when expressed from at least one endogenous COL2 gene locus of the non-human animal where a COL2 polypeptide with the amino acid replacement R360Q or X360Q or homologous substitution or retention is capable of co-expression from the non-human animal genome and / or is co-expressed in the non-human animal cell or in said at least one cell of the non-human animal along with said following polypeptides. In a further non-limiting embodiment, the non-human genome, cell, or animal comprises a COL2 gene sequence with a mutation leading to amino acid replacement D266E or homologous substitution or retention and a further mutation leading to amino acid replacement R360Q or homologous substitution or retention when expressed from at least one endogenous COL2 gene locus of the non-human animal where a COL2 polypeptide with the amino acid replacements D266E or homologous substitution or retention and R360Q or homologous substitution or retention is capable of coexpression from the non-human animal genome and / or is co-expressed in the non-human animal cell or in said at least one cell of the non-human animal along with said following polypeptides.

[0021] In a non-limiting embodiment, the non-human animal genome, non-human animal cell, or non-human animal further comprises an NCF1 gene sequence that is humanized. In a non-limiting embodiment, the NCF1 gene sequence that is humanized comprises a mutation leading to amino acid replacement R90H or X90H or homologous substitution or retention when expressed from at least one endogenous NCF1 gene locus of the non-human animal where an NCF1 polypeptide with the amino acid replacement R90H or homologous substitution or retention is capable of co-expression from the non-human animal genome and / or is co-expressed in the non-human animal cell or in said at least one cell of the non-human animal along with said following polypeptides.

[0022] In a further non-limiting embodiment, the non-human animal comprises a COL2 gene sequence with a mutation leading to amino acid replacement D266E and a further mutation leading to amino acid replacement R360Q and a NCF1 gene sequence with a mutation leading to amino acid replacement R90H where a COL2 polypeptide with the amino acid replacements D266E and R360Q and an NCF1 polypeptide with the amino acid replacement R90H are capable of co-expression from the non-human animal genome and / or are co-expressed in the non-humananimal cell or in said at least one cell of the non-human animal along with said following polypeptides.

[0023] In a non-limiting embodiment, any one or more of the nucleotide sequences of a human DRA, DRB1, CD74, CD4, and CD223 genetic fragment that have respectively replaced nucleotide sequences of corresponding genetic fragments in said at least one endogenous DRA, DRB1, CD74, CD4, and CD223 gene loci encode a fragment consisting essentially of the extracellular part of the respective expressed polypeptide. In a further embodiment, any one or more of the nucleotide sequences encode a fragment consisting of the extracellular part of the respective expressed polypeptide. In a further embodiment, they encode no more than the extracellular part of the respective expressed polypeptide.

[0024] In a non-limiting embodiment, the nucleotide sequence of the human DRA genetic fragment comprises at least exons 2 and 3 of a human DRA gene and replaces at least exons 2 and 3 in the at least one endogenous MHCII a gene locus and the nucleotide sequence of the human DRB1 genetic fragment comprises at least exons 2 and 3 of a human DRB1 gene and replaces at least exons 2 and 3 in the at least one endogenous MHCII 13 gene locus. In a nonlimiting embodiment, at least aa residues 29 to 203 of the expressed MHCII 13 polypeptide replace aa residues 29 to 203 that would have been expressed from the endogenous MHCII a gene locus. In a non-limiting embodiment, at least aa residues 35 to 217 of the expressed MHCII 13 polypeptide replace aa residues 33 to 215 that would have been expressed from the endogenous MHCII 13 gene locus.

[0025] In another non-limiting embodiment, the nucleotide sequence of the human CD74 genetic fragment comprises at least exons 2 through 8 of a human CD74 gene and replace at least exons 2 through 8 of the at least one endogenous CD74 gene locus. In a non-limiting embodiment, at least aa residues 42 to 293 of the expressed CD74 polypeptide replace aa residues 26 to 276 that would have been expressed from the endogenous CD74 gene locus.

[0026] In another non-limiting embodiment, the nucleotide sequence of the human CD4 genetic fragment comprises at least exons 2 through a first portion of exon 8 of the human CD4 gene and replaces at least exons 2 through a first portion of exon 8 of the at least one endogenous CD4 gene locus. In a non-limiting embodiment, at least aa residues 26 to 396 of the expressed CD4 polypeptide replace aa residues 27 to 394 that would have been expressed from theendogenous CD4 gene locus. In a non-limiting embodiment, an endogenous signal peptide of aa residues 1 to 26 encoded in the at least one CD4 gene locus is expressed in the CD4 polypeptide.

[0027] In another non-limiting embodiment, the nucleotide sequence of the human CD223 genetic fragment comprises at least position 10 of exon 2 through exon 7 of the human CD223 and replaces at least position 10 of exon 2 through exon 7 of the at least one endogenous CD223 gene locus. In a non-limiting embodiment, aa residues 23 through 450 of the expressed CD223 polypeptide replace aa residues 24 through 442 that would have been expressed from the at least one endogenous CD223 gene locus.

[0028] In a non-limiting embodiment, an MHCII protein complex from a dimerization of the expressed MHCII a and 13 polypeptides forms in association with a CLIP peptide from the expressed CD74 polypeptide on a surface of an antigen presenting cell or analogous cell of the non-human animal. In a further non-limiting embodiment, the CLIP peptide comprises residues 91 to 99 of the CD74 polypeptide. In further non-limiting embodiment, a peptide of the mutated COL2 polypeptide, wherein said polypeptide comprises at least the residues of the D266E mutation of the COL2 polypeptide, displaces the CLIP peptide in a binding groove of the MHCII protein complex on the surface of the antigen presenting cell or analogous cell of the non-human animal. In a further non-limiting embodiment, the MHCII protein complex with COL2 peptide on the surface of the antigen presenting cell or analogous cell of the non-human animal associates with the expressed CD4 polypeptide and the expressed CD223 polypeptide on a surface of a T-regulatory cell or analogous cell of the non-human animal. In a further nonlimiting embodiment, the MHCII protein complex with any non-CLIP peptide on the surface of the antigen presenting cell or analogous cell of the non-human animal associates with the expressed CD4 polypeptide and the expressed CD 223 polypeptide on a T-regulatory cell or analogous cell of the non-human animal.

[0029] In a non-limiting embodiment, the CD4 gene locus of the non-human animal and the CD223 gene locus of the non-human animal are found on the same chromosome in the non- human animal. In a further non-limiting embodiment, the CD4 gene locus of the non-human animal and the CD223 gene locus of the non-human animal are linked. In a non-limiting embodiment, the CD4 gene locus and the CD223 gene locus are linked with only about 16 kbbetween the genes and with a theoretical recombination frequency of about 0.01%. In a nonlimiting embodiment, the linkage is strong, with a genetic distance of approximately O.OlcM.

[0030] In a non-limiting embodiment, the nucleotide sequence of the human DRA genetic fragment that has replaced the corresponding nucleotide sequence fragment in the at least one endogenous MHCII a gene locus of the non-human animal encodes a site in the expressed MHCII a polypeptide that interacts with a corresponding site in the expressed MHCII 13 polypeptide where the corresponding site is expressed from the nucleotide sequence of the human DRB1 allele genetic fragment that has replaced the nucleotide sequence of the corresponding genetic fragment in the at least one endogenous MHCII 13 gene locus of the non- human animal. In a non-limiting embodiment, the site in the MHCII a polypeptide that interacts with a corresponding site in the MHCII 13 polypeptide is involved in formation and stabilization of the MHCII protein complex and further in maintaining the structure of the MHCII protein complex to enable interaction of the MHCII protein complex with the CD74 molecule for formation of the protein complex between these molecules. In a further non-limiting embodiment, the nucleotide sequence of the human DRA genetic fragment that has replaced the corresponding genetic fragment in the at least one endogenous MHCII a gene locus of the non- human animal encodes polypeptide features involved in CLIP peptide dissociation from the MHCII protein complex and subsequent exchange of the CLIP peptide with a target peptide.

[0031] In a further non-limiting embodiment, the MHCII protein complex further comprises interaction sites among the portion of the polypeptide expressed from the nucleotide sequence of the human DRA genetic fragment that has replaced the nucleotide sequence of the corresponding genetic fragment in the at least one endogenous MHCII a gene locus of the non-human animal and the portion of the polypeptide expressed from the nucleotide sequence of the human CD223 genetic fragment that has replaced the nucleotide sequence of the corresponding genetic fragment in the at least one endogenous CD223 gene locus of the non-human animal. In a further non-limiting embodiment, the MHCII protein complex further comprises interaction sites among the portion of polypeptide expressed from the nucleotide sequence of a human DRB1 genetic fragment that has replaced the nucleotide sequence of the corresponding genetic fragment in the at least one endogenous MHCII 13 gene locus of the non-human animal and the portion of polypeptide expressed from the nucleotide sequence of a human CD223 genetic fragment thathas replaced the nucleotide sequence of the corresponding genetic fragment in the at least one endogenous CD223 gene locus of the non-human animal.

[0032] In a further non-limiting embodiment, the nucleotide sequence of the human DRA genetic fragment that has replaced the nucleotide sequence of the corresponding genetic fragment in the at least one endogenous MHCII a gene locus of the non-human animal comprises a nucleotide sequence encoding an al domain and a nucleotide sequence encoding an a2 domain. In a further non-limiting embodiment, the nucleotide sequences encoding the al domain and the a2 domain comprise modifications wherein the nucleotide sequences share 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more identity with the corresponding endogenous DRA genetic fragment. In other embodiments, the nucleotide sequences encoding the Bl domain or the B2 domain of a DRB1 genetic fragment share 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more identity with the corresponding endogenous DRB1 genetic fragment

[0033] In a further non-limiting embodiment, the Bl domain is the structural part of the peptide binding cleft and interacts with DRA. In a further non-limiting embodiment, the Bl domain comprises modifications as compared to that encoded by a human DRB1 allele that do not affect the MHCII B polypeptide structure, the Bl domain comprises modifications as compared to that encoded by a human DRB1 allele that are not directly involved in peptide binding, the Bl domain comprises modifications as compared to that encoded by a human DRB1 allele that are not directly involved in peptide binding and do not alter hydrogen bond formation, and / or the Bl domain comprises modifications as compared to that encoded by a human DRB1 allele that do not change the function of the MHCII complex.

[0034] In other embodiments, any domain of the invention (including, for example, any domain of MHCII, CD74, CD4, CD223, COL2, or NHCF1) comprises modifications as compared to that encoded by a corresponding human allele of the domain that do not affect the structure of the polypeptide of the invention, that are not directly involved in binding activities of the domain and / or do not alter hydrogen bond formation, and / or that do not change the function of the MHCII / CD74 endosomal process and / or the MHCII / CD4 / CD223 / antigenic peptide / TCR complex (see, e.g., Fig. 1).

[0035] In a non-limiting embodiment, the nucleotide sequence of the human DRB1 genetic fragment that has replaced the nucleotide sequence of the corresponding genetic fragment in theat least one endogenous MHCII B gene locus comprises a nucleotide sequence encoding a B2 domain.

[0036] In a non-limiting embodiment, the expressed a2 domain comprises sites that interact with the expressed CD4 polypeptide. In a non-limiting embodiment, the expressed B2 domain comprises sites that interact with the expressed CD4 polypeptide.

[0037] In non-limiting embodiments, the human DRA genetic fragment codes for an extracellular part of the DRA, the DRA extracellular part comprises an al domain, the DRA extracellular part comprises a a2 domain, and / or the DRA extracellular part comprises an al domain and an a2 domain. In a non-limiting embodiment, the DRB 1 extracellular part comprises a human DRB1 Bl extracellular part, a human DRB1 B2 extracellular part, and / or a human DRB1 Bl domain and a human DRB1 B2 domain.

[0038] In some embodiments, the human DRA extracellular part coded from the at least one endogenous MHCII a gene locus is operably linked to an endogenous non-human MHCII a promoter with non-limiting regulatory elements and / or the human DRB1 allele extracellular part coded from the at least one endogenous MHCII B gene locus is operably linked to an endogenous non-human MHCII B promoter with non-limiting regulatory elements. In some embodiments, any domain of the invention (including, for example, any domain of MHCII, CD74, CD4, CD223, COL2, and NHCF1) is operably linked to a corresponding endogenous non-human promoter with non-limiting regulatory elements.

[0039] In a non-limiting embodiment, the human genetic fragment of CD74, CD4, and / or CD223 that has replaced a nucleotide sequence of a corresponding endogenous genetic fragment shares 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more identity with the corresponding endogenous genetic fragment, respectively. In a non-limiting embodiment, the humanized COL2 and / or NCF1 gene sequence shares 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more identity with the corresponding endogenous COL2 and / or NCF1 gene sequence, respectively.

[0040] In a non-limiting embodiment, the non-human animal is a mouse. In a further nonlimiting embodiment, the at least one endogenous MHCII gene locus of the non-human animal comprises an endogenous mouse MHCII Ea ectodomain gene locus. In a further non-limiting embodiment, the endogenous mouse MHCII Ea ectodomain gene locus is a C57BL / 6N (B6N) mouse endogenous mouse MHCII Ea ectodomain gene locus. In a further non-limitingembodiment, the non-human animal is the C57BL / 6N (B6N) mouse and an MHCII Ea gene promoter is repaired.

[0041] In a non-limiting embodiment, the exons 2 and 3 encoded by the human genomic ectodomain DR sequence is the extracellular part of the 0 chain of DR. In a further non-limiting embodiment, the extracellular part of the 0 chain of the EB (the mouse DRB) is replaced by the 0 chain of MHCII (DRB 1*04: 01, DRBl*04:02, DRBl*04:05, or other allele). In a further nonlimiting embodiment, mouse MHCII (E alpha) Ea-ps-targeted ES cells are double-targeted to humanize the Ebl (E beta) locus with human exons 2-3 from the DRB1 *04:01, DRB1 *04:02, or DRB 1*04: 05 gene. In a further non-limiting embodiment, the non-human animal expresses both DRBl*04:01 / Ebl, DRBl*04:02 / Ebl, or DRBl*04:05 / Ebl and DRA / Ea-ps human-mouse chimeric proteins.

[0042] In a non-limiting embodiment, the non-human animal further comprises a congenic fragment from the original Mus musculus musculus genome (denoted Cia9i) containing the FcR gene region. In a non-limiting embodiment, the congenic fragment from the original Mus musculus musculus genome (denoted Cia9i) containing the FcR gene region correlates with a more natural selected physiology in the mouse. In a further non-limiting embodiment, the non- human animal is a mouse comprising a DRB1 allele selected from the group consisting of 0401, 0402, and 0405. In a further non-limiting embodiment, the mouse expresses a phenotype demonstrating an increase in susceptibility to arthritis.

[0043] In a non-limiting embodiment, the animal is a mammal, a primate, a ruminant, and / or a rodent. In a non-limiting embodiment, the animal is a monkey, a horse, a pig, a rabbit, a goat, a sheep, a chicken, and / or a rodent. In a further non-limiting embodiment, the rodent is a rat and / or a mouse. In a non-limiting embodiment, the non-human animal expresses a humanized MHCII protein complex and also expresses a murine MHCII Ab protein complex. In a non-limiting embodiment, the expressed MHCII a polypeptide and the MHCII 13 polypeptide both comprise a transmembrane domain and a cytoplasmic domain selected from the group consisting of a mouse MHCII E polypeptide, a mouse MHCII A polypeptide, and a mouse MHCII Aqpolypeptide.

[0044] In a non-limiting embodiment, an expressed functional humanized MHCII polypeptide is not expressed from an endogenous MHCII gene locus of the non-human animal. In other embodiments, an expressed functional humanized CD74, CD4, CD223, COL2, and / orNHCF1 polypeptide is not expressed from a respective endogenous CD74, CD4, CD223, COL2, and / or NHCF1 gene locus of the non-human animal.

[0045] In still another aspect of some embodiments, disclosed herein are targeting nucleic acid constructs useful for modifying a rodent genome (e.g., in a rodent ES cell) for making a modified rodent. In a further aspect of some embodiments, disclosed herein is use of a disclosed rodent as a rodent model of human diseases associated with autoimmune disease, including and not limited to RA, MS, and insulin-dependent diabetes mellitus. In still a further aspect of some embodiments, disclosed herein is a method of assessing the therapeutic efficacy of a candidate compound for treating a disease associated with autoimmune disease, including and not limited to RA, MS, and insulin-dependent diabetes mellitus. In a non-limiting embodiment, the method comprises assessing the therapeutic efficacy of a vaccine against RA, MA, or insulin-dependent diabetes comprising initiating a model disease, administering the vaccine, and quantifying disease progression in the non-human animal to assess the therapeutic efficacy of the vaccine. In other embodiments, the vaccine is a DRB1 / COL2 vaccine.

[0046] A second non-limiting aspect of the disclosure provides a method of making a non- human animal genome, a non-human animal cell, and / or a non-human animal of the disclosure comprising, in no particular order: replacing at least one nucleotide sequence of at least one endogenous MHCII a gene locus in a non-human animal genome, animal cell, or animal with a nucleotide sequence of a human DRA genetic fragment; replacing at least one nucleotide sequence of at least one endogenous MHCII B gene locus in said non-human genome, cell, or animal with a nucleotide sequence of a human DRB1 allele genetic fragment; replacing at least one nucleotide sequence of at least one endogenous CD4 gene locus in said non-human genome, cell, or animal with a nucleotide sequence of a human CD4 genetic fragment; replacing at least one nucleotide sequence of at least one endogenous CD223 gene locus in said non-human genome, cell, or animal with a nucleotide sequence of a human CD223 genetic fragment; and replicating said non-human genome or growing said non-human cell or animal to make said non- human genome, cell, or animal.

[0047] In a non-limiting embodiment, further replacing at least one endogenous COL2 gene sequence in an endogenous COL2 gene locus of the non-human genome, cell, or animal with a mutated COL2 gene sequence with amino acid replacement D266E and / or amino acidreplacement R360Q and / or replacing at least one endogenous NCF1 gene sequence in an endogenous NCF1 gene locus of the non-human genome, cell, or animal with a mutated NCF1 gene sequence with the amino acid replacement R90H.

[0048] A non-limiting embodiment provides, in no particular order: in a first non-human animal, replacing at least one nucleotide sequence of a corresponding genetic fragment in at least one endogenous MHCII a gene locus of the non-human animal with a nucleotide sequence of a human DRA genetic fragment; in a second non-human animal, replacing at least one nucleotide sequence of a corresponding genetic fragment in at least one endogenous MHCII 13 gene locus of the non-human animal with a nucleotide sequence of a human DRB1 allele genetic fragment; in a third non-human animal, replacing at least one nucleotide sequence of a corresponding genetic fragment in at least one endogenous CD74 gene locus of the non-human animal with a nucleotide sequence of a human CD74 genetic fragment; crossing the first non-human animal with the second non-human animal resulting in a first progeny; crossing the first progeny with the third non-human animal resulting in a second progeny; and selecting said second progeny as said non-human animal.

[0049] A non-limiting embodiment provides, in no particular order: in a first non-human animal, replacing at least one nucleotide sequence of a corresponding genetic fragment in at least one endogenous MHCII a gene locus of the non-human animal with a nucleotide sequence of a human DRA genetic fragment; in a second non-human animal, replacing at least one nucleotide sequence of a corresponding genetic fragment in at least one endogenous MHCII 13 gene locus of the non-human animal with a nucleotide sequence of a human DRB1 allele genetic fragment; in a third non-human animal, replacing at least one nucleotide sequence of a corresponding genetic fragment in at least one endogenous CD74 gene locus of the non-human animal with a nucleotide sequence of a human CD74 genetic fragment; in a fourth non-human animal, replacing at least one nucleotide sequence of a corresponding genetic fragment in at least one endogenous CD4 gene locus of the non-human animal with a nucleotide sequence of a human CD4 genetic fragment; in a fifth non-human animal, replacing at least one nucleotide sequence of a corresponding genetic fragment in at least one endogenous CD223 gene locus of the non- human animal with a nucleotide sequence of a human CD223 genetic fragment; crossing the first non-human animal with the second non-human animal resulting in a first progeny; crossing thefirst progeny with the third non-human animal resulting in a second progeny; crossing the second progeny with the fourth non-human animal resulting in a third progeny, crossing the third progeny with the fifth non-human animal resulting in a fourth progeny, and selecting said fourth progeny as said non-human animal.

[0050] A further non-limiting embodiment provides using any other means resulting in any one or more of said corresponding genetic fragments being present in said loci in said non- human animal resulting in said non-human animal. Said means may include and are not limited to genetic manipulation of targeted (gene modified) ES cells, or any other means of bringing the corresponding genetic fragments present in said loci into the same individual non-human animal.

[0051] In a further non-limiting embodiment, in a sixth non-human animal, mutating at least one COL2 gene sequence domain of the sixth non-human animal R360Q and / or D266E, in a seventh non-human animal, mutating at least one NCF1 gene sequence domain of the seventh non-human animal R90H, crossing said fourth progeny with said sixth non-human animal resulting in a fifth progeny, crossing said fifth progeny with said seventh non-human animal resulting in a sixth progeny, and selecting said sixth progeny as said non-human animal. In a non-limiting embodiment, in the sixth non-human animal, the at least one COL2 gene sequence domain of the sixth non-human animal is mutated R360Q and / or D266E.

[0052] A further non-limiting embodiment provides a method of making a non-human animal of the disclosure comprising, in no particular order: replacing exons 2 and 3 of at least one ectodomain of at least one endogenous MHCII a gene sequence domain of the non-human animal with a human DRA ectodomain gene sequence containing exons 2 and 3; replacing exons 2 and 3 of at least one ectodomain of at least one endogenous MHCII 13 gene sequence domain of the non-human animal with any human DRB1 ectodomain gene sequence containing exons 2 and 3; replacing exons 2 to 8 of at least one endogenous gene sequence of CD74 of the non-human animal with a human CD74 gene sequence of at least exons 2 to 8; replacing exons 2 through a first portion of exon 7 of at least one endogenous gene sequence domain of CD4 of the non- human animal with exons 2 through a first portion of exon 7 of the gene sequence domain of CD74; replacing at least position 10 of exon 2 through exon 7 of at least one endogenous gene sequence domain of CD223 of the non-human animal with at least position 10 of exon 2 through exon 7 of the gene sequence domain of CD223; mutating at least one COL2 gene sequencedomain of the non-human animal R360Q and D266E; and mutating at least one NCF1 gene sequence domain of the non-human animal R90H. In a non-limiting embodiment, the any human DRB1 allele is selected from the group consisting of DRBl*04:01, DRBl*04:02, and DRBl*04:05. In a further non-limiting embodiment, the non-human animal is a mouse. In a further non-limiting embodiment, the mouse comprises a Cia9i congenic fragment from the original Mus musculus musculus genome containing the FcR region.

[0053] A third non-limiting aspect of the present disclosure provides a method of making an embryonic stem (ES) cell of a non-human animal wherein said ES cell is potent for production of a humanized MHCII protein complex capable of association with a CLIP peptide from expressed humanized CD74 polypeptide on a surface of an antigen presenting cell or analogous cell of the non-human animal, wherein a non-CLIP peptide is capable of displacing the CLIP peptide in a binding groove of the humanized MHCII protein complex on the surface of an antigen presenting cell or analogous cell arising from said ES cell, and wherein the MHCII protein complex with a non-CLIP peptide on the surface of the antigen presenting cell or analogous cell of the non- human animal is capable of association with an expressed humanized CD4 polypeptide and an expressed humanized CD223 polypeptide on a T-regulatory cell or analogous cell arising from said ES cell. In a non-limiting embodiment, the method comprises the following steps in no particular order: replacing at least one nucleotide sequence of at least one endogenous MHCII a gene locus in said ES cell with a nucleotide sequence of a human DRA genetic fragment; replacing at least one nucleotide sequence of at least one endogenous MHCII B gene locus in said ES cell with a nucleotide sequence of a human DRB1 allele genetic fragment; replacing at least one nucleotide sequence of at least one endogenous CD74 gene locus in said ES cell with a nucleotide sequence of a human CD74 genetic fragment; replacing at least one nucleotide sequence of at least one endogenous CD4 gene locus in said ES cell with a nucleotide sequence of a human CD4 genetic fragment; replacing at least one nucleotide sequence of at least one endogenous CD223 gene locus in said ES cell with a nucleotide sequence of a human CD223 genetic fragment; and growing said embryonic stem (ES) cell of said non-human animal to make said embryonic stem cell.

[0054] In a non-limiting embodiment, further replacing at least one endogenous COL2 gene sequence in an endogenous COL2 gene locus of said ES cell at least with a mutated COL2 genesequence with amino acid replacement D266E and / or amino acid replacement R360Q and replacing at least one endogenous NCF1 gene sequence in an endogenous NCF1 gene locus of said ES cell at least with a mutated NCF1 gene sequence with the amino acid replacement R90H wherein said ES cell is potent for production of a mutated COL2 protein and for production of mutated NCF1 protein, wherein a peptide of said mutated COL2 polypeptide is capable of displacing the CLIP peptide in a binding groove of the humanized MHCII protein complex on the surface of an antigen presenting cell or analogous cell arising from said ES cell, and wherein said mutated NCF1 protein is capable of association with the MHCII protein complex to mediate a lower reactive oxygen response (ROS) in cells arising from said ES cell thereby activating MHCII restricted autoreactive T cells in cells arising from said ES cell.

[0055] A non-limiting embodiment of the third non-limiting aspect of the present disclosure provides: replacing exons 2 and 3 of at least one ectodomain of at least one endogenous MHCII a gene sequence domain; exons 2 and 3 of at least one ectodomain of at least one endogenous MHCII B gene sequence domain; exons 2 to 8 of at least one endogenous gene sequence domain of CD74; at least exons 2 through a first portion of exon 7 of at least one endogenous gene sequence domain of CD4; at least position 10 of exon 2 through exon 7 of at least one endogenous gene sequence domain of CD223 of the ES cell each with the corresponding human domain; mutating at least one COL2 gene sequence domain of the ES cell at least with R360Q and D266E; and mutating at least one NCF1 gene sequence domain of the ES cell at least with R90H.

[0056] In a non-limiting embodiment, the DRB1 allele is DRBl*04:01, DRBl*04:02, DRBl*04:05, DRBl*0404, DRB1 *01:01, or DRB1* 10:01. In a further non-limiting embodiment, the non-human animal is a mouse. In a further non-limiting embodiment, the mouse comprises a Cia9i congenic fragment from the original Mus musculus musculus genome containing the FcR gene region.

[0057] A fourth non-limiting aspect of the present disclosure provides a non-human animal model capable of producing antibodies to citrullinated proteins (ACPAs). In a non-limiting embodiment, the ACPAs are antibodies associated with RA. In a non-limiting embodiment, the ACPAs mimic human antibodies. In some embodiments, the ACPAs structurally recognize the target in a similar way, are functionally promiscuous, and / or are functionally either arthritogenicand / or protective. In a non-limiting embodiment, the produced antibodies include LN13-C12, LN20-D9, and / or SP2-D3. A non-limiting embodiment of the present disclosure provides use of said non-human animal model to produce said ACPAs.

[0058] A fifth non-limiting aspect of the present disclosure provides use of any one of the physiologic non-human animals of the disclosure as a human model of immunology. A nonlimiting embodiment provides use of a physiologic non-human animal comprising humanized MHCII, CD74, CD4, CD223, COL2, and / or NCF1 genes for modeling at least one disease. Nonlimiting diseases include those arising in haplotypes containing PTPN22, NCF1, NCF4, PADI4, FCGR2B, TNFRSF14, IL6R, PTPRC, STAT4, CD28, CTLA4, IL2, IL3, CSF1, NFKBIE, TNFAIP, CCR6, CDK6, IRF5, TRAF1, C5, IL2RA, TRAF6, RAG1, CD5, ATM, CDK2, CDK4, SH2B4, SH2B3, IKZF3, CSF3, PTPN2, ICAM1, TYK2, CD40, IFNGR2, ICOSLG, AIRE, IL2RB, IRAKI as well as diseases arising in key regulatory genes (including but not limited to LAT, CD45, VISTA) in the human immune response. A non-limiting embodiment provides use of the physiologic non-human animal for development of at least one therapy for at least one immunologic disease. Some embodiments provide use for development of at least one therapy for any one or more of the listed diseases. A non-limiting disease is selected from RA, MS, and insulin-dependent diabetes mellitus. In some embodiments, the non-human animal is any listed animal such as a mouse.BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Fig. 1 illustrates the humanized MHCII (HLA), CD74, CD4 and CD223 proteins.

[0060] Fig. 2 illustrates the knock-in of human HLA-DRA, HLA-DRB1, CD74, CD223 and CD4.

[0061] Fig. 3 illustrates the functional importance of humanized CD74 in concert with humanized MHCII.

[0062] Fig. 4 illustrates COL2+PAD4 induction of severe arthritis determined by human DRBl*0401.

[0063] Fig. 5 illustrates antibodies from Parker mice are specific for citrullinated peptides.

[0064] Fig. 6 illustrates expansion of Tri cells in Primus (HLA-DRA, HLA-DRBl*04:01, hCD74, hCD4, hCD223) mice after COL2 immunization.

[0065] Fig. 7 illustrates a DR*0401-COL2 vaccine is more effective in Primus and less effective in Parker.

[0066] Fig. 8 illustrates functional NCF1 is needed for a vaccination effect on arthritis in Parker mice.

[0067] Fig. 9 illustrates COL2 R360Q mutation regulates development of arthritis.

[0068] Fig. 10 illustrates a summary of specific non-limiting and exemplary mouse strains disclosed herein.

[0069] Fig. 11 illustrates crossing of non-limiting and exemplary disclosed mouse strains with different genetic modifications.

[0070] Fig. 12 illustrates an observed higher frequency of Tri cells in all disclosed mouse strains with humanized CD4 and CD223 and higher frequency of Tri cells in Primus mouse (HLA-DRA, HLA-DRBl*04:01, hCD74, hCD4, hCD223) with additional humanized COL2 (P.COL2.266e) as compared to Primus mouse without humanized COL2.DETAILED DESCRIPTION OF THE INVENTION

[0071] The present disclosure provides genetically modified non-human animals that coexpress humanized Major Histocompatibility Complex Class II region (MHCII) polypeptide, humanized CD74 (invariant chain or li) polypeptide, humanized CD4 polypeptide, humanized CD223 (LAG3) polypeptide, modified COL2 polypeptide, and / or modified NCF1 polypeptide as well as cells, tissues, embryos and cell production systems co-expressing two or more of the polypeptides. The disclosure further provides methods of making and using the non-human animals, cells, tissues, embryos, and cell production systems. In a non-limiting embodiment, a humanized molecule is a chimeric non-human animal molecule with a human-derived ectodomain, some part of a human-derived ectodomain, or some human-derived extracellular part of the molecule. Also disclosed are targeting nucleic acid constructs useful for modifying a non-human animal genome (e.g., in a non-human animal ES cell) for making a modified non- human animal model of human disease and a method of assessing the therapeutic efficacy of a candidate compound for treating a human disease.

[0072] Co-expressed humanized MHCII proteins interact with humanized CD74 and humanized CD4 and CD223 proteins and may additionally further interact with modified COL2 polypeptide and modified NCF1 polypeptide resulting in till-now unachievable andunpredictable restoration of humanized physiologic interaction in a non-human system. Humanization of the ectodomains or extracellular parts of these molecules further restores physiological interactions in vivo, where interacting molecules expressing human ectodomains or extracellular parts regulate MHCII function.

[0073] Surprisingly, interaction among human-derived MHCII, CD74, CD4, and CD223 molecules in the current humanized non-human model is herein demonstrated to be critical to physiologic MHCII function including a normal expression and function of human MHCII on antigen presenting cells (for example thymic epithelial cells and B cells) and T cells (for example, activated CD44+T cells in the periphery). This interaction is also important for mimicking typical human traits, including a not-before-created human-typical immune response leading to production of ACPAs. Furthermore, the herein-disclosed humanized non-human models provide a more human-like allelic and gender susceptibility to arthritis, human-like infectious tolerance, and human-like T cell expression of DR*04:01 and other alleles. These interactions and resulting physiological phenomena are observed to be important to modeling of human immunology and disease.

[0074] This stands in contrast to interactions between human and mouse derived immunological proteins and individual and non-integrated humanized proteins in mice in the prior art, which have failed to obtain physiological function and have failed to mimic human immunology and disease through non-physiologic thymus and antigen presenting cell expression of MHC, aberrant B cell expression, and non-human-like arthritis expressions, among other anomalous artefacts. The present disclosure surprisingly allows direct in vivo studies of autoimmune diseases such as RA and related human genes and mutations such as human regulatory molecules and self-target antigens (e.g. COL2) while avoiding deleterious artefacts in non-human models.

[0075] In a further surprise, additional humanization of NCF1 (the cytosolic subunit of neutrophil NADPH oxidase enzyme producing superoxide anion and the major polymorphic locus associated with autoimmune disease besides MHCII) now allows studies of the functional role of different MHCII alleles (e.g. DRBl*0401, DRBl*0402, DRBl*0405, DRBl*0404, DRBl*01:01, and DRBl*10:01) and NCFl alleles (e.g. R90H) by facilitating physiologic interaction. Likewise, mutation in the COL2 gene leading to replacement of aspartic acid at triplehelical position 266 with glutamic acid (COL2.D266E), for example, leads to a humanization of the C0L2 protein allowing studies of immune tolerance of key importance — in particular, for autoimmune disease involving arthritis. Moreover, mutation in the COL2 gene leading to replacement of arginine at triple helical position 360 with glutamine (COL2.R360Q), for example, leads to modification of the major B cell epitope for COL2 specific suppressor B cells of herein-demonstrated key importance for studies of immune tolerance in autoimmune diseases like RA.

[0076] Still another critical and surprising finding is that a well-defined genetic background, naturally selected (such as the Mus.musculus.musculus gene background) provides collective physiologic interaction of all these humanized genes.

[0077] Together, these technical advances provide not-before-seen physiologic expression of MHCII on thymus cells and other antigen presenting cells, unique activation of B cells producing antibodies to citrullinated proteins (ACPAs) associated with human RA, human-like allelic susceptibility to arthritis and robust T cell responses to unmodified COL2 peptide, and humanlike gender differences in immune response in mouse models like CIA, among other physiologic phenomena, all allowing for not-before-achieved immunologic human disease modeling in nonhuman animals. Previous attempts in the art at humanizing mouse models have failed to achieve these results, in part because previous attempts have not recognized or predicted the critical interacting rolls of each of the polypeptides of the disclosure.Definitions

[0078] Unless defined otherwise and clearly indicated or clearly apparent from the context in which a term or phrase is used, the terms and phrases in this disclosure reflect their meaning in the art. The term “humanized” means targeted replacement of genomic regions on a non-human animal genome with orthologous human sequences including targeted replacement of genomic regions on a mouse genome with orthologous human sequences. The term “locus” means a specific, fixed position on a chromosome where a particular haplotype, gene, or genetic marker is located. Unless otherwise made clear from the language context, the term “locus” when associated with terms like “gene” may include promoters and regulatory elements. The term “part” in this context means a specified stretch of chromosome or expressed molecule. The term “haplotype” means an evolutionarily conserved chromosomal fragment containing linked geneticvariants that are naturally selected together. The term “allele” means one of two or more alternative forms of a gene or other homologous DNA sequence that can have the same place on homologous chromosomes. The term “polymorphism” is a mutation (or variant) that is common in a population, usually defined as present in greater than 1% of the population.

[0079] The terms “corresponding” or “corresponds to” or similar when referring to a nucleotide sequence fragment or gene nucleotide sequence or the like and its relationship to an endogenous nucleotide sequence fragment or gene nucleotide sequence or gene locus, which may or may not include promoters and regulatory elements, means orthologous nucleotide sequences that share homology and some function. The terms “corresponding” or “corresponds to” or similar when referring to an interaction among amino acid sequences or polypeptide functional motifs means sites among two or more amino acid sequences or polypeptide functional motifs that at least transiently physically interact with one another, stick to one another, bind to one another, including hydrogen bonding, or similar.

[0080] The terms “homology” or “homologous” in reference to nucleic acid and amino acid sequences, means comparable sequences when optimally aligned share at least about 75%, 80%, 90%, 95%, or 97% or greater of nucleotides or amino acids. Targeting constructs may contain homology arms such that homologous recombination will occur between a targeting construct and a targeted endogenous sequence. Homology means that a functional framework is maintained as encoded within a nucleic acid sequence or expressed in an amino acid sequence while possible variations within the respective sequences are allowed such that the expressed amino acid sequence maintains an acceptable level of its function in a given system. The term “identity” used in connection with disclosed and embodied sequences is the amount of amino acid residues or nucleic acid bases that match exactly between two different sequences. Sequence identity may be determined by one of skill in the art utilizing various algorithms. Respective lengths are understood in the art to depend on given details of sequences. A chimeric sequence in comparison to a human sequence compares the chimeric portion to the human sequence. Comparing a sequence from its N-terminus to its C-terminus is also employed for identity. Identities may be determined, for example, with a ClustalW v. 1.83 slow alignment with open gap penalty 10.0, extend gap penalty 0.1.

[0081] The term “replace” or “replacement” or similar in reference to gene replacement refers to replacing all or a portion of an endogenous gene with a homologous or orthologous nucleic acid sequence by placing exogenous genetic material at an endogenous genetic locus. The term “insert” or “insertion” or similar in reference to inserting genetic fragment(s) into a gene refers to inserting a homologous or orthologous nucleic acid sequence into an endogenous genetic locus by placing exogenous genetic material into the endogenous genetic locus. None, some, or all the homologous or orthologous portions of the endogenous genetic locus into which the homologous or orthologous nucleic acid is inserted may be removed. One of ordinary skill in the art understands the purpose of the insertion is to create functional expression of the gene reflecting functional expression of the inserted homologous or orthologous nucleic acid sequence. A “homologous substitution or retention” or similar results in an amino acid residue(s) that is functionally the same as an intended residue(s). For example, R360Q could be homologously substituted or retained from an individual non-human animal or group of nonhuman animals in the following examples: X360Q, Q360Q, R360N, K360Q, or K360N, etc. If the homologous position in any given non-human animal is different from the given position, the homologous position receives the substitution or retention. For example, if R360Q is the intended change but the animal is homologous to R360 at R359, then the substitution from R to Q should be at position 359.

[0082] Amino acid residue numbering for a given polypeptide, protein, or expressed gene are given in canonical numbering. “X” refers to an amino acid residue position filled with any amino acid.

[0083] A “variant” of a polypeptide (e.g. MHC, CD74, CD4, CD223) is any type of variant of genes or molecules in a specific locus that varies in a species. In a more general sense, variant is any type of variable in a gene or molecule in a given population.

[0084] An “MHCII” complex or protein or similar includes a complex between an MHCII a polypeptide and an MHCII 13 polypeptide. An “MHCII” polypeptide includes an MHCII a polypeptide alone or MHCII 13 polypeptide alone. An MHCII complex may additionally further complex with other molecules including CD74, CD4, and CD223.

[0085] A “DR complex”, “DR protein,” “E complex,” “E protein,” “A complex,” “A protein,” “E complex,” “E protein,” or “EA protein,” and similar refer to a complex between aand B polypeptides. DR are human MHCII molecules or components of human MHCII molecules. A and E are mouse MHCII molecules or components of mouse MHCII molecules.

[0086] The term “analogous cell,” when used to describe cell types, includes any cell that shares at least one function in the immune system of an animal with the cell type to which it is referenced as an analog or “analogous cell” such that a polypeptide on, in, or otherwise associated with the “analogous cell” will signal or initiate an action in the immune system similar to the reference cell to which it is analogous.

[0087] The term “linked” or similar refers to genes that are relatively linearly close together on a single chromosome where close alleles are preponderantly (more often than not) inherited, that is, recombination frequency is less than 50%. Linkage is of course a relative estimate. The whole extended human MHC region is estimated to have a recombination rate of 0.72 cM per Mb. It could vary between different haplotypes. To approximate this, it would then be 0.5 cM, i.e., a recombination frequency of 0.5%.

[0088] The term “operably linked” refers to positioning on a chromosome that results in a particular function of a gene or combination of genes. For example, a promoter, enhancer, or silencer sequence, etc. (regulatory sequences) is often operably linked to a nucleic acid sequence encoding a protein. This may result in proper transcriptional regulation of the protein. In a chimeric or humanized protein, various elements may be operably linked to retain proper expression, processing, and folding, among other functions.

[0089] An “antigen-presenting cell” includes cells such as dendritic cells, mononuclear phagocytes, some endothelial cells, thymic epithelial cells, and B cells.

[0090] The term “conservative,” when used to describe amino acid residue substitution, includes one amino acid exchanged into another amino acid that has similar properties where the exchanged residues have side chain R groups with similar chemical properties such as charge or hydrophilicity / hydrophobicity. This type of replacement is expected in the art to be a functional replacement, not substantially changing the functional properties of interest of a protein. A functional replacement may be, for example, a replacement that does not disable an MHCII molecule from presenting a peptide. A nucleotide sequence modification that introduces a nucleotide change encoding a conservative amino acid substitution may be conservative. One of ordinary skill in the art understands how to make conservative substitutions in the nucleic acidsequences and polypeptides of the disclosure and conservative substitutions are contemplated within the disclosure.

[0091] A “functional” polypeptide means a polypeptide demonstrating a desired biological activity associated with a native protein. Functional endocellular activity in this disclosure generally refers to expression, transport, and signaling, and extracellular activity in this disclosure generally refers to binding and signaling.

[0092] Amino acid groupings that reflect similar chemical properties through side chains are basic side chains (including lysine, arginine, and histidine); aliphatic side chains (including alanine, glycine, leucine, valine, and isoleucine); aliphatic-hydroxyl side chains (including threonine and serine); amide-containing side chains (including glutamine and asparagine); acidic side chains (including aspartic acid and glutamic acid); aromatic side chains (including phenylalanine, tyrosine, and tryptophan); and sulfur-containing side chains (including cysteine and methionine). Examples of conservative amino acid substitution groups would include: valine for leucine for isoleucine, alanine for valine, phenylalanine for tyrosine, lysine for arginine, glutamate for aspartate, and asparagine for glutamine and vice versa. A substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis, can be a conservative amino acid substitution in certain non-limiting embodiments. Through the PAM250 log-likelihood matrix, a conservative substitution may be made in a nonlimiting embodiment that has a positive value. A substitution that is a moderately conservative substitution with a nonnegative value in the PAM250 log-likelihood matrix may likewise be made in a non-limiting embodiment.

[0093] Any nucleotide sequence encoding a human or humanized MHCII polypeptide, any human or humanized CD74 polypeptide, any human or humanized CD4 polypeptide, any human or humanized CD223 polypeptide, and any COL2 polypeptide or NCF1 polypeptide comprising conservative amino acid substitutions in the amino acid sequence is likewise embodied.

[0094] Due to the known degeneracy of the genetic code, nucleic acids other than specific nucleic acids herein disclosed may encode the polypeptides of the disclosure as understood by the skilled artisan.

[0095] SEQ ID NO: 1 is a DNA sequence listing reflecting the targeted H2-Ea / HLA-DRA locus with the repaired promoter-exon 1 -region from the H2d haplotype (627bp; positions 15001through 15627) including H2-Ea exon 1 (positions 15348 through 15477), and HLA-DRA exon 2 (positions 19642 through 19887) and exon 3 (positions 20379 through 20660). Positions 21206 through 21374 reflect H2-Ea exon 4, and positions 22001 through 22486 reflect H2-Ea exon 5. Ea is the mouse DRA gene and HLA-DRA is the human DRA gene.

[0096] SEQ ID NO: 2 is a DNA sequence reflecting the predicted cDNA protein coding sequence of a humanized / repaired H2-Ea / HLA-DRA gene.

[0097] SEQ ID NO: 3 is a DNA sequence reflecting human HLA-DRA (Transcript: ENST00000613328.1 HLA-DRA-221) coding sequence for comparison with SEQ ID NO: 2.

[0098] SEQ ID NO: 4 is an amino acid sequence reflecting the predicted humanized / repaired H2-Ea / HLA-DRA Protein. The signal peptide runs from position 1 through position 25. Residues encoded by human exons 2 and 3 run from residue 28 through 203. The transmembrane domain runs from residue 217 through 239. The C-terminal intracellular domains run from residue 240 through 255. Residues encoded by mouse exon 1 run from 1 through 27. Residues encoded by mouse exon 4 run from 204 through 255. Six extracellular amino acid residues, running from residue 206 through 210 and including residue 215, coded by the mouse DNA sequence, are nonconserved between the mouse and human.

[0099] SEQ ID NO: 5 is an amino acid sequence reflecting the HLA-DRA protein (Transcript: ENS T00000613328.1 HLA-DRA-221) for comparison with SEQ ID NO: 4. The signal peptide runs from residue 1 through 25. Exon 1 runs from residue 1 through 27, exon 2 and 3 from residue 28 through 203. Exon 4 runs from residue 204 through 254.[000100] SEQ ID NO: 6 is a DNA sequence of the targeted H2-Ebl / HLA-DRBl*04:01 :01 locus for illustration of H2-Ebl exons and human HLA-DRB 1*04:01: 01 exons 2-3. Positions 16426 through 16456 reflect H2-Ebl exon 1. Positions 20067 through 20618 reflect HLA- DRBl*04:01 exon 2 and 3. Positions 21108 through 21345 reflect H2-Ebl exon 4. Positions 21748 through 21771 reflect H2-Ebl exon 5. Positions 22066 through 22391 reflect H2-Ebl exon 6.[000101] SEQ ID NO: 7 is a DNA sequence of the targeted H2-Ebl / HLA-DRBl*04:02:01 locus for illustration of H2-Ebl exons and human HLA-DRBl*04:02:01 exons 2-3. Positions 16433 through 16463 reflect H2-Ebl exon 1. Positions 21946 through 22497 reflect HLA- DRBl*04:02:01 exons 2 and 3. Positions 22987 through 23224 reflect H2 -Ebl exon 4. Positions23627 through 23650 reflect H2-Ebl exon 5. Positions 23945 through 24270 reflect H2-Ebl exon 6. Eb in mouse corresponds to DRB in humans.[000102] SEQ ID NO: 8 is a DNA sequence of the targeted H2-Ebl / HLA-DRB1 *04:05:01 locus for illustration of H2-Ebl exons and human HLA-DRBl*04:05:01 exons 2-3. Positions 16426 through 16456 reflect H2-Ebl exon 1. Positions 21939 through 22490 reflect HLA- DRBl*04:05:01 exons 2 and 3. Positions 22980 through 23217 reflect H2 -Ebl exon 4. Positions 23620 through 23643 reflect H2-Ebl exon 5. Positions 23938 through 24263 reflect H2-Ebl exon 6.[000103] SEQ ID NO: 9 is a DNA sequence of humanized H2-Ebl / HLA-DRB1 containing the cDNA sequence of exons 2-3 of the human allele of HLA-DRBl*04:02:01 for comparison with SEQ ID NO(s): 10 and 11.[000104] SEQ ID NO: 10 is a DNA sequence of humanized H2-Ebl / HLA-DRB1 containing the cDNA sequence of the exons 2-3 of the human allele of HLA-DRBl*04:01:01 for comparison with SEQ ID NO(s): 9 and 11.[000105] SEQ ID NO: 11 is a DNA sequence of humanized H2-Ebl / HLA-DRB1 containing the cDNA sequence of the exons 2-3 of the human allele of HLA-DRBl*04:05:01 for comparison with SEQ ID NO(s): 9 and 10.[000106] SEQ ID NO: 12 is a DNA sequence of the predicted cDNA of the humanized H2- Ebl / HLA-DRBl allele *04:02:01 for comparison with SEQ ID NO(s): 13 and 14.[000107] SEQ ID NO: 13 is a DNA sequence of the predicted cDNA of the humanized H2- Ebl / HLA-DRBl allele *04:01:01 for comparison with SEQ ID NO(s): 12 and 14.[000108] SEQ ID NO: 14 is a DNA sequence of the predicted cDNA of the humanized H2- Ebl / HLA-DRBl allele *04:05:01 for comparison with SEQ ID NO(s): 12 and 13.[000109] SEQ ID NO: 15 is an amino acid residue sequence of humanized H2-Ebl / HLA- DRBl*04:02 predicted protein for comparison with SEQ ID NO(s): 16 and 17.[000110] SEQ ID NO: 16 is an amino acid residue sequence of the humanized H2-Ebl / HLA- DRBl*04:01 predicted protein for comparison with SEQ ID NO(s): 15 and 17.[000111] SEQ ID NO: 17 is an amino acid residue sequence of humanized H2-Ebl / HLA- DRBl*04:05 predicted protein for comparison with SEQ ID NO(s): 15 and 16.[000112] SEQ ID NO: 18 is an amino acid residue sequence of the protein of the translated H2-Ebl gene transcript (ENSMUST00000074557.11 ; H2-Eb 1-201) for comparison with SEQ ID NO(s): 15, 16, and 17.[000113] SEQ ID NO: 19 is a DNA sequence of Cd74 locus for illustration of human CD74 exons and mouse Cd74 exons. Positions 11370 through 11532 reflect mouse Cd74 exon 1. Positions 17119 through 17291 reflect human CD74 exon 2. Positions 17483 through 17562 reflect human CD74 exon 3. Positions 18122 through 18184 reflect human CD74 exon 4. Positions 19263 through 19358 reflect human CD74 exon 5. Positions 19676 through 19763 reflect human CD74 exon 6. Positions 21131 through 21322 reflect human CD74 exon 7. Positions 21818 through 21880 reflect human CD74 exon 8. Positions 22138 through 22632 reflect mouse Cd74 exon 9.[000114] SEQ ID NO: 20 is a DNA sequence of mouse mCd74 Transcript: ENSMUST00000097563.9; Cd74-202) Cd74 protein coding cDNA, for comparison with SEQ ID NO(s): 21 and 22.[000115] SEQ ID NO: 21 is a DNA sequence of human (humCD74; Transcript: ENST00000009530.13; CD74-201) CD74 protein coding cDNA, for comparison with SEQ ID NO(s): 20 and 22.[000116] SEQ ID NO: 22 is a DNA sequence of the predicted humanized hCD74 protein coding cDNA, for comparison with SEQ ID NO(s): 20 and 21.[000117] SEQ ID NO: 23 is an amino acid residue sequence of mouse (mCt / 74) protein sequence, corresponding to the cDNA shown as SEQ ID NO: 20, for comparison with SEQ ID NO(s): 24 and 25.[000118] SEQ ID NO: 24 is an amino acid residue sequence of human CD74 (humCD74) protein sequence, corresponding to the cDNA shown as SEQ ID NO: 21, for comparison with SEQ ID NO(s): 23 and 25.[000119] SEQ ID NO: 25 is an amino acid residue sequence of the predicted humanized CD74 protein, for comparison with SEQ ID NO(s): 23 and 24. Residues encoded by mouse exon 1 run from residue 1 through 25. Residues encoded by human exons 2-8 run from residue 26 through 277. Residues encoded by mouse exon 9 run from residue 278 through 280. Theintracellular domain runs from residues 1 through 30. The transmembrane domain runs from residues 31 through 56. The extracellular domain runs from residues 57 through 280.[000120] SEQ ID NO: 26 is a DNA sequence of the targeted Cd223 (Lag3) locus for illustration of mouse exons and human coding sequences. Positions 1736 through 2147 reflect mouse exon 1. Positions 2541 through 3824 reflect the coding sequence corresponding to human CD223 protein amino acids 23-450 which is preceded with sequence originating from mouse exon 2 at positions 2530 through 2540 and followed by sequence originating from mouse exon 7 at positions 3825 through 3905. Positions 4143 through 4382 reflect mouse exon 8.[000121] SEQ ID NO: 27 is the amino acid residue sequence of the predicted humanized protein CD223, for comparison with SEQ ID NO(s): 28 and 29. The signal sequence runs from residue 1 through 23. Residues corresponding to human CD223 amino acids 23-450 run from residue 24 through 451. The transmembrane domain runs from residue 452 through 472. The intracellular domain runs from residue 473 through 530.[000122] SEQ ID NO: 28 is an amino acid residue sequence of mouse CD223 (LAG3; Transcript: ENSMUST00000032217.2, Lag3-201) protein sequence, for comparison with SEQ ID NO(s): 27 and 29. The signal peptide runs from residue 1 through 23. The extracellular domain runs from residue 24 through 442. Transmembrane domain runs from residue 443 through 463. The intracellular domain runs from residue 464 through 521.[000123] SEQ ID NO: 29 is an amino acid residue sequence of human CD223 (LAG3; Transcript: ENST00000203629.3, LAG3-201) protein sequence, for comparison with SEQ ID NO(s): 27 and 28. Signal peptide runs from residue 1 through 22. The extracellular domain runs from residue 23 through 450. The transmembrane domain runs from residue 451 through 471. The intracellular domain runs from residue 472 through 525.[000124] SEQ ID NO: 30 is a DNA sequence of the targeted Cd4 locus for illustration of mouse exons and human coding sequences. Positions 5539 through 5629 reflect mouse Cd4 exon 2. Positions 5758 through 5783 reflect mouse exon 3. Positions 5784 through 6896 reflect the coding sequence corresponding to human CD4 protein amino acids 26-396. Positions 6897 through 6989 reflect mouse exon 8. Positions 7327 through 7394 reflect mouse exon 9. Positions 7723 through 9276 reflect mouse exon 10.[000125] SEQ ID NO: 31 is the amino acid residue sequence of the predicted humanized protein CD4, for comparison with SEQ ID NO(s): 32 and 33. Residues encoded by the mouse exons run from residue 1 through 26. Residues corresponding to human CD4 protein amino acids 26 to 396 run from residue 27 through 397. The transmembrane domain runs from residue 398 through 421. The intracellular domain runs from residue 422 through 460.[000126] SEQ ID NO: 32 is an amino acid residue sequence of mouse CD4 (mCD4; Transcript: ENSMUST00000024044.7, Cd4-201) protein sequence, for comparison with sequences with SEQ ID NO(s): 31 and 33. The signal peptide runs from residue 1 through 26. The extracellular domain runs from residue 27 through 394. The transmembrane domain runs from residue 395 through 418. The intracellular domain runs from residue 419 through 457. [000127] SEQ ID NO: 33 is an amino acid residue sequence of the human CD4 (humCD4;Transcript: ENST00000011653.9, CD4-201) protein sequence, for comparison with sequences SEQ ID NO(s): 31 and 32. The signal peptide runs from residue 1 through 25. The extracellular domain runs from residue 26 through 396. The transmembrane domain runs from residue 397 through 418. The intracellular domain runs from residue 419 through 458.Genetically modified non-human animals with humanized MH CI I, CD74, CD4, and CD223 [000128] One non-limiting aspect of the present disclosure provides physiologic non-human models (such as primate, rabbit, horse, dog, goat, rat, and mouse) for uncovering how risk alleles in the human MHCII promote autoimmune diseases such as RA. Significant failures in the present state of the art arising from current transgenic technology and non-physiologic expression are overcome by the disclosed physiologic co-expression of interacting polypeptides resulting in marked mitigation of barriers to success (barriers have included random integration and aberrant expression of desired genes, absence of physiological function and expression, and alterations in B-cell function).[000129] A surprising solution to these failures in the art is the herein disclosed introduction of full sets of genes and proteins demonstrated to interact within a given physiologic system, in this case, the MHCII immune system, and within that construct to introduce only very well- defined DNA sequences, leading to well-defined proteins. This solution can only be achieved with the herein-provided extensive and detailed knowledge of the mechanisms involved in any given physiological function or pathology. In immunologic systems, these extensive mechanisms were heretofore unknown.[000130] As an example, gene sequences coding for the extracellular domains of DRBl*04:01 and *04:02 alleles, DRA, and CD74 (invariant chain) may be inserted into C57BL / 6N mice and the COL2 gene (Col2al) of the mice may be mutated to express amino acids mimicking human COL2 (266E) as well as COL2 lacking glycosylation (266E / 264R) in the major T cell epitope site. Additionally, a single nucleotide polymorphism leading to the NCF1.R90H mutation may be introduced to NCF1 and the COL2.R360Q mutation may further be introduced to COL2. These provide a physiologic autoimmune disease non-human model reflecting some of the technological advances in disease modeling disclosed herein.[000131] In a further non-limiting embodiment, the mouse CD223 endogenous extracellular domain (aa.24-442) may be replaced with the human CD223 extracellular domain (aa.23-450) and the murine signal peptide (aa.1-23) of mouse CD 223 may be maintained. The mouse CD4 endogenous extracellular domain (aa.27-394) may be replaced with the human CD4 extracellular domain (aa.26-396) and the murine signal peptide (aa.1-26) of mouse CD4 may be maintained. In a targeting vector, a Neo cassette may be flanked by Frt sites and a Puro cassette may be flanked by F3 sites. DTA may be used for negative selection. C57BL / 6 ES cells may be used for gene targeting.Physiologic expression on both thymus cells and APCs[000132] The herein described DRBl*04:01 knock-in mice display physiologic expression of MHCII on thymus cells, as well as other antigen presenting cells, and physiologic expression of B cells. Physiologic expression of MHCII on thymus cells in transgenic and humanized DRBl*04:01 mice had not been previously observed. Likewise, non-physiologic expression in B cells has led to toxicity resulting in a reduced number of B cells with altered aberrant function in previous models in the art.[000133] In prior model mice with DRB 1*04:01 knock-in of humanized MHCII molecules alone, expression of MHCII has been observed to be subnormal. In the present disclosure, by contrast, knock-in of humanized CD74 (invariant chain) enhanced MHCII expression on thymic epithelial cells, and increased mature B cell numbers in spleen, leading to improved antigen presentation, and increase in both incidence and severity of CIA upon administration of COL2. In addition, DRBl*04:01 expression in knock-in mice led to a higher susceptibility to arthritis, as compared with knock-in mice expressing DRB1 *04:02 (in both male and female mice). Furthermore, additionally humanizing the T cell epitope (COL259-273-giycosyiated at 264) on COL2improved the human model by reducing arthritis susceptibility while nevertheless allowing significant development of arthritis, rendering the model much more phenotypically human. In a further improvement of the model, mice having the T cell epitope without glycosylation were observed to develop arthritis without autoreactive T cells, which is analogous to observations in human disease.Unique activation of B cells producing ACPAs[000134] In the humanized DRB 1*04:01 and CD74 mouse, when arthritis was induced after immunization with COL2 or with minimal amounts of P AD4 complexed with COL2 or other joint proteins (Fig. 5) a unique activation of B cells producing antibodies to citrullinated proteins (ACPAs) occurred, with characteristics typically occurring also in rheumatoid arthritis in humans. Again, this phenomenon more closely reflects human disease progression where such typical ACPAs are understood to be unique to RA and have not previously been seen in animal models. ACPAs in non-human models are therefore herein disclosed as both novel and not predicted in the art. As observed, the ACPA effect is critically dependent on MHCII-mediated activation of T cells and importantly dependent on DRB 1*0401 and not DRB 1*0402 mice. The ACPA effect is also understood to be present in mice expressing DR alleles associated with RA such as DRB 1*0405. The induction of ACPA is associated with development of severe arthritis that surprisingly mimics RA in humans. Monoclonal ACPA derived from the DRBl*0401 mice showed striking similarities with corresponding human antibodies (Fig. 5b) and among these both arthritogenic and protective antibodies could be identified — a phenomenon likewise observed in human ACPA.[000135] One key for the development of an RA-like disease in non-human animals such as primates, goats, rats, and mice, etc., is breakage of tolerance to the cartilage- related self-antigen by the PAD4-cartilage antigen complex. The humanized DRB 1*0401 mice disclosed here have increased levels of natural antibodies reactive with citrullinated peptides and after immunization develop a strong antibody response specific for citrullinated epitopes. Isolation of monoclonal antibodies from these DRB 1*0401 mice revealed antibodies similar to ACPA derived from humans in that they range from being promiscuous to being more private. Testing the antibody function also revealed that the more private antibodies can induce arthritis whereas the more promiscuous ACPAs protect against arthritis. These data again show that physiologic MHCII humanized mouse models of the disclosure mimic RA regarding MHCII allele association witharthritis and autoantibody response. The physiological expression of humanized knock-in mouse models confirms the DRB1 *04:01 allele association with rheumatoid arthritis, providing surprisingly specific useful models for studies of MHCII-associated diseases.Allelic susceptibility and unusually robust T cell response to unmodified peptide [000136] The development of CIA in physiologic disclosed humanized mouse models has further demonstrated a specific association with the DRBl*04:01 and DRBl*04:05 alleles, emphasizing their importance in disease susceptibility and mimicking allelic disease susceptibility in humans.[000137] The herein exemplified physiologic humanized DRBl*04:01 mice display an unusually robust and unexpected T cell response to the unmodified COL2259-273 peptide. Deficient transgenic expression of DR4 in prior art models has led to toxicity and aberrant antigen presentation. For example, a low expression in thymus epithelial cells allows T cells specific to unmodified COL2 to evade thymic tolerance (Raposo 2018, Nature Communications 9(1): 353). In the present disclosed studies, humanized DR4 mice (DRBl*04:01, DRBl*04:02, and DRB1 *04:05) express the ectodomains of human proteins while the remainder of the expressed protein is murine. This enables physiological expression and normal genetic regulation in vivo within a murine context. As a result, the immune response to COL2 becomes more relevant, and when crossed with the D266E mutation, which is understood to activate only autoreactive T cells, tolerance is strengthened. Interestingly, similar to Aqmice, tolerance is less pronounced for T cells reactive to the galactosylated peptide as compared to the nonglycosylated (naked) peptide (Ge C, etal., Ann Rheum Dis 2022;81:480-489; Raposo 2018, Nature Communications 9(1): 353) demonstrating that prevention and immune regulation of selfpeptides are very sensitively regulated and it is critical to have physiologic expression of several proteins involved in MHCII antigen presentation as shown in Romero-Castillo Adv. Sci. 2024. In addition, the co-expression of a humanized CD74 leads to surprising changes in lymphocyte selection (both B cell and T cell) and autoimmune responsiveness (Romero-Castillo Adv Sci 24), making the model even more analogous to humans. Notably, humanized Parker (DRA, DRB 1*0401, CD74) mice were observed to exhibit not only a normalized human MHCII expression in thymus and on peripheral antigen presenting cells, but also a greater susceptibility to CIA than humanized Dunder (DRA, DRB 1*0402, CD74) mice, mimicking allelic disease in humans.[000138] The observed arthritis association with DRBl*04:01 and not with DRBl*0402 is consistent with earlier observations where minor amino acid variations between DRBl*04:01 and DRBl*04:02 are impactful. In particular, the lysine at position 70 in DRBl*04:01 has been associated with the presence of RA and the aspartic acid at position 70 in the DRBl*04:02 has been associated with the absence of RA.For the first time, human-like gender differences in immune response in animal model [000139] Despite their susceptibility to specific autoimmune disease models like CIA, humanized MHCII mice (without humanization of other components of MHCII physiology) have not been able to replicate the complexity of human autoimmune diseases, including RA. An example of this is the changed gender difference in humanized mice at different levels of humanization. The influence of gender on the prevalence of RA is well-established, with women being more susceptible to the disease than men, in this case at a ratio of approximately 3: 1, even though estrogen exposure during pregnancy is likely to ameliorate the disease. This gender difference pattern is likewise observed in many autoimmune diseases. On the other hand, in classical CIA, male mice are more susceptible to CIA compared to females. In a contrasting and surprising result with an example of physiologic humanized mouse disclosed here (Parker / / DRA, DRB 1*0401, CD74), both female and male mice developed arthritis to a similar extent in terms of incidence and severity, moving the model much closer to actual observation of the disease in humans. This is one aspect of the improved model disclosed herein.Modeling disease[000140] The precise environmental triggers and non-MHC single nucleotide polymorphisms that influence RA development have remained largely unknown. Nevertheless, the disclosure here of broadly introduced factors in animal models provides crucial understanding of the disease. Humanization of interacting molecules within the model brings it ever closer to a fuller understanding of human disease by providing a near-approximation of the disease. In vivo models are crucial for understanding the disease and for developing preventive and therapeutic strategies and the physiologic humanization of non-human animals disclosed herein provides surprising and significant enhancements in important mechanics and outcomes that deliver marked advancement in mimicking human disease.[000141] These same models provide mechanisms for understanding diseases arising in and development of therapies available for haplotypes containing PTPN22, NCF1, NCF4, PADI4,FCGR2B, TNFRSF14, IL6R, PTPRC, STAT4, CD28, CTLA4, IL2, IL3, CSF1, NFKBIE, TNFAIP, CCR6, CDK6, IRF5, TRAF1, C5, IL2RA, TRAF6, RAG1, CD5, ATM, CDK2, CDK4, SH2B4, SH2B3, IKZF3, CSF3, PTPN2, ICAM1, TYK2, CD40, IFNGR2, ICOSLG, AIRE, IL2RB, IRAKI as well as diseases arising in and development of therapies available for key regulatory genes (including but not limited to LAT, CD45, VISTA) in the human immune response.[000142] One non-limiting aspect of the present disclosure provides use of a physiologic nonhuman animal model comprising humanized MHCII, CD74, CD4, CD223, COL2, and / or NCF1 genes for modeling disease and use of the physiologic non-human animal for development of at least one therapy for said diseases.Methods of making[000143] The present disclosure provides non-human animals that co-express mutated or humanized MHCII protein complexes with CD74, CD4, CD223, COL2, and / or NCF1 polypeptides along with cells, tissues, embryos, and cell-based production systems that coexpress the polypeptides. Methods and constructs for manufacture, utilizing techniques such as gene targeting by homologous recombination, and site-specific recombination (Overview of Gene Targeting by Homologous Recombination, Current Protocols in Neuroscience 4.29.1- 4.29.13, July 2007) and / or CRISPR / Cas9 (Cold Spring Harb Protoc; doi: 10.1101 / pdb.prot090704) for genome engineering and production of genetically engineered animals are also provided as well as methods for utilizing the animals and cell-based production systems for investigation of rheumatoid arthritis and other immunologic phenomena in humans.[000144] Method steps (of manufacture, making, producing, growing, treating, or similar) in this disclosure may be understood to encompass steps taken in any order that achieves the stated outcome of the method. Steps of any process in this disclosure may be understood to encompass steps taken in any order that achieves the stated outcome of the process.Detailed Description of Drawings[000145] Fig. 1 illustrates schematically (not in scale) humanized MHCII (HLA), CD74, CD4 and CD223 proteins and their physiologic interactions. The knock-in of HLA-DRB1 alleles *04:01, *04:02 and *04:05, together with humanized HLA-DRA and CD74, when expressed together with the humanized CD4 and CD223, resulted in mice called Primus, Secundus andQuintus, respectively. In the figure, black fill illustrates a domain of mouse origin and white fill illustrates a domain of human origin. CLIP represents the CD74-derived peptide.[000146] Fig. 2 illustrates the knock-in of human HLA-DRA, HLA-DRB1, CD74, CD223 and CD4. Fig. 2(a): illustrates the C57BL6 / J H2-Ea-ps and H2-Ebl loci humanized with HLA- DRA and HLA-DRB1. C57BL6 / J H2-Ea, which is annotated as a pseudogene (ps) due to a 627bp deletion in haplotype H2b, was targeted to express HLA-DRA. The gene targeted allele generated by homologous recombination in ES cells contained the repaired promoter-exon 1- region from H2d haplotype, humanized fragment replacing the mouse genomic sequence around exons 2-3, and a neomycin selection cassette flanked with FRT sites for selection. Double targeting resulted in the double targeted ES cells with HLA-DRB1 / H2-Ebl-HLA-DRA / H2-Ea- ps loci, since the targeting was done into the already H2-Ea-ps-targeted ES cells to target the H2- Ebl locus to express HLA-DRB1. Exons 2-3 originate from the human HLA-DRB 1 *04:01 :01 cDNA and were preceded with the hygromycin resistance cassette flanked with loxP2272 sites. Mouse exons are shown in black fill and human sequences in white fill. Election cassettes were removed by FLP- and Cre-mediated recombination by breeding. Fig. 2b illustrates the knock-in of human CD74 with targeted exons 2-8 of mouse CD74 gene replaced with the orthologous human sequence. The targeted allele contained a neomycin selection cassette flanked by FRT sites and the cassette was removed by Flp-mediated recombination. Fig. 2(c) illustrates humanized Cd223 locus. Mouse Lag-3 (Cd223) was targeted to knock-in human LAG3 (CD223) CDS corresponding to aa. 23-450. Fig. 2(d) illustrates humanized Cd4 locus. The LAG-3 (CD223) targeted ES cells were used for 2nd targeting for CD4 humanization. Mouse Cd4 was targeted to knock-in human CD4 CDS (aa. 26-396). The targeted alleles respectively contained the neomycin (Neo) and puromycin (Puro) selection cassettes which were removed by Flp mediated recombination leaving behind an FRT and an F3 site, respectively. The schematic presentations of Fig. 2 are not in scale.[000147] Fig. 3 illustrates the functional importance of humanized CD74 (hCD74) in concert with humanized MHCII (*0401 ) in Parker mice (0401+hCD74). Fig. 3a illustrates MHCII expression on thymus medullary epithelial cells and thymus cortical epithelial cells with and without hCD74 demonstrating that hCD74 is needed for normal expression of human MHCII in mice. B6N mice (n =6) were used as negative control for HLA-DR expression. mCD74 (n = 7)indicates a mouse expressing HLA-DRB 1*04:01 but with mouse CD74, whereas hCD74 (n = 7) indicates mice expressing human HLA-DRB 1*04: 01 together with human invariant chain (human CD74) but no mouse CD74. Statistics employed a 2- way ANOVA with Sidak’s multiple comparisons test. Fig. 3b illustrates expression of hCD74 needed for arthritis development (CIA). Inflammation in the paws was monitored over 73 days. Number of arthritic mice / total number of mice are indicated in parenthesis. Data from two pooled experiments. Results are expressed as mean ± SEM. Statistics employed a two-tailed Mann- Whitney test. While not illustrated, humanized mice with humanized DRBl*04:05 and humanized CD74 (Wayne) were observed to develop arthritis similarly to Parker mice (DRBl*0401 / CD74).[000148] While also not illustrated, in previously disclosed non-physiologic transgenic mice HLA-DR, expression in TECs was low and a majority of the medullary TECs lacked HLA-DR expression. This likely resulted in suboptimal T cell selection in the thymus, leading to the escape of autoreactive T cells that would normally be deleted entirely thus impeding proper establishment of self-tolerance of T cells in thymus. Further, CD74 knock-in enhanced antigen presentation by increasing both mature antigen presenting cells (B cells and dendritic cells) and HLA-DR expression in B cells and macrophages (Romero-Castillo Adv. Sci. 2024). Unaffected immune cell populations in the presence of CD74 (li) in naive mice further demonstrated that the naive immune system of humanized mice is undisturbed by human CD74. Further, anti-COL2 antibody titers of total IgG (anti-kappa) in serum from *04:01 mCD74 and *04:01 hCD74 mice at the arthritis endpoint did not differ but the frequency of COL2259-273 T cells among the CD4+CD44+cells from spleen of *04:01 hCD74 mice at arthritis endpoint was significantly higher than in *04:01 mCD74 mice.[000149] The humanized mice of the disclosure likewise display human-like immune responses related to the *04:01 and *04:02 alleles. In response to challenge with non-modified COL2259-273 (no modification to the lysine at 264), antigen-specific IFN-y T cell response in strains expressing the *04:01 allele (Parker and Primus) exhibited a higher response upon COL2259-273 peptide stimulation compared to strains expressing the protective *04:02 allele (Dunder and Secundus). In addition, strains containing human COL2 (Parker. COL2.266E and P(Primus).COL2.266E) presented undetectable response to the non-modified peptide due to a predictable strong tolerance.[000150] Fig. 4 illustrates induction of severe arthritis after immunization with COL2 complexed with a minimal amount of human (h) PAD4 or mouse (m) PAD4 at a ratio of 500: 1 (COL2xPAD4), which is dependent on the expression of DRB 1 *04:01 . Fig. 4a illustrates induction of more severe arthritis in Parker mice immunized with COL2xhPAD4 or COL2xmPAD4 as compared with immunization with only COL2. Fig. 4b illustrates PAD4xCOL2 induction of severe arthritis in Parker (*04:01 , hCD74) but not in Dunder (*04:02, hCD74) mice. Statistics were determined using 2- way ANOVA with Sidak’s multiple comparisons test. The number of arthritic mice / total number of mice are indicated in parenthesis. [000151] While not illustrated, histological examination of ankle joints of B6N and Parker mice immunized with PAD4xCOL2 demonstrated that cell infiltration and cartilage / bone destruction were pronounced in Parker mice as compared to wildtype (B6N) and Dunder mice. Likewise, anti-COL2 antibody titer of total IgG (anti-kappa) from serum accompanied the disease course and were present in all groups while induction of ACPA was also observed in Parker with silica challenge in the lung demonstrating rheumatoid arthritis-like disease and immunization of Parker mice with bCOL2xmPAD4 induced robust antibody titers against citrullinated peptides, mirroring observations in RA. Further, ACPA response was observed in Parker but not Dunder or B6N mice and ACPA after immunization with PAD4xenolase was observed in Parker and not Dunder mice.[000152] Fig. 5 illustrates antibodies derived from Parker mice specific for citrulline when expressed on different peptides. Fig. 5a illustrates cyclic citrullinated COL2 peptide-reactive pathogenic monoclonal antibodies found in Parker mice. Antibodies LN13-C12, LN20-D9, and SP2-D3, as well as a blank sample without an antibody were subjected to bead-based flow immunoassay where their binding to nine (1-9) different cyclic peptides with a citrulline or a corresponding peptide with an arginine in place of a citrulline, were determined and quantified as median fluorescence intensity (MFI). Fig. 5b illustrates a crystal structure of a monoclonal promiscuous ACPA (LN20-D9) derived from the Parker mouse, binding a COL2 citrullinated peptide. It essentially interacts only with the citrulline sidechain as a typical ACPA from human RA (compare Ge et al., Nat rev Rheumatol vol. 15 Aug 2019). These promiscuous antibodies have been observed to be protective.[000153] While not illustrated, monoclonal ACPA derived from Parker mice were protective against arthritis in susceptible strains. Likewise, immunization with COL2xmPAD4 in Parker.266E (COL2266E) mice induced autoimmune arthritis mimicking RA and T cell responses to COL2 or PAD4 peptides (frequency specific T cells measured by tetramer staining) after immunization with bCOL2+mPAD4, bCOL2+hPAD4, or bCOL2. Immunization of Parker.266E mice with bCOL2xmPAD4 activated T cell responses but with a relatively decreased number of Tregs.[000154] Fig. 6 illustrates that the expansion of Tri cells in Primus mice (DRA, DRBl*04:01, hCD74, hCD4, hCD223) is a key event for mediating peripheral immune tolerance and protection against autoimmune arthritis. Fig. 6a illustrates expansion of Tri (LAG3 CD49) cells in Primus mice after COL2 immunization as compared with Parker mice (HLA-DRA; HLA-DRB 1 *04:01 ; hCD74). Fig. 6b illustrates Primus mice are relatively protected against arthritis as compared with Parker mice. Number of arthritic mice / total number of mice are indicated in parenthesis. The reduction of the clinical score may be attributed to the expansion of Tri cells since they are known to mediate immune tolerance through IL- 10 production, leading to decreased synovial inflammation and joint destruction. Fig. 6c illustrates transfer of CD4 T cells from Primus mice into Parker mice ameliorates arthritis severity. Dependence on CD223 for even greater amelioration is observed. Results are expressed as mean ± SEM. Statistics in Fig. 6a and b were determined with a two-tailed Mann- Whitney test and in Fig. 6c with a 2-way ANOVA with Sidak’s multiple comparisons test. While not illustrated, a level of at least 100 ug / ml anti-COL2 IgG antibody is associated with arthritis. Likewise, it was observed that there were no differences in T, B and myeloid cell subsets in spleen between naive Parker and Primus mice.[000155] Despite the observation of higher numbers of Tri cells in Primus mice compared to Parker, no differences in CD4, CD44, nor conventional Treg (Foxp3+ CD25+) frequencies after COL2 immunization were observed. Likewise, as demonstrated, Primus mice immunized with COL2 exhibited an increase in Tri cells. While no differences were observed in CD2 expression among total CD4 T cells, a significant reduction in CD2 expression was noted specifically in Tri cells from the spleens of COL2-immunized Primus mice compared to Parker mice. Moreover, Tri cells from Primus mice appeared to be less anergic than those from Parker mice, as indicatedby lower PD-1 expression and a reduced frequency of anergic T cells characterized by a doublepositive population with high expression of both CD73 (CD73Ahi) and Folate Receptor 4 (FR4Ahi). Additionally, following COL2 immunization, Primus mice showed an increased B cell population with attenuated activation, evidenced by lower CD86 and CD95 expression, and a reduced number of myeloid cells, including macrophages (CD1 lb+Ly6C71ow) and neutrophils (CD1 lb+Ly6G+), compared to Parker mice.[000156] Likewise, T cells from COL2 immunized Primus mice were observed to induce a CD223 -dependent activation of Tri in naive Parker recipients.[000157] Importantly, T cells in wild-type mice do not express MHCII molecules, whereas activated T cells in humans do, although the precise function of this expression remains unclear. In contrast, the herein disclosed humanized mice exhibit a human-like expression pattern, with activated T cells in naive Primus mice expressing human MHCII. Specifically, 22% of CD4+CD44+ T cells in these mice express the human MHCII molecule DR*04:01. This unique expression pattern in Primus mice mimics human physiology rather than that of mice, providing a valuable model for studying T cell biology and the role of MHCII, despite the ongoing development of understanding of MHCII expression on T cells.[000158] The expansion of a subset of Tri in Primus mice has also been observed to be dependent on immune activation that may not be antigen specific. Antigen-specific Tregs are understood to transfer suppressive function to other non-specific Tregs. This has earlier been shown in Aq-expressing mice (Batsalova, Jour, of Immunol. 2010; 185, 2701-2709), a mechanism coined as infectious tolerance (Shixin, Science 1993 Feb 12;259(5097):974-7). Parker and Primus were immunized with bCOL2 or ovalbumin (OVA). The expansion of Tri in Primus mice was similar for both antigens as compared relatively to both wild-type and Parker mice, demonstrating even more surprising human-like immune responses.[000159] Fig. 7 illustrates that a DR*0401 -COL2 vaccine is more effective in Primus (DRA, DRBl*0401, hCD74, hCD4, hCD223) and less effective in Parker (DRA, DRBl*0401, hCD74). CD4 and CD223 are related costimulatory molecules on the T cell surface and are understood to operate in concert. They both bind MHCII molecules and humanization of both MHCII and CD4 / CD223 increases physiological interaction and is demonstrated to be of critical importance for the activation of T cells. Thus, these molecules are critical for translation of the function oftolerogenic vaccines, which are understood to operate by modifying T cell function, for example, to become regulatory T cells. Mice were immunized with C0L2 and Complete Freund’s Adjuvant (C0L2 / CFA) and subsequently injected with a vaccine (recombinant DRB 1 *04:01 covalently linked to the COL2259-273 peptide). Development of arthritis was followed by macroscopic inspections. The data demonstrate DR*0401-COL2 vaccine complex is more effective in Primus mice (Fig. 7b) as compared with Parker mice (Fig. 7a). The number of arthritic mice / total number of mice are indicated in parenthesis. Results are expressed as mean ± SEM. Statistics were determined with a two-tailed Mann- Whitney test.[000160] Fig. 8 illustrates functional NCF1 is needed for a vaccination effect on arthritis in Parker mice. The DRB1*0401-COL2 vaccine did not ameliorate arthritis in NCF1 -deficient Parker mice but did ameliorate arthritis in NCF1 -competent Parker mice. The mice were immunized with COL2 / CFA and subsequently injected with DRB1*0401-COL2 vaccine or DRBl*0401 / mCLIP as a control. Development of arthritis was followed by macroscopic inspections. The data demonstrate DRB1*0401-COL2 vaccine complex is markedly less effective in NCF1 -deficient Parker mice as compared with NCF1 -competent Parker mice. The number of arthritic mice / total number of mice are indicated in parenthesis. Results are expressed as mean ± SEM. Statistics were determined by using 2-way ANOVA with Sidak’s multiple comparisons test.[000161] Fig. 9 illustrates the COL2R360Q mutation regulates development of arthritis. Fig. 9a illustrates B10.Q.Cia9i (R360(WT)) and B10.Q.Cia9i.360Q (R360Q) mice immunized with COL2 / CFA and followed for the development of arthritis. The mutated Cl epitope (R360Q) on COL2 led to more chronic CIA. In the CAIA model (anti-cartilage antibody induced arthritis) demonstrated in Fig. 9b, the reverse effect is seen. In Fig. 9b, mice were injected with CB20 antibody binding to the wild type Cl epitope on COL2 on day 1. On day 14, arthritogenic anti- COL2 antibodies M2139 and ULI were further injected. Wild type mice were observed to develop more severe arthritis. One of the arthritis-inducing antibodies (CB20) is understood to react with the Cl epitope. Incidence of arthritis is shown in parentheses.[000162] Fig. 10 illustrates a summary of specific non-limiting and exemplary strains disclosed herein, their modified genetics, genetic background and breeding strategy. Except for the *R360Q and *R90H strains, all strains are on C57BL / 6N background corrected with theCia9i chromosomal fragment. They have been and can be used with and without indicated genetic modifications. The *R360Q and *R90H strains illustrate a B10Q background but may be switched to the standard C57BL / 6N.Cia9i background in a non-limiting embodiment. Physiology may be improved when switched to the standard C57BL / 6N.Cia9i background, including a higher inflammatory response due to a normalized FC-gamma receptor locus.[000163] Fig. 11 illustrates crossing of exemplary and non-limiting mouse strains with different genetic modifications. Introduction of the Cia9i fragment into the *04:01 +CD74 mice generated Parker mice. Dunder (DRBl*04:02) and Wayne (DBR1 *04:05) were generated similarly. Parker, Dunder and Wayne mice, and all their progeny, contain the Cia9i fragment from the B6N.Cia9i mice. Crossing Parker with the mouse with humanized CD4 (hCD4) and humanized CD223 (hCD223) resulted in Primus mice. In similar crosses with hCD4 / hCD223 mice, Dunder (DRBl*04:02) and Wayne (DBRl*04:05) generated Secundus and Quintus mice, respectively. Mice are on the B6N genetic background.[000164] Fig. 12 illustrates an observed higher frequency (percentage) of Tri+(CD49+LAG3+) cells in naive spleen of all disclosed mouse strains with humanized CD4 and CD223 where Parker, Dunder, and Parker. COL2.266E mice (all lacking humanized CD4 and CD223) demonstrate statistically lower frequency of Tri cells as compared to Primus, Secundus, and Primus with humanized COL2 (labeled P.COL2.266E) (all having humanized CD4 and CD223). Further, a higher frequency of Tri cells was observed in Primus mouse with additional humanized COL2 (P.COL2.266e) as compared to Primus mouse without humanized COL2 as well as the *0402 allelic Secundus mouse (HLA-DRA, HLA-DRBl*04:02, hCD74, hCD4, hCD223).[000165] Non-limiting examples are provided herein. Many modifications and variations can be made, as will be apparent to a person of ordinary skill in the art upon reading and understanding the disclosure. Functional equivalents disclosed and enabled within the scope of the disclosure will be apparent to the skilled artisan from the descriptions. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.EXAMPLESExample 1: Establishment of Parker: a mouse with humanized DRA, DRBl*0401, CD74 and Cia9i on C57B1 / 6N background.[000166] The Parker mouse (humanized DRA, DRB1 *0401, CD74) on a C57BL6 / N mouse corrected with a m.musculus FcR containing haplotype on chromosome 1 (Cia9i) has been tested for physiologic expression and function of human MHCII (DR). Replacement of the DRA / DRB extracellular domains are shown in Fig 1. The remaining mouse DQA / DQB (i.e. forming the mouse MHCII Ab molecule) was left intact and served as a control. Replacement of the extracellular domain of the mouse CD74 (invariant chain, li) with the corresponding human CD74 sequence, is also illustrated in Fig 1. Naive mice were found to express normal levels of MHCII in cell types expected to do so physiologically (macrophages, dendritic cells, B cells and thymic epithelial cells), in contrast to earlier used human MHCII transgenic mice. It could be shown that co-expression with human CD74 is one key for full and normal MHCII expression. An example is given in Fig 3, showing expression in thymic epithelial cells, a cell critical for the selection of T cells. Physiologic expression of MHCII is essential for normal differentiation of immune cells and it is here shown that the combination of human MHCII and human CD74 expression allows development of an immune system with normal composition of different immune cells. Higher numbers of matured B cells (CD93‘ CD19+B220+as compared to CD19+B220+and CD93+) and enhanced antigen presentation (IL-2 after co-culture of native-COL2- specific T hybridoma (3H8) together with splenocytes from either *04:01 or *04:01 hCD74 with or without COL2259-273 peptide at different concentrations) observed in *04:01 hCD74 mice demonstrated that CD74 is needed for a normal cellular composition and function of the immune system (Romero-Castillo Adv. Sci. 2024).[000167] Further, unaffected immune cell populations observed in the presence of humanized CD74 in naive mice demonstrated that the naive immune system is undisturbed by humanized CD74. In particular, observation of B cell development in the bone marrow with different B cell stages (immature (IgM+ IgD-) pre and pro (IgM- IgD-), pre (CD2+ cKit-) and pro (CD2- cKit+) B cells) *04:01 and *04:01 hCD74 mice provided similar populations. Also, T cells in the lymph nodes, spleen, and thymus from *04:01 and *04:01 hCD74 mice were similar. FACS gating was employed.[000168] To test the functionality of the immune system, the mice were immunized, by introducing an antigen together with an adjuvant intradermally and antigen presentation function and immune responses were found to be normal by the co-expression of human MHCII and CD74. To test if the human MHCII has a functional role under extreme conditions, Parker, Dunder, and B6N mice were immunized with COL2, leading to the gold standard model for RA, collagen induced arthritis (CIA). There is however confusion on the model in the scientific community as COL2 is prepared from tissue using porcine pepsin and contaminants are often the target for the T cells rather than COL2, which in fact is the case for B6 mice expressing Ab. These disclosed studies nevertheless provide the advantage of having co-expression of Ab. Likewise, to clarify the subject, COL2 was compared with pepsin-free COL2 to make sure T cell responses to COL2 were being studied.[000169] Full development of CIA in the mouse model required co-expression of humanized MHCII and humanized CD74. Importantly, mice expressing DRB 1*0401 (i.e. Parker) were fully susceptible but mice expressing DRBl*04:02 (i.e. Dunder) were not susceptible. This corresponds to the RA epidemiology in humans in which DRB1 *04:01 rather than DRB1 *04:02 is associated with disease. The mice were tested in fully specific pathogen free conditions and were also tested in more conventional conditions. To test whether the human MHCII + CD74 could control immune tolerance, immune responses to COL2 were uniquely observed. COL2 is a major matrix protein expressed not only in the joint cartilage but also in central lymphoid organs, thymus and bone marrow. It is known that certain MHCII alleles (Aqin the mouse and DRBl*04:01 in humans) bind a single posttranslational modified peptide from COL2 located at the position 259-273, numbered from the start of the triple helical structure of the COL2 molecule.[000170] There is one difference in this major epitope between mouse and humans, position 266 is E (glutamic acid) in humans and D (aspartic acid) in the mouse and this difference has a major impact on immune tolerance and susceptibility to CIA. A Parker mouse with the human COL2 variant, 266E, was therefore constructed (Parker.COL2.266E). Parker. COL2.266E (P.266E) had a stronger immune tolerance but could still develop CIA, thus mimicking the situation in humans. Importantly, the arthritis in P.266E mice developed in a chronic relapsing pattern as in RA in humans. Remarkably, the P.266E mouse made a T cell response against thegalactosylated C0L2, as in humans with the MHCII allele 0401, and in mice with the MHCII allele Aq, in contrast to prior art human MHCII transgenic mice (having only the humanized MHCII allele) which develop a response almost exclusively to non-modified COL2 (Romero- Castillo Adv. Sci. 2024). To test the importance of the galactosylation of the lysine (K) at position 264, the lysine (K) at position 264 was replaced with arginine (R). As expected, P.264R mice made a completely non-self COL2 response leading to severe arthritis due to production of pathogenic antibodies rather than autoreactive T cells. Physiological co-expression and interaction of these molecules provide the sensitive context and environment needed to study immune responses in vivo. In summary, the Parker mouse is a surprising model for studies of human MHCII function and the addition of specific mutations of COL2 allows further in-depth studies on the pathophysiologic function of the immune system. The crossing of the strains is illustrated in Fig 11.Example 2: The induction of a rheumatoid arthritis-like disease in Parker mice.[000171] Although CIA is used as a gold standard model for RA it does not fully mimic the human disease, particularly in wild-type and current state-of-the art transgenic mice. As one example, in similarity with all hitherto known animal models, CIA does not develop the typical autoantibodies which define RA, i.e., antibodies to citrullinated protein antigens. Likewise, in classical ACPA (anti-citrullinated protein antibody) and CCP2 (anti-cyclic citrullinated peptide 2) assays, it was observed that naive Parker (h0401 / hCD74 / Cia9i) mice, in contrast to Dunder mice (h0402 / hCD74 / Cia9i), develop low titers of reactive antibodies. To better mimic the human situation, Parker mice were challenged intratracheally with silicon and found that lower titers of specific ACPA, not cross-reactive with corresponding arginine controls, were produced. To facilitate the production of ACPA, the mice were immunized with complexes made with the PAD4 enzyme (an enzyme which transforms arginine sidechains to citrulline) together with COL2 in a ratio of 1:500, i.e., with minimal amounts of PAD4. Immunization of Parker mice with PAD4xCOL2 led to severe and chronic arthritis, more pronounced than induced with COL2 or citrullinated COL2. The same immunization of Dunder mice did not result in severe and chronic arthritis. Importantly, similar to the arthritis effect, increased levels of specific ACPA were seen in the blood of Parker mice (and not Dunder mice), which reflects observation of the same phenomenon in RA in humans. Observations were similar if COL2 was replaced in Parker mice with other antigens known to be present in the joints, for example cartilage oligomericmatrix protein (COMP), glucose 6 phosphoisomerase (GPI) or alpha-enolase (EN01), including responses after immunization with PAD4xEN01. Monoclonal antibodies were isolated from this response. They were shown to have specificities typical for ACPA in RA and could either be pathogenic or protective, similar to monoclonal antibodies derived from human RA. The induction of an ACPA response as well as an arthritogenic immune response was linked to expression of DRBl*0401 and not DRBl*0402, as observed in humans.[000172] To be useful for studies of the human disease, the pathogenic immune response of the model needs to be endogenous. It is herein demonstrated that mouse-derived PAD4 complexed with COL2 induced autoimmunity, ACPA, and arthritis in the disclosed mice. This is observed even in P.266E mice with COL2 mutated to human (Parker. COL2.266E). These data show that the mechanism is autoimmunity that in many respects mimics outcomes in humans. The observed arthritis was associated with an autoreactive T cell response, effecting regulatory T cells. As a result, the animal model provides a new use for investigating the basic etiology of RA, i.e., how an autoimmune response leading to production of ACPA is initially triggered and develops into pathogenicity. No previous model contemplated or provided this unique and surprisingly elevated level of investigative specificity.Example 3: Establishment of Primus: a mouse with human DRA, DRBl*0401, CD74, CD4, CD223 and Cia9i on C57B1 / 6N background.[000173] The Primus mouse (DRA, DRB 1*0401, hCD74, hCD4, hCD223) on a C57BL6 / N mouse corrected with a m. musculus FcR containing haplotype was made using the methods disclosed herein and surprisingly provided an even more precise model for the human immune system. The CD4 and CD223 molecules are closely related and coded from a linked chromosomal location with only about 16 kb between the genes and with a theoretical recombination frequency of about 0.01%. These molecules are expressed on T cells but interact with MHCII on the antigen presenting cells. CD4 is understood to strengthen T cell responses whereas CD223 is understood to produce reverse inhibitory regulatory signals. Through surprising insight, the extracellular domains of CD4 and CD223 were exchanged with human sequences allowing for further increased physiologic regulation of T cell activation through interaction with human MHCII where the closely linked CD4 and CD223 genes are likely coselected within a haplotype.[000174] Naive Primus mice express a normal immuno-cellular distribution and phenotype as compared with Parker — as analyzed with flow cytometric analysis of the common lymphocyte and leucocyte populations. Immunization with COL2 showed the development of severe arthritis albeit with slightly lower severity than Parker, understood to result from regulatory influence by the more physiologically normal CD4 / CD223 interaction. Importantly and surprisingly, the Primus mice showed an acute and longstanding expansion of a subset of peripherally activated regulatory T cells (Tri), after COL2 immunization (Fig. 6). A comparison of the cellular populations showed distinguishing differences between Primus and Parker mice (Fig. 6). Transfer of T cells from COL2 immunized Primus mice induced a CD223 (LAG3) dependent activation of Tri cells in recipient Primus mice, lowering the autoimmune antibody response and suppressing the development of arthritis, an effect that could be blocked by treatment with antibodies to CD223 (Fig. 6(b) and (c)). As the Primus mouse was designed to mimic humans better regarding immune tolerance and the induction of Tregs, an RA vaccine (a complex of DRBl*04:01 with the glycosylated COL2259 -273 peptide), similar to a corresponding murine prototype, was tested on these strains. The Primus mouse was clearly more susceptible to the vaccination effect, blocking the development of arthritis more efficiently than in Parker mice (Fig. 7). Although the immune response to COL2 is provided as one example, the Tri normalized effect of the Primus versus Parker mice was observed as a general phenomenon dependent on the immune response rather than the specific antigen. This was demonstrated using ovalbumin (OVA) as an alternative antigen where the expansion of Tri in Primus mice was similar for both COL2 and OVA.Example 4: Establishment of humanized mouse strains with alleles other than 0401.[000175] In parallel with Parker and Primus, both with the DRBl*04:01 allele, strains with other DR alleles were established. The *04:02 allele is known to be associated with protection of RA and the DRA / DRBl*04:02+CD74 on C57B16 / N mice corrected with Cia9i was established and named Dunder. DRA / DRBl*04:02+CD74+CD4+LAG3 on C57B16 / N mice corrected with Cia9i was established and named Secundus. The *04:05 allele is common in East Asia and known to be associated with RA. A DRADRB1*O4:O5+CD74 on C57B16 / N mice corrected with Cia9i was established and named Wayne. DRA / DRBl*04:05+CD74+CD4+LAG3 on C57B16 / N mice corrected with Cia9i was established and named Quintus.[000176] Expression of *04:05 but not *04:02 in Wayne and Quintus resulted in observed development of severe arthritis in the CIA model.Example 5: Mouse tools for studies of the MHCII restricted immune response in humans [000177] Disclosed various mouse strains, non-limitingly summarized in Fig. 10, provide surprising new tools for experimental studies of the human MHCII restricted immune response. This will have importance in most human diseases involving an immune component. It is of particular importance in disease associated with MHCII polymorphism. A clear example is rheumatoid arthritis.[000178] Two keys allowing the expression of human genes to be physiological in the mouse are emphasized: 1) The human genes should not disturb their genetic environment; and 2) The expressed human proteins should interact properly with other humanized or endogenous proteins. Surprisingly, both criteria are fulfilled in the presently-described strains. In addition, physiologic expression is advanced when genes are expressed in a mouse with a standard genetic background — selected for millions of years and not mixed up by artificial breeding. The C57B1 / 6 / N strain with the correction of the FcR containing fragment on chromosome 1 was therefore chosen for the exemplified embodiments. With the fulfillment of these conditions, additional genetic modifications are made available for use.[000179] One example for use in a non-human (primate, horse, goat, rat, mouse, etc. model in combination with DRA, DRBl*0401, hCD74, hCD4, and / or hCD223 (as well as non-limiting COL2 variants) is the NCF1 single nucleotide polymorphism leading to an amino acid replacement of position 90, the NCF1.R90H mutation. This is highly polymorphic in both experimental animals and in humans and the polymorphism determines the level of the induced reactive oxygen species (ROS) response. This ROS response is understood to be regulatory in many pathways including the MHCII restricted T cell response. Observed data demonstrate that the vaccination effect in Parker (vaccine complex of DRB1*DR.O4O1 with the glycosylated COL2259-273 peptide) is regulated by NCF1 in that expression of the NCF1.90H allele prohibits the protective effect of the vaccine (Fig. 8). Another example for use in a non-human model in combination with DRA, DRB 1*0401, hCD74, hCD4, and / or hCD223 (as well as non-limiting NCF1 and non-limiting COL2259-273 variants) is the COL2 mutation leading to an amino acid replacement of arginine at position 360 to glutamine (COL2.R360Q). This blocks the major Bcell epitope on COL2 and allows studies of antigen specific B suppressor cells. These COL2 specific B suppressor cells occur at similar frequency in several mammals, including humans, in frequency of 1 / 10.000 B cells and are likely to play a key role in immune tolerance and therefore for the understanding of autoimmune diseases. Fig. 9 demonstrates that mice with the 360Q, which lack COL2 specific B suppressor cells, develop a more severe chronic CIA.[000180] The humanized mouse strains, with the Primus / Secundus / Quintus strains as nonlimiting strains, physiologically express the human MHCII molecules and are useful for studies of the MHCII restricted immune response including interaction with other genetic modifications in downstream pathways. For the first time, it is now possible to study the human MHCII associated immune response conclusively in vivo. These disclosures provide models properly reflecting the human MHCII response allowing testing and analysis of drugs targeting the immune response. They also allow the study of interactions with other human polymorphic genes (including and not limited to haplotypes containing PTPN22, NCF1, NCF4, PADI4, FCGR2B, TNFRSF14 , IL6R, PTPRC, STAT4, CD28, CTLA4, IL2, IL3, CSF1,NFKBIE, TNFAIP, CCR6, CDK6, IRF5, TRAF1, C5, IL2RA, TRAF6, RAG1, CD5, ATM, CDK2, CDK4, SH2B4, SH2B3, IKZF3, CSF3, PTPN2, ICAM1, TYK2, CD40, IFNGR2, ICOSLG, AIRE, IL2RB, IRAKI) as well as key regulatory genes (including and not limited to LAT, CD45, VISTA) in the human immune response.[000181] The humanized animal strains disclosed herein likewise enable a method of assessing the therapeutic efficacy of a candidate compound for treating a disease associated with autoimmune disease, including and not limited to RA, MS, and insulin-dependent diabetes mellitus. Induction of models of any one of these diseases in the disclosed non-human humanized animal allows the testing of therapies against the model disease in physiologic conditions.Example 6: Use of materials and cells from the humanized mouse for in vitro studies [000182] Cells with controlled homozygous expression of human receptors are provided from the humanized non-human animals including mammals, primates, and rodents, such as mice.These include DRBl*0401, DRBl*0402, DRBl*0405, DRBl*0404, DRBl*01:01, DRBl*10:01, CD74, CD4 and CD223. Also provided are intracellular unique mutations in NCF1 and COL2.[000183] Unique monoclonal antibodies are isolated from humanized strains.[000184] The humanized strains provide a unique source of T cells restricted in their response by human MHCII alleles (DRB 1*0401, DRB 1*0402, DRB 1*0405, DRB 1*0404, DRB1 *01 :01, DRBl*10:01) and also in expression of human CD4 and CD223. As an example, T cells from Primus mice are used to study the role of human MHCII restricted T cell checkpoint regulation by CD223.

Claims

Claims1. A non-human animal comprising a nucleotide sequence of a human DRA genetic fragment that has replaced a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous MHCII a gene locus of the non-human animal, a nucleotide sequence of a human DRB1 allele genetic fragment that has replaced a nucleotide sequence of a corresponding genetic fragment that comprises an extracellular part of at least one endogenous MHCII 13 gene locus of the non-human animal, a nucleotide sequence of a human CD74 genetic fragment that has replaced a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous CD74 gene locus of the non-human animal, a nucleotide sequence of a human CD4 genetic fragment that has replaced a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous CD4 gene locus of the non-human animal, and a nucleotide sequence of a human CD223 genetic fragment that has replaced a nucleotide sequence of a corresponding genetic fragment that encodes at least an extracellular part of at least one endogenous CD223 gene locus of the non-human animal, where the following polypeptides are co-expressed in at least one cell of the non-human animal, a humanized MHCII a polypeptide expressed from the at least one endogenous MHCII a gene locus of the non-human animal comprising at least an amino acid sequence expressed from the nucleotide sequence of the human DRA genetic fragment, a humanized MHCII 13 polypeptide expressed from the at least one endogenous MHCII 13 gene locus of the non-human animal comprising at least an amino acid sequence expressed from the nucleotide sequence of the human DRB1 allele genetic fragment, a humanized CD74 polypeptide expressed from the at least one endogenous CD74 locus of the non-human animal comprising at least an amino acid sequence expressed from the nucleotide sequence of the human CD74 genetic fragment,a humanized CD4 polypeptide expressed from the at least one endogenous CD4 locus of the non-human animal comprising at least an amino acid sequence expressed from the nucleotide sequence of the human CD4 genetic fragment, and a humanized CD223 polypeptide expressed from the at least one endogenous CD223 locus of the non-human animal containing at least an amino acid sequence expressed from the nucleotide sequence of the human CD223 genetic fragment.

2. The non-human animal of claim 1 , wherein said human DRB 1 allele is selected from the group consisting of DRBl*04:01, DRBl*04:02, and DRBl*04:05.

3. The non-human animal of claims 1 and 2, further comprising a COL2 gene sequence with a mutation leading to amino acid replacement D266E and a further mutation leading to amino acid replacement R360Q when expressed from at least one endogenous COL2 gene locus of the non- human animal where a COL2 polypeptide with the amino acid replacements D266E and R360Q is co-expressed in said at least one cell of the non-human animal along with said following polypeptides.

4. The non-human animal of claims 1 to 3, further comprising an NCF1 gene sequence with a mutation leading to amino acid replacement R90H when expressed from at least one endogenous NCF1 gene locus of the non-human animal where an NCF1 polypeptide with the amino acid replacement R90H is co-expressed in said at least one cell of the non-human animal along with said following polypeptides.

5. The non-human animal of claims 1 to 4, further comprising a COL2 gene sequence with a mutation leading to amino acid replacement D266E and a further mutation leading to amino acid replacement R360Q when expressed from at least one endogenous COL2 gene locus of the non- human animal and an NCF1 gene sequence with a mutation leading to amino acid replacement R90H when expressed from at least one endogenous NCF1 gene locus of the non-human animal where both a COL2 polypeptide with the amino acid replacements D266E and R360Q and anNCF1 polypeptide with the amino acid replacement R90H are co-expressed in said at least one cell of the non-human animal along with said following polypeptides.

6. The non-human animal of claims 1 to 5, wherein any one or more of the nucleotide sequences of said human DRA, DRB1, CD74, CD4, and CD223 genetic fragments that have respectively replaced nucleotide sequences of corresponding genetic fragments in said at least one endogenous MHCII a, MHCII 13, CD74, CD4, and CD223 gene loci encode a fragment consisting essentially of the extracellular part of the respective expressed polypeptide.

7. The non-human animal of claims 1 to 6, wherein said nucleotide sequence of the human DRA genetic fragment comprises at least exons 2 and 3 of a human DRA gene and replace at least exons 2 and 3 in the at least one endogenous MHCII a gene locus of the non-human animal and at least exons 2 and 3 of the human DRB1 allele genetic fragment replace at least exons 2 and 3 of the endogenous MHCII 13 gene locus of the non-human animal.

8. The non-human animal of claims 1 to 7, wherein said nucleotide sequence of the human CD74 genetic fragment comprises at least exons 2 through 8 of a human CD74 gene and replace at least exons 2 through 8 of the at least one endogenous CD74 gene locus of the non-human animal.

9. The non-human animal of claims 1 to 8, wherein said nucleotide sequence of the human CD4 genetic fragment comprises at least exons 2 through a first portion of exon 7 of the human CD4 gene and replace at least exons 2 through a first portion of exon 7 of the at least one endogenous CD4 gene locus of the non-human animal.

10. The non-human animal of claims 1 to 9, wherein said nucleotide sequence of the human CD223 genetic fragment comprises at least position 10 of exon 2 through exon 7 of the human CD223 and replaces at least position 10 of exon 2 through exon 7 of the at least one endogenous CD223 gene locus of the non-human animal.

11. The non-human animal of claims 1 to 10, wherein an MHCII protein complex from a dimerization of the expressed humanized MHCII a and 6 polypeptides forms in association with a CLIP peptide comprising residues 91 to 99 from the expressed humanized CD74 polypeptide on a surface of an antigen presenting cell or analogous cell of the non-human animal, wherein a non-CLIP peptide displaces the CLIP peptide in a binding groove of the MHCII protein complex on the surface of an antigen presenting cell or analogous cell of the non-human animal, and wherein the non-CLIP peptide is a peptide of a mutated COL2 polypeptide, wherein said polypeptide comprises at least the residue of mutation D266E and wherein the MHCII protein complex with COL2 peptide on the surface of the antigen presenting cell or analogous cell of the non-human animal associates with the expressed CD4 polypeptide and the expressed CD223 polypeptide on a surface of a T-regulatory cell or analogous cell of the non-human animal.

12. The non-human animal of claims Ito 11 that is a mouse.

13. A method of making the non-human animal of claims 1 to 12 comprising: replacing at least one nucleotide sequence of at least one endogenous MHCII a gene locus in a non-human animal with a nucleotide sequence of a human DRA genetic fragment; replacing at least one nucleotide sequence of at least one endogenous MHCII 13 gene locus in said non-human animal with a nucleotide sequence of a human DRB1 allele genetic fragment; replacing at least one nucleotide sequence of at least one endogenous CD74 gene locus in said non-human animal with a nucleotide sequence of a human CD74 genetic fragment; and growing said non-human animal to make said non-human animal.

14. The method of making of claim 13, further comprising replacing at least one nucleotide sequence of at least one endogenous CD4 gene locus in said non-human animal with a nucleotide sequence of a human CD4 genetic fragment; replacing at least one nucleotide sequence of at least one endogenous CD223 gene locus in said non-human animal with a nucleotide sequence of a human CD223 genetic fragment; and growing said non-human animal to make said non-human animal.

15. The method of claims 13 and 14 further comprising replacing at least one endogenous COL2 gene sequence in an endogenous COL2 gene locus of the non-human animal with a mutated COL2 gene sequence with amino acid replacement D266E and amino acid replacement R360Q and replacing at least one endogenous NCF1 gene sequence in an endogenous NCF1 gene locus of the non-human animal with a mutated NCF1 gene sequence with the amino acid replacement R90H.

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