Major Histocompatibility Complex Antigens Research Articles

Abstract 7425: UMI-nea: A fast and robust UMI analysis approach to accurately identify and quantify TCR repertoire from targeted RNA sequencing with wide range of input molecules

Abstract The T-cell receptor (TCR) population in humans is comprised of highly diversified heterodimers, regulating the recognition of antigen-major histocompatibility complex. Tremendous TCR sequence diversity is produced by somatic recombination of several TCR gene loci each consisting of multiple gene segments. Next-generation sequencing has enabled comprehensive profiling of the TCR repertoire from different physiological and disease conditions ushering in much interest in using TCR-seq to assess T-cell diversity. However, during NGS library construction and sequencing, errors and enzymatic inefficiencies can compromise the accuracy of the final data, particularly around calling of the VD and VDJ recombined regions and subsequent clonotype assignment. To increase the accuracy of NGS sequencing, Unique Molecular Identifiers (UMIs), consisting of short random nucleotide bases, can be used to mark original molecules in NGS library allowing for error and bias corrections. There are two well studied technical limitations to apply UMIs: 1.) UMI sequences tend to collide when input molecule number is large 2.) UMI sequences are not insulated from PCR and sequencing errors. To address these limitations, many computational approaches had been published. Among them, very few can be used to solve UMI colliding errors and over-simplified error models were implemented for UMI sequencing error handling. Here we report a novel strategy and UMI structure which uses more complex UMIs that is longer and of different length. This results in minimizing UMI collision while maximizing sequencing quality. Our UMI analysis pipeline, “UMI-nea” is able to handle not only substitution errors but also indel errors and UMIs with different lengths. We developed a novel computational framework to parallelly process sequence comparisons to mitigate the elevated computational burden. To account for the varied dispersion of PCR efficiency for different molecules and error bearing UMIs from libraries with different input and with different sequencing depth, we also developed a statistical framework leveraging negative binomial model and single-cell knee plot to set a dynamic threshold for original molecule estimate. We verified UMI-nea with several simulated data and demonstrated that UMI-nea can achieve >99% completeness and homogeneity to recover the original molecule count with various error rates and UMI lengths, outperforming existing tools and methods in comparison. We applied UMI-nea to profile TCR for 8 PBMC samples sequenced on different Illumina platforms with different sequencing depths. We observed >85% reproducibility of clonotype calls on all samples. To test the sensitivity and specificity of UMI-nea, we sequenced pure cell line samples and cell line spike-in samples with different ratios and discovered very high recall and precision rates. Citation Format: Jixin Deng, Jingxiao Zhang, Song Tian, Samuel J. Rulli, Hong Xu, John DiCarlo, Eric Lader. UMI-nea: A fast and robust UMI analysis approach to accurately identify and quantify TCR repertoire from targeted RNA sequencing with wide range of input molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7425.

Tumour immunogenicity goes with the (mitochondrial electron) flow.

Mitochondrial metabolism and electron transport chain (ETC) function are essential for tumour proliferation and metastasis. However, the impact of ETC function on cancer immunogenicity is not well understood. In a recent study, Mangalhara etal. found that inhibition of complex II leads to enhanced tumour immunogenicity, T-cell-mediated cytotoxicity and inhibition of tumour growth. Surprisingly, this antitumour effect is mediated by succinate accumulation affecting histone methylation. Histone methylation promotes the transcriptional upregulation of major histocompatibility complex-antigen processing and presentation (MHC-APP) genes in a manner independent of interferon signalling. Modulating mitochondrial electron flow to enhance tumour immunogenicity provides an exciting new therapeutic avenue and may be particularly attractive for tumours with reduced expression of MHC-APP genes or dampened interferon signalling.

Electrophile Scanning Reveals Reactivity Hotspots for the Design of Covalent Peptide Binders.

Protein-protein interactions (PPIs) are intriguing targets in drug discovery and development. Peptides are well suited to target PPIs, which typically present with large surface areas lacking distinct features and deep binding pockets. To improve binding interactions with these topologies and advance the development of PPI-focused therapeutics, potential ligands can be equipped with electrophilic groups to enable binding through covalent mechanisms of action. We report a strategy termed electrophile scanning to identify reactivity hotspots in a known peptide ligand and demonstrate its application in a model PPI. Cysteine mutants of a known ligand are used to install protein-reactive modifiers via a palladium oxidative addition complex (Pd-OAC). Reactivity hotspots are revealed by cross-linking reactions with the target protein under physiological conditions. In a model PPI with the 9-mer peptide antigen VL9 and major histocompatibility complex (MHC) class I protein HLA-E, we identify two reactivity hotspots that afford up to 87% conversion to the protein-peptide conjugate within 4 h. The reactions are specific to the target protein in vitro and dependent on the peptide sequence. Moreover, the cross-linked peptide successfully inhibits molecular recognition of HLA-E by CD94-NKG2A possibly due to structural changes enacted at the PPI interface. The results illustrate the potential application of electrophile scanning as a tool for rapid discovery and development of covalent peptide binders.

Antigen-specific T cell activation through targeted delivery of in-situ generated antigen and calcium ionophore to enhance antitumor immunotherapy

Recent advances in adoptive T-cell therapy have delivered impressive therapeutic outcomes by instigating enduring anti-tumor responses. Nonetheless, achieving specific T-cell activation remains a challenge due to several factors. Some cancer cells evade T-cell recognition due to the scarcity of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC) presentation. Notably underestimated is the impact of waning T-cell receptor (TCR) expression and the constrained formation of immune synapses (IS) between dendritic cells (DCs) and T cells, impairing T-cell activation. Addressing these complexities, we introduce a pioneering approach featuring the deployment of a gel implant. This implant establishes an on-site antigen reservoir, efficiently targets DCs in lymph nodes, and facilitates calcium ion (Ca2+) delivery. Engineered with controlled swelling, poroelasticity, and resilience, the gel is suitable for surgical implantation. Its ample encapsulation capacity accommodates both photosensitizers and nanoparticles. Upon in situ photothermal irradiation, the gel generates tumor-specific antigens. Furthermore, cationic albumin nanoparticles (cNPs) co-loaded with monophosphoryl lipid A (MPLA) and ionomycin are released, guiding antigens to tumor-draining lymph nodes for DCs maturation. This meticulous process fosters the formation of IS thereby amplifying antigen-specific T-cell activation.

Immune Dynamics Associated with Patient Outcomes Identified By Multimodal Single-Cell Analysis in Multiple Myeloma Patients Receiving Talquetamab Monotherapy

Background: Talquetamab (Tal) is a CD3xGPRC5D bispecific antibody (BiAb) that redirects endogenous T cells to mediate killing of GPRC5D-expressing multiple myeloma (MM) cells. In the recent phase I/II MonumenTAL-1 trial (NCT03399799), Tal showed ≥ 70% response rate when given at the recommended phase 2 doses (RP2D) in heavily pretreated MM patients (Chari A et al., NEJM 2022). However, there is still a need to identify predictive biomarkers and better understand the immune dynamics associated with treatment response and durability with this novel agent. Methods:We identified 30 patients who received Tal monotherapy and had bone marrow (BM) and peripheral blood (PB) samples collected at baseline and PB collected at cycle 3 day 1 (C3D1). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed to understand transcriptional changes and cell surface proteomics. Total cells were isolated by Ficoll separation from PB and BM samples and were then analyzed separately and only samples of good quality were included in the analysis. Samples were filtered for good quality based on total cell count: at least 1000 cells with more than 500 unique molecular identifiers (UMIs) or genes and less than 4-5 standard deviations above median for UMI, genes and mitochondrial transcripts. A total of 60,851 BM cells (15 pts at baseline) and 299,996 PB cells (23 pts at baseline and 17 pts on C3D1) were analyzed. Downstream analysis was performed using the Seurat R package. All patients included in this study had signed consent for an IRB-approved institutional sub-study (GCO # 18-00456). Results: The 30 patients had a median age of 66.2 years and 57% were female. They received a median of 6 prior lines of therapy, with 83% being triple-class refractory and 50% penta-drug refractory. Only 2 patients were previously treated with anti-BCMA CAR-T cell therapy and none were treated with other BiAbs. 43% of patients had high-risk cytogenetics (including t(4;14), t(14;16) and 17p deletion) and 13% had extramedullary disease at time of initiating therapy. Ten patients (30%) received the recommended phase 2 dose (RP2D) of Tal (0.4 mg/kg weekly or 0.8 mg/kg every other week). Patients were followed for a median of 32.5 months and had a median progression-free survival (PFS) of 6.7 months. 22/30 patients (73%) achieved a partial response (PR) or better, and 14 patients had a short PFS of <150 days. There was no association between the prevalence of different immune cell subsets and depth of response, but rather with PFS. In the baseline BM, a longer PFS was associated with a higher percentage of CD8+ cytotoxic T cells (central memory p=0.00031, effector memory p=0.0022, EMRA p=0.014) and a trend towards higher CD4+ cytotoxic T cells (p=0.072). Of note, we did not find the same associations in the baseline PB samples, which may indicate that the PB may not always reflect the tumor microenvironment in the BM. When we next looked at immune changes in the PB of patients with longer PFS, we found that they exhibited higher percentage of NK cells (p=0.0018 for CD56 low, p=0.04 for CD56 high) and Gamma Delta T cells (p=0.04) at C3D1. Gamma Delta T cells are novel players that warrant further investigation, as they only constitute 1-5% of the T cell repertoire yet are involved in early immune response and do not require major histocompatibility complex (MHC) antigen presentation to function. Profiling of activation and exhaustion markers of these immune subsets is underway. Conclusion: Our findings highlight that the baseline BM immune repertoire and the PB immune changes during Tal treatment may predict long term outcomes. Further in vitro mechanistic studies of T cell function and fitness and validation of these results in independent cohorts will be critical to develop these into predictive biomarkers of long-term response to Tal.

Megakaryocytes Direct CD4 + T Cell Responses Via Major Histocompatibility Complex Class II Antigen Presentation

Megakaryocytes (MKs) are rare, large, hematopoietic cells that canonically have the role of producing platelets. Advances in single-cell RNA-sequencing (scRNA-seq) have revealed a previously under-appreciated degree of MK heterogeneity suggesting that both murine and human MKs may perform functions beyond platelet production. Among other inflammatory molecules, these studies identified that select MK subsets express major histocompatibility complex (MHC II) antigen presentation-associated transcripts and display an upregulation of antigen processing pathways. Together, these findings suggest a potential immunoregulatory function of select MK subsets. However, functional validation substantiating these transcriptional signatures and the proposed MK immunoregulatory capacities has lagged, and the expression of MHC II in bone marrow MKs remains controversial. In our studies, we aimed to both quantify and functionally assess immunomodulatory bone marrow MKs. Using flow cytometry and high-resolution confocal imaging, we showed that MHC class II is expressed on ~20% of MKs at various stages of differentiation. Of the cell populations identified, MHC class II expression was observed in CD41+ and vWF+ platelet-biased hematopoietic stem cells (HSCs), c-Kit+CD150+CD41+ MK progenitors, immature CD41+ MKs and mature CD41+ CD42d+ MKs. Intriguingly, we also observed that MHC class II expression was dramatically upregulated, (2.5-fold increase in expression) in mature MKs and MK progenitors of aged mice. Additionally, MHCII expression was significantly correlated with vWF and CD41 expression in long-term HSCs, with 60-70% of MHCII+ HSCs co-expressing both of these markers. To further validate MHC class II expression in the MK lineage, we utilized two unique murine MHCII transgenic reporter models. The first was an MHCII-EGFP knock-in mouse in which endogenous class II product is replaced by GFP, and the second was a Cd74tdT reporter mouse in which the invariant chain (CD74) can be visualized via tdTomato upon MHC class II pathway activation. In both models, approximately 20-30% of MKs were MHCII positive, consistent with antibody staining in wild-type mice. To substantiate the immunoregulatory function of primary murine MKs, we assayed antigen presentation capacity and ability to stimulate T-cells in vitro. We observed that bone marrow derived MKs express basal levels of multiple co-stimulatory receptors including CD80, CD83, CD86, and CD40 in addition to MHC class II. We also observed significant upregulated of these receptors upon TLR stimulation (TLR3, TLR4 TLR9) in a dose dependent manner. Notably, co-culture of MKs with CD4+ T cells resulted in naive T cell activation and proliferation. To verify that these results extended to human MKs, we examined HLA class II expression in MKs differentiated from CD34+ cells obtained from human bone marrow. Comparable with the murine studies, we found that human MKs also upregulated class II pathway molecules, suggesting that human MKs may also engage with the adaptive immune system. Together, our results support the hypothesis that there is an immunomodulatory sub-population of MKs that can be distinguished by MHCII surface expression, engages in class II antigen presentation, and can activate CD4+ T cells to prime adaptive immune responses. Future studies will investigate the in vivo role of these immunomodulatory functions of MK and how they may be exploited to alter T cell differentiation in the context of inflammatory, infectious, or age-associated thrombotic diseases.

Diagnostic significance of blood lymphocyte activation markers in pre-eclampsia.

The adaptive and innate immune system is important in both initiating and preventing functional disorders during pregnancy, one of which is pre-eclampsia. The research aims to conduct the comparative quantification of selected subpopulations of peripheral blood immunoregulatory cells in pregnant women with pre-eclampsia in the third trimester. The marker receptors CD4, CD8, CD95, CD25, and CD27 and the marker antigen HLA-DR were considered. The screening was performed by flow cytometry with dual phenotyping using phycoerythrin- and fluorescein-isothiocyanate-labeled monoclonal antibodies. Data processing consisted in calculating a likelihood value to assess the statistical significance of the difference between the samples. A statistically significant decrease in the subpopulation titer of T and B lymphocytes with marker receptors CD4, CD8, and CD19 was found in pre-eclampsia patients. In the CD4 carrier T-lymphocyte population, there was an increased expression of the CD25/CD95 activation and apoptosis markers. In the CD8 T-killer population, a decreased representation of the CD27/CD25/CD95 markers of differentiation, activation, and apoptosis was deterministic. The expression pattern of the major histocompatibility complex antigen HLA-DR did not change significantly in normality and pathology. The titer of peripheral natural killer cells carrying the CD56 marker increased in patients with various degrees of disease severity, while the number of CD16 natural killer remained at the level of the control group. The research results suggest that a change in the ratio of the above receptors is a diagnostic indicator for pre-eclampsia.

Manipulating mitochondrial electron flow enhances tumor immunogenicity.

Although tumor growth requires the mitochondrial electron transport chain (ETC), the relative contribution of complex I (CI) and complex II (CII), the gatekeepers for initiating electron flow, remains unclear. In this work, we report that the loss of CII, but not that of CI, reduces melanoma tumor growth by increasing antigen presentation and T cell-mediated killing. This is driven by succinate-mediated transcriptional and epigenetic activation of major histocompatibility complex-antigen processing and presentation (MHC-APP) genes independent of interferon signaling. Furthermore, knockout of methylation-controlled J protein (MCJ), to promote electron entry preferentially through CI, provides proof of concept of ETC rewiring to achieve antitumor responses without side effects associated with an overall reduction in mitochondrial respiration in noncancer cells. Our results may hold therapeutic potential for tumors that have reduced MHC-APP expression, a common mechanism of cancer immunoevasion.

Major histocompatibility complex (MHC) antigens polymorphism and alloimmunization study in thalassemia patients with febrile non-hemolytic transfusion reaction (FNHTR).

HLA alloimmunization is one of the most troublesome consequence of regular transfusion which is itself a mainstay measure to provide longevity to the thalassemia patients. Febrile non-hemolytic transfusion reaction (FNHTR) is one of the most common complication which might be related to the HLA alloimmunization. Here, we studied the HLA antigenic system and alloimmunization rate in the Iranian β-thalassemia patients who suffered from FNHTR compare to the β-thalassemia patients without FNHTR. Total of 60 β-thalassemia patients with FNHTR (case group) and 20 β-thalassemia patients without FNHTR (control group) randomly have been selected and enrolled in the study. All were tested for HLA-A and -B loci by PCR-SSP method and also for the presence of anti-lymphocyte antibodies by LIFT method. Comparisons between two groups were performed by Pearson's χ2 test. Totally, a significant predominance was noted for two HLA alleles, HLA-A*24 (P=0.029) and B*55 (P=0.034) which have higher prevalence in control group. Although no significant association was found between the presence of anti-leukocyte antibodies and the development of FNHTR, the HLA-A*32 (P=0.047) allele was considered as possible genetic markers in the susceptibility to the development of anti-leukocyte antibodies. Here some evidences about the possible role of HLA polymorphism in susceptibility to FNHTR are provided. Those results indicated that HLA-A*24 and HLA-B*55 might play protective role on inducing FNHTR in β-thalassemia patients. Further studies which investigate the allele level of HLA-I alongside with specific reactivity of HLA-I antibodies might reveal more deep data about these phenomena.

Abstract LB099: Organoid-based drug efficacy evaluation model for immunotherapy

Abstract The endogenous immune system of patients are known to be activated through immuno-oncology drug, which activates the patient’s own immune system to target and attack tumors. Due to the relatively low occurrence of side effects and sustained success, these therapeutic techniques have gradually replaced the traditional cancer treatment regimen. The creation of an in-vitro screening platform for pre-evaluating the efficacy of immunotherapeutic drug candidates was highly essential despite the fact that many of the new emerging therapies had failed clinical trials.An in-vitro platform mirroring the interaction between a patient's unique Major Histocompatibility Complex (MHC) and T-cell receptor (TCR) is necessary for predicting the precise efficacy of immunotherapies. The tumor cells' MHC antigen molecule attaches to the T-cell receptor (TCR), allowing T-cells to manifest their tumor-killing activities as a result. The link between immunological checkpoint (ICP) in tumor cells and their receptors in T cells allows tumor cells to evade the effects of T-cell-mediated tumor death.We would like to introduce our "ODISEI" platform, a robust efficacy evaluation tool that enables the recapitulation of a patient's unique immune system, utilizing tumor organoids and PBMC from the same donors. Using PD-1/PD-L1 inhibiting antibodies, the enhanced functionality of our ODISEI platform as an effective evaluation platform for immunotherapy drugs was thoroughly examined. Additionally, we created several ODISEI platforms in order to recreate the precise interactions between tumor organoids and various immune sub-populations (T-cells, macrophages, regulatory T-cells, and dendritic cells). Our ODISEI platforms were validated by utilizing a variety of different drug candidates and evaluating their ability to eradicate tumors upon interaction with various reactive immune cells and tumors. As a result, our highly innovative ODISEI platform can be used to screen a variety of different immunotherapeutic drug candidates and distinguish their precise efficacy. Citation Format: Boeun Lee, Woo Kyeom Yang, Sarang Kim, Hee-Ra Lee, Donghyeon Kim, Jongman Yoo. Organoid-based drug efficacy evaluation model for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB099.

Interferons and Resistance Mechanisms in Tumors and Pathogen-Driven Diseases-Focus on the Major Histocompatibility Complex (MHC) Antigen Processing Pathway.

Interferons (IFNs), divided into type I, type II, and type III IFNs represent proteins that are secreted from cells in response to various stimuli and provide important information for understanding the evolution, structure, and function of the immune system, as well as the signaling pathways of other cytokines and their receptors. They exert comparable, but also distinct physiologic and pathophysiologic activities accompanied by pleiotropic effects, such as the modulation of host responses against bacterial and viral infections, tumor surveillance, innate and adaptive immune responses. IFNs were the first cytokines used for the treatment of tumor patients including hairy leukemia, renal cell carcinoma, and melanoma. However, tumor cells often develop a transient or permanent resistance to IFNs, which has been linked to the escape of tumor cells and unresponsiveness to immunotherapies. In addition, loss-of-function mutations in IFN signaling components have been associated with susceptibility to infectious diseases, such as COVID-19 and mycobacterial infections. In this review, we summarize general features of the three IFN families and their function, the expression and activity of the different IFN signal transduction pathways, and their role in tumor immune evasion and pathogen clearance, with links to alterations in the major histocompatibility complex (MHC) class I and II antigen processing machinery (APM). In addition, we discuss insights regarding the clinical applications of IFNs alone or in combination with other therapeutic options including immunotherapies as well as strategies reversing the deficient IFN signaling. Therefore, this review provides an overview on the function and clinical relevance of the different IFN family members, with a specific focus on the MHC pathways in cancers and infections and their contribution to immune escape of tumors.

Membrane-anchored DNA nanojunctions enable closer antigen-presenting cell-T-cell contact in elevated T-cell receptor triggering.

How the engagement of a T-cell receptor to antigenic peptide-loaded major histocompatibility complex on antigen-presenting cells (APCs) initiates intracellular signalling cascades in T cells is not well understood. In particular, the dimension of the cellular contact zone is regarded as a determinant, but its influence remains controversial. This is due to the need for appropriate strategies for manipulating intermembrane spacing between the APC-T-cell interfaces without involving protein modification. Here we describe a membrane-anchored DNA nanojunction with distinct sizes to extend, maintain and shorten the APC-T-cell interface down to 10 nm. Our results suggest that the axial distance of the contact zone is critical in T-cell activation, presumably by modulating protein reorganization and mechanical force. Notably, we observe the promotion of T-cell signalling by shortening the intermembrane distance.

Atomistic basis for the differential load response by the γδ and the chimeric Vγδ-Cαβ T-cell receptors.

αβ and γδ T cells perform vital functions in adaptive and transitional immunity, respectively, and they are both equipped with antigen-sensing T cell receptors. Prototypical recognition of sparse antigenic peptide-loaded major histocompatibility complex molecule (pMHC) by the αβ T-cell receptor (TCRαβ) is assisted by load, through catch bond formation. On the other hand, the contrasting abundance of γδ T cell receptor (TCRγδ) ligands suggests that the recognition may involve a mechanism that is less sensitive to load, as demonstrated in our recent studies. To elucidate structural basis for the load response, we performed a total of 12 µs all-atom molecular dynamics simulations of the 9C2 TCRγδ complexed with the nonclassical MHC molecule CD1d holding a sulfatide lipid antigen JLS (PDB 4LHU). Simulations were performed either without load or with 4.4 to 27.8 pN loads. Contrary to the case of TCRαβ, we did not find any noticeable stabilization of the TCRγδ-CD1d-JLS interface with applied load, supporting an absence of catch bond. We also studied a chimeric Vγδ-Cαβ TCR where the constant domains of PDB 4LHU were replaced with those of N15 TCRαβ (PDB 1NFD). Comparisons between the two systems in terms of interfacial contact dynamics and domain motion reveal how the constant domains can allosterically modulate the interface with the ligand in a load-dependent manner.

Activated autophagy of innate immune cells during the early stages of major trauma.

Trauma-induced immune dysfunction has been a major barrier to achieving reduced mortality, which is poorly understood. Autophagy is a crucial catabolic mechanism of immune cells during times of stress. Few studies have investigated the immune regulatory effects induced by autophagy after trauma. Here, we use single-cell transcriptomics analysis in a major trauma cohort to demonstrate the dominant role of autophagy in innate immune cells during the early stages of major trauma. Single-cell transcriptional profiling of peripheral blood mononuclear cells (PBMCs) was performed, which were sampled from three control participants and five major trauma patients within 6 hours of injury. In detail, after single-cell RNA-sequence data processing, cell type annotation and cluster marker identification were performed. A genetic toolbox with 604 autophagy-related genes was used to monitor the autophagy levels in immune cells. In addition, all transcriptome RNA sequencing data obtained from PBMCs in a cohort of 167 major trauma patients were downloaded from gene expression omnibus (GEO) datasets (GSE36809). Key deregulated biological processes and important autophagic hub genes involved in immune cells were identified by weighted gene co-expression network analysis and gene ontology enrichment analysis. A total of 20,445 differentially expressed genes were identified and five co-expression modules were constructed. Enrichment analysis indicated that activated autophagy is the most important biological process during the early stages of major trauma, and JMY (autophagy-related genes) were identified as hub genes. The single-cell transcriptional profiling of PBMCs demonstrated that all components of adaptive immune cells were significantly decreased, whereas components of innate immune cells (monocytes and neutrophils) were significantly increased in major trauma patients compared with control participants. Activated autophagy was detected in monocytes and neutrophils by monitoring the dynamic transcriptional signature of the autophagy-related genetic toolbox. Biological process analysis shows that antigen uptake, processing presentation, and major histocompatibility complex (MHC) class II protein complex assembly pathways were up-regulated in autophagy-positive monocytes, whereas antigen processing and presentation of endogenous antigen and type I interferon signaling pathways were up-regulated in autophagy-positive neutrophils during the early stages of major trauma. Our study demonstrated that autophagy is a biological process crucial to the development of immune disorders in the early stages of major trauma. Furthermore, the results of our study generated a comprehensive single-cell immune landscape for major trauma patients, in which we determined that autophagy profoundly affects the main functions of innate immune cells and provides insight into the cellular basis of immune dysregulation after major trauma.

A Novel Finding of an HLA Allele's and a Haplotype's Relationship with SARS-CoV-2 Vaccine-Associated Subacute Thyroiditis.

Subacute thyroiditis (SAT) is a thyroid disease associated with viral infections. Its relationship with major histocompatibility complex (MHC) antigens was shown before. SAT cases triggered by different types of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have been reported. In this study, human leukocyte antigen (HLA) genotypes of 27 SAT patients (13 vaccine-associated (V-SAT) and 14 non-SARS-CoV-2-infection non-vaccine-associated (non-V-SAT)) were compared with those of 362 healthy donors. HLA analyses were performed with low-resolution DNA-based sequence-specific oligonucleotide or sequence-specific primer methods. Statistical analyses were performed using IBM SPSS Statistics 25 and Stata/MP 14.1 with the hapipf function. Allele and haplotype frequencies were estimated by PyPop and gene[RATE] tool programs. The allele frequencies of HLA-A*11, HLA-B*35, and HLA-C*04 were higher in the patient groups. Both the allele frequency of HLA-A*11 and the haplotype frequency of A*11-B*35-C*04 were higher in the V-SAT group. The A*11-B*35-C*04 haplotype, including all three loci of MHC class I genes, is shown to be associated with the disease for the first time, especially in the V-SAT group. This finding will contribute to a better understanding of the etiopathogenesis of vaccine-associated SAT and the role of HLA genotypes in the functioning mechanisms of the SARS-CoV-2 vaccines.

The High Mobility Group A1 Chromatin Regulator Drives Immune Evasion during MPN Progression By Repressing Genes Involved in Antigen Presentation

Introduction: Myeloproliferative Neoplasms (MPN) are blood diseases caused by mutations activating JAK2 in hematopoietic stem cells (HSCs) which lead to clonal expansion and overproduction of myeloid lineages. MPN patients are at increased risk for transformation to bone marrow fibrosis (myelofibrosis) and acute myeloid leukemia (AML), both of which are associated with poor clinical outcomes. Importantly, targetable mechanisms to prevent progression remain elusive. The High Mobility Group A1 (HMGA1) gene encodes chromatin regulators which are enriched in hematopoietic stem cells and aberrantly overexpressed in MPN with highest levels after transformation to myelofibrosis and acute leukemia (Li & Kim et al Blood 2022, Xian et al Nature Commun 2017). We recently discovered that HMGA1 is required for progression to leukemia in murine models of JAK2V617F MPN leukemia. Further, loss of just a single Hmga1 allele prevents progression to myelofibrosis in JAK2V617F transgenic mice. Emerging evidence suggests that immune escape represents a fundamental pathway involved in progression in diverse tumors. We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by dysregulating gene networks involved in immune evasion, and, 2) identify mediators of immune escape that could be disrupted in therapy. Methods: To identify gene networks regulated by HMGA1 in JAK2V617F MPN, we performed multi-omics sequencing analyses, including RNAseq, chromatin immunoprecipitation (ChIPseq), and assays of chromatin accessibility (ATACseq) from JAK2V617Fmutant AML cell lines (DAMI, SET-2) + HMGA1 depletion. HMGA1 gene expression was inactivated using CRISPR/Cas9 or short hairpin RNAs (shRNA) targeting 2 different sequences. We used gene set enrichment analysis to dissect molecular mechanisms underlying HMGA1. Transcriptional networks were validated at the level of mRNA and protein via quantitative RT-PCR and flow cytometry. To identify drugs to disrupt HMGA1 immune evasion networks, we applied the Broad Institute Connectivity Map (CMAP) and cytotoxicity assays. To validate these pathways in human MPN, we assessed gene expression in independent MPN patient cohorts. Results: To elucidate transcriptional networks governed by HMGA1 during MPN progression to leukemia, we integrated RNAseq, ChIPseq, and ATACseq in JAK2V617F mutant AML cells (DAMI, SET-2) which revealed that HMGA1 represses genes involved in antigen presentation (Interferon γ response networks), including genes encoding major histocompatibility complex (MHC) class I and II antigens. Silencing HMGA1 via CRISPR or shRNA results in up-regulation of MHC class I and II antigen genes, with greatest induction of MHC class II genes HLA-DRA/DRB1. Similarly, HMGA1 depletion increases cell surface expression of HLA-DR antigens by flow cytometry. Further, inhibiting HMGA1 in JAK2V617Fmutant AML cells results in up-regulating the class II major histocompatibility complex transactivator (CIITA) and invariant chain (CD74), which function in inducing MHC class II gene expression and chaperoning structure assembly, respectively. To identify underlying mechanisms, we intersected ChIPseq with ATACseq, which revealed HMGA1 occupancy downstream of the HLA-DRB1 locus, including regions at +6.27kb, +6.7kb, +14kb, and +15kb from the transcription start site. Further, preliminary results suggest that HMGA1 recruits repressive histone marks to repress HLA-DRB1 at these sites. To target HMGA1 networks, we queried CMAP, which identified the histone deacytelyase inhibitor (HDACi), entinostat. Strikingly, entinostat not only up-regulates MHC class II genes and antigens, but is also cytotoxic in JAK2V617F mutant AML cells. Finally, HMGA1 depletion enhances sensitivity to entinostat, which synergizes with JAK inhibitor, Ruxolitinib, to further induce MHC gene expression. Conclusions: We discovered a novel epigenetic program whereby HMGA1 drives immune evasion during MPN progression by binding to chromatin and modulating transcriptional networks to repress MHC antigen presentation. Most importantly, HMGA1 immune evasion networks are dysregulated in human MPN after progression to AML. Together, our studies reveal a new paradigm whereby HMGA1 down-regulates MHC antigens during MPN progression, suggesting that targeting HMGA1 networks with entinostat could activate an immune attack and prevent MPN progression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transplant immunology I: mechanisms of rejection in solid organ transplants

Non-identical transplants (allogeneic) have been a modern medicine milestone; however, the major pitfall for completed succeed is the recognition of foreign organ by the immune system. The myriad variations in sequence of the molecules of the major histocompatibility complex (MHC) or human leukocyte antigen (HLA) between individuals are the major cause of allograft rejection. Thus, the recognition by the B or T lymphocytes of the receptor occurs in the hypervariable regions of the HLA molecules that triggers to the graft 3 types of rejection: hyperacute, acute, and chronic. In addition, the rejection could be cause antibody-mediated (ABMR), T-cell-mediated (TCMR), or both, which depends on the main branch is cause of rejection. This review will explore the mechanisms mentioned above and give an introductory insight into transplant immunology; thus, preparer the reader to delve into further reviews in a Histocompatibility or Immunogenetics laboratory purpose.

Inverted direct allorecognition triggers early donor-specific antibody responses after transplantation.

The generation of antibodies against donor-specific major histocompatibility complex (MHC) antigens, a type of donor-specific antibodies (DSAs), after transplantation requires that recipient's allospecific B cells receive help from T cells. The current dogma holds that this help is exclusively provided by the recipient's CD4+ T cells that recognize complexes of recipient's MHC II molecules and peptides derived from donor-specific MHC alloantigens, a process called indirect allorecognition. Here, we demonstrated that, after allogeneic heart transplantation, CD3ε knockout recipient mice lacking T cells generate a rapid, transient wave of switched alloantibodies, predominantly directed against MHC I molecules. This is due to the presence of donor CD4+ T cells within the graft that recognize intact recipient's MHC II molecules expressed by B cell receptor-activated allospecific B cells. Indirect evidence suggests that this inverted direct pathway is also operant in patients after transplantation. Resident memory donor CD4+ T cells were observed in perfusion liquids of human renal and lung grafts and acquired B cell helper functions upon in vitro stimulation. Furthermore, T follicular helper cells, specialized in helping B cells, were abundant in mucosa-associated lymphoid tissue of lung and intestinal grafts. In the latter, more graft-derived passenger T cells correlated with the detection of donor T cells in recipient's circulation; this, in turn, was associated with an early transient anti-MHC I DSA response and worse transplantation outcomes. We conclude that this inverted direct allorecognition is a possible explanation for the early transient anti-MHC DSA responses frequently observed after lung or intestinal transplantations.

Molecular Modeling Guided Drug Designing for the Therapeutic Treatment of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory disorder that can cause destructive joint disease, significant disability, and increased mortality. RA is the most frequent of all chronic inflammatory joint diseases, and its prevalence frequency in Pakistan is 1.6 per thousand people. Different cytokines and receptors were involved in the triggering of RA, including interleukin-6 (ILR-6), major histocompatibility complex (MHC) antigen human leukocyte (HLA-DR) receptor, and CD20. Several studies illustrated RA as an inherent immune response and triggered due to the “shared epitope.” Therefore, the involvement of all these receptors (IL-6, HLA-DR, and CD20) leads to the neurological, ocular, respiratory, cardiac, skin, and hematological manifestations that have been considered a potential therapeutic target for drug design. Various herbal, natural, and synthetic source inhibitors of interleukin-6 (IL-6), human leukocyte (HLA-DR), and CD20 were studied and reported previously. Reported inhibitors are compared to elucidate the best inhibitor for clinical trials, leading to the orally active drug. In this study, a computer-aided drug designing approach disclosed the potential inhibitors for all receptors based on their distinct binding affinity. Moreover, drug suitability was carried out using Lipinski’s rule by considering the adsorption, distribution, metabolism, and excretion (ADME) of ligands. Results elucidated “calycosin 7-O-glucoside” and “angeliferulate” as putative ligands for IL-6 and HLA-DR, respectively. However, the pharmacokinetic properties (ADMET) revealed angeliferulate as an effete ligand for the biological system compared to calycosin 7-O-glucoside. Based on docking, drug toxicity profiling or pharmacokinetics, and MD simulation stability, this study highlights orally active therapeutic inhibitors to inhibit the activity of pivotal receptors (IL6, HLA-DR, and CD20) of RA in humans. After clinical trials, the resultant inhibitors could be potential therapeutic agents in the drug development against RA.

Abstract 5632: Combinatorial epigenetic targeting of PRC2 complexes upregulates MHC antigen presentation components in melanoma and gastrointestinal cancer cells

Abstract Low tumor immunogenicity is associated with immune escape, immunotherapy resistance, and poor patient survival [1-3]. Major histocompatibility complex (MHC) antigen presentation pathways (APPs) play a crucial role in promoting tumor T-cell recognition and initiating antitumoral immune responses. However, epigenetic silencing of APPs is a common mechanism in various cancers, including melanoma and gastrointestinal malignancies [3,4]. In the current investigation, mouse melanoma (B16/OVA) and human colon cancer (LOVO and HCT116), small intestine cancer (HUTU80), and normal colonic epithelial (CCD841) cell lines were treated with epigenetic drug candidates. As reported [5], a decrease in histone H3K27 methylation marks via polycomb repressive complex 2 (PRC2) inhibitors, as well as altered histone acetylation status from histone deacetylase 3 (HDAC3)-specific inhibition, increased the expression of APP components, based on immunoblotting and RT-qPCR experiments. Normal colonic epithelial cells showed resistance to the treatments, indicating cancer cell-specific effects of the test agents. In B16/OVA cells, flow cytometry and enzyme linked immunosorbent assays corroborated that HDAC3 and/or PRC2 inhibitor combinations increased cell surface occupancy of MHC-I complexes and increased CD8+ T-cell activation. These findings have implications for the clinical application of next-generation combinatorial epigenetic agents [6,7], targeted towards increased tumor immunogenicity and enhanced efficacy of new immunoepigenetic strategies. Supported in part by NCI grant CA122959, by the John S. Dunn Foundation, and by a Chancellor’s Research Initiative.