Mouse Major Histocompatibility Complex Class Research Articles

Therapy targeting antigen-specific T cells by a peptide-based tolerizing vaccine against autoimmune arthritis

A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exogenous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (Aq-galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dominant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.

An Immunoinformatics Approach to Design a Potent Multi-Epitope Vaccine against Asia-1 Genotype of Crimean-Congo Haemorrhagic Fever Virus Using the Structural Glycoproteins as a Target.

Crimean-Congo haemorrhagic fever (CCHF), caused by Crimean-Congo haemorrhagic fever virus (CCHFV), is a disease of worldwide importance (endemic yet not limited to Asia, Middle East, and Africa) and has triggered several outbreaks amounting to a case fatality rate of 10-40% as per the World Health Organization. Genetic diversity and phylogenetic data revealed that the Asia-1 genotype of CCHFV remained dominant in Pakistan, where 688 confirmed cases were reported between the 2012-2022 period. Currently, no approved vaccine is available to tackle the viral infection. Epitope-based vaccine design has gained significant attention in recent years due to its safety, timeliness, and cost efficiency compared to conventional vaccines. In the present study, we employed a robust immunoinformatics-based approach targeting the structural glycoproteins G1 and G2 of CCHFV (Asia-1 genotype) to design a multi-epitope vaccine construct. Five B-cells and six cytotoxic T-lymphocytes (CTL) epitopes were mapped and finalized from G1 and G2 and were fused with suitable linkers (EAAAK, GGGS, AAY, and GPGPG), a PADRE sequence (13 aa), and an adjuvant (50S ribosomal protein L7/L12) to formulate a chimeric vaccine construct. The selected CTL epitopes showed high affinity and stable binding with the binding groove of common human HLA class I molecules (HLA-A*02:01 and HLA-B*44:02) and mouse major histocompatibility complex class I molecules. The chimeric vaccine was predicted to be an antigenic, non-allergenic, and soluble molecule with a suitable physicochemical profile. Molecular docking and molecular dynamics simulation indicated a stable and energetically favourable interaction between the constructed antigen and Toll-like receptors (TLR2, TLR3, and TLR4). Our results demonstrated that innate, adaptive, and humoral immune responses could be elicited upon administration of such a potent muti-epitope vaccine construct. These results could be helpful for an experimental vaccinologist to develop an effective vaccine against the Asia-1 genotype of CCHFV.

Angiogenesis in patient-derived xenografts of odontogenic myxoma.

Previously, by employing 3D organotypic tissue culture and patient-derived xenograft (PDX) model, oral myxoma response to a MAPK/MEK inhibitor was observed. Gross examination of the tumour fragments obtained after 55days of PDX grafting revealed increased capsule vascularization. Microscopic analyses showed blood capillaries intermixed with myxoma cells, but the origin of these capillaries, from mice or humans, was not established. This study aimed to investigate whether the endothelial cells observed in the myxoma PDX model are derived from the mouse or from the primary human tumour. Immunohistochemistry was performed on five tumour fragments from the PDX of myxoma after 55days of implantation in mice. Immunopositivity for antibodies against human (HLA-ABC) and mouse (H2 Db/H2-D1) major histocompatibility complex class I (MHCI) was assessed in the endothelial cells. The endothelial cells in the PDX fragments revealed a membrane staining for the human MHCI protein in the PDX tumour and adjacent connective tissue capsule, indicating that capillaries were derived from the human tumour fragment. Considering the probable human origin of the endothelial cells from capillary blood vessels in the myxoma PDX, we conclude that this PDX model is an interesting model to study myxoma angiogenesis.

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis.

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Here, step-by-step instructions are described to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control samples, from mouse (EL4 and A20), and human (JY) cell lines more specifically. The various reagents and specific antibodies are thoroughly described to isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. The protocol can be used to establish and standardize an immunopeptidomics workflow, as well as to benchmark new protocols. Moreover, the protocol represents a great starting point for any non-experts in addition to foster the intra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

Peptide vaccination directed against IDO1-expressing immune cells elicits CD8+ and CD4+ T-cell-mediated antitumor immunity and enhanced anti-PD1 responses

BackgroundThe tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which subverts T-cell immunity at multiple levels, is itself subject to inherent T-cell reactivity. This intriguing deviation from central tolerance has been interpreted...

Regulatory CD4+ T Cells Recognize Major Histocompatibility Complex Class II Molecule-Restricted Peptide Epitopes of Apolipoprotein B.

CD4+ T cells play an important role in atherosclerosis, but their antigen specificity is poorly understood. Immunization with apolipoprotein B (ApoB, core protein of low density lipoprotein) is known to be atheroprotective in animal models. Here, we report on a human APOB peptide, p18, that is sequence-identical in mouse ApoB and binds to both mouse and human major histocompatibility complex class II molecules. We constructed p18 tetramers to detect human and mouse APOB-specific T cells and assayed their phenotype by flow cytometry including CD4 lineage transcription factors, intracellular cytokines, and T cell receptor activation. Apolipoprotein E-deficient ( Apoe-/-) mice were vaccinated with p18 peptide or adjuvants alone, and atherosclerotic burden in the aorta was determined. In human peripheral blood mononuclear cells from donors without cardiovascular disease, p18 specific CD4+ T cells detected by a new human leukocyte antigen-antigen D related-p18 tetramers were mostly Foxp3+ regulatory T cells (Tregs). Donors with subclinical cardiovascular disease as detected by carotid artery ultrasound had Tregs coexpressing retinoic acid-related orphan receptor gamma t or T-bet, which were both almost absent in donors without cardiovascular disease. In Apoe-/- mice, immunization with p18 induced Tregs and reduced atherosclerotic lesions. After peptide restimulation, responding CD4+ T cells identified by Nur77-GFP (green fluorescent protein) were highly enriched in Tregs. A new mouse I-Ab-p18 tetramer identified the expansion of p18-specific CD4+ T cells on vaccination, which were enriched for interleukin-10-producing Tregs. These findings show that APOB p18-specific CD4+ T cells are mainly Tregs in healthy donors, but coexpress other CD4 lineage transcription factors in donors with subclinical cardiovascular disease. This study identifies ApoB peptide 18 as the first Treg epitope in human and mouse atherosclerosis.

Protective Effects of Interferon-tau Against Lipopolysaccharide-Induced Embryo Implantation Failure in Pregnant Mice.

Interferon-tau (IFN-τ), a novel type I interferon, is produced by trophoblast cells in ruminants. Previous studies have confirmed that IFN-τ could induce immunological tolerance in humans and other species. However, there are few reports on whether IFN-τ has a protective effect on embryo implantation failure caused by excessive inflammation at the maternal-fetal interface. In our study, a mouse model of lipopolysaccharide (LPS)-induced implantation failure was successfully established, and we investigated the protective effects of IFN-τ. First, we showed that IFN-τ increased the number of implanted embryos in LPS-treated pregnant mice. Subsequently, quantitative real-time polymerase chain reaction (qPCR) and ELISA results showed that several inflammatory cytokines [IL-1β and tumor necrosis factor-alpha (TNF-α)] whose expression was upregulated by LPS were reversed by IFN-τ treatment. Furthermore, we performed Western blotting and found that IFN-τ restrained the LPS-induced phosphorylation of IκBα and NF-κB p65. Moreover, qPCR and immunohistochemistry analyses showed that IFN-τ decreased the LPS-induced expression of mouse major histocompatibility complex (MHC) class I genes (H-2K and H-2D) in LPS-treated pregnant mice. Taken together, these results suggest that IFN-τ has a protective effect in LPS-induced implantation failure by downregulating MHC class I genes expression and inhibiting the production of inflammatory cytokines.

Graft-infiltrating PD-L1hi cross-dressed dendritic cells regulate antidonor T cell responses in mouse liver transplant tolerance.

These findings suggest that graft-infiltrating PD-L1hi CD-DCs may play a key role in the regulation of alloimmunity and in the induction of liver transplant tolerance. (Hepatology 2018;67:1499-1515).

Human TCR-MHC coevolution after divergence from mice includes increased nontemplate-encoded CDR3 diversity.

For thymic selection and responses to pathogens, T cells interact through their αβ T cell receptor (TCR) with peptide-major histocompatibility complex (MHC) molecules on antigen-presenting cells. How the diverse TCRs interact with a multitude of MHC molecules is unresolved. It is also unclear how humans generate larger TCR repertoires than mice do. We compared the TCR repertoire of CD4 T cells selected from a single mouse or human MHC class II (MHC II) in mice containing the human TCR gene loci. Human MHC II yielded greater thymic output and a more diverse TCR repertoire. The complementarity determining region 3 (CDR3) length adjusted for different inherent V-segment affinities to MHC II. Humans evolved with greater nontemplate-encoded CDR3 diversity than did mice. Our data, which demonstrate human TCR-MHC coevolution after divergence from rodents, explain the greater T cell diversity in humans and suggest a mechanism for ensuring that any V-J gene combination can be selected by a single MHC II.

Engineering chimeric human and mouse major histocompatibility complex (MHC) class I tetramers for the production of T-cell receptor (TCR) mimic antibodies.

Therapeutic monoclonal antibodies targeting cell surface or secreted antigens are among the most effective classes of novel immunotherapies. However, the majority of human proteins and established cancer biomarkers are intracellular. Peptides derived from these intracellular proteins are presented on the cell surface by major histocompatibility complex class I (MHC-I) and can be targeted by a novel class of T-cell receptor mimic (TCRm) antibodies that recognise similar epitopes to T-cell receptors. Humoural immune responses to MHC-I tetramers rarely generate TCRm antibodies and many antibodies recognise the α3 domain of MHC-I and β2 microglobulin (β2m) that are not directly involved in presenting the target peptide. Here we describe the production of functional chimeric human-murine HLA-A2-H2Dd tetramers and modifications that increase their bacterial expression and refolding efficiency. These chimeric tetramers were successfully used to generate TCRm antibodies against two epitopes derived from wild type tumour suppressor p53 (RMPEAAPPV and GLAPPQHLIRV) that have been used in vaccination studies. Immunisation with chimeric tetramers yielded no antibodies recognising the human α3 domain and β2m and generated TCRm antibodies capable of specifically recognising the target peptide/MHC-I complex in fully human tetramers and on the cell surface of peptide pulsed T2 cells. Chimeric tetramers represent novel immunogens for TCRm antibody production and may also improve the yield of tetramers for groups using these reagents to monitor CD8 T-cell immune responses in HLA-A2 transgenic mouse models of immunotherapy.

Development of an Enhanced VETO cells for the generation of Alloantigen-specific Tolerance

Abstract Development of specific immunotherapies to prevent graft rejection and graft-versus-host disease offers the promise of selectively deleting only allospecific immune responses. Veto activity is the ability of a cell to specifically suppress/delete only T cells directed against antigens of the veto cells themselves, but not against third-party antigens. In the present study, we reasoned that providing TCR-like signaling to allogeneic major histocompatibility complex (MHC) molecules on the surface of CD8+CTLs might result in enhanced veto function. To generate the donor antigen specific veto cells, we have utilized the Chimeric Antigen Receptor (CAR) T cell approach, in which CD8+CTLs express an MHC alloantigen fused to signaling molecule apparatuses. We fused mouse MHC class I alloantigen (H-2Dd) to the hinge and trans-membrane domains of the mouse CD8 chain plus intracellular signaling sequences derived from mouse 4-1BB, and CD3-ζ chain creating various CAR constructs. These CARs were cloned into retroviral vectors. Ex-vivo transduced CD8+T cells (FVB-Dd) were adoptively transferred in the recipient parental mice strain (FVB-H-2q). 4–6 weeks post adoptive transfer, we detected low levels of Dd+ CD8+T cells in lymph node and spleen, ranging from 2–3% of total CD8+T cells. Dd expressing skin grafts from 3604 transgenic mice (H-2Dd transgenic mice on FVB-H-2q background) were transplanted on recipient mice and preliminary data suggests that skin graft survival was extended for 4 months (usual rejection in naïve mice is by 15–18 days).Veto activity of these Dd+CD8+T cells will be examined by in vitro Dd specific CTL assays, proliferation assays.

Induction of antigen specific transplantation tolerance using chimeric antigen receptor type T cells engineered to kill allospecific T cells by a gene therapy immunotherapeutic approach (TRAN2P.968)

Abstract Development of specific immunotherapies to prevent graft rejection and graft-versus-host disease offers the promise of selectively deleting only allospecific immune responses. Veto activity is the ability of a cell to specifically suppress/delete only T cells directed against antigens of the veto cells themselves, but not against third-party antigens. In the present study, we reasoned that providing TCR-like signaling to allogeneic major histocompatibility complex (MHC) molecules on the surface of CD8+CTLs might result in enhanced veto function. To generate the donor antigen specific veto cells, we have utilized the Chimeric Antigen Receptor (CAR) T cell approach, in which CD8+CTLs express an MHC alloantigen fused to signaling molecule apparatuses. We fused mouse MHC class I alloantigen (H-2Dd) to the hinge and trans-membrane domains of the mouse CD8 chain plus intracellular signaling sequences derived from mouse 4-1BB, and CD3-ζ chain creating various CAR constructs. These CARs were cloned into retroviral vectors. Ex-vivo transduced T cells (FVB-Dd) were adoptively transferred in the recipient parental mice strain (FVB-H-2q) which do not express Dd. Two weeks post-adoptive transfer, we detected low levels of Dd+CD8+ T cells in lymph node and spleen, ranging from 0.2-1.2% of total CD8+ T cells. Veto activity of these Dd+CD8+T cells will be examined by in vitro Dd specific CTL assays, proliferation assays, and rejection of Dd skin grafts in parental strain (FVB) mice.

Anti-metastatic effects of DNA vaccine encoding single-chain trimer composed of MHC I and vascular endothelial growth factor receptor 2 peptide.

Vascular endothelial growth factor receptor 2 (VEGFR2)-mediated signaling is the key rate-limiting step in angiogenesis. VEGFR2 serves as the most important target of anti-angiogenic therapy for cancers. Single-chain trimer (SCT) comprising antigen peptide, β2-microglobulin (β2m), and major histocompatibility complex (MHC) class I heavy chain was a particularly powerful strategy involved in the increase of the potency of DNA vaccine against tumors and infections. In the present study, we constructed an SCT-encoding VEGFR2 antigen peptide [aa400-408, also known as kinase insert domain-containing receptor (KDR2)], β2m, and mouse MHC class I heavy chain H-2Db [pcDNA3.1(+)-KDR2-β2m-H-2Db, or SCT-KDR2]. The constructed SCT-KDR2 DNA was efficiently expressed in the human A293 embryonic kidney cell line. Intradermal immunization of C57BL/6 mice with SCT-KDR2 DNA was able to successfully break self-immunological tolerance and induce robust cytotoxic T‑lymphocyte (CTL) response to VEGFR2, leading to marked suppression of tumor cell‑induced angiogenesis and metastasis in murine models of B16 melanoma and 3LL Lewis lung carcinoma. Taken together, the results showed that VEGFR2-targeted SCT vaccination is an effective modality that can be utilized in anti-angiogenic active immunotherapy for various types of cancer.

Targeted DNA vaccines eliciting crossreactive anti-idiotypic antibody responses against human B cell malignancies in mice.

BackgroundTherapeutic idiotypic (Id) vaccination is an experimental treatment for selected B cell malignancies. A broader use of Id-based vaccination, however, is hampered by the complexity and costs due to the individualized production of protein vaccines. These limitations may be overcome by targeted DNA vaccines encoding stereotyped immunoglobulin V regions of B cell malignancies. We have here investigated whether such vaccines might elicit cross-reactive immune responses thus offering the possibility to immunize subsets of patients with the same vaccine.MethodsFusion vaccines targeting patient Id to mouse Major Histocompatibility Complex (MHC) class II molecules (chimeric mouse/human) or chemokine receptors (fully human) on antigen-presenting cells (APC) were genetically constructed for two Chronic Lymphocytic Leukemia (CLL) patients and one prototypic stereotyped B-cell receptor (BCR) commonly expressed by Hepatitis C Virus (HCV)-associated Non Hodgkin Lymphoma (NHL). The A20 murine B lymphoma cells were engineered to express prototypic HCV-associated B cell lymphoma BCR. Anti-Id antibody responses were studied against stereotyped and non-stereotyped BCRs on CLL patients’ cells as well as transfected A20 cells.ResultsDNA vaccination of mice with Id vaccines that target APC elicited increased amounts of antibodies specific for the patient’s Id as compared with non targeted control vaccines. Anti–Id antibodies cross-reacted between CLL cells with closely related BCR. A20 cells engineered to express patients’ V regions were not tumorigenic in mice, preventing tumor challenge experiments.ConclusionsThese findings provide experimental support for use of APC-targeted fusion Id DNA vaccines for the treatment of B cell lymphoma and CLL that express stereotyped BCRs.

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.

Targeted idiotype‐fusion DNA vaccines for human multiple myeloma: preclinical testing

A homodimeric fusion DNA vaccine targeting idiotype (Id) to antigen-presenting cells (APC) induced robust tumor protection in a mouse model of multiple myeloma (MM). Similar Id vaccine molecules were generated for four patients with MM with three main objectives: (i) do the vaccine molecules induce bona fide anti-Id immune responses in mice? (ii) does targeting of the vaccine molecules to APC enhance immune responses? (iii) can anti-Id antibodies, generated as by-product in vaccinated mice, be used to establish sensitive assays for complete remission (CR) prior to patient vaccination? Chimeric vaccine molecules targeting patient Id to mouse major histocompatibility complex (MHC) class II molecules were genetically constructed for four patients with MM. DNA vaccination of mice with chimeric vaccines targeting patient Id to mouse MHC class II molecules elicited antibodies specific for the patient's myeloma protein. Targeting MHC class II greatly enhanced anti-Id responses. Mouse anti-Id antibodies were used to establish myeloma protein-specific enzyme-linked immunosorbent assays (ELISAs) that were between 75 and 1500 times more sensitive than conventional serum protein electrophoresis and immunofixation. These results pave the way for testing targeted DNA Id vaccines in patients in CR. Id- and patient-specific ELISA could be established affording evaluation of CR depth beyond current serological methods.

Chromogranin A is an autoantigen in type 1 diabetes

Autoreactive CD4(+) T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4(+) T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-A(g7) in an atypical manner, occupying only the carboxy-terminal half of the I-A(g7) peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.

Effect of a tapasin mutant on the assembly of the mouse MHC class I molecule H2‐Kd

Major histocompatibility complex (MHC) class I heavy chain/beta(2)m heterodimers assemble with antigenic peptides through interactions with peptide-loading complex proteins, including tapasin and ERp57. In human cells, a cysteine residue within tapasin (C95) has been shown to form a covalent bond with ERp57. In this study, we focused on the effect of this tapasin amino-acid residue in mouse cells expressing the MHC class I molecule H2-K(d). We showed that a large disulfide-bonded complex was present in the mouse cells that included ERp57, tapasin, and K(d). Furthermore, in mouse cells, unlike human cells, we found that tapasin mutated at C95 can participate in a non-covalent complex with ERp57. Comparison of our findings to earlier findings with a human molecule (HLA-B(*)4402) also revealed that a tapasin C95 mutation has a stronger effect on the maturation and stability of K(d) than HLA-B(*)4402. Overall, our results characterize the influence of this tapasin cysteine residue on the stable surface expression of a mouse MHC class I molecule and reveal differences in tapasin C95 interactions and effects between mouse and human systems.

Comparative analysis of the impact of a free cysteine in tapasin on the maturation and surface expression of murine MHC class I allotypes

Tapasin is a key molecule in the major histocompatibility complex (MHC) class I peptide-loading complex, interacting with several other proteins in the complex. An amino acid substitution at a free cysteine position in tapasin has been shown to disrupt the covalent association of tapasin with ERp57. In this study, we mutated the free cysteine in mouse tapasin, and analysed the effects on the cell surface expression of the mouse MHC class I molecules K(d) and K(b). The C95S substitution in mouse tapasin increased the proportion of open forms relative to folded forms for both types of MHC class I molecules at the cell surface. Furthermore, the C95S substitution resulted in increased association of tapasin with folded K(d). Overall, our studies with these mouse MHC class I allotypes have revealed that the free cysteine 95 in mouse tapasin influences stable expression at the plasma membrane for both MHC class I allotypes, and have shown that tapasin's interaction with folded K(d) is elevated by the C95S substitution in tapasin.