T-cell Receptor Gene Research Articles

Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia.

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively. As an alternative to using low thresholds, we show that IG/TR gene rearrangements in subpopulations of cancer cells can be discriminated from background IG/TR gene rearrangements in normal B-and T-cells through a combination of flow cytometry cell sorting and multiple displacement amplification (MDA)-based whole genome amplification (WGA) prior to the NGS. Using this approach to investigate the clonal evolution in a BCP-ALL patient with double relapse, clonal TR rearrangements were found in sorted leukemic cells at the time of second relapse that could be identified at the time of diagnosis, below 1% of the total sequence reads. These data emphasize that caution should be exerted when interpreting rare sequences in NGS experiments and show the advantage of employing the flow sorting of malignant cell populations in NGS clonality assessments.

De novo genome assembly depicts the immune genomic characteristics of cattle

Immunogenomic loci remain poorly understood because of their genetic complexity and size. Here, we report the de novo assembly of a cattle genome and provide a detailed annotation of the immunogenomic loci. The assembled genome contains 143 contigs (N50 ~ 74.0 Mb). In contrast to the current reference genome (ARS-UCD1.2), 156 gaps are closed and 467 scaffolds are located in our assembly. Importantly, the immunogenomic regions, including three immunoglobulin (IG) loci, four T-cell receptor (TR) loci, and the major histocompatibility complex (MHC) locus, are seamlessly assembled and precisely annotated. With the characterization of 258 IG genes and 657 TR genes distributed across seven genomic loci, we present a detailed depiction of immune gene diversity in cattle. Moreover, the MHC gene structures are integrally revealed with properly phased haplotypes. Together, our work describes a more complete cattle genome, and provides a comprehensive view of its complex immune-genome.

Clinicopathologic characterization of cutaneous adult T-cell leukemia/lymphoma: A single tertiary care center experience in the United States.

Adult T-cell leukemia/lymphoma (ATLL) is a rare aggressive T-cell leukemia/lymphoma associated with human T lymphotropic virus type 1 infection. The patients might present with skin rash before, at, or after the diagnosis. The dermatopathologic finding might be diagnostically very challenging. We retrospectively identified 110 patients with ATLL at a single institution in a 19-year period, with 19 patients having skin biopsies. Clinical, dermatopathologic, immunophenotypic, and molecular findings were studied. The cohort included 13 skin-first (5 acute, 5 lymphomatous, 2 chronic, 1 smoldering), 6 skin-second (4 acute, 1 lymphomatous, 1 smoldering), and 91 patients without skin biopsy. Some nonphotoprotected areas of body such as the forearm and lower lip were also seen. Skin manifestations included papular (5), erythroderma (1), nodulotumoral (3), plaques (1), patches (1), and a combination of skin rashes (2). Histopathologic findings included large pleomorphic cells, angiocentrism, epidermal infiltration with large Pautrier-like microabscesses, and folliculotropism. Fifteen (78.9%) cases showed CD4+/CD7-/CD25+. Next-generation sequencing study was conducted on 5 patients using either blood or bone marrow samples, revealing multiple genetic mutations across multiple signaling pathways. Pleomorphic large, atypical cells with CD4+/CD25+/CD7- immunophenotype from a non-"bathing trunk" location, especially in a patient from endemic regions, raise suspicion for ATLL. T-cell receptor gene rearrangement is almost always positive, and the neoplasm usually demonstrates multiple mutations by next-generation sequencing study.

Mini-TCRs: Truncated T cell receptors to generate T cells from induced pluripotent stem cells

Allogeneic Tcell platforms utilizing induced pluripotent stemcell (iPSC) technology exhibit significant promise for the facilitation of adoptive immunotherapies. While mature Tcell receptor (TCR) signaling plays a crucial role in generating Tcells from iPSCs, the introduction of exogenous mature TCR genes carries a potential risk of causing graft-versus-host disease (GvHD). In this study, we present the development of truncated TCRα and TCRβ chains, termed mini-TCRs, which lack variable domains responsible for recognizing human leukocyte antigen (HLA)-peptide complexes. We successfully induced cytotoxic T lymphocytes (CTLs) from iPSCs by employing mini-TCRs. Combinations of TCRα and TCRβ fragments were screened from mini-TCR libraries based on the surface localization of CD3 proteins and their ability to transduce Tcell signaling. Consequently, mini-TCR-expressing iPSCs underwent physiological Tcell development, progressing from the CD4 and CD8 double-positive stage to the CD8 single-positive stage. The resulting iPSC-derived CTLs exhibited comparable cytokine production and cytotoxicity in comparison to that of full-length TCR-expressing T lymphocytes when chimeric antigen receptors (CARs) were expressed. These findings demonstrate the potential of mini-TCR-carrying iPSCs as a versatile platform for CAR Tcell therapy, offering a promising avenue for advancing adoptive immunotherapies.

Genetic alterations and MRD refine risk assessment for KMT2A-rearranged B-cell precursor ALL in adults: a GRAALL study

AbstractKMT2A-rearranged (KMT2A-r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is widely recognized as a high-risk leukemia in both children and adults. However, there is a paucity of data on adults treated in recent protocols, and the optimal treatment strategy for these patients is still a matter of debate. In this study, we set out to refine the prognosis of adult KMT2A-r BCP-ALL treated with modern chemotherapy regimen and investigate the prognostic impact of comutations and minimal residual disease (MRD). Of 1091 adult patients with Philadelphia-negative BCP-ALL enrolled in 3 consecutive trials from the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL), 141 (12.9%) had KMT2A-r, with 5-year cumulative incidence of relapse (CIR) and overall survival (OS) rates of 40.7% and 53.3%, respectively. Molecular profiling highlighted a low mutational burden in this subtype, reminiscent of infant BCP-ALL. However, the presence of TP53 and/or IKZF1 alterations defined a subset of patients with significantly poorer CIR (69.3% vs 36.2%; P = .001) and OS (28.1% vs 60.7%; P = .006) rates. Next, we analyzed the prognostic implication of MRD measured after induction and first consolidation, using both immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements and KMT2A genomic fusion as markers. In approximately one-third of patients, IG/TR rearrangements were absent or displayed clonal evolution during the disease course, compromising MRD monitoring. In contrast, KMT2A-based MRD was highly reliable and strongly associated with outcome, with early good responders having an excellent outcome (3-year CIR, 7.1%; OS, 92.9%). Altogether, our study reveals striking heterogeneity in outcomes within adults with KMT2A-r BCP-ALL and provides new biomarkers to guide risk-based therapeutic stratification.

A significant proportion of classic Hodgkin lymphoma recurrences represents clonally unrelated second primary lymphoma

AbstractDespite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is underinvestigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements was performed in paired cHL diagnoses and recurrences among 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal Ig rearrangements were detected by next-generation sequencing (NGS) in 69 of 120 (58%) diagnoses and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24 of 34 patients (71%). Clonally unrelated cHL was observed in 10 of 34 patients (29%) as determined by IG-NGS clonality assessment and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of >2 years, ∼60% of patients with cHL for whom the clonal relationship could be established showed a second primary cHL. Clonal TCR gene rearrangements were identified in 14 of 125 samples (11%), and TCL-associated gene mutations were detected in 7 of 14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged >50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based Ig/TCR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.

Gastrointestinal ALK-positive anaplastic large cell lymphoma: a clinicopathological analysis of five cases

Objective: To investigate the clinicpathological characteristics of ALK-positive anaplastic large cell lymphoma (ALCL) of the gastrointestinal tract, and to discuss its diagnosis and differential diagnosis. Methods: Five cases of gastrointestinal ALK-positive ALCL diagnosed and treated in Xijing Hospital of the Fourth Military Medical University, between 2011 and 2019 were collected. There were three male and two female patients, aged 5-42 years (mean 25 years). These patients clinically presented with fever and night sweats, weight loss, abdominal pain, abdominal mass, ulcers, bleeding, or intestinal obstruction, and underwent surgical resection of the tumors or endoscopic biopsy. The clinical manifestations, auxiliary examinations, histopathological characteristics, immunophenotypes and genetic alterations were analyzed. Results: In this cohort, one case was common type, two cases were monomorphic variant of common type, and two cases were small cell variant. The tumor cells in all cases expressed ALK, CD30, and one or more T lymphocyte markers, while all the markers of B lymphocyte and plasmacyte were negative. Clonality analysis showed that two cases had clonal T cell receptor (TCR) and immunoglobulin (Ig) gene rearrangement, one case had no clonal TCR but Ig gene rearrangement, and one case had no clonal TCR and Ig gene rearrangements. During the 4 to 67 months' follow-up, two patients died of the disease, two were alive with free of disease and one had a relapse. Conclusions: ALK-positive ALCL of the gastrointestinal tract is extremely rare, and has poor prognosis. Lymphoma originating from this site with CD30 and ALK-positive phenotypes may be considered to be ALCL; however differentiation from other tumors that had anaplastic features, expressed CD30 and or ALK, in particular, ALK positive large B-cell lymphoma is necessary.

Context-dependent T-cell Receptor Gene Repertoire Profiles in Proliferations of T Large Granular Lymphocytes.

T cell large granular lymphocyte (T-LGL) lymphoproliferations constitute a disease spectrum ranging from poly/oligo to monoclonal. Boundaries within this spectrum of proliferations are not well established. T-LGL lymphoproliferations co-occur with a wide variety of other diseases ranging from autoimmune disorders, solid tumors, hematological malignancies, post solid organ, and hematopoietic stem cell transplantation, and can therefore arise as a consequence of a wide variety of antigenic triggers. Persistence of a dominant malignant T-LGL clone is established through continuous STAT3 activation. Using next-generation sequencing, we profiled a cohort of 27 well-established patients with T-LGL lymphoproliferations, aiming to identify the subclonal architecture of the T-cell receptor beta (TRB) chain gene repertoire. Moreover, we searched for associations between TRB gene repertoire patterns and clinical manifestations, with the ultimate objective of discriminating between T-LGL lymphoproliferations developing in different clinical contexts and/or displaying distinct clinical presentation. Altogether, our data demonstrates that the TRB gene repertoire of patients with T-LGL lymphoproliferations is context-dependent, displaying distinct clonal architectures in different settings. Our results also highlight that there are monoclonal T-LGL cells with or without STAT3 mutations that cause symptoms such as neutropenia on one end of a spectrum and reactive oligoclonal T-LGL lymphoproliferations on the other. Longitudinal analysis revealed temporal clonal dynamics and showed that T-LGL cells might arise as an epiphenomenon when co-occurring with other malignancies, possibly reactive toward tumor antigens.

TCR Sequencing in Mouse Models of Allorecognition Unveils the Features of Directly and Indirectly Activated Clonotypes.

Allorecognition is known to involve a large number of lymphocytes carrying diverse T-cell receptor repertoire. Thus, one way to understand allorecognition and rejection mechanisms is via high-throughput sequencing of T-cell receptors. In this study, in order to explore and systematize the properties of the alloreactive T-cell receptor repertoire, we modeled direct and indirect allorecognition pathways using material from inbred mice in vitro and in vivo. Decoding of the obtained T-cell receptor genes using high-throughput sequencing revealed some features of the alloreactive repertoires. Thus, alloreactive T-cell receptor repertoires were characterized by specific V-gene usage patterns, changes in CDR3 loop length, and some amino acid occurrence probabilities in the CDR3 loop. Particularly pronounced changes were observed for directly alloreactive clonotypes. We also revealed a clustering of directly and indirectly alloreactive clonotypes by their ability to bind a single antigen; amino acid patterns of the CDR3 loop of alloreactive clonotypes; and the presence in alloreactive repertoires of clonotypes also associated with infectious, autoimmune, and tumor diseases. The obtained results were determined by the modeling of the simplified allorecognition reaction in inbred mice in which stimulation was performed with a single MHCII molecule. We suppose that the decomposition of the diverse alloreactive TCR repertoire observed in humans with transplants into such simple reactions will help to find alloreactive repertoire features; e.g., a dominant clonotype or V-gene usage pattern, which may be targeted to correct the entire rejection reaction in patients. In this work, we propose several technical ways for such decomposition analysis, including separate modeling of the indirect alloreaction pathway and clustering of alloreactive clonotypes according to their ability to bind a single antigen, among others.

Serological and Molecular Characterization of Hepatitis C Virus-Related Cryoglobulinemic Vasculitis in Patients without Cryoprecipitate.

Prolonged B cells stimulation due to the Hepatitis C virus (HCV) can result in autoimmunity, stigmatized by rising levels of cryoglobulins (CGs), the rheumatoid factor (RF), and free light chains (FLC) of immunoglobulins (Ig) associated with a range of symptoms, from their absence to severe cryoglobulinemic vasculitis and lymphoma. Here, we aimed to identify an immunological signature for the earliest stages of vasculitis when cryoprecipitate is still not detectable. We firstly analyzed the IgG subclasses, FLC, and RF in 120 HCV-RNA-positive patients divided into four groups according to the type of cryoprecipitate and symptoms: 30 asymptomatic without cryoprecipitate (No Cryo), 30 with vasculitis symptoms but without CGs that we supposed were circulating but still not detectable (Circulating), 30 type II and 30 type III mixed cryoglobulinemia (Cryo II and Cryo III, respectively). Our results revealed that patients with supposed circulating CGs displayed a pattern of serological parameters that closely resembled Cryo II and Cryo III, with a stronger similarity to Cryo II. Accordingly, we analyzed the groups of Circulating and Cryo II for their immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements, finding a similar mixed distribution of monoclonal, oligoclonal, and polyclonal responses compared to a control group of ten HCV-RNA-negative patients recovered from infection, who displayed a 100% polyclonal response. Our results strengthened the hypothesis that circulating CGs are the origin of symptoms in HCV-RNA-positive patients without cryoprecipitate and demonstrated that an analysis of clonal IGH and TCR rearrangements is the best option for the early diagnosis of extrahepatic complications.

Anti-myeloma efficacy of CAR-iNKT is enhanced with a long-acting IL-7, rhIL-7-hyFc

AbstractMultiple myeloma (MM), a malignancy of mature plasma cells, remains incurable. B-cell maturation antigen (BCMA) is the lead protein target for chimeric antigen receptor (CAR) therapy because of its high expression in most MM, with limited expression in other cell types, resulting in favorable on-target, off tumor toxicity. The response rate to autologous BCMA CAR-T therapy is high; however, it is not curative and is associated with risks of cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome. Outcomes in patients treated with BCMA CAR-T cells (CAR-Ts) may improve with allogeneic CAR T-cell therapy, which offer higher cell fitness and reduced time to treatment. However, to prevent the risk of graft-versus-host disease (GVHD), allogenic BCMA CAR-Ts require genetic deletion of the T-cell receptor (TCR), which has potential for unexpected functional or phenotype changes. Invariant natural killer T cells (iNKTs) have an invariant TCR that does not cause GVHD and, as a result, can be used in an allogeneic setting without the need for TCR gene editing. We demonstrate significant anti-myeloma activity of BCMA CAR-iNKTs in a xenograft mouse model of myeloma. We found that a long-acting interleukin-7 (IL-7), rhIL-7-hyFc, significantly prolonged survival and reduced tumor burden in BCMA CAR-iNKT–treated mice in both primary and re-challenge settings. Furthermore, in CRS in vitro assays, CAR-iNKTs induced less IL-6 than CAR-Ts, suggesting a reduced likelihood of CAR-iNKT therapy to induce CRS in patients. These data suggest that BCMA CAR-iNKTs are potentially a safer, effective alternative to BCMA CAR-Ts and that BCMA CAR-iNKT efficacy is further potentiated with rhIL-7-hyFc.

A current and future perspective on T cell receptor repertoire profiling.

T cell receptors (TCR) play a vital role in the immune system's ability to recognize and respond to foreign antigens, relying on the highly polymorphic rearrangement of TCR genes. The recognition of autologous peptides by adaptive immunity may lead to the development and progression of autoimmune diseases. Understanding the specific TCR involved in this process can provide insights into the autoimmune process. RNA-seq (RNA sequencing) is a valuable tool for studying TCR repertoires by providing a comprehensive and quantitative analysis of the RNA transcripts. With the development of RNA technology, transcriptomic data must provide valuable information to model and predict TCR and antigen interaction and, more importantly, identify or predict neoantigens. This review provides an overview of the application and development of bulk RNA-seq and single-cell (SC) RNA-seq to examine the TCR repertoires. Furthermore, discussed here are bioinformatic tools that can be applied to study the structural biology of peptide/TCR/MHC (major histocompatibility complex) and predict antigenic epitopes using advanced artificial intelligence tools.

Integrative time-serial networks for genome-wide lncRNA-mRNA interactions reveal interferon-inducible antiviral and T-cell receptor regulations against PRRSV infection

Porcine reproductive and respiratory syndrome virus (PRRSV) infection severely affects the swine industry each year. Although the host mechanisms against PRRSV infection have been identified in key target tissues through whole transcriptome sequencing, specific molecular regulators have not been elucidated. Long non-coding RNA (lncRNA) expression is highly specific and could thus be used to effectively identify PRRSV-specific candidates. Here, we identified novel lncRNAs in lungs, bronchial lymph nodes, and tonsils after PRRSV infection and constructed phenotype-based integrative co-expression networks using time-series differentially expressed (DE) lncRNAs and mRNAs. After the analyses, a total of 309 lncRNA-mRNA interactions were identified. During early host innate signalling, interferon-inducible and interferon genes were positively regulated by specific lncRNA. Moreover, T-cell receptor genes in lung adaptive immune signalling were negatively regulated by specific lncRNA. Collectively, our findings provide insights into the genome-wide lncRNA-mRNA interactions and dynamic regulation of lncRNA-mediated mechanisms against PRRSV infection.

Modeling cell-mediated immunity in human type 1 diabetes by engineering autoreactive CD8+ T cells.

The autoimmune pathogenesis of type 1 diabetes (T1D) involves cellular infiltration from innate and adaptive immune subsets into the islets of Langerhans within the pancreas; however, the direct cytotoxic killing of insulin-producing β-cells is thought to be mediated primarily by antigen-specific CD8+ T cells. Despite this direct pathogenic role, key aspects of their receptor specificity and function remain uncharacterized, in part, due to their low precursor frequency in peripheral blood. The concept of engineering human T cell specificity, using T cell receptor (TCR) and chimeric antigen receptor (CAR)-based approaches, has been demonstrated to improve adoptive cell therapies for cancer, but has yet to be extensively employed for modeling and treating autoimmunity. To address this limitation, we sought to combine targeted genome editing of the endogenous TCRα chain gene (TRAC) via CRISPR/Cas9 in combination with lentiviral vector (LV)-mediated TCR gene transfer into primary human CD8+ T cells. We observed that knockout (KO) of endogenous TRAC enhanced de novo TCR pairing, which permitted increased peptide:MHC-dextramer staining. Moreover, TRAC KO and TCR gene transfer increased markers of activation and effector function following activation, including granzyme B and interferon-γ production. Importantly, we observed increased cytotoxicity toward an HLA-A*0201+ human β-cell line by HLA-A*02:01 restricted CD8+ T cells engineered to recognize islet-specific glucose-6-phosphatase catalytic subunit (IGRP). These data support the notion of altering the specificity of primary human T cells for mechanistic analyses of autoreactive antigen-specific CD8+ T cells and are expected to facilitate downstream cellular therapeutics to achieve tolerance induction through the generation of antigen-specific regulatory T cells.

Changes in Thymic Size and Immunity Are Associated with Bronchopulmonary Dysplasia.

Preterm infants with bronchopulmonary dysplasia (BPD) are at increased risk for dysfunctional immune responses in the postnatal period. This study aimed to verify the hypothesis that thymic function is altered in infants with BPD and changes in the expression of thymic function-related genes affect thymic development. Included in the study were infants who had a gestational age ≤32 weeks and survived to a postmenstrual age of ≥36 weeks. The clinical features and thymic size were comparatively studied between infants with and without BPD. Thymic function and the expression of thymic function-related genes were determined in BPD infants at birth, week 2, and 4 of life. The thymic size was ultrasonographically assessed in terms of the thymic index (TI) and thymic weight index (TWI). T-cell receptor excision circles (TRECs) and gene expression were quantitatively determined by real-time quantitative reverse transcription polymerase chain reaction. Compared to non-BPD infants, their BPD counterparts had a shorter GA, lower birth weight, lower Apgar scores at birth, and were more likely to be of the male gender. BPD infants had an elevated incidence of respiratory distress syndrome and sepsis. TI was 1.73 ± 0.68 versus 2.87 ± 0.70 cm3 and TWI was 1.38 ± 0.45 versus 1.72 ± 0.28 cm3/kg in the BPD group versus the non-BPD group (p < 0.05). In BPD infants, no significant changes were observed in thymic size, lymphocyte counts, and TREC copy numbers at the first 2 weeks (p > 0.05), but they all exhibited a significant increase at week 4 (p < 0.05). BPD infants presented a trend toward increased expression of transforming growth factor-β1 and decreased expression of forkhead box protein 3 (Foxp3) from birth to week 4 (p < 0.05). Nonetheless, no significant difference was found in IL-2 or IL-7 expression at all time points (p > 0.05). For preterm infants with BPD, reduced thymic size at birth might be associated with impaired thymic function. Thymic function was developmentally regulated in the BPD process. · For preterm infants with BPD, reduced thymic size at birth might be associated with impaired thymic.. · BPD infants had an elevated incidence of respiratory distress syndrome and sepsis.. · Thymic function was developmentally regulated in the BPD process..

Detection of mutant antigen-specific T cell receptors against multiple myeloma for T cell engineering

Multiple myeloma (MM) remains an incurable hematological neoplasm. Neoantigen-specific Tcell receptor (TCR)-engineered T(TCR-T) cell therapy is a potential alternative treatment. Particularly, TCRs derived from a third-party donor may cover broad ranges of neoantigens, whereas TCRs in patients suffering from immune disorders are limited. However, the efficacy and feasibility of treating MM have not been evaluated thoroughly. In this study, we established a system for identifying immunogenic mutant antigens on MM cells and their corresponding TCRs using healthy donor-derived peripheral blood mononuclear cells (PBMCs). Initially, the immune responses to 35 candidate peptides predicted by the immunogenomic analysis were investigated. Peptide-reactive T lymphocytes were enriched, and subsequently, TCR repertoires were determined by single-cell TCR sequencing. Eleven reconstituted TCRs showed mutation-specific responses against 4 peptides. Particularly, we verified the HLA-A∗24:02-binding QYSPVQATF peptide derived from COASY S55Y as the naturally processed epitope across MM cells, making it a promising immune target. Corresponding TCRs specifically recognized COASY S55Y+HLA-A∗24:02+ MM cells and augmented tumoricidal activity. Finally, adoptive cell transfer of TCR-T cells showed objective responses in the xenograft model. We initiatively proposed the utility of tumor mutated antigen-specific TCR genes to suppress MM. Our unique strategy will facilitate further identification of neoantigen-specific TCRs.

Elucidating the role of TCR expression in Mycosis Fungoides: A Preliminary Analysis of Skin of Color biospecimens from Howard University Department of Dermatology

Abstract Mycosis fungoides (MF) is the most prevalent Primary Cutaneous T Cell Lymphoma, comprising up to 60% of known cases. It is primarily a disease of resident memory T-lymphocytes that undergo phenotypic changes as a result of several gene alterations including ones coding for T-cell receptor (TCR) expression. The upregulation and downregulation of TCRs may give insight into the complex etiology of MF. There is a clear discrepancy in the morbidity and mortality of MF in the skin of color (SOC) population due to a myriad of factors including delayed diagnoses, biological differences, and limited research. Differences in the physiology of skin of color can cause atypical presentations, often mimicking other pigmentary and inflammatory dermatoses, causing delays in diagnosis and higher mortality. Therefore, further investigation into the molecular physiology of MF is warranted to better understand the disease presentation, progression, and appropriate therapies for SOC patients. Previous studies have identified the most common TCRs present in predominantly White cohorts including TCRVb3, TCRVb5, TCRVb6, TCRVb7, TCRVb20 and TCRVb21. Using MF biopsies from SOC patients at Howard University Dermatology, we analyzed lesional TCR gene expression and compared it to healthy SOC patients with the goal of considering novel therapeutic targets and analyzing how these molecular findings impact the diagnosis and treatment. Preliminary data shows that TRBC1 and TRBC2 are enriched in MF SOC patients whereas TRBV7–8 was enriched in the healthy margins. Because TCRs can be used as a biomarker to identify MF, this knowledge also has the potential to aid in the early diagnosis of MF, thus improving the disparity in outcomes seen in patients of color. Sachi Desse - recipient of the Dermatology Foundation DRSA Jillian M. Richmond - recipient of the Skin of Color Society Research Grant Angel S. Byrd - recipient of the American Skin Association Milstein Research Scholar Award for Melanoma/Non-Melanoma Skin Cancer

Specific Genetic Polymorphisms Contributing in Differential Binding of Gliadin Peptides to HLA-DQ and TCR to Elicit Immunogenicity in Celiac Disease.

Immunogenicity of gliadin peptides in celiac disease (CD)is majorly determined by the pattern of molecular interactions with HLA-DQ and T-cell receptors (TCR). Investigation of the interactions between immune-dominant gliadin peptides, DQ protein, and TCR are warranted to unravel the basis of immunogenicity and variability contributed by the genetic polymorphisms. Homology modeling of HLA and TCR done using Swiss Model and iTASSER, respectively. Molecular interactions of eight common deamidated immune-dominant gliadin with HLA-DQ allotypes and specific TCR gene pairs were evaluated. Docking of the three structures was performed with ClusPro2.0 and ProDiGY was used to predict binding energies. Effects of known allelic polymorphisms and reported susceptibility SNPs were predicted on protein-protein interactions. CD susceptible allele, HLA-DQ2.5 was shown to have considerable binding affinity to 33-mer gliadin (ΔG = -13.9; Kd = 1.5E-10) in the presence of TRAV26/TRBV7. Higher binding affinity was predicted (ΔG = -14.3, Kd = 8.9E-11) when TRBV28 was replaced with TRBV20 paired with TRAV4 suggesting its role in CD predisposition. SNP rs12722069 at HLA-DQ8 that codes Arg76α forms three H-bonds with Glu12 and two H-bonds with Asn13 of DQ2 restricted gliadin in the presence of TRAV8-3/TRBV6. None of the HLA-DQ polymorphisms was found to be in linkage disequilibrium with reported CD susceptibility markers. Haplotypic presentations of rs12722069-G, rs1130392-C, rs3188043-C and rs4193-A with CD reported SNPs were observed in sub-ethnic groups. Highly polymorphic sites of HLA alleles and TCR variable regions could be utilized for better risk prediction models in CD. Therapeutic strategies by identifying inhibitors or blockers targeting specific gliadin:HLA-DQ:TCR binding sites could be investigated.

Abstract 4071: Identification of myeloma-specific T cell receptors by functional single cell interaction analyses

Abstract Innovative immunotherapy approaches such as adoptive transfer of chimeric antigen receptor (CAR) T cells or tumor infiltrating lymphocytes (TILs) have shown great success in the treatment of solid tumors and hematological malignancies. Although treatment of multiple myeloma with CAR T cells can induce deep responses, relapses frequently occur due to antigen escape and limited CAR T cell persistence. TCR-engineered T cells may mediate sustained antitumor effects upon recognition of intracellular targets, thereby significantly increasing the range of relevant target antigens. In our project, we propose to identify T cell receptors (TCRs) specifically targeting autologous myeloma cells. Tumor-reactive T cells were identified using the Berkeley Lights Lightning platform, allowing simultaneous functional analysis of up to 1500 individual T cell/target cell interactions per run. Reactive T cells have been identified upon detection of secreted chemokines (IFNγ, TNFα, IL-2) and by measurement of CD137 surface expression. Tumor-reactive T cells showing various cytokine secretion patterns and CD137 expression profiles could be detected in each myeloma patient (7 to 26 of approx.1200 cells tested per patient). Individual tumor reactive T cells have been isolated for TCR sequencing. Recovered TCR genes will be cloned and overexpressed in autologous T cells for functional validation and analysis of tumor derived neoepitope specificity. In summary, we present a pipeline allowing identification of myeloma-recognizing T cells and recovery of bona fide tumor-reactive TCRs eligible for patient-individualized T cell therapy. Citation Format: Tim R. Wagner, Eren Boğa, Patrick Schmidt, Wolfram Osen, Michael Platten, Hartmut Goldschmidt, Marc S. Raab, Mirco J. Friedrich, Stefan B. Eichmüller. Identification of myeloma-specific T cell receptors by functional single cell interaction analyses. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4071.

Abstract 6547: Features of the TCR repertoire associate with patient’s clinical and molecular characteristics in acute myeloid leukemia

Abstract Allogeneic hematopoietic stem cell transplant (allo-HSCT) remains the most effective strategy for high risk patients with acute myeloid leukemia (AML). Leukemia specific neoantigens when presented by the major histocompatibility complexes (MHCs) are recognized by the T cell receptors (TCR) triggering the graft versus leukemia effect. Unique TCR signature is generated by a complex rearrangement process of an array of variable (V), diversity (D), and joining (J) exons to form a functional receptor with complementary determining regions 3 responsible for the interaction between the TCR and the peptide-MHC. The generated TCR repertoire undergoes dynamic changes with the onset and progression of leukemia and with the course of treatment. To better understand how the TCR repertoire impacts disease progression and patient’s clinical outcome, we leveraged RNA-seq data from The Cancer Genome Atlas (TCGA) to extract the TCR sequences using TRUST4 and MIXCR and examined the association between features of the TCR repertoire in patients with AML and their clinical and molecular characteristics. Data were analyzed by normalizing the number of unique clones to the number of total RNA-seq reads specific for each sample and one minus peripheral blood blast percentage. Both TRUST4 and MIXCR data showed a strong correlation between the number of unique clones for TCRA and TCRB per patient (r=0.944, p &amp;lt;0.001 for TRUST4, r=0.941, p &amp;lt;0.001 for MIXCR). Only 1389 TCRA clones (28.9%; TRUST4: 3450, MIXCR: 2747) and 1327 TCRB (31.3%; TRUST4: 3986, MIXCR: 1584) unique clonotypes were shared by the two computational tools. Nevertheless, we were able to identify the majority of the known TRAV and TRBV segments. Our analysis shows that the normalized number of unique TCRA and TCRB clones were lower in patients carrying FLT3 mutations (TRUST4: TCRA: p=0.013; TCRB: p=0.058; MIXCR: TCRA: p=0.043; TCRB: p=0.030), and higher in patients carrying IDH1 mutations (TRUST4: TCRA: p=0.037; TCRB: p=0.038; MIXCR: TCRA: p=0.037; TCRB: p=0.002) compared with patients carrying the wild type gene. No significant difference was observed for DNMT3A, NPM1, TET2, RUNX1, CEBPA IDH2, NRAS, WT1 and TP53 compared with wild type. Although the normalized number of unique TCR clones was not significantly associated with patient’s overall survival, we found that patients that shared more than five TCRB clones with other patients had an improved overall survival compared with patients that shared less than 5 clones (median OS: 55.4 vs 17 months, p=0.058). Despite the limitations of low TCR gene coverage associated with RNAseq data and the small sample size, we were able to find associations between TCR repertoire and clinical and molecular features in patients with AML using two different computational tools. This study further supports the need for TCR-Seq analyses to provide the depth needed to characterize the TCR repertoire in patients with AML. Citation Format: Mateusz Pospiech, Mukund Tamizharasan, Yu-Chun Wei, Advaith M. Kumar, Mimi Lou, Joshua Milstein, Houda Alachkar. Features of the TCR repertoire associate with patient’s clinical and molecular characteristics in acute myeloid leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6547.