T-cell Receptor Alpha Beta Research Articles

High-Throughput Identification of Graft-Vs-Leukemia TCRs in AML Patient Samples after Allogeneic Stem Cell Transplantation

For patients with intermediate and high-risk AML/MDS, allogeneic stem cell transplantation (aSCT) is a potentially curative therapy. Efficacy is driven by the graft-versus-leukemia (GvL) response, in which donor-derived T cells recognize peptide:MHC antigens presented by leukemia cells, and subsequently kill them. The antigens that drive GvL responses are attractive therapeutic targets for engineered cell therapies and/or biologics. However, only a small number of GvL antigens have been identified, limiting the therapeutic potential of targeted immunotherapies. Deep interrogation of the T cell repertoires of patients who have responded favorably to aSCT presents an opportunity to identify bona fide GvL antigens, but high-throughput methods need to be developed to do this effectively. A popular method to identify antigen-specific T cells is fluorescent peptide:MHC tetramer staining, whereby T cells whose T cell receptors (TCR) bind a particular peptide:MHC antigen become fluorescent and identifiable by flow cytometry. However, a major challenge to identifying GvL T cells using tetramers is that the prediction of immunogenic antigens - that is, those that induce a T cell response in vivo - is poor; studies of solid tumors suggest that only ~1% of predicted MHC-binding neoantigens are immunogenic in patients. In AML/MDS aSCT patients, there are thousands of peptides that are predicted to bind MHCI and represent potential GvL antigens, but due to practical limitations of patient blood and bone marrow sample quantities, only a small subset can be screened by flow cytometry. Other higher throughput methods using heavy-metal tagged peptide:MHC tetramers followed by mass cytometry, or DNA-barcoded tetramers followed by bulk sequencing have been employed to increase antigen screening scale, but these methods are limited in sensitivity and destroy the T cell without providing TCR sequences. To overcome these limitations and rapidly identify GvL T cells in patient samples, we developed a single cell sequencing based method that enables the screening of thousands of peptide:MHC antigens simultaneously, using a single patient sample. We generated peptide: MHCI monomers using UV-mediated peptide exchange and generated large panels of combinatorial DNA-barcoded peptide:MHCI tetramers at microscale. We integrated our methodology into the 10X Genomics Single Cell Immune Profiling workflow to enable simultaneous recovery of TCR antigen-specificity, paired TCR alpha beta sequences, and single cell RNA-Seq data. These methods were used to screen post-transplant AML patient blood and bone marrow, and we identified expanded GvL T cell clonotypes that target novel minor histocompatibility antigens, overexpressed “aberrant self” proteins, and other leukemia associated antigens. We generated primary CD8 T cells that retrovirally express these TCRs and demonstrate that they are potent and have the expected antigen specificities. The methods described are highly sensitive, specific, efficient, and scalable, and enable high confidence identification of antigen specific clonotypes in patient samples. They markedly expand the throughput compared to existing methods, and simultaneously yield paired alpha beta TCR sequences, facilitating a full characterization of the antigen-TCR drivers of GvL. Furthermore, these methods have broad utility beyond SCT, as they are applicable to studying T cell responses to other immune settings, including anti-cancer, autoimmune, and anti-microbial responses.

An integrated cytomorphology and sequencing analysis for hepatosplenic alpha/beta T-cell lymphoma.

A 47-year-old male with a past medical history of diabetes mellitus under the control of oral hypoglycemic medications developed nausea and intermittent fever for weeks. Physical examination showed splenomegaly (4 cm below the costal margin) without significant lymphadenopathy. Laboratory data showed pancytopenia (Hemoglobin: 11 g/dl, White blood cells: 1.53 k/μl, Platelet: 47 k/μl), elevations of liver function tests (totalbilirubin: 1.38 mg/dl, direct bilirubin: 0.65 mg/dl, alanine transaminase (ALT): 56 U/L, aspartate transaminase (AST): 103 U/L, gamma-glutamyl transferase (GGT): 515 U/L, alkaline phosphatase (ALP): 260 U/L) and serum lactate dehydrogense (LDH) level (311 U/L, 1.1X). Flow cytometry of peripheral blood revealed that the abnormal lymphoid cells accounted for 4.3% of total nucleated cells, larger-than-normal lymphocytes, with positive expression of CD2, CD3, CD7, CD8, CD56 and negative for CD5 and CD45. Bone marrow (BM) aspirate revealed abundant lymphoma cells, med- to large-sized, with pleomorphic nuclei, prominent nucleoli, and coarse cytoplasmic granules (Figure 1). BM biopsy showed hypercellularity and the CD3+ lymphoma cells were negative for CD45, CD34, CD117, TdT, CD79a, and Epstein-Barr virus-encoded RNA (EBER) staining, with the pattern of sinusoidal infiltration (Figure S1). BM flow cytometry gating on the lymphoma cells confirmed the positive expression of CD2, CD3, CD7, CD8, CD56, T-cell receptor (TCR) alpha-beta chains, perforin, granzyme B, and negative for CD34, CD4, CD5, and TCR gamma-delta chains (Figure 2), consistent with natural killer (NK)/T lymphoma involvement. Cytogenetic analysis of the bone marrow was normo-karyotypic. Fluorodeoxyglucose-positron emission tomography (FDG-PET) scans revealed hypermetabolism in hepatosplenomegaly (maximum standardized uptake value (SUVmax) = 7.72 in spleen), bilateral cervical nodes, right supraclavicular nodes, right paratracheal nodes (SUVmax = 4.64), L4 (SUVmax = 5.03) and T9 vertebral bodies (SUVmax = 6.3) (Figure 3). Next-generation sequencing of BM TCR gamma gene VJ-recombination was indicative of monoclonality of T cells (59.52% of total reads) (Figure 4). This hepatosplenic T-cell lymphoma, alpha-beta type, Ann Arbor stage IV was thus diagnosed. He received three cycles of chemotherapy L-CHOEP (L-asparaginase, cyclophosphamide, doxorubicin, vincristine, etoposide, prednisolone) followed by a myeloablative matched sibling peripheral stem cell transplant. He remained free from relapse 14 months post-transplant with a normal hemogram and normal-metabolic FDG-PET scans. We acknowledge the service provided by the Department of Pathology, National Taiwan University Hospital. The authors declare no conflict of interest. We obtained informed consent from the patient. We have IRB approval from Research Ethics Committee A National Taiwan University Hospital with IRB number: 201910033RSB (Case number: 01–0019). Data are available in the article's Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Tetraploid Ancestry Provided Atlantic Salmon With Two Paralogue Functional T Cell Receptor Beta Regions Whereof One Is Completely Novel.

Protective cellular immune responses have been difficult to study in fish, due to lack of basic understanding of their T cell populations, and tools to study them. Cellular immunity is thus mostly ignored in vaccination and infection studies compared to humoral responses. High throughput sequencing, as well as access to well assembled genomes, now advances studies of cellular responses. Here we have used such resources to describe organization of T cell receptor beta genes in Atlantic salmon. Salmonids experienced a unique whole genome duplication approximately 94 million years ago, which provided these species with many functional duplicate genes, where some duplicates have evolved new functions or sub-functions of the original gene copy. This is also the case for T cell receptor beta, where Atlantic salmon has retained two paralogue T cell receptor beta regions on chromosomes 01 and 09. Compared to catfish and zebrafish, the genomic organization in both regions is unique, each chromosomal region organized with dual variable- diversity- joining- constant genes in a head to head orientation. Sequence identity of the chromosomal constant sequences between TRB01 and TRB09 is suggestive of rapid diversification, with only 67 percent as opposed to the average 82-90 percent for other duplicated genes. Using virus challenged samples we find both regions expressing bona fide functional T cell receptor beta molecules. Adding the 292 variable T cell receptor alpha genes to the 100 variable TRB genes from 14 subgroups, Atlantic salmon has one of the most diverse T cell receptor alpha beta repertoire of any vertebrate studied so far. Perhaps salmonid cellular immunity is more advanced than we have imagined.

Abstract LB094: First of its kind T cell receptors and functional proteomics detected from the same single cells to advance cancer immunology discovery

Abstract T cell receptor repertoire diversity and T cell cytokine production are the important attributes for the assessment of effective T cell immunity against cancer and pathogens, yet existing single-cell genomics platforms cannot interrogate TCR sequences and proteomic cytokines simultaneously. IsoPlexis’ functional proteomics has demonstrated the values in cell therapy and cancer immunology against blood cancers and solid tumors. The ability to connect the T cell receptor diversity to the most potent single cells enables a wide variety of applications for tumor infiltrating lymphocytes, personalized neoantigen TCR’s, cancer immunology in terms of understanding antigen specificity, as well as self-potency. IsoPlexis single-cell TCR Duomic platform provide a unique measurement for connecting T cell receptor sequences to those T cell cytokine profiles, for the first time. Human PBMC-derived T cell line established from healthy donor was stimulated with PMA/ionomycin (2ul/ml, Biolegend) for 6hrs, stained and resuspended in Reverse transcription premix containing reverse transcriptase with buffer and protein phosphatase inhibitor at 1 x 106/ml and then loaded on Duomic chips. Ran on IsoLight overnight, cDNA was collected from the chip followed by cleanup and amplification, proceeded with TCR amplification and sequencing on NextSeq 1000/2000. Proteomic data from single cells was automatically generated and analyzed by IsoSpeak software. The single-cell TCR sequencing data on the IsoPlexis TCR Duomics platform shows that 78.9% cells have TCR alpha chain recombination and 94.4% cells have TCR beta chain recombination. Totally 77.7% cells have combination on both alpha and beta chain. VDJ Usage Plot further displays the top10 most common V (D) J combination flow between T cell receptor alpha chain and beta chain among the single cells, revealing the diversity of TCR repertoire at the single-cell level. In addition, the proteomics data identifies the dominant proteins among the single cells are Granzyme B, GM-CSF, IFN-g and TNF-a. Furthermore, we define 3 distinct cell clusters based on functional proteins and an association of VDJ distribution with these cell clusters, suggesting the occurrence of V(D)J sequences in the most common reconstructed alpha and beta sequence. IsoPlexis single-cell TCR Duomics platform, for the first time, enables simultaneous profiling of functional proteomics and TCR repertoires/TCR clonotypes as well as identification of specific TCR V(D)J combination with functional cell clusters across the same individual T cells. The combination of these two analytes at the single cells provides a key new modality of tracking the most potent and also the antigen specific immune cells together to fight cancer and infectious disease, for the first time. Citation Format: Jennifer Yang, Jing Zhou, Sean Mackay. First of its kind T cell receptors and functional proteomics detected from the same single cells to advance cancer immunology discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB094.

Prediction of immunodominant CD4+ SARS-CoV-2 epitopes with TCR repertoire sequencing data

Abstract The T cell receptor (TCR) is a hypervariable molecule defining the specificity of T cells for peptide-MHC complex. Defining exact epitopes is crucial to profile T cell responses in COVID-19 and yet very few MHC-II restricted SARS-CoV-2 epitopes are currently known. Here, we propose a reverse epitope discovery technique, which, instead of using large pools of peptides to identify reactive T cells, utilizes TCR repertoire sequencing data as the means to predict immunodominant epitopes. The core idea of the approach is to combine information from large, publicly available TCR repertoire datasets: TCRbeta repertoires from cohorts of COVID-19 patients and controls, and paired TCR repertoires of single T cells activated by SARS-CoV-2 peptides. Our pipeline allows us to predict the HLA-restriction of public SARS-CoV-2 specific TCR clonotypes, which in turn allows us to predict binding to a specific peptide. We applied this approach to single cell TCR repertoires of CD4+ T cells from COVID-19 patients, and predicted six MHC-II restricted immunodominant epitopes and alpha-beta TCR motifs recognising them and tested our predictions experimentally. We further applied this technique to bulk TCRalpha repertoires from T follicular helper cells from draining lymph nodes of healthy donors after BNT162b2 mRNA vaccination. We found that the DPB1*04 restricted S167–180 epitope is responsible for the largest public CD4+ response, and recognition of this epitope is driven by a semi-invariant TCR alpha chain. This finding led to generation of the DPB1*04 S167–180 tetramer, allowing to track SARS-CoV-2 specific CD4 cells in peripheral blood and lymph node samples with flow cytometry. This work was partially supported by R01AI136514 grant. This work was partially supported by R01AI136514 grant.

Graft-Vs-Host Disease (GvHD) Prophylaxis with Post-Transplant Cyclophosphamide (PTCy) and Thymoglobulin (ATG) Are More Important Determinants of Cytomegalovirus (CMV) Reactivation after Allogeneic Hematopoietic Cell Transplantation (HCT) Than Ex-Vivo T-Cell Depletion in the Era of Pre-Emptive Therapy

Background:Cytomegalovirus (CMV) is a common cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT) despite major advances in diagnostic techniques and antiviral prophylactic strategies. The relative impacts of donor/recipient CMV serologic status, disease-specific and transplant-related prognostic factors on the risk of CMV reactivation and survival are undefined.Methods:We studied the outcome of 199 patients (median age, 46 years; range 17-71 years) receiving allogeneic HCT at National University Cancer Institute of Singapore (NCIS) between January 2016 and December 2020. Their hematologic diseases included AML (n=92), ALL (n=46), MDS (n=19), lymphomas (n=19), MPN (n=7) and others (n=16) such as refractory myelomas and aplastic anemias. The conditioning regimens used were either myeloablative (n=80) or reduced intensity conditioning (n=119) prior to an allograft from different donor sources. T-cell depletion (TCD) was used for GVHD prophylaxis in 124 patients; and this included post-transplant cyclophosphamide (PTCy, n=31), ex-vivo T-cell receptor alpha-beta / CD45RA depletion (TCRab/CD45RA) (ex-vivo TCD, n=31) for haploidentical HCT, or thymoglobulin (ATG, n=62) for matched unrelated donor (MUD) HCT.Results:With a median follow-up duration of 15.6 months (range, 0.2-63.6 months), 136 (68.3%) patients had CMV reactivation (median onset, 27.5 days) while 6 (3.0 %) patients developed clinically significant CMV disease, such as colitis, retinitis and encephalitis. The cumulative incidences of CMV reactivation within the first 100 days among the recipients of matched unrelated donor (MUD) (n=60), mismatched related donor or unrelated donor (MMRD/MMUD) (n=60), umbilical cord blood (UCB) (n=18) and matched related donor (MRD) (n=61) HCT were 71.6 %, 61.7 %, 50.0 % and 32.7 %, respectively (p<0.001).There were no statistically significant differences in overall survival (OS, p=0.830) and disease-free survival (DFS, p=0.983) at 5 years between CMV-seropositive (D+/R+ or D-/R+, n=181) and CMV-seronegative recipients (D-/R- or D+/R-, n=18). There were also no significant differences in the cumulative incidences of CMV reactivation within 100 days (p=0.879), CMV end-organ disease (p=0.522) and non-relapse mortality (NRM, p=0.202), respectively. HCT-CI score of ≥1 (p=0.005) and the use of reduced intensity conditioning regimen (p<0.001) were associated with a higher NRM at 2 years. There was also a trend towards higher NRM among patients with peak CMV DNA titers of above 1000 IU/ml, but this did not reach statistical significance (p=0.188).The secondary objective of this study was to determine the risk factors associated with CMV reactivation within the first 100 days post-transplant. There was no statistically significant impact of the donor or recipient CMV serostatus (p=0.790) on the risk of CMV reactivation. In multivariable analysis, the use of any T-cell depletion (p<0.001) was a significant predictor of CMV reactivation. In a subset analysis comparing the 3 different methods of TCD, the use of ATG (p=0.004) and PTCy (p=0.005) was found to be associated with an increased risk of CMV reactivation, but not in patients receiving ex-vivo TCD (p=0.184) (Figure 1). Notably, patients receiving ex-vivo TCD haploidentical HCT was not associated with a higher risk of CMV reactivation as compared to the recipients of MRD HCT without any TCD.Conclusions:Our study concluded that CMV serologic status did not affect the incidence of CMV reactivation, NRM, OS and DFS in patients undergoing allogeneic HCT. The use of PTCy and ATG for GVHD prophylaxis, remains the most important risk factor for CMV reactivation in the era of pre-emptive therapy and hence, the need for aggressive prevention strategies in this vulnerable group of patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Recurrent Fevers and Painful Subcutaneous Nodules: A Case of Subcutaneous Panniculits-like T-Cell Lymphoma

Background: Subcutaneous Panniculitis-Like T-Cell Lymphoma is a cytotoxic T-cell lymphoma that infiltrates subcutaneous adipose tissue and rarely involves the lymph nodes. SPTCL accounts for less than 1% of non-Hodgkin’s lymphomas. Lesions are commonly localized to the trunk and lower extremities and may be relapsing and remitting in nature. Systemic symptoms such as fever and night sweats are common. SPTCL may closely mimic cellulitis, and other causes of skin and soft tissue infections. Definitive diagnosis is made with tissue biopsy showing atypical lymphocytes rimming adipocytes. There is no current standard of treatment. Prognosis is favorable with a 5-year survival rate as high as 91%. Case Report: A 40-year-old Chinese female presented with a two-week history of recurrent fevers and painful subcutaneous nodules and plaques, initially thought to be infectious in nature. Tissue sampling demonstrated CD4+ and CD8+ T-cells rimming adipocytes with a high Ki-67 proliferation index and an alpha beta T-cell receptor subtype. A diagnosis of SPTCL was established and the patient was treated with multiagent chemotherapy regimen consisting of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP therapy), which resulted in resolution of her constitutional symptoms and cutaneous. Conclusion: SPTCL is a challenging diagnosis due to the wide array of etiologies of panniculitides and its close clinical resemblance to soft tissue infection. Early tissue biopsy should be employed to expedite management.

Analysis of Genomic Landscape of Large Granular Lymphocyte Leukemia Reveals Etiologic Insights

Introduction: Large Granular Lymphocyte (LGL) leukemia is a rare lymphoproliferative disorder characterized by clonal expansion of either CD3+ cytotoxic T cells expressing T cell receptor (TCR) alpha beta or CD3- natural killer (NK) cells. A less frequent CD3+ T cell subtype expresses TCR gamma delta (GD). Here, we present the molecular landscape of known LGL types (T, NK, GD) from analysis of the largest patient cohort assembled to date. An integrative analysis of genomic datasets from all LGL subtypes is necessary to more precisely define the shared and unique etiology of this rare disorder. Methods: We collected paired saliva and PBMC samples and related clinical information from 116 LGL leukemia patients after informed consent. The assembled cohort consisted of 93 T-LGL, 11 NK-LGL, and 12 GD T-LGL patients. Genomic analyses were performed on the leukemic (PBMC) and germline (saliva) samples after whole exome sequencing (WES) and transcriptome sequencing (RNAseq, PBMC only). Results: Somatic mutations were detected in the previously described potential drivers STAT3 (n=56), TNFAIP3 (n=9), and PIK3R1 (n=4). We also identified somatic mutations in CDH8 (n=3) and CCL22 (n=4), which we postulate as putative drivers based on mutational clustering. CDH8 was mutated in all three LGL subtypes, but CCL22 somatic mutations were only observed in NK-LGL patients. We observed that STAT3 and CCL22 together account for 64% (7/11) of NK-LGL cases. STAT3 is the most recurrently mutated gene in LGL leukemia, yet concurrent molecular and clinical features are incompletely defined. Interestingly, patients with STAT3 mutations have a higher mutational burden (P=0.0006) compared to those with wild-type (WT) STAT3. This effect is independent of the age of the patient, which correlates with the mutational burden (R=0.26, P=0.0039) and agrees with the finding that the dominant mutational signatures in this cohort exhibit clock-like properties. We also observed that patients with STAT3 mutations are enriched (P=0.0273) for additional mutations in chromatin modifier enzymes such as KMT2D, TET2, DNMT3A, and SETD1B (Figure 1). We found that ~10% of the samples exhibit broad somatic copy-number aberrations, and a patient with somatic mutations in STAT3 and KMT2D displayed high-level microsatellite instability. STAT3 mutations were also significantly associated with increased expression of genes involved in apoptosis, complement activation, and interferon cytokine signaling compared to STAT3 WT (FDR &amp;lt; 0.05). Early-onset LGL patients with age 51 years or less (n=28), as defined by the bottom quartile of the cohort, displayed no differential enrichment of the somatic driver genes. Interestingly, the age of the patient was significantly associated with absolute neutrophil counts (ANC) (P = 0.0068), with younger patients exhibiting lower neutrophil counts, even after adjusting for the presence of STAT3 mutation, as it is associated with lower ANC. As neutropenia is a hallmark feature of LGL leukemia and often a trigger for initiating therapy, the association of young age with lower neutrophil counts and lower somatic mutational burden suggests other mechanisms may be involved. Focusing on the germline variants, we found that 17 of the patients (14.6%) had at least one pathogenic or likely pathogenic germline variant with known oncogenic association as annotated using CharGer. 5 patients had pathogenic mutations in known tumor suppressors including FANCC (n=1), BRCA1 (n=1), PALB2 (n=1), MUTYH (n=1), and SDHA (n=1), while 1 patient had pathogenic mutations in ALK, a known oncogene. Conclusions: We report on the genomic analyses done on whole exome and RNA-seq data from the largest cohort assembled for LGL leukemia to date. We show that the presence of STAT3 mutation is significantly associated with an increase in mutation burden and additional somatic mutations in chromatin modifiers, hinting at potential pathogenic mechanisms within STAT3 mutated patients. By combining LGL subtypes in our analysis, we were able to identify CDH8 as a putative driver that is present in T, NK, and GD subtypes. Additionally, we report CCL22 mutations specific to NK-LGL leukemia, however did not detect any subtype specific mutations in GD T-LGL. We found that about 15% of the patients carry at least one pathogenic germline variant with known oncogenic associations. These findings highlight emerging etiologic insights into this rare disorder. Disclosures Feith: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Loughran:Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees.

Abstract B62: Evaluation of systemic RNA-based cancer vaccine induced T-cell responses via mouse T-cell receptor (TCR) profiling

Abstract Identification of biomarkers is pivotal towards the proper development of cancer immunotherapies, in particular for designing the most effective regimens and identifying patient subgroups most likely to benefit from a given treatment. T-cell receptor (TCR) repertoire emerged as a promising biomarker in this respect, as it allows measurement of the dynamics of the T cell response both in time and in different compartments. In this work, we developed and evaluated an NGS-based mouse TCR profiling method (TCR alpha and TCR beta) for discovery and validation of biomarkers related to the antitumoral T-cell response in different compartments upon systemic administration of an RNA-based cancer vaccine (RNA-lipoplex). The in-house established mouse TCR profiling protocol was able to detect blood-spiked known-TCR sequences down to a frequency of 0,001% spiked cells for both TCR alpha and TCR beta chains and the relative frequencies of detected TCRs in the tumor correlated well with the degree of T-cell infiltration into the same tumors assessed by flow cytometry. Comparative analysis of changes in the TCR repertoire between blood and tumor upon repetitive vaccination with RNA-lipoplexes encoding immunodominant epitope of chicken ovalbumin (OVA257-264) revealed that richness as well as diversity of endogenous TCRs were generally lower in B16-OVA tumors compared to blood and decreased with further vaccination. Interestingly, top clones overlapped increasingly in blood and tumor until after the second vaccination, but drifted apart after the third vaccination, with the TCR repertoire in the tumor narrowing down. This overlap was less evident when mice were inoculated with B16-F10 tumors. Top clones within the same compartment were found to be highly variable between individual animals, complicating comparative analyses. Monitoring intra-individual TCR repertoire changes in the blood of a given mouse over time, however, indicated a tendency for decreasing richness and diversity following a single RNA vaccination, which may be pointing at repertoire adaptation and antigen focus. Supporting this notion, tumors isolated at late time-points exhibited a lower diversity than those investigated at earlier time-points and at baseline. In contrast to RNA vaccination, richness and diversity were reduced in response to treatment with an agonistic anti-CD40 antibody in blood and spleen but were similar among tumors. Consequently, richness and diversity in the periphery may not necessarily be predictive of corresponding repertoire changes in the tumor microenvironment. Citation Format: Lena Mareen Kranz, Barbara Schrörs, Thomas Bukur, Christian Albrecht, Lorieta Leppin, Martin Suchan, Marta Faryna, Eileen Powalsky, Rainer Hipfel, Özlem Türeci, Ugur Sahin, Mustafa Diken. Evaluation of systemic RNA-based cancer vaccine induced T-cell responses via mouse T-cell receptor (TCR) profiling [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B62.

Conformational Dynamics of T-Cell Receptor Chassis for Mechanosensing

AlphaBeta T-cell receptors (TCRαβ) are the major components of the adaptive immune system, sensing foreign antigens displayed on the surface of mammalian host cells altered by infection or other transformations with a remarkably high signal to noise ratio. Despite their high sensitivity, the affinity of each to the antigen peptide-bound major histocompatibility complex (pMHC) is low in thermal equilibrium, typically in the micromolar range. An emerging concept that reconciles the conundrum of high sensitivity versus low affinity is that the TCRαβ functions as a mechanosensor. Mechanical load on the TCRαβ-pMHC complex enhances the bond lifetime via activation of a catch bond pathway, a mechanism which is largely uncharacterized in structural terms. Past studies focused on behaviors of contacts directly involving TCRαβ and pMHC surfaces. However, such localized interactions alone cannot account for the catch bond since there is an allostery that modulates the catch bond behavior through TCR subdomains distal to the TCRαβ-pMHC interface. Thus, understanding of the conformational behavior of the entire TCR protein is required. Here we perform atomistic molecular dynamics simulations of TCRαβ as well as TCR gamma-delta (TCRγδ), receptors of cognate and innate T-cell immunity subsets, respectively. We also study a TCRγδ-αβ chimera that has the antigen-binding variable domains of TCRγδ with the membrane-proximal TCRαβ constant domains. By building ball-and-stick models of TCRs based on simulation trajectories, we examine relative motions of the four subdomains of each TCR type. We find that interdomain motion affects the organization of the pMHC-binding complementarity determining loops. These results suggest a TCR catch bond mechanism whereby an applied load alters the conformational dynamics of the subdomains and in turn, controls the TCR-pMHC interaction including resultant bond lifetime.

Abstract 3567: Novel approaches to high-affinity TCR isolation for clinical translation enabled by single cell RNA sequencing

Abstract The success of T cell therapy for treatment of leukemia and melanoma provides rationale to pursue similar approaches for other cancers, particularly solid tumors. Merkel cell carcinoma, a highly aggressive skin cancer, is a model tumor for developing immunotherapies, as it is immune sensitive and most cases require obligate expression of conserved viral antigens. Like many solid tumor antigens, Merkel cell polyomavirus (MCPyV) oncoproteins are intracellular and thus not accessible to CAR-T cells, but can be targeted with T cell receptor (TCR) based therapy. TCRs for therapeutic translation for cellular immunotherapy must have sufficient affinity to bind the limited number of peptide-MHC molecules commonly expressed on tumor cells. Isolating high affinity natural TCRs offers a safety advantage to mutating lower affinity TCRs since the former TCRs have undergone selection in the thymus. However, identifying such TCRs by limited dilution cloning has been constrained not only by the time and effort required but also by the rarity of the highest affinity clones. Single cell RNA sequencing techniques allow for TCR identification from tiny, clonally diverse samples. We hypothesized this technique would be useful for isolating high-affinity TCRs from the peripheral repertoire of healthy donors or tumor bearing patients and allow for rapid clinical translation. We derived TCRs specific for the HLA-A*0201 restricted MCPyV viral epitope KLLEIAPNC from two sources: 1) the peripheral repertoire of 6 healthy HLA-A*02:01 donors stimulated 3 times in vitro with KLL peptide, and 2) polyclonal MCPyV-specific T cells that were adoptively transferred in a previous clinical trial and shown to localize to tumor and mediate a durable complete remission in a 60-year-old man with metastatic MCC. A total of 20,000 T cells were loaded onto the 10x Genomics 5' V(D)J scRNAseq platform; 14,022 cells (70.1%) were recovered, of which 11,417 (81% of recovered cells) yielded productive TCR alpha-beta pairing. Recovery was similar between healthy peripheral repertoire and patient TIL sources. TCR gene segments were codon optimized and assembled into a PRRLSIN lentiviral vector that is currently in clinical use. Initial screening in TCR-transduced cells revealed 5 TCRs that strongly bind KLL-pMHC tetramer independent of the CD8 co-receptor, including TCRs identified from both healthy donors and the immunotherapy responder. The candidate TCRs selected for further pre-clinical analysis produced inflammatory cytokines and specifically lysed HLA matched fibroblasts expressing physiologic levels of endogenously presented MCPyV oncoproteins. Safety analyses are ongoing. In summary, single cell RNA sequencing allows for the rapid identification from small, polyclonal human samples of functional highly avid paired TCRs, despite their presence at low frequency, thus facilitating rapid therapeutic translation. Citation Format: Megan S. McAfee, Kelly Paulson, Thomas Schmitt, Natalie Miller, Daniel Hunter, Jason Bielas, David Koelle, Valentin Voillet, Raphael Gottardo, Phillip Greenberg, Paul Nghiem, Aude Chapuis. Novel approaches to high-affinity TCR isolation for clinical translation enabled by single cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3567.

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of 'unconventional' T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity. Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women's Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR

Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.

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Abnormal immunophenotype of the T-cell-receptor beta Chain in follicular-helper T cells of angioimmunoblastic T-cell lymphoma.

Analysis for 24 variable regions of the T-cell-receptor beta chain by flow cytometry (Vbeta) is a new technique to detect clonal alpha-beta T lymphocytes and is characteristically performed on peripheral blood. Angioimmunoblastic T-cell lymphoma (AITL) has increased neoplastic follicular-helper T cells (FHT), which often express CD10; but nonneoplastic, CD10-positive T cells may be associated with reactive lymphadenopathy and with B-cell lymphomas. This study documents the utility of Vbeta analysis of FHT in specimens of AITL from blood, from marrow, and from lymph nodes. The electronic medical record in the flow cytometry laboratory was searched for specimens that were analyzed by flow cytometry for Vbeta and that were involved by AITL. Flow cytometry was performed for the following antigens: T-cell-associated proteins, CD10, CD56, CD94, CD161, killer-cell immunoglobulin-like receptors, alpha-beta T-cell receptor, gamma-delta T-cell receptor, and Vbeta. Five patients had six specimens of blood (two), of bone marrow (one), or of lymph nodes (three). Immunophenotypic aberrances were detected for antigens: CD2 (2/6), CD3 (6/6), CD4 (1/6), CD5 (1/6), CD7 (5/6), and CD45 (2/6). All abnormal T-cell populations expressed CD4, and most expressed CD10 (5/6). Four specimens were clonally restricted for Vbeta. Two specimens lacked the alpha-beta T-cell receptor and Vbeta. Vbeta analysis by flow cytometry can be used to detect clonal alpha-beta FHT in AITL, which may be difficult to diagnose with early involvement. Abnormal Vbeta expression on CD10-positive T cells confirms that FHT are the neoplastic cells.

Specific Gamma Delta T Cells for Cellular Immunotherapy of EBV-Associated Diseases after Allo-HSCT By T-Cell Receptor Gene Modification

Epstein-Barr virus (EBV) infection may result in a spectrum of diseases in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially post-transplant lymphoproliferative disease (PTLD). Furthermore, the clinical course of EBV-associated diseases often superimposes with graft-versus-host disease (GVHD) and that increases the difficulty of treatment. Adoptive immunotherapy with EBV-specific cytotoxic T lymphocytes (EBV-CTL) is recommended as one of the first-line therapy for PTLD. Engineering of T cell receptor (TCR)-transferred T lymphocytes could be an attractive strategy to obtain sufficient T cells with an antigen specificity of choice. But TCR-modified alpha beta T cells are restricted by major histocompatibility (MHC) class I expression and low affinity. Because gamma delta T cells (GD T cells) recognize target cells independent of human leukocyte antigen (HLA), the alpha beta TCR engineering of GD T cells forms a feasible strategy to generate antigen-specific effector T cells without HLA restriction. And regulatory GD T cells (GD Tregs) (one of the novel subset of GD T cells), which express Foxp3 and primarily belong to CD27+CD25high phenotype, might have application prospect in the treatment of GVHD. In order to investigate a novel strategy with potential therapeutic benefits in EBV-associated diseases after allo-HSCT, we obtained the monoclonal EBV-CTL clone HLA-A*0201/GLCTLVAML from the PROIMMUNE company (U.K.) and detected peptide-specific CTL activity in chromium release assays, then identified the EBV-CTL associated - TCR Valpha 15 and TCR Vbeta 1 chain genes, and subsequently cloned into the eukaryotic expression vector pIRES to construct the recombinant plasmid; We transferred the EBV specific recombinant plasmid to GD T cells and GD Tregs (generated in vitro by stimulating with anti-TCR gamma delta in the presence of TGF-beta and IL-2). The TCR gene-transduced groups had special TCR Vbeta 1 expression in fluorescence detection, quantitative PCR and western Blot analysis. TCR gene-transduced GD T cells had significant cytotoxicity against the EBV+ target cell lines (including toledo cells (HLA-A2 positive) (38.17±0.86%) and daudi cells (HLA-A2 negative) (61.74±1.84%)) compared with empty vector transfected GD T cells (toledo cells (30.15±0.89%); daudi cells (53.91±4.33%)) (P<0.001, P=0.006). TCR gene-transduced GD Tregs also had significant cytotoxicity against toledo cells (56.21±5.34%) and daudi cells (68.41±2.30%) compared with empty vector transfected GD Tregs (toledo cells (48.47±0.35%); daudi cells (56.23±2.04%)) (P=0.012, P<0.001). Both TCR gene-transduced GD T cells and GD Tregs had high levels of IFN-gamma in culture supernatants. Moreover, TCR gene-transduced GD Tregs had high levels of IL-10 in culture supernatants. Interestingly, TCR-modified GD Tregs showed significant suppressive effect on autologous CD4+ responder cells in CCK8 detection and CFSE assays. In conclusion, GD T cells or GD Tregs co-transduced with TCR alpha beta genes (from EBV CTL clone) could acquire antiviral capacity. Immunomodulatory function of GD Tregs could remain after TCR gene transfection. TCR modified GD Tregs might be an alternative and attractive strategy with potential therapeutic benefits in EBV-associated diseases after allo-HSCT, especially with GVHD. DisclosuresWu:National Natural Science Foundation of China (No. 81200388), the Science and Technology Planning Project of Guangdong Province of China (No. 2012B031800403), the project of the Zhujiang Science & Technology Star of Guangzhou(No. 2013027): Research Funding. Li:National Natural Science Foundation of China (No. 81200388), the Science and Technology Planning Project of Guangdong Province of China (No. 2012B031800403), the project of the Zhujiang Science & Technology Star of Guangzhou(No. 2013027): Research Funding.

CD4 positive/CD8 negative/CD56 positive T cell large granular lymphocyte proliferations; clonal disorders of uncertain significance

Clonal expansions of CD4 positive T large granular lymphocytes with or without co-expression of CD8 are extremely uncommon. These typically display an indolent clinical course and are said to be associated with other solid malignancies. We present the clinicopathologic features and immunophenotypic analyses of two rare cases of CD4 positive T large granular lymphocyte proliferation with expression of natural killer cell associated antigen CD56. Both had high positive anti cytomegalovirus (CMV) IgG titres and persistent lymphocytosis with a relatively benign course at 15 and 13 months of follow up, respectively. Case 1 A 58 year old Indian man presented with fever and cough of one week duration. On examination, he was febrile with crackles in the right lower zone. Hematological investigations revealed mild neutropenia and a monotypic CD3 positive/CD4 positive/CD8 negative/CD56 positive/T cell receptor alpha-beta positive large granular lymphocyte population representing 50% of blood and bone marrow lymphocytes. Clonality was demonstrated using a T cell receptor gamma polymerase chain reaction. A chest roentgenogram showed features suggestive of bronchopneumonia. Patient was treated with broad spectrum antibiotics and antipyretics with improvement. At follow up of 15 months, he continued to have persistent large granular lymphocytosis. Case 2 A 38 year old Indian woman presented with generalized weakness and malaise of two months duration. On examination, she was afebrile with no lympadenopathy or hepatosplenomegaly. A routine hemogram showed absolute lymphocytosis. Flow cytometric evaluation was performed on peripheral blood affirmed the cells to be CD4 positive T large granular lymphocytes, with similar findings to the first case. At follow up of 13 months, patient was otherwise asymptomatic but continued to have persistent CD4 positive large granular lymphocytosis. Flow cytometric immunophenotyping remains an essential tool for the diagnosis of T cell proliferations and helped to clinch the diagnosis in these cases. The aim of this case series is to present clinical and immunophenotypic features of these rare CD4 positive T large granular lymphocyte proliferations that fulfil the WHO defining criteria, however are better regarded as clonal expansions rather than as leukemias in view of their indolent and non progressive nature.

Haploidentical T-cell alpha beta receptor and CD19–depleted stem cell transplant for Wiskott-Aldrich syndrome

Haploidentical T-cell alpha beta receptor and CD19–depleted stem cell transplant for Wiskott-Aldrich syndrome

Pairing of T‐cell receptor chains via emulsion PCR

Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains--both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here, we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.

Ascorbic Acid Promotes Increased mRNA Expression of Rearranged TCR-Alpha and TCR-Beta Genes During In Vitro T Cell Development.

AbstractAbstract 2623 Introduction:A complete understanding of factors that drive T cell differentiation from hematopoietic progenitors remains a fundamental goal of hematology. T cells are key regulators and effectors in defense against infections and malignancies. Aberrations in T cell regulation play causative roles in autoimmune and graft-versus-host disease. The OP9-DL1 stromal cell line enables investigation of lymphocyte development in vitro. Lymphocyte progenitors (KLS, Thy1.1 -) harvested from murine adult bone marrow and seeded onto the OP9-DL1 stromal line can be followed through stages of maturation by immunophenotyping. We previously demonstrated that addition of a stabilized form of vitamin C, phospho-ascobate (pAsc), to culture media promoted T lineage differentiation. To address the mechanism of this effect, we employed quantitative RT-PCR and spectratyping analysis to examine mRNA expression of rearranged complementarity-determining region 3 (CDR3) polymorphisms in T cell receptor (TCR) beta variable (BV) and alpha variable (AV) genes in the presence or absence of pASC. Methods:Lymphocyte progenitor cells (KLS, Thy1.1-) were sorted from adult mouse bone marrow and 1000–2000 progenitors were seeded per well in a 24 well plate coated with OP9-DL1 stromal cells. Cultures were supplemented with IL-7 (5 ng/ml), Flt3 ligand (5 ng/mL) and SCF (100 ng/mL) plus or minus pAsc (100 mcg/mL). Cells were passaged, counted and reseeded with fresh media and supplements twice a week over a 21 day period. Immunophenotype and viability were evaluated by flow cytometry. Markers for T cell development included CD44, CD25, CD3, CD4, CD8, TCR beta chain and TCR gamma-delta chains. Total RNA from cultured cells was isolated at day 21, reverse transcribed to cDNA, and analyzed by RT-PCR for differential expression of BV and AV genes using gene-specific primers for BV1, BV4, BV8.2, BV13, AV2, and AV8 with corresponding beta constant (BC) and alpha constant (AC) primers. For spectratyping, RT-PCR amplicons were generated using BV or AV gene-specific primers for BV1, BV4, BV8.2, BV13, AV1, AV2, AV5, AV8, AV10, AV13, AV16, AV18, and AV19 with corresponding BC and AC primers. These products were then re-amplified with the same gene-specific primers but with fluorochrome-labeled nested BC or AC primers. Spectratype analysis was performed on labeled amplicons by capillary electrophoresis. Results:T cell differentiation was markedly advanced by the addition of pAsc, with the majority of cells co-expressing CD4/CD8 and TCR beta/CD3. Transfection of a functionally rearranged TCR beta gene failed to rescue cells cultured without pAsc to the double positive stage; similar results were obtained with bone marrow cells derived from TCR alpha-beta transgenic donor mice. Cells cultured with pAsc demonstrated an average 5 fold increase (5.08 ± 0.40) in expression of BV genes and an average 13 fold increase (13.46 ± 2.18) of AV genes. pAsc did not induce alterations in the spectratype distributions of BV amplicons compared those generated under non-pAsc conditions, nor to distributions derived from thymic cDNA. However, spectratype distributions of AV amplicons generated under pAsc conditions more closely resembled those derived from thymic and lymph node cDNA than distributions generated from non-pAsc conditions. Conclusions:In our in vitro model, the addition of pAsc promotes robust differentiation of adult mouse bone marrow progenitors to T cells co-expressing CD4/CD8 and alpha-beta TCR. However, the mechanism by which pAsc exerts its effect remains elusive. We suspect that pAsc enhances an already pre-programmed process. The fact that transfection of a functional TCR beta gene fails to rescue differentiation, coupled with our observation that pAsc has no effect on BV spectratypes suggests that enhancement of beta selection is not involved. Rather, the AV spectratyping data suggest that pAsc exerts its effect temporally near alpha gene rearrangement, possibly via enhancement of the TCR signal transduction cascade. Further work will include PCR microarray analysis encompassing multiple signal transduction cascades involved in hematopoietic progenitor differentiation. These findings will help develop a model for future mechanistic studies and for ex vivo expansion of immune cells for therapeutic use. Disclosures:No relevant conflicts of interest to declare.