Pease, Larry R.

Abstract The expansion and contraction of mammalian class I multigene families raises the issue as to what determines the loss or retention of family members. We propose that accumulating changes in regulatory regions result in the loss of expression of the gene products during times critical to selection, leading to the extinction of misregulated genes. The structures of promoter regions of MHC class I genes in nonhuman primates support this view. The B promoters are more homogeneous and contain regulatory elements also found in the promoters of the homologous class I genes of more distant mammals, whereas the A locus promoters were significantly more heterogeneous, have fewer sequence motifs related to known transcription factor-binding sites and have accumulated nucleotide substitutions within one of the widely conserved class I promoter elements. These findings are consistent with the view that the more polymorphic B locus is the principal MHC locus encoding functional class I Ag-presenting molecules whereas the less polymorphic A locus is assuming a secondary role as a consequence of promoter defects.

Abstract Peptide binding is known to influence the conformation of the surface of class I molecules as detected with mAbs and TCR. A new conformationally sensitive epitope on the mouse class I molecule Kb is defined by mAb AF6-88.5. The recognized structure is affected by amino acid substitutions in any of the three external domains of the class I heavy chain and, in addition, is influenced by the substitution of human for mouse β2-microglobulin. Interestingly, the epitope for this Ab is not affected by mutations within the peptide-binding cleft or by the nature of the peptide bound. These findings indicate that the effect of a change in one domain of class I can radiate to other parts of the molecule. Furthermore, the existence of conformationally sensitive structures outside of the peptide-binding site suggests the possibility that class I molecules may change their structure in response to binding by receptors and ligands such as the TCR and the coligand CD8. Such structural changes may represent signals that can influence cellular activation events.

Abstract Malignant melanoma is the most aggressive form of skin cancer, accounting for 10,000 deaths annually. Largely resistant to conventional cytotoxic chemotherapy and radiation, immune checkpoint blockade (ICB) therapies have revolutionized patient care, accounting for partial or complete responses in up to 70% of patients. An important aspect of cancer elimination is activation of the cytotoxic T lymphocyte (CTL) response. Recent data suggest that ICB mediated broadening of the peripheral blood CTL repertoire correlates with good clinical outcomes. Thus, recent studies in cancer immunotherapy have focused on targeting tumor neoantigens (tNeoAg) with cancer vaccines for use in combination with ICB. Computational algorithm-driven predictions of immunogenic tNeoAgs yield vast numbers of potential peptide targets, only a fraction of which may be immunogenic in patients. This likely contributed to the modest success of cancer vaccines targeting predicted, high-avidity tNeoAgs, emphasizing the importance of identifying true tumor rejection antigens, including sub-dominant antigens, outside the scope of predictive models. In addition to promoting T cell-mediated tumor rejection, ICB often comes at the expense of treatment-induced immune-related adverse effects (irAE) that frequently require discontinuation of treatment. Modulation of ICB towards antitumor immunity and away from autoimmunity may dramatically improve the therapeutic index of modern cancer therapy. This appears feasible considering the clinical observation that antitumor efficacy does not correlate with type/severity of irAE, suggesting that the mechanisms of both processes, though likely related (CTL mediated), may not be identical. We aim to identify and separate non-cross-reacting antigenic targets mediating tumor rejection from those mediating irAEs to enable therapeutic interventions that maximize the efficacy of ICB, expanding tumor-specific CTLs with vaccines while minimizing irAEs through desensitization. We have access to samples from responding/nonresponding melanoma patients subjected to ICB with varying degrees of irAEs. We have designed an experimental approach that combines established mass spectrometry and sequencing techniques to identify peptides and matching TCR clones with a novel strategy that targets the TCR-associated CD3 complex to allow inclusion of subdominant antigens in our studies. T-cell “co-potentiation” is achieved when anti-CD3 monovalent Fabs induce a conformational change in the CD3 complex that sustains the T-cell response to weak antigenic stimulation. We have successfully used anti-human CD3 Fabs to co-potentiate in vitro the activation and subsequent response to weak peptide-HLA/TCR interactions of human T cells found in PBMCs isolated from healthy donors. Our preliminary data suggest T-cell co-potentiation may allow identification of ICB-induced CTL clones specific for subdominant tNeoAgs and irAE targets in patients with melanoma undergoing active immunotherapy. Citation Format: Laura Elsbernd, Alfreda Nelson, Hien Huynh, Michele Hoffmann, Christopher Parks, Wendy Nevala, Shari Sutor, Larry Pease, Adam Schrum, Diana Gil, Svetomir N. Markovic. Co-potentiation of human T cells to identify subdominant tumor neoantigens from melanoma patients responding to immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B78.

AbstractTheiler's murine encephalomyelitis virus causes a chronic demyelinating disease in susceptible strains of mice that is similar to human multiple sclerosis. Several nonmajor histocompatibility complex–linked genes have been implicated as determinants of susceptibility or resistance to either demyelination or virus persistence. In this study, we used linkage analysis of major histocompatibility complex identical H-2d (DBA/2J × B10.D2) F2 intercross mice to identify loci associated with susceptibility to virus-induced demyelinating disease. In a 20-cM region on chromosome 14, we identified four markers, D14Mit54, D14Mit60, D14Mit61, and D14Mit90 that are significantly associated with demyelination. Because two peaks were identified, one near D14Mit54 and one near D14Mit90, it is possible that two loci in this region are involved in controlling demyelination.

Abstract Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(−) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Abstract Immunosuppression of unknown aetiology is a hallmark feature of glioblastoma and is characterized by decreased CD4 T-cell counts and downregulation of major histocompatibility complex class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for glioblastoma. We recapitulated the immunosuppression observed in glioblastoma patients in the C57BL/6 mouse and investigated the aetiology of low CD4 T-cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of brain cancer, including mice harbouring GL261 glioma, B16 melanoma, and in a spontaneous model of diffuse intrinsic pontine glioma. In addition to thymic involution, we determined that tumour growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC II expression on blood leucocytes, and a modest increase in bone marrow resident CD4 T cells. Using parabiosis we report that thymic involution, declines in peripheral T-cell counts, and reduced major histocompatibility complex class II expression levels were mediated through circulating blood-derived factors. Conversely, T-cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is non-steroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the immunosuppression was not unique to cancer itself, but rather occurs in response to brain injury. Non-cancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that brain cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity.

Abstract Theiler's murine encephalomyelitis virus (TMEV) infection of the CNS is cleared in C57BL/6 mice by a CD8 T cell response restricted by the MHC class I molecule H-2Db. The identity and function of the APC(s) involved in the priming of this T cell response is (are) poorly defined. To address this gap in knowledge, we developed an H-2Db LoxP-transgenic mouse system using otherwise MHC class I–deficient C57BL/6 mice, thereby conditionally ablating MHC class I–restricted Ag presentation in targeted APC subpopulations. We observed that CD11c+ APCs are critical for early priming of CD8 T cells against the immunodominant TMEV peptide VP2121-130. Loss of H-2Db on CD11c+ APCs mitigates the CD8 T cell response, preventing early viral clearance and immunopathology associated with CD8 T cell activity in the CNS. In contrast, animals with H-2Db–deficient LysM+ APCs retained early priming of Db:VP2121-130 epitope–specific CD8 T cells, although a modest reduction in immune cell entry into the CNS was observed. This work establishes a model enabling the critical dissection of H-2Db–restricted Ag presentation to CD8 T cells, revealing cell-specific and temporal features involved in the generation of CD8 T cell responses. Employing this novel system, we establish CD11c+ cells as pivotal to the establishment of acute antiviral CD8 T cell responses against the TMEV immunodominant epitope VP2121-130, with functional implications both for T cell–mediated viral control and immunopathology.

Abstract The BALB/c-neuT mouse has been a faithful model for investigating metastatic breast cancer. Spontaneous breast tumors from BALB/c-neuT mice are readily transplantable orthotopically into syngeneic or rat neuT negative BALB/c WT hosts facilitating controlled studies of developing breast cancers. When introduced into the breast ducts of BALB/c WT hosts, lung metastases form similarly to how they appear in mice with spontaneous tumors. Tumor grafts grow out with comparable kinetics and induce similar systemic immune abnormalities in the spleens and lymph nodes of both lines of mice as seen in mice with spontaneous tumors. Factors inducing these abnormal immune cell configurations are not yet known. Immunization of BALB/c WT mice against the rat Her2 antigen can induce both T cell and antibody mediated rejection of the transplanted tumors, but these approaches have not been equally successful in syngeneic, rat neuT+ positive hosts, indicating tolerance to rat Her2 is a significant barrier to the induction of immunity against these breast tumors. The MHC encodes class I and class II antigen presenting molecules which are known to be highly polymorphic in both structure and function. We introduced the H-2b MHC haplotype into the MMTV-NeuT model to assess the influence of a different set of antigen presenting molecules on tumor biology. In BALB.B-neuT mice spontaneous tumors appeared with frequencies and in numbers similar to those seen in the BALB/c model. In anything, lung metastases are more frequent and more aggressive in BALB.B-neuT mice. Again, the tumors were readily transplantable into the breast fat pads of syngeneic BALB.B-neuT hosts which developed systemic immune abnormalities very similar to those observed in BALB/c -neuT or BALB/c WT mice. However, BALB.B WT hosts initially accepted the grafts, but subsequently rejected them after they were established. Mice that were in the process of rejecting the tumors retained normal cellularity within their spleens and lymph nodes indicating the importance of an ongoing immune response and/or tumor rejection in shaping the microenvironment in secondary lymphoid organs. Adoptive transfer of spleen cells from BALB.B WT mice which had rejected established tumor conferred resistance to the tumor grafts as long as B cells were included (P<0.01), indicating active humoral immunity was important to tumor rejection. A mechanistic understanding or how antibody-mediated immunotherapy works in patients is incomplete. This new model provides an opportunity to address this question. The Her2 ecto-domain is known to be shed from tumor cells. We propose a model in which the ecto-domain of rat neuT is shed, but is insufficiently immunogenic in BALB/c (H-2d) hosts, but highly immunogenic in BALB.B (H-2b) mice. Consistent with this model is the prediction using the Immune Epitope and Data Base Resource of the presence of a potentially immunogenic IAb-restricted class II epitope within the ecto-domain of rat Her2 neuT oncogene, but no such epitope restricted by IAd or IEd was identified. This provides a compelling hypothesis explaining why grafted spontaneous tumors grow in BALB/c, but not in BALB.B WT mice. An important prediction from this model is that linkage of a potent class II epitope to the Erbb2-encoded ecto-domain may provide a basis for a potent vaccine against Her2 positive breast tumors which should be a strategy adaptable to any MHC genotype. It also will be of interest to determine whether the presence of an antibody reactive against established tumor restores the normal cellular composition of secondary lymphoid organs which have been perturbed by established breast tumors. Citation Format: Sarah Anne Castro, Alvaro Pena, Khashayarsha Khazaie, Larry R. Pease. An immunogenic mouse model using BALB.B-MMTV-neuT breast cancers reveals spontaneous protective humoral immunity [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-15.

Abstract Breast tumors develop under environmental pressures with phenotypically variant cells generated by mutation and epigenetic changes providing the substrate for clonal selection. Chromosomal mutations are a feature of spontaneous breast tumors in the BALB-neuT mouse model. However, there is little evidence that specific changes in chromosomal structure or ploidy confer selective advantage in these spontaneous tumors. The prominent exception is a consistent loss of chromosome 4, a little understood feature common to several mouse tumors. We previously measured heterozygosity in spontaneous breast tumors from FVB X BALB-neuT F1 mice using Illumina’s Golden Gate assay. In addition to the expected gain and loss of heterozygosity (LOH) evident throughout the genomes of a cohort of spontaneous tumors, the assay also detected wide-spread stochastic pseudo-“LOH” at 600 discrete loci spread throughout the genome in unique patterns among the tumors. Several of these positions were examined by sequence analysis revealing no deviations from the WT sequences, suggesting the detected “LOH” may have been generated by epigenetic modification of DNA which altered sequence detection. While, epigenetically modified DNA templates recapitulated the observed “LOH” signals, no canonical CpG motifs were present in the majority of the 600 loci probed, suggesting that an unusual DNA modification could be responsible for the unexpected wide-spread stochastic structural changes in the breast tumor DNA. We next assessed whether these putative epigenetic changes in DNA structure might impact gene regulation, and have reported that the stochastic pattern observed as “LOH” in DNA was recapitulated in the transcriptomes in unique patterns among the tumors. A direct correlation between the number of “LOH” variants and the down regulation of hundreds of non-polymorphic genes in the transcriptome also was noted. Furthermore, pathway analyses of the genes exhibiting changes in allelic ratios in these tumors revealed significant enrichments within gene networks regulating tissue homeostasis and antigen presentation, providing strong evidence that the perturbations in gene expression translated into selectable tumor cell phenotypes. We now have extended this study to examine the relationship between the magnitude of change in the expression of genes mapping within the pathways regulating tissue homeostasis (Molecular Mechanisms of Cancer). A remarkable feature of the flagged genes is that the magnitude of change in gene expression was not great in each case, yet the biological consequences were strongly reflected in the evolutionary history of the tumors. Importantly, the polymorphisms marking the parental alleles are mostly silent, not altering the structure of the encoded products. Therefore magnitude and timing of gene expression are the likely determinants of phenotypic variation. Each tumor contained several outliers within the pathways regulating tissue homeostasis, suggesting that the integration of multiple small perturbations in the expression of genes comprising functional networks could influence the biology properties of the tumor cells. Overall expression of the loci marked by allelic outliers was significantly below the average expression found among tumors in the cohort highlighting the importance of down regulation of one allele in the establishment of selectable traits. We find a similar direct correlation of multiple small changes in the transcriptome of normal lymphocytes with immune response phenotypes, suggesting this principle of integration of multiple small deviations in gene expression applies widely to the phenotypes of normal cells, tumors, and by extension to organismic traits. Citation Format: Larry R Pease, Sara J Felts, Adam D Scheid, Xiaojia Tang, Krishna R Kalari. Phenotypes of breast tumor cells and normal lymphocytes are determined by the integration of minor changes in expression of multiple genes: A new dimension in quantitative inheritance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-03.