Mutant MHC Class Research Articles

Abstract A110: Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Abstract Mutations are regarded as ideal targets for cancer immunotherapy. As neoepitopes with strict lack of expression in any healthy tissue, they are expected to be safe and could bypass the central tolerance mechanisms. Recent advances in nucleic acid sequencing technologies have revolutionized the field of genomics, allowing the readily targeting of mutated neoantigens for personalized cancer vaccination. We demonstrated in three independent murine tumor models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that unexpectedly the immunogenic mutanome is pre-dominantly recognized by CD4+ T cells. RNA vaccination with such MHC class II restricted immunogenic mutations leads to infiltration of CD4+ and CD8+ T cells into the tumor, reduces intratumoral regulatory T cells and ultimately confers strong anti-tumor activity. Encouraged by these findings we set up a process comprising mutation detection by exome sequencing, selection of vaccine targets by solely bioinformatical prioritization of mutated epitopes predicted to be abundantly expressed and presented on MHC class II molecules. Synthetic mRNA vaccines encoding multiple of these prioritized mutated epitopes induce potent tumor control and complete rejection of established aggressively growing tumors in mice. Moreover, we demonstrate that CD4+ T cell neoepitope vaccination primes CTL responses against an independent immunodominant antigen in tumor bearing mice indicating orchestration of antigen spread. Our findings reveal that cancer mutation based MHC class II restricted epitopes are attractive vaccination targets and provide the preclinical proof of concept for an integrated process from tumor sample to a cancer vaccine customized to the unique repertoire of each patient`s tumor. Citation Format: Mathias Vormehr, Sebastian Kreiter, Niels van de Roemer, Mustafa Diken, Martin Löwer, Fulvia Vascotto, Jan Diekmann, Sebastian Boegel, Barbara Schroers, Arbel D. Tadmor, Özlem Türeci, Ugur Sahin. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A110.

Distinct immunological activities of four variants of one mutation-generated neoepitope of a mouse fibrosarcoma (TUM10P.1023)

Abstract Passenger mutations in cancer cells can give rise to mutant MHC Class I restricted epitopes, also referred to as neoepitopes. One such neoepitope, Tnpo3 (SYMLQALCI), has been described for the BALB/c Meth A fibrosarcoma, by our laboratory1. Immunization with a peptide corresponding to the Tnpo3 neoepitope elicits antigen specific CD8+ T cells, which are capable of mediating specific rejection of Meth A. Interrogating the site of mutation within the Tnpo3 neoepitope with NetMHC predicts three other variants of Tnpo3 having the same amino terminus but different carboxyl termini. Of these four Tnpo3 variants, three elicit functional CD8+ T cells upon immunization, and of these, only one elicits tumor rejection. Here, the structural and cellular immunological aspects of the differential activities of these four neoepitopes are investigated. 1Duan F, Duitama J, Al Seesi S, Ayres CM, Corcelli SA, Pawashe AP, Blanchard T, McMahon D, Sidney J, Sette A, Baker BM, Mandoiu II, Srivastava PK. Genomic and Bioinformatic Profiling of Mutational Neoepitopes Reveals New Rules to Predict Anticancer Immunogenicity." J Exp Med. 211 (2014): 2231-248. PMID: 25245761

Pig Artery Patch Transplantation (Tx) as a Surrogate For Heart Tx in Baboons.

Background: Pig heart Tx in baboons is time-consuming, expensive, and associated with complications. Graft failure can result from the development of (i) elicited anti-pig antibodies or (ii) thrombotic microangiopathy in the graft. We have developed a simple substitute model of pig artery patch Tx and have compared the results with those following heart Tx. Methods: Baboons received artery patch Tx into the wall of the abdominal aorta) (Gp 1; n=28) or heterotopic heart Tx (Gp 2; n=17) from GTKO pigs (+/- transgenes aimed at complement or coagulation regulation [n=22] +/- T cell regulation [a human mutant MHC class II transactivator, CIITA, n=9]). Nine recipients (patch 4, heart 5) received no immunosuppressive therapy (IS). The remainder (patch 24, heart 12) received IS based on blockade of either the CD40:CD154 pathway (anti-CD154 or anti-CD40 mAbs) or the CD28:B7 pathway (CTLA4-Ig - abatacept or belatacept) or of both pathways (anti-CD40mAb+belatacept). Monitoring was by MLR, anti-pig IgM and IgG, histopathology, and various coagulation (platelet count, fibrinogen, D-dimer) and inflammatory (C-reactive protein, CRP) parameters. Results: In both models, (i) with no IS, grafts induced a significant T cell response; (ii) anti-CD154mAb prevented a T cell response, whereas CTLA4-Ig did not; and (iii) anti-CD40mAb+belatacept prevented T cell proliferative and elicited antibody responses and cellular infiltration of the graft. Expression of CIITA in a patch graft reduced the elicited antibody response. Despite heparin therapy, thrombocytopenia, fall in fibrinogen, and increases in D-dimer and CRP were documented in baboons with heart grafts that did not express a human coagulation-regulatory transgene, but only increases in D-dimer and CRP were seen after patch Tx. Treatment with anti-CD40mAb+belatacept did not prevent the rise of D-dimer or CRP in either model. An IL-6R antagonist prevented a rise in CRP after patch Tx. Conclusions: Outcomes of patch and heart Tx are similar. Expression of CIITA reduces the elicited antibody response to a patch graft. To replace anti-CD154mAb, blockade of both the CD28 and CD154 pathways is essential. Patch Tx provides some (but not all) information on the coagulation and inflammatory responses to a heart graft. IL-6R blockade may reduce the inflammatory response. DISCLOSURE:Ayares, D.: Employee, Revivicor.

Increased numbers and suppressive activity of regulatory CD25+CD4+ T lymphocytes in the absence of CD4 engagement by MHC class II molecules

Mechanisms of central and peripheral tolerance prevent autoimmunity. Regulatory T cells inhibit the activation of potentially auto-reactive T cells in peripheral lymphoid organs. In transgenic mice in which all MHC class II molecules are incapable of binding to CD4, class II MHC-restricted T cells preferentially differentiated into immunosuppressive, regulatory T cells. In these mutant MHC class II transgenic mice, a subset of CD4+ T cells constitutively expressed moderately elevated levels of CD25 and potently inhibited interleukin-2 secretion by T cells from normal mice in a cell-to-cell, contact-dependent manner. Immunosuppressive activity depended on activation of the regulatory T cells. Thus, CD25+CD4+ T cells from mutant MHC class II transgenic mice resembled phenotypically and functionally a major subset of natural regulatory T cells in normal mice, but were two to three-times more abundant. These results further clarify the mechanisms that govern the differentiation and maintenance of CD25+CD4+ regulatory T cells, and present avenues for immunomodulation.

T cell receptor-independent CD4 signalling: CD4–MHC class II interactions regulate intracellular calcium and cyclic AMP

CD4 is a coreceptor on T helper (Th) cells that interacts with MHC class II molecules (MHCII). The mechanisms mediating the effects of CD4 on responses by T helper cells to stimulation of the antigen-specific T cell receptor (TCR) are still poorly understood. Here, we demonstrate T cell costimulation via CD4 signalling independent of T cell receptor-mediated signals. Incubation of T helper cells with peptide mimetics of the CD4-binding region on the MHC class II β2 domain caused intracellular calcium mobilization in the absence of antigen or other T cell receptor stimuli. Engagement of CD4 by peptide mimetics or wild-type MHC class II, but not by mutant MHC class II molecules incapable of engaging CD4, inhibited the T cell receptor-mediated increase in cyclic AMP (cAMP) concentrations in T helper cells. CD4-mediated signals activated cyclic AMP phosphodiesterases (PDEs) and inhibited adenylyl cyclase. Full activation and clonal expansion of antigen-stimulated T helper cells required the CD4-mediated regulation of cyclic AMP. Our results suggest a costimulatory mechanism of CD4 function that acts on the second messengers, calcium and cyclic AMP.

Mutant MHC class I molecules define interactions between components of the peptide-loading complex.

Class I histocompatibility molecules, consisting of a heavy chain, beta2-microglobulin and peptide, are assembled in the endoplasmic reticulum (ER) with the assistance of several molecular chaperones and accessory proteins. Peptide binding occurs when assembling class I molecules associate with a loading complex consisting of the transporter associated with antigen processing (TAP) peptide transporter, tapasin, ERp57 and calreticulin (CRT)/calnexin. To assess the physical organization of this complex, we generated a series of mutants in the murine H-2Dd heavy chain and assessed their association with components of the complex. Seven mutations, clustered between amino acids 122 and 136 in the heavy chain alpha2 domain plus one mutation at position 222 in the alpha3 domain, resulted in loss of interaction with tapasin. Association with TAP was always lost simultaneously, supporting the view that tapasin acts as an obligatory bridge between class I molecules and TAP. Compared with previous studies on the HLA-A2 molecule, some differences in points of tapasin interaction were observed. Failure of the H-2Dd mutants to bind tapasin resulted in low cell-surface expression and altered intracellular transport. Most mutants retained a substantial degree of peptide loading, consistent with the view that although tapasin may promote peptide binding to class I, it is not required. A surprising observation was that all mutants lacking tapasin interaction retained normal association with CRT. This contrasts with previous observations on other class I molecules and, combined with differences in tapasin interaction, suggests that the organization of the ER peptide-loading complex can vary depending on the specific class I molecule examined.

The Human CD8 Coreceptor Effects Cytotoxic T Cell Activation and Antigen Sensitivity Primarily by Mediating Complete Phosphorylation of the T Cell Receptor ζ Chain

Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes.

Selection and Function of CD4+ T Lymphocytes in Transgenic Mice Expressing Mutant MHC Class II Molecules Deficient in Their Interaction with CD4

Interactions of the T cell coreceptors, CD4 and CD8, with MHC molecules participate in regulating thymocyte development and T lymphocyte activation and differentiation to memory T cells. However, the exact roles of these interactions in normal T cell development and function remain unclear. CD4 interacts with class II MHC7 molecules via several noncontiguous regions in both the class II MHC alpha- and beta-chains. We have introduced a double mutation that disrupts interaction with CD4 into the I-A(beta)k gene and used this construct to generate transgenic mice expressing only mutant class II MHC. Although CD4+ thymocytes matured to the single-positive stage in these mice, their frequency was reduced by threefold compared with that of wild-type transgenics. Positive selection of CD4+ T cells in the mutant transgenic mice may have been mediated by TCRs with a higher than usual affinity for class II MHC/Ag complexes. In A(beta)k mutant transgenics, peripheral CD4+ lymphocytes promoted B cell differentiation to plasma cells. These CD4+ T cells also secreted IFN-gamma in response to various stimuli (e.g., protein Ag, bacterial superantigen, and alloantigen), but were deficient in IL-2 secretion. Interactions between CD4 and class II MHC molecules appeared to regulate lymphokine production, with a strong bias toward IFN-gamma and against IL-2 in the absence of these interactions. Our results have implications for the manipulation of T cell-dependent immune responses.

Impaired Th2 Subset Development in the Absence of CD4

Prior studies in CD4-deficient mice established the capacity of T helper (Th) lineage cells to mature into Th1 cells. Unexpectedly, challenge of these mice with Nippostrongylus brasiliensis, a Th2-inducing stimulus, failed to result in the development of Th2 cells. Additional studies were performed using CD4 + or CD4 −CD8 − (double-negative) T cell receptor (TCR) transgenic T cells reactive to LACK antigen of Leishmania major. Double-negative T cells were unable to develop into Th2 cells in vivo, and, unlike CD4 + T cells, could not be primed for interleukin-4 production in vitro. Similarly, CD4 + TCR transgenic T cells primed on antigen-presenting cells expressing mutant MHC class II molecules unable to bind CD4 did not differentiate into Th2 cells. These data suggest that interactions between the TCR, MHC II–peptide complex and CD4 may be involved in Th2 development.

Variable influence of MHC polymorphism on the recognition of bacterial superantigens by T cells.

Superantigen recognition by some T cells is strongly influenced by polymorphic residues of the MHC class II molecule, suggesting that the TCR contacts the class II molecule during superantigen engagement. However, the degree of MHC preference varies with V beta expression. For example, the recognition of staphylococcal enterotoxin B (SEB) by murine V beta 14+ T cell hybridomas is strongly influenced by MHC polymorphism, whereas the recognition of SEB by V beta 8.2+ hybridomas appears to be independent of MHC polymorphism. One possible explanation for this difference is that V beta 14+ TCR may have a lower avidity for SEB such that the stability of the TCR/SEB/MHC complex is more strongly influenced by potential TCR/MHC interactions. To investigate this possibility, we examined the MHC preference of SEB recognition by murine V beta 8.2+/CD4+ T cell hybridomas in which we altered the strength of SEB recognition. First, we compared the recognition of wild-type SEB and mutant SEB, which only weakly activates T cells. Second, we compared recognition of SEB presented by I-E and I-A molecules, which differ in their affinity for SEB. In both cases, the degree of MHC preference was significantly increased when the strength of SEB recognition was reduced. Furthermore, we used mutant MHC class II molecules to show that the degree of MHC preference was controlled by residues of the class II molecule predicted to interact with the TCR during SEB recognition. Taken together, these data support the idea that there is a TCR/MHC interaction during SEB recognition, and the influence of this interaction varies with the overall avidity of the TCR/SEB/MHC complex.

A conservative mutation in a class I MHC molecule outside the peptide binding groove stimulates responses to self peptides.

A transgenic mouse has been made that expresses a mutant MHC class I H-2Kb molecule with glutamic acid at position 65 (E65) in place of glutamine. The side chain at position 65, on the outward face of the alpha-helix of the alpha 1 domain of the class I molecule, interacts with the TCR, and not with the peptide binding groove. The transgenic mouse, on a DBA/2 background, mounts Kb,E65-restricted Ag-specific responses to conventional Kb-restricted Ag such as OVA and vesicular stomatitis virus, and shows strong alloreactivity to wild-type Kb. The transgenic mouse also mounts a primary in vitro alloreactive response directed to a mutant molecule with aspartic acid at position 65 (D65). This response is relatively weak, probably because of the structural similarities between aspartic and glutamic acid side chains; both have carboxylic termini, and the aspartic acid side chain is shorter by a single secondary carbon. The alloreactive CTL lines elicited by this conservative change are cross-reactive among several position-65 variants of H-2Kb. Individual CTL clones are specific for self peptides that can be extracted from cells expressing Kb,E65, and from purified wild-type Kb molecules, and that are recognized in the context of the D65 residue. Thus, the smallest variance from self in a class I molecule, even outside the peptide binding groove, can be antigenic.

Unique biochemical properties of a mutant MHC class I molecule, H-2K sml

This study describes serological and biochemical properties of a novel MHC class I molecule. The mutant H-2K sml molecule was discovered in a mouse because of loss of reactivity of its peripheral blood lymphocytes to monoclonal antibodies. This mutation in the H-2K s molecule is the first in vivo mutation described that has altered an amino acid residue (amino acid 107) distant from the regions generally considered to be peptide or TCR contacts. Cell surface expression of the mutant molecules remains high but the Arg107 to Trp substitution appears to alter the native protein conformation, markedly decreasing cell surface association with beta 2-microglobulin light chains and conferring a loss of recognition by K s specific antibodies.