Multiple germline kappa light chains generate anti-insulin B cells in nonobese diabetic mice

The highly selective nature of organ-specific autoimmune disease is consistent with a critical role for adaptive immune responses against specific autoantigens. In type 1 diabetes mellitus, autoantibodies to insulin are important markers of the disease process in humans and nonobese diabetic (NOD) mice; however, the Ag-specific receptors responsible for these autoantibodies are obscured by the polyclonal repertoire. NOD mice that harbor an anti-insulin transgene (Tg) (V(H)125Tg/NOD) circumvent this problem by generating a tractable population of insulin-binding B cells. The nucleotide structure and genetic origin of the endogenous kappa L chain (Vkappa or IgL) repertoire that pairs with the V(H)125Tg were analyzed. In contrast to oligoclonal expansion observed in systemic autoimmune disease models, insulin-binding B cells from V(H)125Tg/NOD mice use specific Vkappa genes that are clonally independent and germline encoded. When compared with homologous IgL genes from nonautoimmune strains, Vkappa genes from NOD mice are polymorphic. Analysis of the most frequently expressed Vkappa1 and Vkappa9 genes indicates these are shared with lupus-prone New Zealand Black/BINJ mice (e.g., Vkappa1-110*02 and 9-124) and suggests that NOD mice use the infrequent b haplotype. These findings show that a diverse repertoire of anti-insulin B cells is part of the autoimmune process in NOD mice and structural or regulatory elements within the kappa locus may be shared with a systemic autoimmune disease.     

B cell specificity contributes to the outcome of diabetes in nonobese diabetic mice.

Type I diabetes mellitus (TIDM) is an autoimmune disorder characterized by T cell-mediated destruction of insulin-producing beta cells in the pancreas. In the nonobese diabetic (NOD) model of TIDM, insulitis and diabetes are dependent on the presence of B lymphocytes; however, the requirement for specificity within the B cell repertoire is not known. To determine the role of Ag-specific B cells in TIDM, V(H) genes with different potential for insulin binding were introduced into NOD as H chain transgenes. VH125 H chain combines with endogenous L chains to produce a repertoire in which 1-3% of mature B cells are insulin specific, and these mice develop accelerated diabetes. In contrast, NOD mice harboring a similar transgene, VH281, with limited insulin binding develop insulitis but are protected from TIDM. The data indicate that Ag-specific components in the B cell repertoire may alter the course of TIDM.     

Impaired activation of islet-reactive CD4 T cells in pancreatic lymph nodes of B cell-deficient nonobese diabetic mice

Despite the impressive protection of B cell-deficient (muMT(-/-)) nonobese diabetic (NOD) mice from spontaneous diabetes, existence of mild pancreatic islet inflammation in these mice indicates that initial autoimmune targeting of beta cells has occurred. Furthermore, muMT(-/-) NOD mice are shown to harbor a latent repertoire of diabetogenic T cells, as evidenced by their susceptibility to cyclophosphamide-induced diabetes. The quiescence of this pool of islet-reactive T cells may be a consequence of impaired activation of T lymphocytes in B cell-deficient NOD mice. In this regard, in vitro anti-CD3-mediated stimulation demonstrates impaired activation of lymph node CD4 T cells in muMT(-/-) NOD mice as compared with that of wild-type counterparts, a deficiency that is correlated with an exaggerated CD4 T cell:APC ratio in lymph nodes of muMT(-/-) NOD mice. This feature points to an insufficient availability of APC costimulation on a per T cell basis, resulting in impaired CD4 T cell activation in lymph nodes of muMT(-/-) NOD mice. In accordance with these findings, an islet-reactive CD4 T cell clonotype undergoes suboptimal activation in pancreatic lymph nodes of muMT(-/-) NOD recipients. Overall, the present study indicates that B cells in the pancreatic lymph node microenvironment are critical in overcoming a checkpoint involving the provision of optimal costimulation to islet-reactive NOD CD4 T cells.     

Induction of lymph follicle formation with several mitogens and adjuvants in the mouse popliteal lymph node

The formation of lymph follicles in draining popliteal nodes was investigated in young adult male mice which had been injected in the rear footpad with several mitogens and adjuvants, and killed after 3-21 days. PPD (100 micrograms-1 mg) and PHA (25-500 micrograms) induced germinal centers in association with existing follicles and mild plasmacytosis, but failed to produce new follicles in draining nodes. Endotoxin LPS (50-200 micrograms), Con A (50 micrograms-1 mg) and PWM (50 micrograms-1 mg) induced germinal centers within existing follicles and plasmacytosis, and also produced new follicles which soon developed germinal centers. Both Freund's complete and incomplete adjuvants (FCA and FICA, 25 microliters) induced virtually no germinal centers and plasmacytosis, but produced a significant number of new primary follicles. Poly (A, U) (600 micrograms) produced neither germinal centers nor plasmacytosis, and did not induce new follicles. Analysis of the distribution of lymphoid cells which had incorporated 3H-thymidine in the draining nodes at 3 days after the injection of test substances indicated that PPD, PHA, LPS, Con A and PWM preferentially stimulated in vivo the same types of lymphocytes as they do in vitro. FCA triggered lymphocyte activation in the deep cortex, whereas Poly (A, U) appeared not to stimulate lymphocytes in vivo. In further experiments, induction of lymph follicles with artificially precipitated PPD and PHA was studied. The draining nodes treated with alum-precipitated PPD or PHA were found to produce a significant number of new follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identical beta cell-specific CD8(+) T cell clonotypes typically reside in both peripheral blood lymphocyte and pancreatic islets

A major issue regarding T cell responses in autoimmunity is how the repertoire compares between the periphery and target organ. In type 1 diabetes, the status of at-risk or diabetic individuals can be monitored by measuring beta cell-specific T cells isolated from PBL, but whether these T cells accurately reflect the repertoire residing in the pancreatic islets is unclear. The TCR repertoire of disease-relevant, tetramer-sorted CD8(+) T cells was examined at the single-cell level in PBL, pancreatic lymph nodes (PLN), and the islets of individual NOD mice. CDR3alpha and CDR3beta sequences demonstrated that the same repertoire of T cells in PBL was detected in the islets and PLN, although the frequency of specific clonotypes varied. Albeit infrequent, clonotypes that were prevalent in the islets but not found in PBL were also detected. beta cell Ag immunization expanded immunodominant PBL clonotypes present in the islets and PLN. These results show that insight into repertoire profiles of islet-infiltrating T cells can be obtained from PBL.     

Antibody repertoire development in fetal and neonatal pigs. VII. Characterization of the preimmune kappa light chain repertoire

Combinatorial diversity is highly restricted in the preimmune porcine H chain repertoire compared with that in humans and mice. This raised the question of whether similar restriction characterized the preimmune L chain repertoire. In this study we present evidence that >90% of all expressed Vkappa genes in the porcine preimmune repertoire belong to three subfamilies of Vkappa genes that share 87% sequence similarity with human IGKV2. This porcine Vkappa family also shares sequence similarity with some, but not all, Vkappa genes from sheep. Hybridization with sperm DNA and sequence analyses of polynucleotides from overlapping bacterial artificial chromosome clones suggest swine possess approximately 60 IGVK2 genes. The latter method also revealed that certain IGKV2 subfamilies are not expressed in the preimmune repertoire. Six members of an IGVK1 family were also expressed as part of the preimmune repertoire, and these shared 87% sequence similarity with human IGVK1. Five Jkappa segments, complete with recombination signal sequences and separated by approximately 300 nt, were identified approximately 3 kb upstream of a single Ckappa. Surprisingly, Jkappa2 accounted for >90% of all framework region 4 sequences in the preimmune repertoire. These findings show that swine use approximately 10 IGVK2 genes from three of six subfamilies and preferentially one Jkappa segment to generate their preimmune kappa repertoire. These studies, like those of porcine Ig constant regions and MHC genes, also indicate unexpected high sequence similarity with their human counterparts despite differences in phylogeny and the mechanism of repertoire diversification.     

Anti-serpin Antibody-mediated Regulation of Proteases in Autoimmune Diabetes

Secretion of anti-serpin B13 autoantibodies in young diabetes-prone nonobese diabetic mice is associated with reduced inflammation in pancreatic islets and a slower progression to autoimmune diabetes. Injection of these mice with a monoclonal antibody (mAb) against serpin B13 also leads to fewer inflammatory cells in the islets and more rapid recovery from recent-onset diabetes. The exact mechanism by which anti-serpin activity is protective remains unclear. We found that serpin B13 is expressed in the exocrine component of the mouse pancreas, including the ductal cells. We also found that anti-serpin B13 mAb blocked the inhibitory activity of serpin B13, thereby allowing partial preservation of the function of its target protease. Consistent with the hypothesis that anti-clade B serpin activity blocks the serpin from binding, exposure to exogenous anti-serpin B13 mAb or endogenous anti-serpin B13 autoantibodies resulted in cleavage of the surface molecules CD4 and CD19 in lymphocytes that accumulated in the pancreatic islets and pancreatic lymph nodes but not in the inguinal lymph nodes. This cleavage was inhibited by an E64 protease inhibitor. Consequently, T cells with the truncated form of CD4 secreted reduced levels of interferon-γ. We conclude that anti-serpin antibodies prevent serpin B13 from neutralizing proteases, thereby impairing leukocyte function and reducing the severity of autoimmune inflammation.     

NK T cell-induced protection against diabetes in V alpha 14-J alpha 281 transgenic nonobese diabetic mice is associated with a Th2 shift circumscribed regionally to the islets and functionally to islet autoantigen

The onset of autoimmune diabetes is related to defective immune regulation. Recent studies have shown that NK T cells are deficient in number and function in both diabetic patients and nonobese diabetic (NOD) mice. NK T cells, which are CD1d restricted, express a TCR with an invariant V alpha 14-J alpha 281 chain and rapidly produce large amounts of cytokines. V alpha 14-J alpha 281 transgenic NOD mice have increased numbers of NK T cells and are protected against diabetes onset. In this study we analyzed where and how NK T cells interfere with the development of the anti-islet autoimmune response. NK T cells, which are usually rare in lymph nodes, are abundant in pancreatic lymph nodes and are also present in islets. IL-4 mRNA levels are increased and IFN-gamma mRNA levels decreased in islets from diabetes-free V alpha 14-J alpha 281 transgenic NOD mice; the IgG1/IgG2c ratio of autoantibodies against glutamic acid decarboxylase is also increased in these mice. Treatment with IL-12 (a pro-Th1 cytokine) or anti-IL-4 Ab abolishes the diabetes protection in V alpha 14-J alpha 281 NOD mice. The protection from diabetes conferred by NK T cells is thus associated with a Th2 shift within islets directed against autoantigen such as glutamic acid decarboxylase. Our findings also demonstrate the key role of IL-4.     

B cell-specific S1PR1 deficiency blocks prion dissemination between secondary lymphoid organs

Many prion diseases are peripherally acquired (e.g., orally or via lesions to skin or mucous membranes). After peripheral exposure, prions replicate first upon follicular dendritic cells (FDC) in the draining lymphoid tissue before infecting the brain. However, after replication upon FDC within the draining lymphoid tissue, prions are subsequently propagated to most nondraining secondary lymphoid organs (SLO), including the spleen, by a previously underdetermined mechanism. The germinal centers in which FDC are situated produce a population of B cells that can recirculate between SLO. Therefore, we reasoned that B cells were ideal candidates by which prion dissemination between SLO may occur. Sphingosine 1-phosphate receptor (S1PR)1 stimulation controls the egress of T and B cells from SLO. S1PR1 signaling blockade sequesters lymphocytes within SLO, resulting in lymphopenia in the blood and lymph. We show that, in mice treated with the S1PR modulator FTY720 or with S1PR1 deficiency restricted to B cells, the dissemination of prions from the draining lymph node to nondraining SLO is blocked. These data suggest that B cells interacting with and acquiring surface proteins from FDC and recirculating between SLO via the blood and lymph mediate the initial propagation of prions from the draining lymphoid tissue to peripheral tissues.     

First signature of islet beta-cell-derived naturally processed peptides selected by diabetogenic class II MHC molecules

The diversity of Ags targeted by T cells in autoimmune diabetes is unknown. In this study, we identify and characterize a limited number of naturally processed peptides from pancreatic islet beta-cells selected by diabetogenic I-A(g7) molecules of NOD mice. We used insulinomas transfected with the CIITA transactivator, which resulted in their expression of class II histocompatibility molecules and activation of diabetogenic CD4 T cells. Peptides bound to I-A(g7) were isolated and examined by mass spectrometry: some peptides derived from proteins present in secretory granules of endocrine cells, and a number were shared with cells of neuronal lineage. All proteins to which peptides were identified were expressed in beta cells from normal islets. Peptides bound to I-A(g7) molecules contained the favorable binding motif characterized by acidic amino acids at the P9 position. The draining pancreatic lymph nodes of prediabetic NOD mice contained CD4 T cells that recognized three different natural peptides. Furthermore, four different peptides elicited CD4 T cells, substantiating the presence of such self-reactive T cells. The overall strategy of identifying natural peptides from islet beta-cells opens up new avenues to evaluate the repertoire of self-reactive T cells and its role in onset of diabetes.     

Rotavirus Activates Lymphocytes from Non-Obese Diabetic Mice by Triggering Toll-Like Receptor 7 Signaling and Interferon Production in Plasmacytoid Dendritic Cells

It has been proposed that rotavirus infection promotes the progression of genetically-predisposed children to type 1 diabetes, a chronic autoimmune disease marked by infiltration of activated lymphocytes into pancreatic islets. Non-obese diabetic (NOD) mice provide a model for the human disease. Infection of adult NOD mice with rhesus monkey rotavirus (RRV) accelerates diabetes onset, without evidence of pancreatic infection. Rather, RRV spreads to the pancreatic and mesenteric lymph nodes where its association with antigen-presenting cells, including dendritic cells, induces cellular maturation. RRV infection increases levels of the class I major histocompatibility complex on B cells and proinflammatory cytokine expression by T cells at these sites. In autoimmunity-resistant mice and human mononuclear cells from blood, rotavirus-exposed plasmacytoid dendritic cells contribute to bystander polyclonal B cell activation through type I interferon expression. Here we tested the hypothesis that rotavirus induces bystander activation of lymphocytes from NOD mice by provoking dendritic cell activation and proinflammatory cytokine secretion. NOD mouse splenocytes were stimulated with rotavirus and assessed for activation by flow cytometry. This stimulation activated antigen-presenting cells and B cells independently of virus strain and replicative ability. Instead, activation depended on virus dose and was prevented by blockade of virus decapsidation, inhibition of endosomal acidification and interference with signaling through Toll-like receptor 7 and the type I interferon receptor. Plasmacytoid dendritic cells were more efficiently activated than conventional dendritic cells by RRV, and contributed to the activation of B and T cells, including islet-autoreactive CD8⁺ T cells. Thus, a double-stranded RNA virus can induce Toll-like receptor 7 signaling, resulting in lymphocyte activation. Our findings suggest that bystander activation mediated by type I interferon contributes to the lymphocyte activation observed following RRV infection of NOD mice, and may play a role in diabetes acceleration by rotavirus.     

Schistosoma mansoni: the effect of regional lymphadenectomy on the level of protection induced in mice by radiation-attenuated cercariae

The lymph nodes which drain the sites of percutaneous vaccination with optimally irradiated cercariae of Schistosoma mansoni were surgically excised in studies to determine their role in the induction of protective immunity. Lymphadenectomy of the axillary and inguinal nodes which drain the abdominal exposure site, or of the cervical node which drains the aural site of exposure, five days prior to vaccination reduced the levels of resistance by two-thirds. Excision of these nodes on Days 5, 10, 15, or 20 postvaccination also significantly reduced the levels of immunity induced, though ablation was less effective at later times. Removal of lymph nodes not draining the site of vaccination had no effect on the induction of resistance. We interpret the results as indicating that successful vaccination of mice against S. mansoni requires the presentation of antigen to lymphocytes in local lymph nodes draining the vaccination site, rather than distant lymphoid organs such as the spleen.     

Up-regulation of CD1d expression restores the immunoregulatory function of NKT cells and prevents autoimmune diabetes in nonobese diabetic mice

The immunoregulatory function of NKT cells is crucial for prevention of autoimmunity. The prototypical NKT cell Ag alpha-galactosylceramide is not present in mammalian cells, and little is known about the mechanism responsible for NKT cell recruitment and activation. Up-regulation of CD1d, the NKT cell restriction molecule, expressed on mononuclear cells infiltrating the target organ, could represent the physiological trigger for NKT cells to self-contain T cell immunity and to prevent autoimmune disease. Recognition of CD1d, either by itself or bound to self-ligands (selfCD1d), could drive NKT cells toward an immunoregulatory phenotype. Hence, ineffective NKT cell-mediated immunoregulation in autoimmune-prone individuals including nonobese diabetic (NOD) mice could be related to defective signals that regulate CD1d expression at time and site of autoimmunity. To test this hypothesis, we transgenically overexpressed CD1d molecules under the control of the insulin promoter within the pancreatic islets of NOD mice (insCD1d). Recognition of overexpressed CD1d molecules rescued NKT cell immunoregulatory function and prevented autoimmune diabetes in insCD1d transgenic NOD mice. Protection from diabetes was associated with a biased IL-4-secreting cytokine phenotype of NKT cells and alteration of the cytokine microenvironment in the pancreatic lymph nodes of transgenic mice. The net effect was a reduced development of the autoimmune T cell repertoire. Our findings suggest that up-regulation of CD1d expression during inflammation is critical to maintain T cell homeostasis and to prevent autoimmunity.     

CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.     

Pancreas-infiltrating Th1 cells and diabetes develop in IL-12-deficient nonobese diabetic mice.

IL-12 and IL-12 antagonist administration to nonobese diabetic (NOD) mice accelerates and prevents insulin-dependent diabetes mellitus (IDDM), respectively. To further define the role of endogenous IL-12 in the development of diabetogenic Th1 cells, IL-12-deficient NOD mice were generated and analyzed. Th1 responses to exogenous Ags were reduced by approximately 80% in draining lymph nodes of these mice, and addition of IL-12, but not IL-18, restored Th1 development in vitro, indicating a nonredundant role of IL-12. Moreover, spontaneous Th1 responses to a self Ag, the tyrosine phosphatase-like IA-2, were undetectable in lymphoid organs from IL-12-deficient, in contrast to wild-type, NOD mice. Nevertheless, wild-type and IL-12-deficient NOD mice developed similar insulitis and IDDM. Both in wild-type and IL-12-deficient NOD mice, approximately 20% of pancreas-infiltrating CD4+ T cells produced IFN-gamma, whereas very few produced IL-10 or IL-4, indicating that IDDM was associated with a type 1 T cell infiltrate in the target organ. T cell recruitment in the pancreas seemed favored in IL-12-deficient NOD mice, as revealed by increased P-selectin ligand expression on pancreas-infiltrating T cells, and this could, at least in part, compensate for the defective Th1 cell pool recruitable from peripheral lymphoid organs. Residual Th1 cells could also accumulate in the pancreas of IL-12-deficient NOD mice because Th2 cells were not induced, in contrast to wild-type NOD mice treated with an IL-12 antagonist. Thus, a regulatory pathway seems necessary to counteract the pathogenic Th1 cells that develop in the absence of IL-12 in a spontaneous chronic progressive autoimmune disease under polygenic control, such as IDDM.     

TCR bias of in vivo expanded T cells in pancreatic islets and spleen at the onset in human type 1 diabetes

Autoreactive T cells, responsible for the destruction of pancreatic β cells in type 1 diabetes, are known to have a skewed TCR repertoire in the NOD mouse. To define the autoreactive T cell repertoire in human diabetes, we searched for intraislet monoclonal expansions from a recent onset in human pancreas to then trace them down to the patient's peripheral blood and spleen. Islet infiltration was diverse, but five monoclonal TCR β-chain variable expansions were detected for Vβ1, Vβ7, Vβ11, Vβ17, and Vβ22 families. To identify any sequence bias in the TCRs from intrapancreatic T cells, we analyzed 139 different CDR3 sequences. We observed amino acid preferences in the NDN region that suggested a skewed TCR repertoire within infiltrating T cells. The monoclonal expanded TCR sequences contained amino acid combinations that fit the observed bias. Using these CDR3 sequences as a marker, we traced some of these expansions in the spleen. There, we identified a Vβ22 monoclonal expansion with identical CDR3 sequence to that found in the islets within a polyclonal TCR β-chain variable repertoire. The same Vβ22 TCR was detected in the patient's PBMCs, making a cross talk between the pancreas and spleen that was reflected in peripheral blood evident. No other pancreatic monoclonal expansions were found in peripheral blood or the spleen, suggesting that the Vβ22 clone may have expanded or accumulated in situ by an autoantigen present in both the spleen and pancreas. Thus, the patient's spleen might be contributing to disease perpetuation by expanding or retaining some autoreactive T cells.     

T and B lymphocyte migration into syngeneic tumors.

The migration of splenic T and B lymphocytes into syngeneic tumors undergoing immunologic rejection was investigates. Spleen cells were obtained from normal BALC/c mice or BALB/c mice bearing tumors induced by murine sarcoma virus (MSV). Either whole spleen cells or immunoabsorbent purified T and B cells were radiolabeled with sodium chromate-51 and injected i.v. into normal or MSV inducted-tumor bearing syngeneic recipients. Twenty-four hours later the recipient mice were sacrificed and radioactivity was assessed for tumor, contralateral normal muscle, the lymph nodes draining the tumor and contralateral draining lymph nodes, peripheral lymph nodes, spleen, and liver. Both T and B lymphocytes from either normal or MSV tumor-bearing animals show greatly increased migration into the tumor when compared with normal muscle. Migration of T cells from both normal and MSV tumor bearers was 30 times that of migration to normal muscle. B cells from tumor-bearing mice, on the other hand, localized in the tumor itself only 50% as frequently as did B cells from normal animals. In addition, T cells from MSV tumor bearers were found in the highest proportion in the lymph node draining the tumor site. We conclude that T and B lymphocytes from either normal or tumor-bearing mice migrate to a syngeneic tumor undergoing immunologic rejection. In contrast, the migration of both T and B cells from tumor-bearing animals was decreased to the peripheral lymph nodes at the time of maximum tumor growth.     

Vκ polymorphisms in NOD mice are spread throughout the entire immunoglobulin kappa locus and are shared by other autoimmune strains

The diversity of immunoglobulin (Ig) and T cell receptor (TCR) genes available to form the lymphocyte repertoire has the capacity to produce a broad array of both protective and harmful specificities. In type 1 diabetes (T1D), the presence of antibodies to insulin and other islet antigens predicts disease development in both mice and humans, and demonstrate that immune tolerance is lost early in the disease process. Anti-insulin T cells isolated from T1D-prone non-obese diabetic (NOD) mice use polymorphic TCRα chains, suggesting that the available T cell repertoire is altered in these autoimmune mice. To probe whether insulin-binding B cells also possess polymorphic V genes, Ig light chains were isolated and sequenced from NOD mice that harbor an Ig heavy chain transgene. Three insulin-binding Vκ genes were identified, all of which were polymorphic to the closest germline sequence matches present in the GenBank database. Additional analysis of over 300 light chain sequences from multiple sources, including germline DNA, shows that polymorphisms are spread throughout the entire NOD Igκ locus, as these polymorphic sequences represent 43 distinct Vκ genes which belong to 14 Vκ families. Database searches reveal that a majority of polymorphic Vκ genes identified in NOD are identical to Vκ genes isolated from SLE-prone NZBxNZW F1 or MRL strains of mice, suggesting that a shared Igκ haplotype may be present. Predicted amino acid changes preferentially occur in CDR, and thus could alter antigen recognition by the germline B cell repertoire of autoimmune versus non-autoimmune mouse strains.     

Colocalization of antigen-specific B and T cells within ectopic lymphoid tissue following immunization with exogenous antigen

Chronic inflammation promotes the formation of ectopic lymphoid tissue morphologically resembling secondary lymphoid tissues, though it is unclear whether this is a location where Ag-specific immune responses develop or merely a site of lymphocyte accumulation. Ectopic lymphoid tissue formation is associated with many humoral autoimmune diseases, including lupus induced by tetramethylpecadentane in mice. We examined whether an immune response to 4-hydroxy-3-nitrophenyl acetyl-keyhole limpet hemocyanin (NP-KLH) and NP-OVA develops within ectopic lymphoid tissue ("lipogranulomas") induced by tetramethylpecadentane in C57BL/6 mice. Following primary immunization, NP-specific B cells bearing V186.2 and related heavy chains as well as lambda-light chains accumulated within ectopic lymphoid tissue. The number of anti-NP-secreting B cells in the ectopic lymphoid tissue was greatly enhanced by immunization with NP-KLH. Remarkably, the H chain sequences isolated from individual lipogranulomas from these mice were diverse before immunization, whereas individual lipogranulomas from single immunized mice had unique oligo- or monoclonal populations of presumptive NP-specific B cells. H chain CDR sequences bore numerous replacement mutations, consistent with an Ag-driven and T cell-mediated response. In mice adoptively transferred with OT-II or DO11 T cells, there was a striking accumulation of OVA-specific T cells in lipogranulomas after s.c. immunization with NP-OVA. The selective colocalization of proliferating, Ag-specific T and B lymphocytes in lipogranulomas from tetramethylpecadentane-treated mice undergoing primary immunization implicates ectopic lymphoid tissue as a site where Ag-specific humoral immune responses can develop. This has implications for understanding the strong association of humoral autoimmunity with lymphoid neogenesis, which may be associated with deficient censoring of autoreactive cells.     

Altered availability of PD-1/PD ligands is associated with the failure to control autoimmunity in NOD mice

Costimulation via the PD-1 and B7-H1/B7-DC pathway regulates immunity. We investigated whether the PD-1/PD-L pathway is impaired in autoimmune diabetes. A progressive increase in the expression of PD-1 and B7-H1/B7-DC on T cells and APC, respectively, was observed in the pancreatic lymph nodes of female non-obese diabetic (NOD) mice as they developed diabetes. A significantly decreased expression of PD-1 and B7-H1/B7-DC on T cells and APC, respectively, was observed in the periphery of prediabetic NOD mice versus non-diabetic C57BL/6 strain. NOD islets also displayed a reduced capacity to upregulate B7-H1 following exposure to inflammatory cytokines. In vivo blocking studies in NOD/B7-2KONOD mice revealed that B7-H1 and B7-DC positively costimulate autoreactive CD4 and CD8 T cells and may co-operate with B7-2 to augment priming and expansion of naïve autoreactive T cells. In summary, these data suggest that diabetes susceptibility in NOD mice is associated with altered PD-1/PD-L availability.