Concomitant Disruption of CD4 and CD8 Genes Facilitates the Development of Double Negative αß TCR+ Peripheral T Cells That Respond Robustly to Staphylococcal Superantigen.

Mature peripheral double negative T (DNT) cells expressing αß TCR but lacking CD4/CD8 coreceptors play protective as well as pathogenic roles. To better understand their development and functioning in vivo, we concomitantly inactivated CD4 and CD8 genes in mice with intact MHC class I and class II molecules with the hypothesis that this would enable the development of DNT cells. We also envisaged that these DNT cells could be activated by bacterial superantigens in vivo as activation of T cells by superantigens does not require CD4 and CD8 coreceptors. Because HLA class II molecules present superantigens more efficiently than murine MHC class II molecules, CD4 CD8 double knockout (DKO) mice transgenically expressing HLA-DR3 or HLA-DQ8 molecules were generated. Although thymic cellularity was comparable between wild type (WT) and DKO mice, CD3+ αß TCR+ thymocytes were significantly reduced in DKO mice, implying defects in thymic-positive selection. Splenic CD3+ αß TCR+ cells and Foxp3+ T regulatory cells were present in DKO mice but significantly reduced. However, the in vivo inflammatory responses and immunopathology elicited by acute challenge with the staphylococcal superantigen enterotoxin B were comparable between WT and DKO mice. Choric exposure to staphylococcal enterotoxin B precipitated a lupus-like inflammatory disease with characteristic lympho-monocytic infiltration in lungs, livers, and kidneys, along with production of anti-nuclear Abs in DKO mice as in WT mice. Overall, our results suggest that DNT cells can develop efficiently in vivo and chronic exposure to bacterial superantigens may precipitate a lupus-like autoimmune disease through activation of DNT cells.

Passive therapy with humanized anti-staphylococcal enterotoxin B antibodies attenuates systemic inflammatory response and protects from lethal pneumonia caused by staphylococcal enterotoxin B-producing Staphylococcus aureus.

Drugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.

Airway Memory CD4(+) T Cells Mediate Protective Immunity against Emerging Respiratory Coronaviruses.

Two zoonotic coronaviruses (CoVs)-SARS-CoV and MERS-CoV-have crossed species to cause severe human respiratory disease. Here, we showed that induction of airway memory CD4(+) T cells specific for a conserved epitope shared by SARS-CoV and MERS-CoV is a potential strategy for developing pan-coronavirus vaccines. Airway memory CD4(+) T cells differed phenotypically and functionally from lung-derived cells and were crucial for protection against both CoVs in mice. Protection was dependent on interferon-γ and required early induction of robust innate and virus-specific CD8(+) T cell responses. The conserved epitope was also recognized in SARS-CoV- and MERS-CoV-infected human leukocyte antigen DR2 and DR3 transgenic mice, indicating potential relevance in human populations. Additionally, this epitope was cross-protective between human and bat CoVs, the progenitors for many human CoVs. Vaccine strategies that induce airway memory CD4(+) T cells targeting conserved epitopes might have broad applicability in the context of new CoVs and other respiratory virus outbreaks.

Identifying a Small Molecule Blocking Antigen Presentation in Autoimmune Thyroiditis.

We previously showed that an HLA-DR variant containing arginine at position 74 of the DRß1 chain (DRß1-Arg74) is the specific HLA class II variant conferring risk for autoimmune thyroid diseases (AITD). We also identified 5 thyroglobulin (Tg) peptides that bound to DRß1-Arg74. We hypothesized that blocking the binding of these peptides to DRß1-Arg74 could block the continuous T-cell activation in thyroiditis needed to maintain the autoimmune response to the thyroid. The aim of the current study was to identify small molecules that can block T-cell activation by Tg peptides presented within DRß1-Arg74 pockets. We screened a large and diverse library of compounds and identified one compound, cepharanthine that was able to block peptide binding to DRß1-Arg74. We then showed that Tg.2098 is the dominant peptide when inducing experimental autoimmune thyroiditis (EAT) in NOD mice expressing human DRß1-Arg74. Furthermore, cepharanthine blocked T-cell activation by thyroglobulin peptides, in particular Tg.2098 in mice that were induced with EAT. For the first time we identified a small molecule that can block Tg peptide binding and presentation to T-cells in autoimmune thyroiditis. If confirmed cepharanthine could potentially have a role in treating human AITD.

Superantigen-Producing Staphylococcus aureus Elicits Systemic Immune Activation in a Murine Wound Colonization Model.

Staphylococcus aureus, the most common cause of wound infection, produces several exotoxins, including superantigens (SAgs). SAgs are the potent activators of the immune system. Given this unique property, we hypothesized that SAgs produced by S. aureus in wounds would have local, as well as systemic immunologic effects. We tested our hypothesis using a novel staphylococcal skin wound infection model in transgenic mice expressing HLA-DR3. Skin wounds were left uninfected or colonized with S. aureus strains producing SAgs or an isogenic strain not producing any SAg. Animals with wounds challenged with SAg-producing S. aureus had increased morbidity and lower serum IL-17 levels compared to those challenged with the SAg non-producing S. aureus (p = 0.027 and p = 0.032, respectively). At Day 8 following microbial challenge, compared to mice with uninfected wounds, the proportion of Vß8⁺CD4⁺ T cells was increased, while the proportion of Vß8⁺CD8⁺ T cells was decreased only in the spleens of mice challenged with SAg-producing S. aureus (p < 0.001). No such changes were measured in mice challenged with SAg non-producing S. aureus. Lungs, livers and kidneys from mice challenged with SAg-producing, but not SAg non-producing, S. aureus showed inflammatory changes. Overall, SAg-mediated systemic immune activation in wounds harboring S. aureus may have clinical implications.

A Central Role for HLA-DR3 in Anti-Smith Antibody Responses and Glomerulonephritis in a Transgenic Mouse Model of Spontaneous Lupus.

MHC, especially HLA-DR3 and HLA-DR2, is one of the most important genetic susceptibility regions for systemic lupus erythematosus. Human studies to understand the role of specific HLA alleles in disease pathogenesis have been hampered by the presence of strong linkage disequilibrium in this region. To overcome this, we produced transgenic mice expressing HLA-DR3 (DRß1*0301) and devoid of endogenous class II (both I-A and I-E genes, AE(0)) on a lupus-prone NZM2328 background (NZM2328.DR3(+)AE(0)). Both NZM2328 and NZM2328.DR3(+)AE(0) mice developed anti-dsDNA and glomerulonephritis, but anti-dsDNA titers were higher in the latter. Although kidney histological scores were similar in NZM2328 and NZM2328.DR3(+)AE(0) mice (7.2 ± 4.3 and 8.6 ± 5.7, respectively, p = 0.48), the onset of severe proteinuria occurred earlier in NZM2328.DR3(+)AE(0) mice compared with NZM2328 mice (median, 5 and 9 mo respectively, p < 0.001). Periarterial lymphoid aggregates, classic wire loop lesions, and occasional crescents were seen only in kidneys from NZM2328.DR3(+)AE(0) mice. Interestingly, NZM2328.DR3(+)AE(0) mice, but not NZM2328 mice, spontaneously developed anti-Smith (Sm) Abs. The anti-Sm Abs were seen in NZM2328.DR3(+)AE(0) mice that were completely devoid of endogenous class II (AE(-/) (-)) but not in mice homozygous (AE(+/+)) or heterozygous (AE(+/-)) for endogenous MHC class II. It appears that only HLA-DR3 molecules can preferentially select SmD-reactive CD4(+) T cells for generation of the spontaneous anti-Sm immune response. Thus, our mouse model unravels a critical role for HLA-DR3 in generating an autoimmune response to SmD and lupus nephritis in the NZM2328 background.

Superantigens produced by catheter-associated Staphylococcus aureus elicit systemic inflammatory disease in the absence of bacteremia.

SAgs, produced by Staphylococcus aureus, play a major role in the pathogenesis of invasive staphylococcal diseases by inducing potent activation of the immune system. However, the role of SAgs, produced by S. aureus, associated with indwelling devices or tissues, are not known. Given the prevalence of device-associated infection with toxigenic S. aureus in clinical settings and the potency of SAgs, we hypothesized that continuous exposure to SAgs produced by catheter-associated S. aureus could have systemic consequences. To investigate these effects, we established a murine in vivo catheter colonization model. One centimeter long intravenous catheters were colonized with a clinical S. aureus isolate producing SAgs or isogenic S. aureus strains, capable or incapable of producing SAg. Catheters were subcutaneously implanted in age-matched HLA-DR3, B6, and AE(o) mice lacking MHC class II molecules and euthanized 7 d later. There was no evidence of systemic infection. However, in HLA-DR3 transgenic mice, which respond robustly to SSAgs, the SSAg-producing, but not the nonproducing strains, caused a transient increase in serum cytokine levels and a protracted expansion of splenic CD4(+) T cells expressing SSAg-reactive TCR Vß8. Lungs, livers, and kidneys from these mice showed infiltration with CD4(+) and CD11b(+) cells. These findings were absent in B6 and AE(o) mice, which are known to respond poorly to SSAgs. Overall, our novel findings suggest that systemic immune activation elicited by SAgs, produced by S. aureus colonizing foreign bodies, could have clinical consequences in humans.

The viral transcription group determines the HLA class I cellular immune response against human respiratory syncytial virus.

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design.

Superantigens in Staphylococcus aureus isolated from prosthetic joint infection.

Staphylococcus aureus is a common cause of prosthetic joint infection (PJI). The prevalence of superantigens (SAgs) among PJI-associated S. aureus is unknown. Eighty-four S. aureus isolates associated with PJI isolated between 1999 and 2006 were studied. SAg genes, sea, seb, sec, sed, see, seg, seh, sei, and tst, were assayed by PCR. Seventy-eight (92.9%) isolates carried at least 1 SAg gene studied, with 61 (72.6%) harboring more than 1. seg was most commonly (70.2%), and seh was least frequently (4.8%) detected. tst-positive isolates were associated with early infection and increased erythrocyte sedimentation rate at diagnosis (P=0.006 and P=0.021, respectively). seg and sei were associated with methicillin resistance (P=0.008 and P=0.002, respectively). A majority of PJI-associated isolates studied produced biologically active SAgs in both planktonic and biofilm growth modes. SAg genes are prevalent in S. aureus causing PJI.

Systemic inflammatory response elicited by superantigen destabilizes T regulatory cells, rendering them ineffective during toxic shock syndrome.

Life-threatening infections caused by Staphylococcus aureus, particularly the community-acquired methicillin-resistant strains of S. aureus, continue to pose serious problems. Greater virulence and increased pathogenicity of certain S. aureus strains are attributed to higher prevalence of exotoxins. Of these exotoxins, the superantigens (SAg) are likely most pathogenic because of their ability to rapidly and robustly activate the T cells even in extremely small quantities. Therefore, countering SAg-mediated T cell activation using T regulatory cells (Tregs) might be beneficial in diseases such as toxic shock syndrome (TSS). As the normal numbers of endogenous Tregs in a typical host are insufficient, we hypothesized that increasing the Treg numbers by administration of IL-2/anti-IL-2 Ab immune complexes (IL2C) or by adoptive transfer of ex vivo expanded Tregs might be more effective in countering SAg-mediated immune activation. HLA-DR3 transgenic mice that closely recapitulate human TSS were treated with IL2C to increase endogenous Tregs or received ex vivo expanded Tregs. Subsequently, they were challenged with SAg to induce TSS. Analyses of various parameters reflective of TSS (serum cytokine/chemokine levels, multiple organ pathology, and SAg-induced peripheral T cell expansion) indicated that increasing the Tregs failed to mitigate TSS. On the contrary, serum IFN-γ levels were increased in IL2C-treated mice. Exploration into the reasons behind the lack of protective effect of Tregs revealed IL-17 and IFN-γ-dependent loss of Tregs during TSS. In addition, significant upregulation of glucocorticoid-induced TNFR family-related receptor on conventional T cells during TSS could render them resistant to Treg-mediated suppression, contributing to failure of Treg-mediated immune regulation.

DRB1*0402 may influence arthritis by promoting naive CD4+ T-cell differentiation in to regulatory T cells.

HLA-DRB1*0401 expression in humans has been associated with a predisposition to developing rheumatoid arthritis (RA) and collagen-induced arthritis (CIA), while HLA-DRB1*0402 is not associated with susceptibility. Here, we determined if mice transgenic (Tg) for human *0401 have a CD4+ T-cell repertoire that predetermines proinflammatory cytokine production. The data show that both *0401 and *0402 Tg mice can produce TH1/TH17 cytokines, although the kinetics of response may be different. However, in the context of antigen-specific responses in a CIA model, *0402 Tg mice generate a TH2 response that may explain their resistance to developing arthritis. In addition, a significant subset of naïve CD4+ T cells from *0402 Tg mice can be activated in polarizing conditions to differentiate into Treg cells that produce IFN-γ. *0401 Tg mice harbor memory CD4+ T cells that differentiate into IL-17(+) cells in various polarizing conditions. Our data suggest that *0401 Tg mice generate a strong immune response to lipopolysaccharide and may be efficient in clearing infection, and may *0401 have been evolutionarily selected for this ability. Autoimmunity, such as RA, could likely be a bystander effect of the cytokine storm that, along with the presence of low Treg-cell numbers in *0401 Tg mice, causes immune dysregulation.

The impact of Staphylococcus aureus-associated molecular patterns on staphylococcal superantigen-induced toxic shock syndrome and pneumonia.

Staphylococcus aureus is capable of causing a spectrum of human illnesses. During serious S. aureus infections, the staphylococcal pathogen-associated molecular patterns (PAMPs) such as peptidoglycan, lipoteichoic acid, and lipoproteins and even intact S. aureus, are believed to act in conjunction with the staphylococcal superantigens (SSAg) to activate the innate and adaptive immune system, respectively, and cause immunopathology. However, recent studies have shown that staphylococcal PAMPs could suppress inflammation by several mechanisms and protect from staphylococcal toxic shock syndrome, a life-threatening systemic disease caused by toxigenic S. aureus. Given the contradictory pro- and anti-inflammatory roles of staphylococcal PAMPs, we examined the effects of S. aureus-derived molecular patterns on immune responses driven by SSAg in vivo using HLA-DR3 and HLA-DQ8 transgenic mice. Our study showed that neither S. aureus-derived peptidoglycans (PGN), lipoteichoic acid (LTA), nor heat-killed Staphylococcus aureus (HKSA) inhibited SSAg-induced T cell proliferation in vitro. They failed to antagonize the immunostimulatory effects of SSAg in vivo as determined by their inability to attenuate systemic cytokine/chemokine response and reduce SSAg-induced T cell expansion. These staphylococcal PAMPs also failed to protect HLA-DR3 as well as HLA-DQ8 transgenic mice from either SSAg-induced toxic shock or pneumonia induced by a SSAg-producing strain of S. aureus.

Superantigen profiling of Staphylococcus aureus infective endocarditis isolates.

The frequency of superantigen production among Staphylococcus aureus isolates associated with endocarditis is not well defined. We tested 154 S. aureus isolates from definite infective endocarditis cases for the presence of staphylococcal enterotoxins A-E, H, and TSST-1 by PCR, enzyme-linked immunosorbent assay, and using an HLA-DR3 transgenic mouse splenocyte proliferation assay. Sixty-three isolates (50.8%) tested positive for at least 1 superantigen gene, with 21 (16.9%) testing positive for more than 2. tst (28.6%) was most common, followed by seb (27%), sea (22.2%), sed (20.6%), see (17.5%), and sec (11.1%). Of 41 methicillin-resistant S. aureus, 21 had superantigen genes, with sed being more frequently detected in this group compared to methicillin-susceptible S. aureus (P < 0.05). Superantigen genes were not associated with mortality (P = 0.81). 75% of PCR-positive isolates induced robust splenocyte proliferation. Overall, more than half of S. aureus isolates causing endocarditis carry superantigen genes, of which most are functional.

Modulation of Immune response by Ultra-violet light in HLA class-II transgenic mice.

Multiple Sclerosis (MS) is a chronic and debilitating disease of the central nervous system linked to both genetic and environmental factors. Among the genetic factors, MHC, especially HLA class-II, is strongly associated with predisposition to MS. Although in vitro studies have helped us understand some aspects of HLA class-II association with the disease, performing in vivo analysis is necessary in order to further understand this correlation. Studying the role of class-II genes in vivo is a difficult task due to the heterogeneity of human population, the complexity of MHC, and the strong linkage disequilibrium among different class-II genes. To overcome this challenge, we generated HLA class-II transgenic mice to study the role of these molecules in MS. Among the environmental factors linked with MS, ultra violet radiation (UVR)/vitamin-D is suggested to have protective effect against the development of the disease. Indeed, genetic studies have shown that presence of susceptible HLA-Class II and decrease in UVR exposure or vitamin D levels together increase risk of MS. Therefore, this study was designed to investigate the direct effect of UVR on immune response using novel humanized HLA-class II transgenic mice. HLA-class II transgenic mice expressing MS susceptible HLA-DR2 allele were treated with different doses of UVR (0.50-3.75 kJ/day) for seven consecutive days. T-cell proliferation, immune cell sub-populations and cytokines levels were analyzed. Our results show that treatment with UVR increased levels of regulatory CD4+FoxP3+ T cells and Gr1+ CD11b+ suppressive macrophages. Thus our study indicates that UVR modulates the immune response towards a tolerogenic phenotype in HLA-transgenic mice immunized with MOG35-55. Therefore, HLA class-II transgenic mice offer a novel tool to decipher the mechanism by which interaction between environmental and genetic factors play a role in predisposition and/or protection against development of MS.

HLA class II (DR0401) molecules induce Foxp3+ regulatory T cell suppression of B cells in Plasmodium yoelii strain 17XNL malaria.

Unlike human malaria parasites that induce persistent infection, some rodent malaria parasites, like Plasmodium yoelii strain 17XNL (Py17XNL), induce a transient (self-curing) malaria infection. Cooperation between CD4 T cells and B cells to produce antibodies is thought to be critical for clearance of Py17XNL parasites from the blood, with major histocompatibility complex (MHC) class II molecules being required for activation of CD4 T cells. In order to better understand the correspondence between murine malaria models and human malaria, and in particular the role of MHC (HLA) class II molecules, we studied the ability of humanized mice expressing human HLA class II molecules to clear Py17XNL infection. We showed that humanized mice expressing HLA-DR4 (DR0401) molecules and lacking mouse MHC class II molecules (EA(0)) have impaired production of specific antibodies to Py17XNL and cannot cure the infection. In contrast, mice expressing HLA-DR4 (DR0402), HLA-DQ6 (DQ0601), HLA-DQ8 (DQ0302), or HLA-DR3 (DR0301) molecules in an EA(0) background were able to elicit specific antibodies and self-cure the infection. In a series of experiments, we determined that the inability of humanized DR0401.EA(0) mice to elicit specific antibodies was due to expansion and activation of regulatory CD4(+) Foxp3(+) T cells (Tregs) that suppressed B cells to secrete antibodies through cell-cell interactions. Treg depletion allowed the DR0401.EA(0) mice to elicit specific antibodies and self-cure the infection. Our results demonstrated a differential role of MHC (HLA) class II molecules in supporting antibody responses to Py17XNL malaria and revealed a new mechanism by which malaria parasites stimulate B cell-suppressogenic Tregs that prevent clearance of infection.

HLA class II molecules influence susceptibility versus protection in inflammatory diseases by determining the cytokine profile.

The MHC in humans encodes the most polymorphic genes, the HLA genes, which are critical for the immune system to clear infection. This can be attributed to strong selection pressure as populations moved to different parts of the world and encountered new kinds of infections, leading to new HLA class II alleles. HLA genes also have the highest relative risk for autoimmune diseases. Three haplotypes, that is, HLA-DR2DQ6, DR4DQ8, and DR3DQ2, account for HLA association with most autoimmune diseases. We hypothesize that these haplotypes, along with their multiple subtypes, have survived bottlenecks of infectious episodes in human history because of their ability to present pathogenic peptides to activate T cells that secrete cytokines to clear infections. Unfortunately, they also present self-peptides/mimics to activate autoreactive T cells secreting proinflammatory cytokines that cause autoimmune diseases.

Two discreet subsets of CD8 T cells modulate PLP(91-110) induced experimental autoimmune encephalomyelitis in HLA-DR3 transgenic mice.

Previously we showed that transgenic mice expressing human HLA-DR3 gene are susceptible to PLP(91-110) induced experimental autoimmune encephalomyelitis (EAE) and can serve as an animal model of multiple sclerosis (MS). HLA-DR3 mice with EAE showed increased number of CD8 T cells indicating their important role in disease pathogenesis. The role of CD8 T cells in MS, an inflammatory demyelinating disease of CNS, has been enigmatic as it has been assigned both regulatory and pathogenic roles. Therefore, to evaluate the role of CD8 T cells, we generated CD8 deficient HLA-DR3 transgenic mice (DR3.CD8(-/-)). Immunization with PLP(91-110) led to more severe EAE in DR3.CD8(-/-) mice compared to HLA-DR3 mice indicating a regulatory role for CD8 T cells. Interestingly, DR3.CD8(-/-) mice with EAE showed decreased CNS pathology compared to DR3 mice thus suggesting a pathogenic role for CD8 T cells. We show that these two subsets of CD8 T cells can be differentiated based on the surface expression of CD122 (IL-2 Rß chain). CD8 T cells expressing CD122 (CD8+CD122+) play a regulatory role while CD8+CD122- T cells act as a pathogenic subset. CD122 expressing CD8 T cells are the regulatory subset of CD8 T cells and regulate the encephalitogenic CD4 T cells through direct modulation of antigen presenting cells and/or through the release of immunoregulatory cytokines such as IL-10, IFNγ and TGFß. We also showed that adoptive transfer of CD8CD122- T cells caused increased spinal cord demyelination indicating that these are pathogenic subset of CD8 T cells. Our study suggests that CD8+ T cells play both regulatory as well as pathogenic role in disease pathogenesis of EAE. A better understanding of these subsets could aid in designing novel therapy for MS patients.

Nonequivalence of classical MHC class I loci in ability to direct effective antiviral immunity.

Structural diversity in the peptide binding sites of the redundant classical MHC antigen presenting molecules is strongly selected in humans and mice. Although the encoded antigen presenting molecules overlap in antigen presenting function, differences in polymorphism at the MHC I A, B and C loci in humans and higher primates indicate these loci are not functionally equivalent. The structural basis of these differences is not known. We hypothesize that classical class I loci differ in their ability to direct effective immunity against intracellular pathogens. Using a picornavirus infection model and chimeric H-2 transgenes, we examined locus specific functional determinants distinguishing the ability of class I sister genes to direct effective anti viral immunity. Whereas, parental FVB and transgenic FVB mice expressing the H-2K(b) gene are highly susceptible to persisting Theiler's virus infection within the CNS and subsequent demyelination, mice expressing the D(b) transgene clear the virus and are protected from demyelination. Remarkably, animals expressing a chimeric transgene, comprised primarily of K(b) but encoding the peptide binding domain of D(b), develop a robust anti viral CTL response yet fail to clear virus and develop significant demyelination. Differences in expression of the chimeric K(b)α1α2D(b) gene (low) and D(b) (high) in the CNS of infected mice mirror expression levels of their endogenous H-2(q) counterparts in FVB mice. These findings demonstrate that locus specific elements other than those specifying peptide binding and T cell receptor interaction can determine ability to clear virus infection. This finding provides a basis for understanding locus-specific differences in MHC polymorphism, characterized best in human populations.

DQB1*0602 rather than DRB1*1501 confers susceptibility to multiple sclerosis-like disease induced by proteolipid protein (PLP).

BACKGROUND: Multiple sclerosis (MS) is associated with pathogenic autoimmunity primarily focused on major CNS-myelin target antigens including myelin basic protein (MBP), proteolipidprotein (PLP), myelin oligodendrocyte protein (MOG). MS is a complex trait whereby the HLA genes, particularly class-II genes of HLA-DR15 haplotype, dominate the genetic contribution to disease-risk. Due to strong linkage disequilibrium in HLA-II region, it has been hard to establish precisely whether the functionally relevant effect derives from the DRB1*1501, DQA1*0102-DQB1*0602, or DRB5*0101 loci of HLA-DR15 haplotype, their combinations, or their epistatic interactions. Nevertheless, most genetic studies have indicated DRB1*1501 as a primary risk factor in MS. Here, we used 'HLA-humanized' mice to discern the potential relative contribution of DRB1*1501 and DQB1*0602 alleles to susceptibility to "humanized" MS-like disease induced by PLP, one of the most prominent and encephalitogenic target-antigens implicated in human MS. METHODS: The HLA-DRB1*1501- and HLA-DQB1*0602-Tg mice (MHC-II(-/-)), and control non-HLA-DR15-relevant-Tg mice were immunized with a set of overlapping PLP peptides or with recombinant soluble PLP for induction of "humanized" MS-like disease, as well as for ex-vivo analysis of immunogenic/immunodominant HLA-restricted T-cell epitopes and associated cytokine secretion profile. RESULTS: PLP autoimmunity in both HLA-DR15-Tg mice was focused on 139-151 and 175-194 epitopes. Strikingly, however, the HLA-DRB1*1501-transgenics were refractory to disease induction by any of the overlapping PLP peptides, while HLA-DQB1*0602 transgenics were susceptible to disease induction by PLP139-151 and PLP175-194 peptides. Although both transgenics responded to both peptides, the PLP139-151- and PLP175-194-reactive T-cells were directed to Th1/Th17 phenotype in DQB1*0602-Tg mice and towards Th2 in DRB1*1501-Tg mice. CONCLUSIONS: While genome studies map a strong MS susceptibility effect to the region of DRB1*1501, our findings offer a rationale for potential involvement of pathogenic DQ6-associated autoimmunity in MS. Moreover, that DQB1*0602, but not DRB1*1501, determines disease-susceptibility to PLP in HLA-transgenics, suggests a potential differential, functional role for DQB1*0602 as a predisposing allele in MS. This, together with previously demonstrated disease-susceptibility to MBP and MOG in DRB1*1501-transgenics, also suggests a differential role for DRB1*1501 and DQB1*0602 depending on target antigen and imply a potential complex 'genotype/target antigen/phenotype' relationship in MS heterogeneity.