Major Histocompatibility Research Articles

MHC heterozygosity limits T cell receptor variability in CD4 T cells.

αβ T cell receptor (TCR) V(D)J genes code for billions of TCR combinations. However, only some appear on peripheral T cells in any individual because, to mature, thymocytes must react with low affinity but not high affinity with thymus expressed major histocompatibility (MHC)/peptides. MHC proteins are very polymorphic. Different alleles bind different peptides. Therefore, any individual might express many different MHC alleles to ensure that some peptides from an invader are bound to MHC and activate T cells. However, most individuals express limited numbers of MHC alleles. To explore this, we compared the TCR repertoires of naïve CD4 T cells in mice expressing one or two MHC alleles. Unexpectedly, the TCRs in heterozygotes were less diverse that those in the sum of their MHC homozygous relatives. Our results suggest that thymus negative selection cancels out the advantages of increased thymic positive selection in the MHC heterozygotes.

DNA structural features and variability of complete MHC locus sequences.

The major histocompatibility (MHC) locus, also known as the Human Leukocyte Antigen (HLA) genes, is located on the short arm of chromosome 6, and contains three regions (Class I, Class II and Class III). This 5Mbp locus is one of the most variable regions of the human genome, yet it also encodes a set of highly conserved and important proteins related to immunological response. Genetic variations in this region are responsible for more diseases than in the entire rest of the human genome. However, information on local structural features of the DNA is largely ignored. With recent advances in long-read sequencing technology, it is now becoming possible to sequence the entire 5Mbp MHC locus, producing complete diploid haplotypes of the whole region. Here, we describe structural maps based on the complete sequences from six different homozygous HLA cell lines. We find long-range structural variability in the different sequences for DNA stacking energy, position preference and curvature, variation in repeats, as well as more local changes in regions forming open chromatin structures, likely to influence gene expression levels. These structural maps can be useful in visualizing large scale structural variation across HLA types, in particular when this can be complemented with epigenetic signals.

Elucidating the immune active state of HR+HER2- MammaPrint High 2 early breast cancer.

506 Background: The 70-gene signature, MammaPrint (MP), classifies patients (pts) with early-stage breast cancer (EBC) as having an UltraLow, Low, High 1 (H1), or High 2 (H2) risk of distant recurrence. I-SPY 2 showed that pts with MP H2, HR+HER2- tumors have significantly higher response rates to neoadjuvant chemotherapy + immunotherapy relative to pts with MP H1 tumors. Expression-based immune deconvolution provides more detailed information about immune cell function beyond conventional methods enumerating tumor infiltrating lymphocytes. To elucidate the underlying biology that mediates immune therapy response, we performed in silico analysis of full transcriptome data to characterize immune cell frequencies and antigen presentation in HR+HER2-, MP High Risk EBC from pts enrolled in FLEX. Methods: The prospective, observational FLEX Study (NCT03053193) includes stage I-III pts with EBC who received MP testing and consented to full transcriptome and clinical data collection. Women with HR+HER2-, and MP High Risk invasive ductal EBC were included (n = 2916). Tumors were stratified into MP H1 or MP H2. The R-package Limma was used to preprocess gene expression data. The gene signature-based method xCell was used to determine immune cell abundances for each group based on enrichment score. Expression of genes involved in “antigen processing and presentation KEGG pathway”, and immune regulation, were evaluated between groups. Differences in immune cell frequency and gene expression were determined by a t-test with an adjusted p-value < 0.05. Results: MP classified 79% (n=2292) of HR+HER2- EBC as H1 and 21% (n=624) as H2. Pts with H2 EBC were more likely to be ≤ 50 years (32%) compared to pts with H1 tumors (23%). Although low ER staining (1-10%) tumors were more frequent in H2 vs H1 (15% vs 1%), a majority of H2 tumors (75%) had ER staining > 10%. Nearly 30% of H2 tumors were Grade 1 or 2, whereas 22% of H1 tumors were Grade 3. H2 tumors had significantly higher frequency of antigen presenting cells (APCs) including activated dendritic cells and macrophages, CD4+ memory T cells, CD8+ T cells, memory B cells and plasma cells relative to H1 tumors. The genes expressing PD-1 and PDL-1 and genes involved in antigen processing, including B2Mand TAP1/2, and presentation, i.e., major histocompatibility (MHC) class I ( HLA-A, -B, -F) and class II molecules ( HLA-DM, -DQ), were significantly upregulated in H2 vs H1. Conclusions: H2 tumors exhibited a heighted immune active state compared to H1 tumors among HR+HER2- EBC pts. The presence of APCs and increased antigen presentation, which are critical in eliciting T- and B-cell activation may explain the improved response rates to immunotherapy observed in H2 tumors. Selecting pts based on grade or ER% alone may exclude pts likely to benefit from immune therapy. These data support ongoing trials evaluating the response and benefit of adding immunotherapy to standard of care treatment regimens in MP H2 tumors. Clinical trial information: NCT03053193 .

Nucleobase adducts bind MR1 and stimulate MR1-restricted T cells.

MR1T cells are a recently found class of T cells that recognize antigens presented by the major histocompatibility complex-I-related molecule MR1 in the absence of microbial infection. The nature of the self-antigens that stimulate MR1T cells remains unclear, hampering our understanding of their physiological role and therapeutic potential. By combining genetic, pharmacological, and biochemical approaches, we found that carbonyl stress and changes in nucleobase metabolism in target cells promote MR1T cell activation. Stimulatory compounds formed by carbonyl adducts of nucleobases were detected within MR1 molecules produced by tumor cells, and their abundance and antigenicity were enhanced by drugs that induce carbonyl accumulation. Our data reveal carbonyl-nucleobase adducts as MR1T cell antigens. Recognizing cells under carbonyl stress allows MR1T cells to monitor cellular metabolic changes with physiological and therapeutic implications.

Construction of an immune-related gene prognostic model with experimental validation and analysis of immune cell infiltration in lung adenocarcinoma.

There is a correlation between tumors and immunity with the degree of immune cell infiltration in tumors being closely related to tumor growth and progression. Therefore, the present study identified immune-related prognostic genes and evaluated the immune infiltration level in lung adenocarcinoma (LUAD). This study performed Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and Gene Set Enrichment Analysis (GSEA) enrichment analyses on differential immune-associated genes. A risk model was created and validated using six immune-related prognostic genes. Reverse transcription-quantitative PCR was used to assess the prognostic gene expression in non-small cell lung cancer cells. Immune cell infiltration in LUAD was analyzed using the CIBERSORT method. Single sample GSEA was used to compare Tumor Immune Dysfunction and Exclusion (TIDE) scores between high and low-risk groups and to assess the activation of thirteen immune-related pathways. Multifactor Cox proportional hazards model analysis identified six prognostic risk genes (S100A16, FURIN, FGF2, LGR4, TNFRSF11A and VIPR1) to construct a risk model. The survival and receiver operating characteristic curves indicated that patients with higher risk scores had lower overall survival rates. The expression levels of prognostic genes S100A16, FURIN, LGR4, TNFRSF11A and VIPR1 were significantly increased in LUAD. B cells naive, plasma cells, T cells CD4 memory activated, T cells follicular helper, T cells regulatory, NK cells activated, macrophages M1, macrophages M2, and Dendritic cells resting cells showed elevated expression in LUAD. The prognostic genes were differentially associated with individual immune cells. Immune-related function scores, such as those for antigen presenting cell (APC) co-stimulation, APC co-inhibition, check-point, Cytolytic-activity, chemokine receptor, parainflammation, major histocompatibility complex-class-I, type-I-IFN-reponse and T-cell-co-inhibition, were higher in the high-risk group compared with the low-risk group. Furthermore, the TIDE score of the high-risk group was significantly lower than the low-risk group. This immune-related gene prognostic model has the potential to predict the prognosis of LUAD patients, supporting the development of a personalized clinical diagnosis and treatment plan.

D-2-hydroxyglutarate supports a tolerogenic phenotype with lowered major histocompatibility class II expression in non-malignant dendritic cells and acute myeloid leukemia cells.

D-2-hydroxyglutarate (D-2-HG) accumulates in patients with acute myeloid leukemia (AML) with mutated isocitrate dehydrogenase (IDH) and in other malignancies. D-2-HG suppresses antitumor T-cell immunity but little is known about potential effects on non-malignant myeloid cells. Here we show that D-2-HG impairs human but not murine dendritic cell differentiation, resulting in a tolerogenic phenotype with low major histocompatibility class II expression. In line with this, IDH-mutated AML blasts exhibited lower expression of HLA-DP and were less susceptible to lysis by HLA-DP-specific T cells. Interestingly, besides its expected impact on DNA demethylation, D-2-HG reprogrammed metabolism towards increased lactate production in dendritic cells and AML. Vitamin C accelerated DNA demethylation, but only the combination of vitamin C and glycolytic inhibition lowered lactate levels and supported major histocompatibility complex class II expression. Our results indicate an unexpected link between the immunosuppressive metabolites 2-HG and lactic acid and suggest a potentially novel therapeutic strategy with combinations of anti-glycolytic drugs and epigenetic modulators (hypomethylating agents) or other therapeutics for the treatment of AML.

The role of natural killer T cells in liver transplantation.

Natural killer T cells (NKTs) are innate-like lymphocytes that are abundant in the liver and participate in liver immunity. NKT cells express both NK cell and T cell markers, modulate innate and adaptive immune responses. Type I and Type II NKT cells are classified according to the TCR usage, while they recognize lipid antigen in a non-classical major histocompatibility (MHC) molecule CD1d-restricted manner. Once activated, NKT cells can quickly produce cytokines and chemokines to negatively or positively regulate the immune responses, depending on the different NKT subsets. In liver transplantation (LTx), the immune reactions in a series of processes determine the recipients' long-term survival, including ischemia-reperfusion injury, alloresponse, and post-transplant infection. This review provides insight into the research on NKT cells subpopulations in LTx immunity during different processes, and discusses the shortcomings of the current research on NKT cells. Additionally, the CD56-expressing T cells are recognized as a NK-like T cell population, they were also discussed during these processes.

Effective γδ T-cell clinical therapies: current limitations and future perspectives for cancer immunotherapy.

γδ T cells are a unique subset of T lymphocytes, exhibiting features of both innate and adaptive immune cells and are involved with cancer immunosurveillance. They present an attractive alternative to conventional T cell-based immunotherapy due, in large part, to their lack of major histocompatibility (MHC) restriction and ability to secrete high levels of cytokines with well-known anti-tumour functions. To date, clinical trials using γδ T cell-based immunotherapy for a range of haematological and solid cancers have yielded limited success compared with in vitro studies. This inability to translate the efficacy of γδ T-cell therapies from preclinical to clinical trials is attributed to a combination of several factors, e.g. γδ T-cell agonists that are commonly used to stimulate populations of these cells have limited cellular uptake yet rely on intracellular mechanisms; administered γδ T cells display low levels of tumour-infiltration; and there is a gap in the understanding of γδ T-cell inhibitory receptors. This review explores the discrepancy between γδ T-cell clinical and preclinical performance and offers viable avenues to overcome these obstacles. Using more direct γδ T-cell agonists, encapsulating these agonists into lipid nanocarriers to improve their pharmacokinetic and pharmacodynamic profiles and the use of combination therapies to overcome checkpoint inhibition and T-cell exhaustion are ways to bridge the gap between preclinical and clinical success. Given the ability to overcome these limitations, the development of a more targeted γδ T-cell agonist-checkpoint blockade combination therapy has the potential for success in clinical trials which has to date remained elusive.

Genetic background of type 1 diabetes mellitus: A review

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the complete lack of insulin secretion due to the immune system’s destruction of insulin-producing pancreatic β-cells. Approximately 50% of the risk factors for T1DM are attributed to genetic susceptibility, with the remaining causes linked to epigenetic modifications and environmental factors. The major gene region involved in T1DM is the major histocompatibility (MHC) complex, which is the first region discovered, conferring 50% of the genetic risk, followed by the insulin (INS), cytotoxic T lymphocyte-associated protein (CTLA4), protein tyrosine phosphatase, non-receptor type 22 (PTPN22), interleukin 2 receptor alpha (IL2RA) and interferon-induced with helicase C domain (IFIH1) genes. The remaining genetic susceptibility has been uncovered through genome-wide association (GWA) studies, which have identified 120 single nucleotide polymorphisms (SNPs) associated with T1DM risk to date. Additionally, non-coding RNAs (ncRNAs) also play a role, affecting the expression of the genes involved in T1DM. Epigenetics bridges the gap between genetic predisposition and environmental factors, which act as a trigger for T1DM in susceptible individuals. In this paper, the major genes involved in the risk of T1DM are reviewed.

Transfer learning improves pMHC kinetic stability and immunogenicity predictions

The cellular immune response comprises several processes, with the most notable ones being the binding of the peptide to the Major Histocompability Complex (MHC), the peptide-MHC (pMHC) presentation to the surface of the cell, and the recognition of the pMHC by the T-Cell Receptor. Identifying the most potent peptide targets for MHC binding, presentation and T-cell recognition is vital for developing peptide-based vaccines and T-cell-based immunotherapies. Data-driven tools that predict each of these steps have been developed, and the availability of mass spectrometry (MS) datasets has facilitated the development of accurate Machine Learning (ML) methods for class-I pMHC binding prediction. However, the accuracy of ML-based tools for pMHC kinetic stability prediction and peptide immunogenicity prediction is uncertain, as stability and immunogenicity datasets are not abundant. Here, we use transfer learning techniques to improve stability and immunogenicity predictions, by taking advantage of a large number of binding affinity and MS datasets. The resulting models, TLStab and TLImm, exhibit comparable or better performance than state-of-the-art approaches on different stability and immunogenicity test sets respectively. Our approach demonstrates the promise of learning from the task of peptide binding to improve predictions on downstream tasks. The source code of TLStab and TLImm is publicly available at https://github.com/KavrakiLab/TL-MHC.

EXTH-70. UTILIZING ONCOLYTIC VIROTHERAPY AS AN APPROACH TO ENHANCE NEOANTIGEN SPECIFIC IMMUNE RESPONSES IN GLIOBLASTOMA

Abstract Despite the success in treating many solid cancers, immunotherapy has not shown significant efficacy in patients with glioblastoma (GBM). The lack of tumor-infiltrating T cells recognizing tumor antigens displayed with major histocompability complex I (MHC I) is one of the reasons for GBM’s evasion from immunotherapy. Our preclinical GBM models and an ongoing clinical trial with oncolytic herpes simplex virus-1 (oHSV) at our institution (NCT03152318) demonstrated that oHSV treatment leads to an increase influx of CD8+ and CD4+ T cells into tumor. However, it remained unclear whether these T cells are specific to the tumor antigens (neoantigens) or reactive to only viral antigens. To determine whether infiltrating CD8 T cells target tumor antigens, we first performed whole-exome and RNA sequencing of CT2A, a preclinical model of GBM impervious to immunotherapies for identification of neoantigens. We screened immunogenicity of identified neoantigens by ELISPOT assay. TILs from the oHSV-treated (but not from control group) reacted to two of the neoantigens that were identified with our pipeline and this activation was similar in magnitude to the reaction to the immunodominant HSV antigen. FACS analyses of TILs seven days after virotherapy showed that oHSV-treated tumor had higher level of TILs than vehicle control tumors, a finding that mirrored what we observed in our phase 1 clinical trial with oHSV. High dose of oHSV treatment not just induces the infiltration of viral specific T cells, but infiltration of T cells targeting neoantigens that were not presented without OV infection. Phosphoproteomic analysis of bulk tumor revealed activation of key inflammatory pathways, including that of T cells receptor signaling upon virus treatment. We evaluated the efficacy of therapeutic neoantigen vaccination in combination with oncolytic intratumoral virotherapy. Survival analysis showed that combination therapy not just delays the tumor progression but also provides survival advantage compared to monotherapies.

Cell Communication‐Mediated Selective Drug Delivery for Epitope‐Specific Deletion of Alloreactive T Cells in Organ Transplant

AbstractAllogeneic grafts are vulnerable to the attack launched by host alloreactive T cells, which accounts primarily for allograft failure in the clinic. Conventional pharmacological intervention mainly involves broad‐acting, nonspecific immunosuppressants that provide limited protection over the graft and may cause deleterious side effects due to insufficient allospecificity. Here, core–shell structured nanoparticles co‐loaded with allogeneic antigen (Ag) and immunosuppressive agent Tacrolimus (Ag@FK506‐NPs) are constructed. Dendritic cells (DCs) pulsed with Ag@FK506‐NPs present alloantigenic epitopes on the cell surface as a bait to capture specific T cells, especially CD8+ T cells, for intercellular FK506 transfer and alloreactive T‐cell deletion. Meanwhile, DCs are maintained in an immature tolerogenic state that induces the functional inactivation of recruited T cells. Compared with free FK506, the adoptive transfer of low immunosuppressant dose (25 000‐fold lower) of Ag@FK506‐NPs pulsed DCs is enough to yield a superior antirejection effect in a major histocompatibility complex‐mismatched murine skin transplant model. Of note, due to the cell–cell communication conferred selective T‐cell suppression, the recipient mouse is able to respond to the subsequent stimulation of an irrelevant Ag, indicating that off‐target toxicity is bypassed. Overall, this work may address some of the limitations of current allograft therapeutics.

Histopathological changes of the dural myeloid cells and lymphatic vessels in a mouse model of sepsis-associated encephalopathy

As a common diffuse encephalopathy caused by sepsis, sepsis-associated encephalopathy (SAE) is closely associated with increased mortality, severe cognition dysfunction and increased cost of health care in patients of sepsis. Accumulating evidence suggests that the dura mater, the outermost meninges of the central nervous system (CNS), plays an important role in CNS immunity, especially with the discovery of meningeal lymphatic vessels (mLVs), as well as a plentiful array of resident or infiltrating immune cells harbored in the dura. Although these findings have significantly enhanced our understanding of the immune function of dura under both steady-state and pathological condition of CNS, whether and how the immune cells and mLVs within dura response to SAE still remains largely unexplored. Here, we established lipopolysaccharide (LPS) intraperitoneal injection-induced SAE model and examined the dural resident immune cells and mLVs. We analysed the histological change in dura by performing hematoxylin and eosin (H&E) and immunofluorescence staining. Results showed that systemic exposure to LPS induced neutrophils recruitment, exudation and gathering around the dural blood vessels. Moreover, resident macrophage altered its shape as well as location, and downregulated major histocompatibility (MHC) class II expression following LPS injection. We also found that LPS exposure induced dorsal meningeal lymphangiogenesis. Together, these findings collectively demonstrated that LPS-induced SAE can stimulate immune cells and mLVs within dura and provided more information about the immune response of the dura in sepsis.

Investigating the human and non-obese diabetic mouse MHC class II immunopeptidome using protein language modelling.

Identifying peptides associated with the major histocompability complex class II (MHCII) is a central task in the evaluation of the immunoregulatory function of therapeutics and drug prototypes. MHCII-peptide presentation prediction has multiple biopharmaceutical applications, including the safety assessment of biologics and engineered derivatives in silico, or the fast progression of antigen-specific immunomodulatory drug discovery programs in immune disease and cancer. This has resulted in the collection of large-scale data sets on adaptive immune receptor antigenic responses and MHC-associated peptide proteomics. In parallel, recent deep learning algorithmic advances in protein language modelling (PLM) have shown potential in leveraging large collections of sequence data and improve MHC presentation prediction. Here, we train a compact transformer model (AEGIS) on human and mouse MHCII immunopeptidome data, including a preclinical murine model, and evaluate its performance on the peptide presentation prediction task. We show that the transformer performs on par with existing deep learning algorithms and that combining datasets from multiple organisms increases model performance. We trained variants of the model with and without MHCII information. In both alternatives, the inclusion of peptides presented by the I-Ag7 MHC class II molecule expressed by the non-obese diabetic (NOD) mice enabled for the first time the accuratein silico prediction of presented peptides in a preclinical type 1 diabetes model organism, which has promising therapeutic applications. The source code is available at https://github.com/Novartis/AEGIS. Supplementary data are available at Bioinformatics online.

138-OR: Characterization and Prediction of Genetic Risk of T1D in Individuals without HLA-DR3 or HLA-DR4

The Major Histocompatibility (MHC) class II haplotypes HLA-DR3 and HLA-DR4 confer substantial genetic risk for type 1 diabetes (T1D), where 90% of individuals with T1D carry at least one copy of DR3 and/or DR4. We sought to understand whether the remaining 10% of T1D individuals without DR3 or DR4 haplotypes (non-DR3/DR4) have differences in genetic risk and whether current risk scores are effective for these individuals. We tested for T1D association using 12,716 non-DR3/DR4 samples from five European ancestry cohorts using genotypes imputed into the Michigan HLA and TOPMed v2 reference panels and identified independent signals using stepwise conditional analyses. There were 21 signals with significant effects on T1D, including 13 at the MHC locus and 8 genome-wide, of which 5 were novel. Across all T1D signals, 14 lead variants exhibited significant heterogeneity in effects on T1D in the non-DR3/DR4 subset compared to the full T1D population, including HLA-B*39 and the PTPN22 locus. Nearly 50% of non-DR3/DR4 T1D would be misclassified by a current T1D genetic risk score (T1D GRS1), which is weighted heavily by DR3/DR4 tag variants. We developed a new GRS using the 21 independent signals and determined whether this GRS could better discriminate T1D in individuals with non-DR3/DR4 haplotypes. The non-DR3/DR4 T1D GRS effectively differentiated T1D case and control samples (AUC=0.87) including in samples not in the initial discovery set (AUC=0.86), and improved prediction compared to GRS1 (AUC=0.52 using all controls, AUC=0.79 using only non-DR3/DR4 controls). These findings reveal a subset of T1D cases with heterogeneity in genetic risk and which are not as well represented by current risk scores, and argue that risk scores developed within heterogeneous subsets may enable even more accurate prediction of T1D. Disclosure C.Mcgrail: None. K.J.Gaulton: Consultant; Genentech, Inc., Stock/Shareholder; Neurocrine Biosciences, Inc., Vertex Pharmaceuticals Incorporated. Funding National Institutes of Health (DK120429, DK122607)

Sex, T Cells, and the Microbiome in Natural ABO Antibody Production in Mice.

"Natural" ABO antibodies (Abs) are produced without known exposure to A/B carbohydrate antigens, posing significant risks for hyperacute rejection during ABO-incompatible transplantation. We investigated anti-A "natural" ABO antibodies versus intentionally induced Abs with regard to the need for T-cell help, the impact of sex, and stimulation by the microbiome. Anti-A was measured by hemagglutination assay of sera from untreated C57BL/6 wild-type (WT) or T cell-deficient mice of both sexes. Human ABO-A reagent blood cell membranes were injected intraperitoneally to induce anti-A Abs. The gut microbiome was eliminated by maintenance of mice in germ-free housing. Compared with WT mice, CD4 + T-cell knockout (KO), major histocompability complex-II KO, and αβ/γδ T-cell receptor KO mice produced much higher levels of anti-A nAbs; females produced dramatically more anti-A nAbs than males, rising substantially with puberty. Sensitization with human ABO-A reagent blood cell membranes did not induce additional anti-A in KO mice, unlike WT. Sex-matched CD4 + T-cell transfer significantly suppressed anti-A nAbs in KO mice and rendered mice responsive to A-sensitization. Even under germ-free conditions, WT mice of several strains produced anti-A nAbs, with significantly higher anti-A nAbs levels in females than males. Anti-A nAbs were produced without T-cell help, without microbiome stimulation, in a sex- and age-dependent manner, suggestive of a role for sex hormones in regulating anti-A nAbs. Although CD4 + T cells were not required for anti-A nAbs, our findings indicate that T cells regulate anti-A nAb production. In contrast to anti-A nAbs, induced anti-A production was T-cell dependent without a sex bias.

Abstract B034: A scoping review of carcinogenic potential in prostate cancer from 40+ years of functional vitamin D and K deficiency

Abstract Introduction: Carcinogenesis is a form of accelerated genetic “Darwinian” evolution. Many cells start genetic change. Tobacco tar, an example of extensively researched carcinogen, demonstrates that it takes chronic exposure over 40+ years, accumulating mutations before death with multiple genetic events. Terminal acceleration often includes loss of major histocompatibility (MHC) antigens. Other co-carcinogens, genetic inheritance and chronic inflammation accelerates this process. Experiments, showing that malignant change induced in vitro is much faster than in vivo, suggest that other factors impact on the pace of malignant evolution. Two such factors (DNA repair and immune surveillance) are the best established. As considerable research is targeting DNA repair gene damage, this abstract focuses on latent immunity (less dependent on MHC system than T cell immunity) and its under-performance in carcinogenesis due to Vitamin D and K deficiency. Literature Review terms: Vitamin D(VD), Vitamin K(VK), Cancer, Carcinogenesis, Latent Immunity. Results: Three pieces of evidence that functional long-term VD and VK deficiency could have oncogenic potential were identified. The first was discovery that patients with chronic inflammation had hypermethylation of the VD receptor resulting in failure of the liver to convert stored VD into its active forms. Cancer patients with higher extent of methylation of VD receptor had worse clinical outcomes. Such deficiency is associated with reduced innate immunity due to macrophage dysfunction and this deficiency could increase microbiome dysbiosis. This suggestion is supported by studies linking PCa with 40+ years of persistent infection with the sun-sensitive microaerophilic bacterium Cutibacterium acnes and VD deficiency is linked with increased cancer but limited benefits in VD trials. The second observation is the demonstration that VK2 (Menaquinone) not VK1 (Phylloquinone) deficiency increased risk of PCa. These authors also showed that undercarboxylated osteocalcin, a biomarker of VK2 deficiency, was also linked with risk of PCa. The third observation was evidence from in-vitro breast cancer cell-line studies that Calcitriol plus K2 was synergistic in cytotoxicity assays. Measuring these effects, through the study of the biological age using methylation-based measures PhenoAge and GrimAge because of their relationship between biological aging and cancer, could be useful to monitor in future trials. Conclusions: Combined VD and VK studies have been found safe and beneficial in bone health. This limited review justifies further exploration of this combination in cancer patients. Using serial epigenetic clock scores is justified, given that these clocks are known to improve from diet and exercise interventions. Functional deficiency of VD and VK could be acting as co-carcinogens through enhanced inflammatory process, a precursor in many cancers including those without known carcinogen involvement such as PCa and also involved in accelerating tumour growth after primary treatment. Citation Format: Tim R.D. Oliver, Belinda Nedjai. A scoping review of carcinogenic potential in prostate cancer from 40+ years of functional vitamin D and K deficiency [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B034.

The role of bystander activation among CD8αα T cells in controlling Mycobacterium tuberculosisinfection

Abstract Classical cytotoxic T cells have a CD8 coreceptor with an αβ heterodimer and recognize peptide antigens via major histocompatibility (MHC) molecules. CD8 T cells with an αα homodimer structure are nonclassical and can recognize non-MHC molecules independently of antigen. CD8αα T cells have a higher propensity to undergo T cell receptor (TCR) independent bystander activation via pro-inflammatory cytokines. Bystander activation of CD8 T cells has shown a protective effect during Mycobacterium tuberculosis (M.tb) infection, but the distinct roles of CD8αα and CD8αβ T cells have not been elucidated. To begin assessing this, we compared the levels of CD8αα and CD8αβ T cell proliferation in response to TCR dependent stimulation using anti-CD3/CD28 beads. We observed that CD8αα T cells had lower levels of proliferation compared to CD8αβ T cells after three days of stimulation. After seven days, a population of CD8αα T cells did not proliferate while nearly all CD8αβ T cells showed high proliferative capacity. We next stimulated healthy peripheral blood mononuclear cells with IL-12, IL-15, and IL-18 to induce bystander activation and found 52.8% of CD8αα T cells secreted granzyme B compared to 25.1% of CD8αβ T cells. Interestingly, CD8αα T cells also had higher levels of CD69 expression than CD8αβ T cells (62.6% and 38.2%, respectively). Finally, we present plans for defining the role of bystander activated CD8αα and CD8αβ T cells during M.tb infection using tonsil organoids. These data reveal that CD8αα T cells have a distinct bimodal response to TCR mediated activation as well as an increased cytotoxic and activated phenotype after bystander activation. We further establish a foundation for future bystander activation studies in M.tb infection. Supported by grants from the NIH (R01 AI146072)

Targeting the NKG2A axis with a TCR mimic antibody containing an active Fc domain promotes anti-tumor immunity

Abstract The NKG2A axis is an emerging immune checkpoint. Blocking antibodies to the NKG2A receptor in combination with cancer vaccines and PD-(L)1 antibodies have been found to enhance anti-tumor immunity in murine models and cancer patients. The ligand for the NKG2A receptor is the non-classical major histocompatibility (MHC) class Ib molecule, Qa-1 bin mice (HLA-E in humans), loaded with leader peptides from classical MHC class Ia molecules to form Qa-1 b/Qdm peptide complexes. Here, we explore the therapeutic value of targeting the NKG2A ligand, not done previously, using our recently described single-domain T-cell receptor mimic (TCRm) antibody, EXX-1. Two syngeneic murine tumor models (A20 and CT26) were used to evaluate the therapeutic potential of EXX-1 and its mode(s) of action. The initial study done showed EXX-1 Fc active (mIgG2a) displayed optimal tumor growth inhibition and prolonged survival time in mice compared to a modified form of EXX-1 containing an inactive Fc domain. These findings suggested that in addition to blocking the NKG2A pathway, EXX-1 Fc active antibody likely induces tumor cell death via antibody dependent cellular cytotoxicity (ADCC). Moreover, mice treated with EXX-1 antibody as single agent therapy showed significantly better survival outcomes compared to the cohort of mice treated with blocking antibody to the NKG2A receptor. This implies that there might be potential benefits of targeting the NKG2a ligand over the NKG2A receptor though this will require further investigation. Additionally, combination therapy using EXX-1 Fc active and PD-(L)1 blocking antibodies revealed markedly improved rates of tumor-free mice compared to mice receiving treatment with EXX-1 Fc active or anti-PD-(L)1 antibody alone.

NLRC5 affects diet-induced adiposity in female mice and co-regulates peroxisome proliferator-activated receptor PPARγ target genes

Nucleotide-binding and oligomerization domain containing 5 (NLRC5) is the key transcriptional regulator of major histocompatibility (MHC) class I genes. Recent observations suggest a role for NLRC5 in metabolic traits and in transcriptional regulation beyond MHC class I genes. To understand the function of NLRC5 in metabolic disease, we subjected Nlrc5 -/- mice to high-fat diet (HFD) feeding. Female Nlrc5 -/- mice presented with higher weight gain and more adipose tissue (AT) compared to wild-type (WT) animals. Mechanistically, we demonstrate that NLRC5 enhanced the expression of peroxisome proliferator-activated receptor (PPAR) γ target genes in human cells. We identify Sin3A and negative elongation factor (NELF) B as two novel NLRC5 interaction partners and show that Sin3A partly modulates the synergistic transcriptional effect of NLRC5 on PPARγ. Collectively, we show that NLRC5 contributes to weight gain in mice, which involves transcriptional enhancement of PPARγ targets by NLRC5 that is co-regulated by Sin3A.