Genetically modified human CD4+ T cells can be evaluated in vivo without lethal graft‐versus‐host disease

Adoptive cell immunotherapy for human diseases, including the use of T cells modified to express an anti‐tumour T‐cell receptor (TCR) or chimeric antigen receptor, is showing promise as an effective treatment modality. Further advances would be accelerated by the availability of a mouse model that would permit human T‐cell engineering protocols and proposed genetic modifications to be evaluated in vivo. NOD‐scid IL2rγ^null (NSG) mice accept the engraftment of mature human T cells; however, long‐term evaluation of transferred cells has been hampered by the xenogeneic graft‐versus‐host disease (GVHD) that occurs soon after cell transfer. We modified human primary CD4⁺ T cells by lentiviral transduction to express a human TCR that recognizes a pancreatic beta cell‐derived peptide in the context of HLA‐DR4. The TCR‐transduced cells were transferred to NSG mice engineered to express HLA‐DR4 and to be deficient for murine class II MHC molecules. CD4⁺ T‐cell‐depleted peripheral blood mononuclear cells were also transferred to facilitate engraftment. The transduced cells exhibited long‐term survival (up to 3 months post‐transfer) and lethal GVHD was not observed. This favourable outcome was dependent upon the pre‐transfer T‐cell transduction and culture conditions, which influenced both the kinetics of engraftment and the development of GVHD. This approach should now permit human T‐cell transduction protocols and genetic modifications to be evaluated in vivo, and it should also facilitate the development of human disease models that incorporate human T cells.     

Human immune system development and survival of non‐obese diabetic (NOD)‐scid IL2rγnull (NSG) mice engrafted with human thymus and autologous haematopoietic stem cells

Immunodeficient mice bearing targeted mutations in the IL2rg gene and engrafted with human immune systems are effective tools for the study of human haematopoiesis, immunity, infectious disease and transplantation biology. The most robust human immune model is generated by implantation of human fetal thymic and liver tissues in irradiated recipients followed by intravenous injection of autologous fetal liver haematopoietic stem cells [often referred to as the BLT (bone marrow, liver, thymus) model]. To evaluate the non‐obese diabetic (NOD)‐scid IL2rγ^null (NSG)–BLT model, we have assessed various engraftment parameters and how these parameters influence the longevity of NSG–BLT mice. We observed that irradiation and subrenal capsule implantation of thymus/liver fragments was optimal for generating human immune systems. However, after 4 months, a high number of NSG–BLT mice develop a fatal graft‐versus‐host disease (GVHD)‐like syndrome, which correlates with the activation of human T cells and increased levels of human immunoglobulin (Ig). Onset of GVHD was not delayed in NSG mice lacking murine major histocompatibility complex (MHC) classes I or II and was not associated with a loss of human regulatory T cells or absence of intrathymic cells of mouse origin (mouse CD45⁺). Our findings demonstrate that NSG–BLT mice develop robust human immune systems, but that the experimental window for these mice may be limited by the development of GVHD‐like pathological changes.     

Accelerated thymopoiesis and improved T‐cell responses in HLA‐A2/‐DR2 transgenic BRGS‐based human immune system mice

Human immune system (HIS) mouse models provide a robust in vivo platform to study human immunity. Nevertheless, the signals that guide human lymphocyte differentiation in HIS mice remain poorly understood. Here, we have developed a novel Balb/c Rag2^?/? Il2rg^?/? Sirpa^NOD (BRGS) HIS mouse model expressing human HLA‐A2 and ‐DR2 transgenes (BRGSA2DR2). When comparing BRGS and BRGSA2DR2 HIS mice engrafted with human CD34⁺ stem cells, a more rapid emergence of T cells in the circulation of hosts bearing human HLA was shown, which may reflect a more efficient human T‐cell development in the mouse thymus. Development of CD4⁺ and CD8⁺ T cells was accelerated in BRGSA2DR2 HIS mice and generated more balanced B and T‐cell compartments in peripheral lymphoid organs. Both B‐ and T‐cell function appeared enhanced in the presence of human HLA transgenes with higher levels of class switched Ig, increased percentages of polyfunctional T cells and clear evidence for antigen‐specific T‐cell responses following immunization. Taken together, the presence of human HLA class I and II molecules can improve multiple aspects of human B‐ and T‐cell homeostasis and function in the BRGS‐based HIS mouse model.     

Generation of β cell‐specific human cytotoxic T cells by lentiviral transduction and their survival in immunodeficient human leucocyte antigen‐transgenic mice

Several β cell antigens recognized by T cells in the non‐obese diabetic (NOD) mouse model of type 1 diabetes (T1D) are also T cell targets in the human disease. While numerous antigen‐specific therapies prevent diabetes in NOD mice, successful translation of rodent findings to patients has been difficult. A human leucocyte antigen (HLA)‐transgenic mouse model incorporating human β cell‐specific T cells might provide a better platform for evaluating antigen‐specific therapies. The ability to study such T cells is limited by their low frequency in peripheral blood and the difficulty in obtaining islet‐infiltrating T cells from patients. We have worked to overcome this limitation by using lentiviral transduction to ‘reprogram’ primary human CD8 T cells to express three T cell receptors (TCRs) specific for a peptide derived from the β cell antigen islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP_265–273) and recognized in the context of the human class I major histocompatibility complex (MHC) molecule HLA‐A2. The TCRs bound peptide/MHC multimers with a range of avidities, but all bound with at least 10‐fold lower avidity than the anti‐viral TCR used for comparison. One exhibited antigenic recognition promiscuity. The β cell‐specific human CD8 T cells generated by lentiviral transduction with one of the TCRs released interferon (IFN)‐γ in response to antigen and exhibited cytotoxic activity against peptide‐pulsed target cells. The cells engrafted in HLA‐A2‐transgenic NOD‐scid IL2rγ^null mice and could be detected in the blood, spleen and pancreas up to 5 weeks post‐transfer, suggesting the utility of this approach for the evaluation of T cell‐modulatory therapies for T1D and other T cell‐mediated autoimmune diseases.     

Functional engraftment of human peripheral T and B cells and sustained production of autoantibodies in NOD/LtSzscid/IL‐2Rγ−/− mice

NOD/LtSzscid/IL‐2Rγ^?/? (NSG) mice have advantages in establishing humanized mouse models. However, transferring human PBMCs into these mice often causes lethal GVH disease. In this study, we discovered an improved method for the engraftment of normal or pathological human PBMCs into NSG mice and examined the subsequent induction of specific immune responses. We sequentially transferred human CD4⁺ memory T (Tm) and B cells obtained from PBMCs of healthy adults or patients with autoimmune diseases into NSG mice. Removing naïve CD4⁺ T cells from the transferred PBMCs allowed successful engraftment without lethal GVH disease. The transferred Tm cells were found to reside mainly in the spleen and the lymphoid nodules, where they expressed MHC class II molecules and produced cytokines, including IL‐21. Surprisingly, the transferred B cells were also well maintained in the lymphoid organs, underwent de novo class‐switch recombination, and secreted all isotypes of human Igs at significant levels. Moreover, transferring patient‐derived Tm and B cells resulted in sustained production of IgM‐rheumatoid factor and antiaminoacyl transfer RNA synthetase Abs in these mice. These results suggest that transfer of Tm and B cells derived from human PBMCs into NSG mice could be a useful method for the study of human autoimmune mechanisms.     

Type 1 diabetes associated HLA‐DQ2 and DQ8 molecules are relatively resistant to HLA‐DM mediated release of invariant chain‐derived CLIP peptides

HLA‐DM is essential for editing peptides bound to MHC class II, thus influencing the repertoire of peptides mediating selection and activation of CD4⁺ T cells. Individuals expressing HLA‐DQ2 or DQ8, and DQ2/8 trans‐dimers, have elevated risk for type 1 diabetes (T1D). Cells coexpressing DM with these DQ molecules were observed to express elevated levels of CLIP (Class II associated invariant chain peptide). Relative resistance to DM‐mediated editing of CLIP was further confirmed by HPLC‐MS/MS analysis of eluted peptides, which also demonstrated peptides from known T1D‐associated autoantigens, including a shared epitope from ZnT8 that is presented by all four major T1D‐susceptible DQ molecules. Assays with purified recombinant soluble proteins confirmed that DQ2‐CLIP complexes are highly resistant to DM editing, whereas DQ8‐CLIP is partially sensitive to DM, but with an apparent reduction in catalytic potency. DM sensitivity was enhanced in mutant DQ8 molecules with disruption of hydrogen bonds that stabilize DQ8 near the DM‐binding region. Our findings show that T1D‐susceptible DQ2 and DQ8 share significant resistance to DM editing, compared with control DQ molecules. The relative resistance of the T1D‐susceptible DQ molecules to DM editing and preferential presentation of T1D‐associated autoantigenic peptides may contribute to the pathogenesis of T1D.     

Co‐expression of HLA‐B7 and HLA‐B27 alleles is associated with B7‐restricted immunodominant responses following influenza infection

It is recognized that host response following viral infection is characterized by immunodominance, but deciphering the different factors contributing to immunodominance has proved a challenge due to concurrent expression of multiple MHC class I alleles. To address this, we generated H2‐K^?/?/D^?/? double‐knockout transgenic mice expressing either one or two human MHC‐I alleles. We hypothesized that co‐expression of different allele combinations figures critically in immunodominance and examined this in influenza‐infected, double Tg MHC‐I mice. In A2/B7 or A2/B27 mice, using ELISpot assays with the A2‐restricted matrix I.58–66, the B7‐restricted NP418–426 or the B27‐restricted NP383–391 influenza A (flu) epitopes, we observed the expected recognition of both peptides for both alleles. In contrast, in flu‐infected B7/B27 mice, a significantly reduced level of B27/NP383‐restricted CTL response was detected while there was no change in the B7/NP418‐restricted CTL response. Flu‐specific tetramer studies revealed a partial deletion of Vβ8.1⁺ NP383/B27‐restricted CD8⁺ T cells, and a diminished Vβ12⁺ CD8⁺ T‐cell expansion in B7/B27 Tg mice. Using HLA Tg chimeric mice, we confirmed these findings. These findings shed light on the immune consequences of co‐dominant expression of MHC‐I alleles for host immune response to pathogens.     

Mouse pancreatic beta cells express MHC class II and stimulate CD4+ T cells to proliferate

Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. Both CD4⁺ and CD8⁺ T cells have been shown to mediate beta‐cell killing. While CD8⁺ T cells can directly recognize MHC class I on beta cells, the interaction between CD4⁺ T cells and beta cells remains unclear. Genetic association studies have strongly implicated HLA‐DQ alleles in human type 1 diabetes. Here we studied MHC class II expression on beta cells in nonobese diabetic mice that were induced to develop diabetes by diabetogenic CD4⁺ T cells with T‐cell receptors that recognize beta‐cell antigens. Acute infiltration of CD4⁺ T cells in islets occurred with rapid onset of diabetes. Beta cells from islets with immune infiltration expressed MHC class II mRNA and protein. Exposure of beta cells to IFN‐γ increased MHC class II gene expression, and blocking IFN‐γ signaling in beta cells inhibited MHC class II upregulation. IFN‐γ also increased HLA‐DR expression in human islets. MHC class II⁺ beta cells stimulated the proliferation of beta‐cell‐specific CD4⁺ T cells. Our study indicates that MHC class II molecules may play an important role in beta‐cell interaction with CD4⁺ T cells in the development of type 1 diabetes.     

Reversal of type 1 diabetes by a new MHC II‐peptide chimera: “Single‐epitope‐mediated suppression” to stabilize a polyclonal autoimmune T‐cell process

Polyclonality of self‐reactive CD4⁺ T cells is the hallmark of several autoimmune diseases like type 1 diabetes. We have previously reported that a soluble dimeric MHC II‐peptide chimera prevents and reverses type 1 diabetes induced by a monoclonal diabetogenic T‐cell population in double Tg mice [Casares, S. et al., Nat. Immunol. 2002. 3 : 383–391]. Since most of the glutamic acid decarboxylase 65 (GAD65)‐specific CD4⁺ T cells in the NOD mouse are tolerogenic but unable to function in an autoimmune environment, we have activated a silent, monoclonal T‐regulatory cell population (GAD65_217–230‐specific CD4⁺ T cells) using a soluble I‐A/GAD65_217–230/Fcγ2a dimer, and measured the effect on the ongoing polyclonal diabetogenic T‐cell process. Activated GAD65_217–230‐specific T cells and a fraction of the diabetogenic (B_9–23‐specific) T cells were polarized toward the IL‐10‐secreting T‐regulatory type 1‐like function in the pancreas of diabetic NOD mice. More importantly, this led to the reversal of hyperglycemia for more than 2 months post‐therapy in 80% of mice in the context of stabilization of pancreatic insulitis and improved insulin secretion by the β cells. These findings argue for the stabilization of a polyclonal self‐reactive T‐cell process by a single epitope‐mediated bystander suppression. Dimeric MHC class II‐peptide chimeras‐like approach may provide rational grounds for the development of more efficient antigen‐specific therapies in type 1 diabetes.     

The induction of autoimmune hepatitis in the human leucocyte antigen‐DR4 non‐obese diabetic mice autoimmune hepatitis mouse model

Autoimmune hepatitis (AIH) is a chronic liver disease characterized by progressive inflammation, female preponderance and seropositivity for autoantibodies such as anti‐smooth muscle actin and/or anti‐nuclear, anti‐liver kidney microsomal type 1 (anti‐LKM1) and anti‐liver cytosol type 1 (anti‐LC1) in more than 80% of cases. AIH is linked strongly to several major histocompatibility complex (MHC) alleles, including human leucocyte antigen (HLA)‐DR3, ‐DR7 and ‐DR13. HLA‐DR4 has the second strongest association with adult AIH, after HLA‐DR3. We investigated the role of HLA‐DR4 in the development of AIH by immunization of HLA‐DR4 (DR4) transgenic non‐obese diabetic (NOD) mice with DNA coding for human CYP2D6/FTCD fusion autoantigen. Immunization of DR4 mice leads to sustained mild liver injury, as assessed biochemically by elevated alanine aminotransferase, histologically by interface hepatitis, plasma cell infiltration and mild fibrosis and immunologically by the development of anti‐LKM1/anti‐LC1 antibodies. In addition, livers from DR4 mice had fewer regulatory T cells (T_regs), which had decreased programmed death (PD)‐1 expression. Splenic T_regs from these mice also showed impaired inhibitory capacity. Furthermore, DR4 expression enhanced the activation status of CD8⁺ T cells, macrophages and dendritic cells in naive DR4 mice compared to naive wild‐type (WT) NOD mice. Our results demonstrate that HLA‐DR4 is a susceptibility factor for the development of AIH. Impaired suppressive function of T_regs and reduced PD‐1 expression may result in spontaneous activation of key immune cell subsets, such as antigen‐presenting cells and CD8⁺ T effectors, facilitating the induction of AIH and persistent liver damage.     

Human peripheral blood CD4 T cell-engrafted non-obese diabetic-scid IL2rγ(null) H2-Ab1 (tm1Gru) Tg (human leucocyte antigen D-related 4) mice: a mouse model of human allogeneic graft-versus-host disease

Graft-versus-host disease (GVHD) is a life-threatening complication of human allogeneic haematopoietic stem cell transplantation. Non-obese diabetic (NOD)-scid IL2rγ(null) (NSG) mice injected with human peripheral blood mononuclear cells (PBMC) engraft at high levels and develop a robust xenogeneic (xeno)-GVHD, which reproduces many aspects of the clinical disease. Here we show that enriched and purified human CD4 T cells engraft readily in NSG mice and mediate xeno-GVHD, although with slower kinetics compared to injection of whole PBMC. Moreover, purified human CD4 T cells engraft but do not induce a GVHD in NSG mice that lack murine MHC class II (NSG-H2-Ab1(tm1Gru), NSG-Ab°), demonstrating the importance of murine major histocompatibility complex (MHC) class II in the CD4-mediated xeno-response. Injection of purified human CD4 T cells from a DR4-negative donor into a newly developed NSG mouse strain that expresses human leucocyte antigen D-related 4 (HLA-DR4) but not murine class II (NSG-Ab° DR4) induces an allogeneic GVHD characterized by weight loss, fur loss, infiltration of human cells in skin, lung and liver and a high level of mortality. The ability of human CD4 T cells to mediate an allo-GVHD in NSG-Ab° DR4 mice suggests that this model will be useful to investigate acute allo-GVHD pathogenesis and to evaluate human specific therapies.     

Role of HLA class II genes in susceptibility/resistance to inflammatory arthritis: studies with humanized mice

Summary: Predisposition to develop rheumatoid arthritis (RA) has been associated with certain human leukocyte antigen (HLA) class II molecules, although the mechanism is still unknown. Various experimental animal models of inflammatory arthritis have been studied to address the role of major histocompatibility complex (MHC) genes in pathogenesis. We have generated transgenic mice expressing HLA class II molecules (DR and DQ) lacking complete endogenous class II molecules to study the interactions involved between class II molecules (DQ and DR) and to define the immunologic mechanisms in inflammatory arthritis. The HLA transgene can positively select CD4⁺ T cells expressing various Vβ T-cell receptors, and a peripheral tolerance is maintained to transgenic HLA molecules. The expression of HLA molecules on various cells in these mice is similar to that known in humans. In this review, we describe collagen-induced arthritis as a model for human inflammatory arthritis using these transgenic mice. The transgenic mice carrying RA-susceptible haplotype develop gender-biased inflammatory arthritis with clinical and histopathological similarities to RA. Our studies show that polymorphism of HLA class II genes determine the predisposition to rheumatoid/inflammatory arthritis and the epistatic interactions between HLA-DQ and HLA-DR molecules dictate the severity, progression, and modulation of the disease.     

Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex

Immunodeficient non-obese diabetic (NOD)-severe combined immune-deficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2rγ^null) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2rγ^null mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 × 10⁶ PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-α signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly.     

Composition of the HLA‐DR‐associated human thymus peptidome

Major histocompatibility complex class II (MHC‐II) molecules bind to and display antigenic peptides on the surface of antigen‐presenting cells (APCs). In the absence of infection, MHC‐II molecules on APCs present self‐peptides and interact with CD4⁺ T cells to maintain tolerance and homeostasis. In the thymus, self‐peptides bind to MHC‐II molecules expressed by defined populations of APCs specialised for the different steps of T‐cell selection. Cortical epithelial cells present peptides for positive selection, whereas medullary epithelial cells and dendritic cells are responsible for peptide presentation for negative selection. However, few data are available on the peptides presented by MHC molecules in the thymus. Here, we apply mass spectrometry to analyse and identify MHC‐II‐associated peptides from five fresh human thymus samples. The data show a diverse self‐peptide repertoire, mostly consisting of predicted MHC‐II high binders. Despite technical limitations preventing single cell population analyses of peptides, these data constitute the first direct assessment of the HLA‐II‐bound peptidome and provide insight into how this peptidome is generated and how it drives T‐cell repertoire formation.     

Peptidomic analysis of type 1 diabetes associated HLA‐DQ molecules and the impact of HLA‐DM on peptide repertoire editing

HLA‐DM and class II associated invariant chain (Ii) are key cofactors in the MHC class II (MHCII) antigen processing pathway. We used tandem mass spectrometry sequencing to directly interrogate the global impact of DM and Ii on the repertoire of MHCII‐bound peptides in human embryonic kidney 293T cells expressing HLA‐DQ molecules in the absence or presence of these cofactors. We found that Ii and DM have a major impact on the repertoire of peptides presented by DQ1 and DQ6, with the caveat that this technology is not quantitative. The peptide repertoires of type 1 diabetes (T1D) associated DQ8, DQ2, and DQ8/2 are altered to a lesser degree by DM expression, and these molecules share overlapping features in their peptide binding motifs that are distinct from control DQ1 and DQ6 molecules. Peptides were categorized into DM‐resistant, DM‐dependent, or DM‐sensitive groups based on the mass spectrometry data, and representative peptides were tested in competitive binding assays and peptide dissociation rate experiments with soluble DQ6. Our data support the conclusion that high intrinsic stability of DQ‐peptide complexes is necessary but not sufficient to confer resistance to DM editing, and provide candidate parameters that may be useful in predicting the sensitivity of T‐cell epitopes to DM editing.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

Loss of central and peripheral CD8+ T-cell tolerance to HFE in mouse models of human familial hemochromatosis

HFE, an MHC class Ib molecule that controls iron metabolism, can be directly targeted by cytotoxic TCR αβ T lymphocytes. Transgenic DBA/2 mice expressing, in a Rag 2 KO context, an αβ TCR that directly recognizes mouse HFE (mHFE) were created to further explore the interface of HFE with the immune system. TCR‐transgenic mHfe WT mice deleted mHFE‐reactive T cells in the thymus, but a fraction of reprogrammed cells were able to escape deletion. In contrast, TCR‐transgenic mice deprived of mHFE molecules (mHfe KO mice) or expressing a C282→Y mutated mHFE molecule – the most frequent mutation associated with human hereditary hemochromatosis – positively selected mHFE‐reactive CD8⁺ T lymphocytes and were not tolerant toward mHFE. By engrafting these mice with DBA/2 WT (mHFE⁺) skin, it was established, as suspected on the basis of similar engraftments performed on DBA/2 mHfe KO mice, that mHFE behaves as an autonomous skin‐associated histocompatibility antigen, even for mHFE‐C282→Y mutated mice. By contrast, infusion of DBA/2 mHFE⁺ mice with naïve mHFE‐reactive transgenic CD8⁺ T lymphocytes did not induce GVHD. Thus, tolerance toward HFE in mHfe WT mice can be acquired at either thymic or peripheral levels but is disrupted in mice reproducing human familial hemochromatosis.     

Microenvironmental stresses induce HLA‐E/Qa‐1 surface expression and thereby reduce CD8+ T‐cell recognition of stressed cells

Hypoxia and glucose deprivation are often observed in the microenvironment surrounding solid tumors in vivo. However, how they interfere with MHC class I antigen processing and CD8⁺ T‐cell responses remains unclear. In this study, we analyzed the production of antigenic peptides presented by classical MHC class I in mice, and showed that it is quantitatively decreased in the cells exposed to either hypoxia or glucose deprivation. In addition, we unexpectedly found increased surface expression of HLA‐E in human and Qa‐1 in mouse tumor cells exposed to combined oxygen and glucose deprivation. The induced Qa‐1 on the stressed tumor model interacted with an inhibitory NKG2/CD94 receptor on activated CD8⁺ T cells and attenuated their specific response to the antigen. Our results thus suggest that microenvironmental stresses modulate not only classical but also nonclassical MHC class I presentation, and confer the stressed cells the capability to escape from the CD8⁺ T‐cell recognition.     

Improved methods for predicting peptide binding affinity to MHC class II molecules

Major histocompatibility complex class II (MHC‐II) molecules are expressed on the surface of professional antigen‐presenting cells where they display peptides to T helper cells, which orchestrate the onset and outcome of many host immune responses. Understanding which peptides will be presented by the MHC‐II molecule is therefore important for understanding the activation of T helper cells and can be used to identify T‐cell epitopes. We here present updated versions of two MHC–II–peptide binding affinity prediction methods, NetMHCII and NetMHCIIpan. These were constructed using an extended data set of quantitative MHC–peptide binding affinity data obtained from the Immune Epitope Database covering HLA‐DR, HLA‐DQ, HLA‐DP and H‐2 mouse molecules. We show that training with this extended data set improved the performance for peptide binding predictions for both methods. Both methods are publicly available at www.cbs.dtu.dk/services/NetMHCII-2.3 and www.cbs.dtu.dk/services/NetMHCIIpan-3.2 .     

Binding of conserved islet peptides by human and murine MHC class II molecules associated with susceptibility to type I diabetes

The major histocompatibility complex (MHC) is the most important susceptibility locus for type I diabetes in humans and NOD mice. NOD mice express a single MHC class II molecule (I-Ag7) which carries a unique beta chain sequence. In humans, DQ alleles that encode DQ8 and DQ2 confer the highest risk for the disease. Soluble DQ8 and I-Ag7 were used to directly compare the binding specificity of these MHC molecules. Peptides from three islet antigens--insulin, GAD 65 and HSP 60--bound to both CQ8 and I-Ag7. These peptides included epitopes that are immunodominant in NOD mice, namely insulin (9-23), GAD (206-220) and HSP 60 (441-460). All of these peptide sequences are highly conserved between the human and murine antigens. The binding specificity of DQ8 and I-Ag7 was similar, but not identical, since two peptides eluted from splenocytes of NOD mice did not bind to DQ8. DQ8 formed long-lived complexes with the majority of these peptides, indicating that DQ8 is not a poor peptide binder. These results demonstrate functional similarities between human and murine MHC class II molecules that confer susceptibility to type I diabetes.