Human MHC Class Research Articles

Activation induced cell death (AICD) of human melanoma antigen-specific TCR engineered CD8 T cells involves JNK, Bim and p53

ABSTRACTObjectives: Adoptive cancer immunotherapy (ACT) with transgenic T cell receptor (TCR) engineered (TCReng) anti-tumor T cells has produced encouraging results, however, efficacy of these approaches need improvement. Since premature activation induced cell death (AICD) of adoptively administered T cells could be a major impediment, we examined the mechanism(s) underlying AICD in TCReng CD8+ cytolytic T lymphocytes (CTL).Methods: AICD in human tumor antigen-specific MHC class I restricted TCR engineered CD8+ CTL was induced by exposing them to cognate peptide epitope.Results: We show that TCReng CD8+ human primary CTL undergo AICD even upon encountering their cognate peptide epitope for the very first time. AICD in TCReng CTL is a death-receptor-independent, JNK activation-driven intrinsic processes, in which p53-mediated mitochondria-centric, non-transcription-dependent pathway plays an essential role. Activated JNK modulates mitochondrial membrane integrity in CTL undergoing AICD by directly interacting with Bcl family protein, Bim, and the mitochondrial membrane pore complex, voltage dependent anion channel (VDAC), leading to the release of caspase-independent death executioner, apoptosis inducing factor (AIF), accumulation of single strand DNA breaks and eventually to cell death.Conclusions: Our findings offer opportunities to interfere with AICD in TCReng CD8+ anti-tumor CTL for sustaining them longer for producing better clinical outcomes.

Kinase Regulation of Human MHC Class I Molecule Expression on Cancer Cells.

The major histocompatibility complex I (MHC-1) presents antigenic peptides to tumor-specific CD8+ T cells. The regulation of MHC-I by kinases is largely unstudied, even though many patients with cancer are receiving therapeutic kinase inhibitors. Regulators of cell-surface HLA amounts were discovered using a pooled human kinome shRNA interference-based approach. Hits scoring highly were subsequently validated by additional RNAi and pharmacologic inhibitors. MAP2K1 (MEK), EGFR, and RET were validated as negative regulators of MHC-I expression and antigen presentation machinery in multiple cancer types, acting through an ERK output-dependent mechanism; the pathways responsible for increased MHC-I upon kinase inhibition were mapped. Activated MAPK signaling in mouse tumors in vivo suppressed components of MHC-I and the antigen presentation machinery. Pharmacologic inhibition of MAPK signaling also led to improved peptide/MHC target recognition and killing by T cells and TCR-mimic antibodies. Druggable kinases may thus serve as immediately applicable targets for modulating immunotherapy for many diseases. Cancer Immunol Res; 4(11); 936-47. ©2016 AACR.

Gene conversion of the major histocompatibility complex class I Caja-G in common marmosets (Callithrix jacchus).

SummaryCurrently, the amount of sequenced and classified MHC class I genes of the common marmoset is limited, in spite of the wide use of this species as an animal model for biomedical research. In this study, 480 clones of MHC class I G locus (Caja‐G) cDNA sequences were obtained from 21 common marmosets. Up to 10 different alleles were detected in each common marmoset, leading to the assumption that the Caja‐G loci duplicated in the marmoset genome. In the investigated population, four alleles occurred more often, giving evidence for higher immunological advantage of these alleles. In contrast to the human non‐classical MHC class I genes, Caja‐G shows high rates of polymorphism at the relevant peptide‐binding sites, despite its phylogenetic relationship to the non‐classical HLA‐G. Our results provide information for better understanding of the immunological properties of the common marmoset and confirm the theory of a gene conversion of the Caja‐G due to its detected plasticity and the absence of any known HLA‐A equivalent.

Comparative genomics of the human, macaque and mouse major histocompatibility complex.

The MHC is a highly polymorphic genomic region that encodes the transplantation and immune regulatory molecules. It receives special attention for genetic investigation because of its important role in the regulation of innate and adaptive immune responses and its strong association with numerous infectious and/or autoimmune diseases. The MHC locus was first discovered in the mouse and for the past 50 years it has been studied most intensively in both mice and humans. However, in recent years the macaque species have emerged as some of the more important and advanced experimental animal models for biomedical research into MHC with important human immunodeficiency virus/simian immunodeficiency virus and transplantation studies undertaken in association with precise MHC genotyping and haplotyping methods using Sanger sequencing and next-generation sequencing. Here, in this special issue on 'Macaque Immunology' we provide a short review of the genomic similarities and differences among the human, macaque and mouse MHC class I and class II regions, with an emphasis on the association of the macaque class I region with MHC polymorphism, haplotype structure and function.

Viral infection causes a shift in the self peptide repertoire presented by human MHC class I molecules.

MHC class I presentation of peptides allows T cells to survey the cytoplasmic protein milieu of host cells. During infection, presentation of self peptides is, in part, replaced by presentation of microbial peptides. However, little is known about the self peptides presented during infection, despite the fact that microbial infections alter host cell gene expression patterns and protein metabolism. The self peptide repertoire presented by HLA-A*01;01, HLA-A*02;01, HLA-B*07;02, HLA-B*35;01, and HLA-B*45;01 (where HLA is human leukocyte antigen) was determined by tandem MS before and after vaccinia virus infection. We observed a profound alteration in the self peptide repertoire with hundreds of self peptides uniquely presented after infection for which we have coined the term "self peptidome shift." The fraction of novel self peptides presented following infection varied for different HLA class I molecules. A large part (approximately 40%) of the self peptidome shift arose from peptides derived from type I interferon-inducible genes, consistent with cellular responses to viral infection. Interestingly, approximately 12% of self peptides presented after infection showed allelic variation when searched against approximately 300 human genomes. Self peptidome shift in a clinical transplant setting could result in alloreactivity by presenting new self peptides in the context of infection-induced inflammation.

A Distinctive Cytoplasmic Tail Contributes to Low Surface Expression and Intracellular Retention of the Patr-AL MHC Class I Molecule

Chimpanzees have orthologs of the six fixed, functional human MHC class I genes. But, in addition, the chimpanzee has a seventh functional gene, Patr-AL, which is not polymorphic but contributes substantially to population diversity by its presence on only 50% of MHC haplotypes. The ancestral AL gene emerged long before the separation of human and chimpanzee ancestors and then subsequently and specifically lost function during human evolution, but was maintained in chimpanzees. Patr-AL is an alloantigen that participates in negative and positive selection of the T cell repertoire. The three-dimensional structure and the peptide-binding repertoire of Patr-AL and HLA-A*02 are surprisingly similar. In contrast, the expression of these two molecules is very different, as shown using specific mAbs and polyclonal Abs made against Patr-AL. Peripheral blood cells and B cell lines express low levels of Patr-AL at the cell surface. Higher levels are seen for 221-cell transfectants expressing Patr-AL, but in these cells a large majority of Patr-AL molecules are retained in the early compartments of the secretory pathway: mainly the endoplasmic reticulum, but also cis-Golgi. Replacing the cytoplasmic tail of Patr-AL with that of HLA-A*02 increased the cell-surface expression of Patr-AL substantially. Four substitutions distinguish the Patr-AL and HLA-A*02 cytoplasmic tails. Systematic mutagenesis showed that each substitution contributes changes in cell-surface expression. The combination of residues present in Patr-AL appears unique, but each individual residue is present in other primate MHC class I molecules, notably MHC-E, the most ancient of the functional human MHC class I molecules.

HLA-DR peptide occupancy can be regulated with a wide variety of small molecules

abstractHLA-DR is the most commonly expressed and likely the most medically important human MHC class II, antigen presenting protein. In a normal immune response, HLA-DR binds to antigenic peptide and the HLA-DR/peptide complex binds to a T-cell receptor, thus contributing to T-cell activation and stimulation of an immune response against the antigen. When foreign antigen is not present, HLA-DR binds endogenous peptide which, under normal conditions does not stimulate an immune response. In most cases, the human peptide is CLIP, but a certain percentage of HLA-DR molecules will be present at the cell surface with other human peptides. We have recently shown that cell surface, CLIP/HLA-DR ratios are a measure of peptide heterogeneity, and in particular, changes in CLIP/HLA-DR ratios represent changes in the occupancy of HLA-DR by other, endogenous peptides. For example, treatment of cells with the HDAC inhibitor, Entinostat, leads to an upregulation of Cathepsin L1 and replacement of Cathepsin L1 senstitive peptides with HLA-DR binding, Cathepsin L1 resistant peptides, an alteration that can be at least partially assessed via assessment of CLIP/HLA-DR cell surface ratios. Here we assay for CLIP/HLA-DR ratios following treatment of immortalized B-cells with a variety of common drugs, almost all of which indicate significant changes in the CLIP/HLA-DR ratios. Furthermore, the CLIP/HLA-DR ratio changes parallel the impact of the drug panoply on cell viability, suggesting that alterations in the HLA-DR peptidome are governed by a variety of mechanisms, rather than exclusively dependent on a dedicated peptide loading process. These results raise questions about how FDA approved drugs may affect the immune response, and whether any of these drugs could be useful as vaccine adjuvants?

Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification.

A key event in the generation of a cellular response against malicious organisms through the endocytic pathway is binding of peptidic antigens by major histocompatibility complex class II (MHC class II) molecules. The bound peptide is then presented on the cell surface where it can be recognized by T helper lymphocytes. NetMHCIIpan is a state-of-the-art method for the quantitative prediction of peptide binding to any human or mouse MHC class II molecule of known sequence. In this paper, we describe an updated version of the method with improved peptide binding register identification. Binding register prediction is concerned with determining the minimal core region of nine residues directly in contact with the MHC binding cleft, a crucial piece of information both for the identification and design of CD4(+) T cell antigens. When applied to a set of 51 crystal structures of peptide-MHC complexes with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped to the epitope binding core. NetMHCIIpan is publicly available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.1 .

HLA-DR*0401 expression in the NOD mice prevents the development of autoimmune diabetes by multiple alterations in the T-cell compartment

Several human HLA alleles have been found associated with type 1 diabetes (T1D), but their precise role is not clearly defined. Herein, we report that a human MHC class II (HLA-DR*0401) allele transgene that has been expressed into NOD (H-2g7I-Enull) mice prone to T1D rendered the mice resistant to the disease. T1D resistance occurred in the context of multi-point T-cell alterations such as: (i) skewed CD4/CD8 T-cell ratio, (ii) decreased size of CD4+CD44high T memory pool, (iii) aberrant TCR Vβ repertoire, (iv) increased neonatal number of Foxp3+ and TR-1+ regulatory cells, and (v) reduced IFN-γ inflammatory response vs. enhanced IL-10 suppressogenic response of T-cells upon polyclonal and antigen-specific stimulation. The T-cells from NOD/DR4 Tg mice were unable to induce or suppress diabetes in NOD/RAG deficient mice. This study describes a multifaceted regulatory function of the HLA-DR*0401 allele strongly associated with the lack of T1D development in NOD mice.

Abstract 1352: Identifying pre-diagnostic breast cancer antigens in transgenic mouse mammary tumor models for preventative vaccine development

Abstract Preventative vaccine therapy may benefit women at high risk for breast cancer but currently it is impossible to identify women who will develop breast cancer prior to tumor development to identify initiating cancer antigens. Immunocompetent transgenic mouse mammary tumor models that are genetically similar to human breast cancer subtypes may identify pre-invasive antigens because mice can be followed longitudinally. Comparing tumor growth characteristics in two mouse models (TgMMTV-neu is genetically similar to luminal B breast cancer and C3(1)tag is genetically similar to triple negative breast cancer) demonstrated that, similar to triple negative breast cancer, C3(1)Tag mice develop tumors earlier and tumors grow more rapidly than TgMMTV-neu tumors. Furthermore, C3(1)Tag tumors have increased CD8+/CD4+ ratio (p<0.05) similar to human triple negative tumors. Using these two mouse models, we identified putative pre-invasive breast cancer antigens present prior to tumor development by serologic analysis of recombinant cDNA expression libraries and serological analysis of chip-arrayed proteins. We identified 65 pre-invasive antigens that were present in mice that would develop cancer but not parental control mice. The goal of this study was to identify which of the 65 antigens were required for human breast cancer survival with the goal to develop a preventative multi-antigen polyepitope breast cancer vaccine. We chose the pre-invasive antigens necessary for human breast cancer tumor cell survival using a high throughput siRNA screen evaluating for increased apoptosis and decreased cell survival in either HER2 positive or triple negative human breast cancer cell lines with decreased expression of the target protein. Five of the antigens were essential for human breast cancer cell survival: VPS35, SERBP1, ARPC2, PDIA6, and KRT8. All of these genes have roles in human cancer progression, and KRT8, SERBP1, and PDIA6 have identified roles in breast cancer pathogenesis. After designing human MHC class II peptides for each of these targets that cover at least 25% of the protein, we evaluated implanted tumor inhibition in the mouse models. Vaccination VPS35 peptides inhibited tumor growth by 47% (p<0.0001), vaccination with ARPC2 peptides inhibited tumor growth by 54% (p<0.0001), and vaccination with SERBP1 peptides inhibited tumor growth by 61% (p<0.0001) in the TgMMTV-neu mice. In C3(1)Tag mice, vaccination with VPS35 peptides inhibited tumor growth by 39% (p<0.0001) and vaccination with SERBP1 peptides inhibited tumor growth by 59% (p<0.0001) but vaccination with ARPC2 peptides did not inhibit tumor growth. These studies have demonstrated that mouse models can be used to identify pre-invasive breast cancer antigens. Further studies will evaluate the use of vaccines containing epitopes from several of the antigens to prevent spontaneous tumors in these mouse models. Citation Format: Sasha E. Stanton, Ekram Gadd, Lauren Rastetter, James Annis, Jianning Mao, John Ladd, Samir Hanash, Mary L. Disis. Identifying pre-diagnostic breast cancer antigens in transgenic mouse mammary tumor models for preventative vaccine development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1352. doi:10.1158/1538-7445.AM2015-1352

The Thermodynamic Mechanism of Peptide-MHC Class II Complex Formation Is a Determinant of Susceptibility to HLA-DM.

Peptides bind MHC class II molecules through a thermodynamically nonadditive process consequent to the flexibility of the reactants. Currently, how the specific outcome of this binding process affects the ensuing epitope selection needs resolution. Calorimetric assessment of binding thermodynamics for hemagglutinin 306-319 peptide variants to the human MHC class II HLA-DR1 (DR1) and a mutant DR1 reveals that peptide/DR1 complexes can be formed with different enthalpic and entropic contributions. Complexes formed with a smaller entropic penalty feature circular dichroism spectra consistent with a non-compact form, and molecular dynamics simulation shows a more flexible structure. The opposite binding mode, compact and less flexible, is associated with greater entropic penalty. These structural variations are associated with rearrangements of residues known to be involved in HLA-DR (DM) binding, affinity of DM for the complex, and complex susceptibility to DM-mediated peptide exchange. Thus, the thermodynamic mechanism of peptide binding to DR1 correlates with the structural rigidity of the complex, and DM mediates peptide exchange by "sensing" flexible complexes in which the aforementioned residues are rearranged at a higher frequency than in more rigid ones.

EBP1 protein modulates the expression of human MHC class II molecules in non-hematopoietic cancer cells.

Many solid tumours including melanoma, glioblastoma, and breast carcinomas express MHC class II molecules (MHC II). The surface expression of these molecules confers to non-hematopoietic tumour cells the role of non-professional antigen presenting cells and the ability to potentially stimulate tumour-specific CD4+ T cell response. We studied EBP1, an ErbB3 binding protein, and the effects of p48 and p42 isoforms on the MHC II expression in U87 glioblastoma, M14 melanoma and MCF7 mammary carcinoma cell lines. We found that overexpression of p48 increases MHC II transcription in U87 and M14, through upregulation of CIITA transactivator and STAT1 phosphorylation. In addition, p48 protein influences MHC II expression by increasing mRNA stability. In melanoma and glioblastoma cell lines, p48 isoform functions as oncogene promoting tumour growth, while p42 isoform, that does not affect MHC II expression, acts as a tumour suppressor by blocking cell growth and inducing apoptosis. In contrast, p48 seems to act as tumour suppressor in breast carcinoma inhibiting proliferation, favouring apoptosis, and inducing a slight increase of MHC II expression similar to p42. Our data highlight the tissue specificity function of EBP1 isoforms and demonstrate that only the oncogene p48 activates MHC II expression in human solid tumours, via STAT1 phosphorylation, in order to affect tumour progression by triggering specific immune response.

Analysis of the genetic architecture of susceptibility to cervical cancer indicates that common SNPs explain a large proportion of the heritability.

The genetic architecture of susceptibility to cervical cancer is not well-understood. By using a genome-wide association study (GWAS) of 1034 cervical cancer patients and 3948 controls with 632668 single-nucleotide polymorphisms (SNPs), we estimated that 24.0% [standard error (SE) = 5.9%, P = 3.19×10(-6)] of variation in liability to cervical cancer is captured by autosomal SNPs, a bit lower than the heritability estimated from family study (27.0%), suggesting that a substantial proportion of the heritability is tagged by common SNPs. The remaining missing heritability most probably reflects incomplete linkage disequilibrium between causal variants and the genotyped SNPs. The variance explained by each chromosome is not related to its length (R (2) = 0.020, P = 0.516). Published genome-wide significant variants only explain 2.1% (SE = 1.5%, P = 0) of phenotypic variance, which reveals that most of the heritability has not been detected, presumably due to small individual effects. Another 2.1% (SE = 1.1%, P = 0.013) of variation is attributable to biological pathways associated with risk of cervical cancer, supporting that pathway analysis can identify part of the hidden heritability. Except for human leukocyte antigen genes and MHC class I polypeptide-related sequence A (MICA), none of the 82 candidate genes/regions reported in other association studies contributes to the heritability of cervical cancer in our dataset. This study shows that risk of cervical cancer is influenced by many common germline genetic variants of small effects. The findings are important for further study design to identify the hiding heritability that has not yet been revealed. More susceptibility loci are yet to be found in GWASs with higher power.

ER egress of invariant chain isoform p35 requires direct binding to MHCII molecules and is inhibited by the NleA virulence factor of enterohaemorrhagic Escherichia coli

Four invariant chain (Ii) isoforms assist the folding and trafficking of human MHC class II (MHCIIs). The main isoforms, Iip33 and Iip35, assemble in the ER into homo- and/or hetero-trimers. The sequential binding of up to three MHCII αβ heterodimers to Ii trimers results in the formation of pentamers, heptamers and nonamers. MHCIIs are required to overcome the p35-encoded di-arginine (RxR) ER retention motif and to allow anterograde trafficking of the complex. Here, we show that inactivation of the RxR motif requires a direct cis interaction between p35 and the MHCII, precluding ER egress of some unsaturated Ii trimers. Interestingly, as opposed to MHCII/p33 complexes, those including p35 remained in the ER when co-expressed with the NleA protein of enterohaemorrhagic Escherichia coli. Taken together, our results demonstrate that p35 influences distinctively MHCII/Ii assembly and trafficking.

F pocket flexibility influences the tapasin dependence of two differentially disease-associated MHC Class I proteins.

The human MHC class I protein HLA-B*27:05 is statistically associated with ankylosing spondylitis, unlike HLA-B*27:09, which differs in a single amino acid in the F pocket of the peptide-binding groove. To understand how this unique amino acid difference leads to a different behavior of the proteins in the cell, we have investigated the conformational stability of both proteins using a combination of in silico and experimental approaches. Here, we show that the binding site of B*27:05 is conformationally disordered in the absence of peptide due to a charge repulsion at the bottom of the F pocket. In agreement with this, B*27:05 requires the chaperone protein tapasin to a greater extent than the conformationally stable B*27:09 in order to remain structured and to bind peptide. Taken together, our data demonstrate a method to predict tapasin dependence and physiological behavior from the sequence and crystal structure of a particular class I allotype. Also watch the Video Abstract.

Initial in vivo experience of pig artery patch transplantation in baboons using mutant MHC (CIITA-DN) pigs

BackgroundIn the pig-to-nonimmunosuppressed baboon artery patch model, a graft from an α1,3-galactosyltransferase gene-knockout pig transgenic for human CD46 (GTKO/CD46) induces a significant adaptive immune response (elicited anti-pig antibody response, increase in T cell proliferation on MLR, cellular infiltration of the graft), which is effectively prevented by anti-CD154mAb-based therapy. MethodsAs anti-CD154mAb is currently not clinically applicable, we evaluated whether it could be replaced by CD28/B7 pathway blockade or by blockade of both pathways (using belatacept+anti-CD40mAb [2C10R4]). We further investigated whether a patch from a GTKO/CD46 pig with a mutant human MHC class II transactivator (CIITA-DN) gene would allow reduction in the immunosuppressive therapy administered. ResultsWhen grafts from GTKO/CD46 pigs were transplanted with blockade of both pathways, a minimal or insignificant adaptive response was documented. When a GTKO/CD46/CIITA-DN graft was transplanted, but no immunosuppressive therapy was administered, a marked adaptive response was documented. In the presence of CD28/B7 pathway blockade (abatacept or belatacept), there was a weak adaptive response that was diminished when compared with that to a GTKO/CD46 graft. Blockade of both pathways prevented an adaptive response. ConclusionAlthough expression of the mutant MHC CIITA-DN gene was associated with a reduced adaptive immune response when immunosuppressive therapy was inadequate, when blockade of both the CD40/CD154 and CD28/B7 pathways was present, the response even to a GTKO/CD46 graft was suppressed. This was confirmed after GTKO/CD46 heart transplantation in baboons.

A bioinformatics tool for epitope-based vaccine design that accounts for human ethnic diversity: Application to emerging infectious diseases

BackgroundPeptide vaccination based on multiple T-cell epitopes can be used to target well-defined ethnic populations. Because the response to T-cell epitopes is restricted by HLA proteins, the HLA specificity of T-cell epitopes becomes a major consideration for epitope-based vaccine design. We have previously shown that CD4+ T-cell epitopes restricted by 95% of human MHC class II proteins can be predicted with high-specificity. MethodsWe describe here the integration of epitope prediction with population coverage and epitope selection algorithms. The population coverage assessment makes use of the Allele Frequency Net Database. We present the computational platform Predivac-2.0 for HLA class II-restricted epitope-based vaccine design, which accounts comprehensively for human genetic diversity. ResultsWe validated the performance of the tool on the identification of promiscuous and immunodominant CD4+ T-cell epitopes from the human immunodeficiency virus (HIV) protein Gag. We further describe an application for epitope-based vaccine design in the context of emerging infectious diseases associated with Lassa, Nipah and Hendra viruses. Putative CD4+ T-cell epitopes were mapped on the surface glycoproteins of these pathogens and are good candidates to be experimentally tested, as they hold potential to provide cognate help in vaccination settings in their respective target populations. ConclusionPredivac-2.0 is a novel approach in epitope-based vaccine design, particularly suited to be applied to virus-related emerging infectious diseases, because the geographic distributions of the viruses are well defined and ethnic populations in need of vaccination can be determined (“ethnicity-oriented approach”). Predivac-2.0 is accessible through the website http://predivac.biosci.uq.edu.au/.

Mutant IDH1: An immunotherapeutic target in tumors

The discovery of driver mutations in cancers has raised interest in their suitability as immunotherapeutic targets. A recent study demonstrates that a point mutation in isocitrate dehydrogenase 1 (IDH1R132H), expressed in gliomas and other tumors, is presented on human MHC class II and induces a mutation-specific CD4+ antitumor T cell response in patients and a syngeneic tumor model in MHC-humanized mice.

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.

IgG1 anti-epidermal growth factor receptor antibodies induce CD8-dependent antitumor activity.

Anti-EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR+ solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibody-dependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab-mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA-A2 and adoptively transferred human HLA-A2+ PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti-EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti-EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8+ T effector cells. This novel mechanism of ADCC conducted by CD8+ T effector cells was restricted to IgG1 anti-EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8+ T effector cell-mediated ADCC was enhanced in the presence of IL-15 and strongly improved after glycosylation of anti-EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.