Changes In T-cell Receptor Repertoire Research Articles

Quantifiable TCR repertoire changes in prediagnostic blood specimens among patients with high-grade ovarian cancer

High-grade ovarian cancer (HGOC) is a major cause of death in women. Early detection of HGOC usually leads to a cure, yet it remains a clinical challenge with over 90% HGOCs diagnosed at advanced stages. This is mainly because conventional biomarkers are not sensitive enough to detect the microscopic yet metastatic early lesions. In this study, we sequence the blood Tcell receptor (TCR) repertoires of 466 patients with ovarian cancer and controls and systematically investigate the immune repertoire signatures in HGOCs. We observe quantifiable changes of selected TCRs in HGOCs that are reproducible in multiple independent cohorts. Importantly, these changes are stronger during stage I. Using pre-diagnostic patient blood samples from the Nurses' Health Study, we confirm that HGOC signals can be detected in the blood TCR repertoire up to 4 years preceding conventional diagnosis. Our findings may provide the basis for future immune-based HGOC early detection criteria.

Abstract 53: T-cell receptor repertoire changes associated with clinical response in patients with B-cell non-Hodgkin’s lymphoma receiving CD19 CAR-T therapy

Abstract Background: Although CAR T-cell therapy has achieved remarkable response rates in adult patients (pts) with relapsed and refractory B-cell non-Hodgkin’s lymphoma (NHL), 20-30% of pts still have primary refractory disease, and 30-40% of pts relapse, often within 6 months of therapy. Given that durable clinical response can be achieved without CAR T cell persistence, we hypothesized that durable remission is correlated with the expansion of tumor-reactive T-cell receptor (TCR) clones following CAR T-cell infusion. To assess this, we examined the TCR repertoire of adult pts with B-NHL who received CD19 CAR T-cell (axi-cel) therapy to identify correlates of clinical response. Methods: Single-cell (sc) RNA sequencing (seq) and scTCR-seq were performed on peripheral blood mononuclear cells from B-NHL pts before lymphodepletion chemotherapy (pre), at peak CAR-T expansion (peak), and one month following CAR-T (M1). scRNAseq and scTCR-seq data were processed using CellRanger v7.0.1, which were then analyzed using Immunarch (v0.9.0) for TCR clonality and sample diversity. Diversity was assessed using the Gini coefficient to indicate clone size inequality (0 = maximum diversity). TCR diversity and phenotype of expanded and emerged TCR clones post CAR-T were compared across clinical response categories: CR (complete remission for at least 6 months), PD1 (primary refractory), and PD2 (relapsed after partial or complete response). Results: We analyzed 107,035 T cells from 77 serial samples of 32 pts (16 CR, 4 PD1, 12 PD2) and 5 healthy controls. Compared to pts with PD1 and PD2, pts with CR had a larger Gini coefficient for TCR clones at M1 relative to Pre and Peak. The Gini coefficient was the largest for tumor recirculating CD8 T effector memory (Tem) and activated CD8 peripheral memory (Tpm) cells at M1 for pts with CR. Examining the repertoire of emerged and expanded TCR clones at Peak and M1 from Pre, pts with PD2, compared to pts with CR, had a higher percentage of activated CD8 T peripheral memory (Tpm) and T regulatory cells (Treg) among the emerged clones and activated CD8 Tpm among the expanded clones. Patients with PD1, relative to patients with CR, had more proliferating and non-activated CD8 Tpm among the expanded and emerged clones. Conclusion: NHL pts with CR after CAR-T had more robust clonal expansion of T cells, particularly tumor recirculating CD8 Tem and activated CD8 Tpm, compared to pts with relapsed or refractory disease. In contrast, pts with relapsed or refractory disease had an expansion of TCR clones with a larger proportion of CD8 Tpm and Tregs. Endogenous tumor-reactive TCR expansion may help predict favorable clinical response following CAR T-cell therapy. Citation Format: Aiman J. Faruqi, Panwen Wang, Radhika Bansal, Andre De Menezes Silva Corraes, Henan Zhang, Zuoyi Shao, Kevin Regan, Eider Moreno Cortes, Arushi Khurana, Nora Bennani, Yucai Wang, Paul Hampel, Jonas Paludo, Patrick Johnston, Hemant Murthy, Madiha Iqbal, Stephen Ansell, Javier Munoz, Allison Rosenthal, Mohamed Kharfan Dabaja, Januario E. Castro, Saad Kenderian, Haidong Dong, Ying Li, Yi Lin. T-cell receptor repertoire changes associated with clinical response in patients with B-cell non-Hodgkin’s lymphoma receiving CD19 CAR-T therapy [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 53.

Multi-omics analysis to reveal the change of peripheral blood TCR repertoire and tumor burden during neoadjuvant chemotherapy combined with sequential PD-1 blockade for patients with non-small cell lung cancers: A single-center, single-arm phase 2 trial.

e20582 Background: Preoperative neoadjuvant chemotherapy combined with immunotherapy has been applied to the treatment of non-small cell lung cancer (NSCLC). However, the optimal dosage of chemotherapy and sequencing for chemotherapy and immunotherapy remains controversial. Methods: In this single-arm phase 2 trial, patients with stage IB to IIIA resectable NSCLC were enrolled and received 2 cycles of dose-limiting platinum-based chemotherapy (albumin-bound paclitaxel or pemetrexed plus cisplatin or carboplatin) on day 1, combined with sintilimab (200mg) on day 5 before undergoing radical surgery. Combination postoperative adjuvant therapy with the same regimen was administered. The primary endpoints were objective response rate (ORR) and major pathological response (MPR). Biopsies and plasma at different timepoint were collected and performed with RNA sequencing, circulating tumor DNA sequencing and T cell receptor (TCR) sequencing. Results: 17 patients were enrolled. After neoadjuvant treatment, patients with completed response, partial responses and stable diseases were 11.8% (2/17), 70.6% (12/17) and 17.6% (3/17), respectively, yielding an ORR of 82.4%. Radiological downstaging was achieved in 82.4% (14/17) of patients. All patients (17/17, 100%) experienced the R0 resection. Pathologic complete response and MPR were identified in 35.2% (6/17) patients and 23.5% (4/17) patients, respectively. Only 1 of the 17 patients (5.9%) experienced grade 3 or greater adverse event. Multi-omics analysis revealed chemotherapy combined with sequential PD-1 blockade results in the increase of dominated TCR clones and the decrease of the tumor burden. Especially during chemotherapy of cycle 1, the dominated TCR clones were significantly increased, and the maxVAF of ctDNA was significantly decreased. The change between the dominated TCR clones and maxVAF of ctDNA was negatively correlated. Conclusions: In patients with stage IB to IIIA resectable NSCLC, the neoadjuvant therapy with dose-limiting platinum-based chemotherapy combined with sequential sintilimab was safer and resulted in significant antitumor activity in patients with resectable NSCLC. The changes in TCR repertoire and ctDNA during treatment were analyzed, which provided evidence for the rationality of neoadjuvant chemotherapy combined with sequential PD-1 blockade. Clinical trial information: ChiCTR2000033092 .

Characterizing the cellular and molecular variabilities of peripheral immune cells in healthy recipients of BBIBP-CorV inactivated SARS-CoV-2 vaccine by single-cell RNA sequencing

ABSTRACT Over 3 billion doses of inactivated vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been administered globally. However, our understanding of the immune cell functional transcription and T cell receptor (TCR)/B cell receptor (BCR) repertoire dynamics following inactivated SARS-CoV-2 vaccination remains poorly understood. Here, we performed single-cell RNA and TCR/BCR sequencing on peripheral blood mononuclear cells at four time points after immunization with the inactivated SARS-CoV-2 vaccine BBIBP-CorV. Our analysis revealed an enrichment of monocytes, central memory CD4+ T cells, type 2 helper T cells and memory B cells following vaccination. Single-cell TCR-seq and RNA-seq comminating analysis identified a clonal expansion of CD4+ T cells (but not CD8+ T cells) following a booster vaccination that corresponded to a decrease in the TCR diversity of central memory CD4+ T cells and type 2 helper T cells. Importantly, these TCR repertoire changes and CD4+ T cell differentiation were correlated with the biased VJ gene usage of BCR and the antibody-producing function of B cells post-vaccination. Finally, we compared the functional transcription and repertoire dynamics in immune cells elicited by vaccination and SARS-CoV-2 infection to explore the immune responses under different stimuli. Our data provide novel molecular and cellular evidence for the CD4+ T cell-dependent antibody response induced by inactivated vaccine BBIBP-CorV. This information is urgently needed to develop new prevention and control strategies for SARS-CoV-2 infection. (ClinicalTrials.gov Identifier: NCT04871932).

Proportional Tumor Infiltration of T Cells via Circulation Duplicates the T Cell Receptor Repertoire in a Bilateral Tumor Mouse Model.

Temporal analysis of the T cell receptor (TCR) repertoire has been used to monitor treatment-induced changes in antigen-specific T cells in patients with cancer. However, the lack of experimental models that allow a temporal analysis of the TCR repertoire in the same individual in a homogeneous population limits the understanding of the causal relationship between changes in TCR repertoire and antitumor responses. A bilateral tumor model, where tumor cells were inoculated bilaterally into the backs of mice, could be used for temporal analysis of the TCR repertoire. This study examined the prerequisite for this strategy: the TCR repertoire is conserved between bilateral tumors that grow symmetrically. Bilateral tumors and draining lymph nodes (dLNs) were collected 13 days after tumor inoculation to analyze the TCR repertoire of CD4+ and CD8+ T cells. The tumor-infiltrating T-cell clones were highly similar between the bilateral tumors and expanded to a similar extent. In addition, the differences of TCR repertoire between the bilateral tumors were equivalent to Intra-tumoral heterogeneity on one side. On the other hand, the similarity of the TCR repertoire in the bilateral dLNs was markedly lower than that in the tumor, suggesting that tumor-reactive T cell clones induced independently in each dLN are mixed during recirculation and then proportionally infiltrated the bilateral tumors. These findings provide the basis for future analysis of temporal and treatment-induced changes in tumor-reactive T cell clones using this bilateral tumor model.

Revisiting the Role of γδ T Cells in Anti-CMV Immune Response after Transplantation

Gamma delta (γδ) T cells form an unconventional subset of T lymphocytes that express a T cell receptor (TCR) consisting of γ and δ chains. Unlike conventional αβ T cells, γδ T cells share the immune signature of both the innate and the adaptive immunity. These features allow γδ T cells to act in front-line defense against infections and tumors, rendering them an attractive target for immunotherapy. The role of γδ T cells in the immune response to cytomegalovirus (CMV) has been the focus of intense research for several years, particularly in the context of transplantation, as CMV reactivation remains a major cause of transplant-related morbidity and mortality. Therefore, a better understanding of the mechanisms that underlie CMV immune responses could enable the design of novel γδ T cell-based therapeutic approaches. In this regard, the advent of next-generation sequencing (NGS) and single-cell TCR sequencing have allowed in-depth characterization of CMV-induced TCR repertoire changes. In this review, we try to shed light on recent findings addressing the adaptive role of γδ T cells in CMV immunosurveillance and revisit CMV-induced TCR reshaping in the era of NGS. Finally, we will demonstrate the favorable and unfavorable effects of CMV reactive γδ T cells post-transplantation.

Subset distinct richness reduction and clonal expansion of human CD4+ and CD8+ αβ TCR repertoires with aging

Abstract A competent T-cell receptor (TCR) repertoire contains TCRs recognizing both novel and experienced antigens. It is widely believed that the human TCR repertoire declines with age, but its precise change in size and clonal distribution with age has not been fully determined. Here, we investigated how the size and content of TCR repertoire changes with age by analyzing the TCR repertoire of human CD4+ and CD8+ T cells and their naïve and memory subsets from 30 healthy adults aged from 25 to 85 at first visit and an average of 9-year follow-up as second visit by RNAseq. First, we calculated abTCR repertoire changes with age and found a profound reduction of the TCRa and TCRb richness in naïve CD8+ T cells and an increase of clonal expansion especially in memory CD8 T cells compared to CD4+ T cells. We also study the degree of overlap of TCRa/b repertoire between naïve and memory T cells in each subject at the same visit and found little overlap of TCRa/b unique sequences between naïve and memory CD4 T cells and ~40% of the total TCRs in CD8 T cells. Moreover, we showed elevated overlap of TCR sequences between two visits in both CD4 and CD8 T cells with memory CD8 T cells as the highest. Finally, we determined the degree of TCR clonotypes shares among different subjects via the analysis of the sharing of TCR sequences in the 30 healthy adults. As results, we observed increased sharing of identical TCR between CD4 and CD8 T cells suggesting that the public clonotypes may share with the common pathogens shared in the population, particularly in the old population. Our findings showed more profound reduction in the CD8+TCR repertoire with age compared to CD4+ TCR repertoire. All together provide evidence of ab TCR sequence-based age-associated changes of T cells and their subsets.

T-cell complexity and density are associated with sensitivity to neoadjuvant chemoradiotherapy in patients with rectal cancer.

Emerging evidence suggests that an increased density of pre-treatment CD8+ tumor-infiltrating lymphocytes (TILs) is associated with good response to chemoradiotherapy (CRT) in patients with locally advanced rectal cancer. However, the significance of T-cell complexity in the clinical setting remains unknown. High-throughput T-cell receptor (TCR) β sequencing was applied to quantify the TCR repertoire of pre-treatment biopsies from 67 patients with advanced rectal cancer receiving preoperative CRT. Diversity index was used to represent the complexity of the TCR repertoire in a tumor. Pre-treatment CD8+ TIL densities were assessed by immunohistochemistry. Changes in TCR repertoire before and after CRT were also analysed in 23 patients. Diversity indices were significantly higher for good responders than for non-responders (P = 0.031). The multivariate analysis revealed that both CD8+ TIL density and TCR diversity index were independently associated with good response to CRT (P < 0.001 and P = 0.049, respectively). Patients who were high for both CD8+ TIL density and TCR diversity (double-high) had markedly better responses to CRT than double-low patients (84.2% vs 16.7%, P < 0.0001). Larger changes in TCR repertoires before and after CRT were correlated with better recurrence-free survival (P = 0.027). The complexity and dynamic change in the TCR repertoire might serve as a useful indicator of response to CRT in combination with CD8+ TIL density in patients with rectal cancer.

The Outcome of Ex Vivo TIL Expansion Is Highly Influenced by Spatial Heterogeneity of the Tumor T-Cell Repertoire and Differences in Intrinsic In Vitro Growth Capacity between T-Cell Clones.

During our efforts to develop tumor-infiltrating lymphocyte (TIL) therapy to counter the devastating recurrence rate in patients with primary resectable pancreatic ductal adenocarcinoma (PDA), we found that PDA TILs can readily be expanded in vitro and that the majority of resulting TIL cultures show reactivity against the autologous tumor. However, the fraction of tumor-reactive T cells is low. We investigated to which extent this was related to the in vitro expansion. We compared the clonal composition of TIL preparations before and after in vitro expansion using T-cell receptor (TCR) deep sequencing. Our findings for PDA were benchmarked to experiments with melanoma TILs. We found that the TIL TCR repertoire changes dramatically during in vitro expansion, leading to loss of tumor- dominant T-cell clones and overgrowth by newly emerging T-cell clones that are barely detectable in the tumor. These changes are primarily driven by differences in the intrinsic in vitro expansion capacity of T-cell clones. Single-cell experiments showed an association between poor proliferative capacity and expression of markers related to antigen experience and dysfunction. Furthermore, we found that spatial heterogeneity of the TIL repertoire resulted in TCR repertoires that are greatly divergent between TIL cultures derived from distant tumor samples of the same patient. Culture-induced changes in clonal composition are likely to affect tumor reactivity of TIL preparations. TCR deep sequencing provides important insights into the factors that govern the outcome of in vitro TIL expansion and thereby a path toward optimization of the production of TIL preparations with high therapeutic efficacy.See related commentary by Lozano-Rabella and Gros, p. 4177.

The dynamic change of cancer genome and T-cell receptor repertoire of an early gastric cancer cohort.

e16553 Background: The dynamics of the cancer genome and T-cell receptor (TCR) repertoire are largely unclear during the development of gastric cancer, especially in the early stage. In this study, we studied the characteristics of genome and tumor microenvironment changes chronologically in 15 early gastric cancer (EGC) patients and illustrated their influence on carcinogenesis. Methods: 75 gastric tissues including precancerous lesions, cancer lesions and paired reference gastric samples, and 29 peripheral blood samples were obtained chronologically from 15 patients. All the lesions were categorized into 3 groups according to its pathology type (group1: inflammation or intestinal metaplasia, group 2: low-grade dysplasia (LGD), group 3: EGC). Deep sequencing of whole-exome and CDR3 chain of TCR of each sample was performed. Tumor mutation burden (TMB), TCR repertoire diversity and clonality were evaluated based on the sequencing data. Results: Heterogeneity existed in cancer genome and TCR repertoire among different patients. During carcinogenesis, TMB increased at first and then decreased, reaching its peak at LGD, which was different from what we have known from other advanced cancer. A detailed analysis showed that only part of the driver gene mutations maintained in this process. Compared with T cells infiltrated in reference samples, tumor-infiltrating T cells shared more clones with peripheral blood T cells. Diversity and clonality of TCR repertoire didn’t show significant change with the development of cancer (Shannon index: group 1 = 6.32, group 2 = 6.06, group 3 = 6.09, p = 0.836; Clonality: group 1 = 0.19, group 2 = 0.19, group 3 = 0.17, p = 0.374). Some CDR3 clones appeared in early-stage and maintained during the carcinogenesis process. Conclusions: Our research is the first to analyze the cancer genome and TCR repertoire changes from both the spatial and temporal perspectives during the development of early gastric cancer. Somatic mutations appeared in the very early stage of gastric cancer and may induce the immune microenvironment changes in gastric mucosa.

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.

Endogenous retroviral proteins provide an immunodominant but not requisite antigen in a murine immunotherapy tumor model

ABSTRACT Clinical observations suggest that responses to cancer immunotherapy are correlated with intra-tumoral T cell receptor (TCR) clonality, tumor mutation burden (TMB) and host HLA genotype, highlighting the importance of host T cell recognition of tumor antigens. However, the dynamic interplay between T cell activation state and changes in TCR repertoire in driving the identification of potential immunodominant antigen(s) remains largely unexplored. Here, we performed single-cell RNA-sequencing on CD8+ tumor-infiltrating T cells (TILs) using the murine colorectal tumor model MC38 to identify unique TCR sequences and validate their tumor reactivity. We found that the majority of clonally expanded TILs are tumor-reactive and their TCR repertoire is unique amongst individual MC38 tumor-bearing mice. Our query identified that multiple expanded TCR clones recognized the retroviral epitope p15E as an immunodominant antigen. In addition, we found that the endogenous retroviral genome encoding for p15E is highly expressed in MC38 tumors, but not in normal tissues, due to epigenetic derepression. Further, we demonstrated that the p15E-specific TILs exhibit an activated phenotype and an increase in frequency upon treatment with anti-41BB and anti-PD-1 combination immunotherapy. Importantly, we showed that although p15E-specific TILs are not required to mount a primary anti-tumor response, they contributed to the development of strong immune memory. Overall our results revealed that endogenous retroviral antigens expressed by tumor cells may represent an important and underappreciated category of tumor antigens that could be readily targeted in the clinic.

Profiling T Cell Receptor Repertoires in Phase I/II Clinical Trials of Donor Treg Infusion for the Treatment of Chronic Graft-Versus-Host Disease

IntroductionUnder the auspices of the EC-Horizon 2020 project “TREGeneration”, we are conducting five clinical phase I/II trials investigating the safety and efficacy of donor regulatory T cell (Treg) infusion in patients with chronic Graft-versus-Host Disease (cGvHD), a serious complication following hematopoietic stem cell transplantation (HSCT). Workpackage 4 evaluates T cell receptor (TCR) repertoire changes by high-throughput sequencing following the infusion of donor Tregs.MethodsFor repertoire analysis, four different T cell subsets (Treg, conventional CD4 T cells - Tcon, total CD4 and CD8 T cells) are FACS sorted from the infused product and from the cGvHD patients' peripheral blood before the infusion and at 10 time-points up to 12 months post infusion (Fig. 1A). To monitor TCR repertoire changes over time, we developed a novel quantitative “DeepImmune” technique for TCRβ amplification and sequencing. We amplify all TCRβ chain family transcripts from total RNA using a normalized primer set including molecular identifiers for unbiased and quantitative determination of TCRβ copy numbers. We have normalized the concentrations of our TCRβ primers using synthetic TCRβ gene sequences, thus minimizing amplification biases during RT-PCR to ≤3-fold differences among gene families. The inclusion of molecular identifiers during the RT-PCR step allows us to computationally remove additional biases introduced during the secondary PCR step for Illumina library preparation. Thus, our method results in ultra-deep characterization of immune receptor repertoires for sensitive detection of differences among samples and sample cohorts. Depending on T cell numbers in the samples, we routinely identify 1,000 - 150,000 clonotypes per sample.ResultsAs first quality control for repertoire analysis, we cluster the top 200 TCRβ clonotypes from the different sequenced T cell subsets for each patient, resulting in heatmaps showing prominent repertoire differences among the analyzed T cell subsets (Fig. 1B; infused patient example shown). Next, we track the fate of infused donor Treg cells in the time course samples of the patients after Treg infusion, allowing us to quantify the number and frequency of TCRβ clonotypes that persist or expand in the patients after the infusion. We report that TCRβ clonotypes specific to the infused Treg cells (which were not present in the patients before Treg infusion) can be detected in the patients' blood after the infusion. Infusion-specific Treg clonotypes can reach up to 10-15% of the Treg repertoire of the patients and usually decline over time (Fig. 1C; boxplot shows clonotype overlap between infusion-specific Treg clonotypes and patient Treg clonotypes from five patients of the Lisbon cohort over 12 months). In the Tcon compartment of the patients, infusion-specific Treg clonotypes typically reach only 1-2% of the Tcon repertoire of the patients and also decline over time (Fig. 1D), similarly to what is observed in the CD8 T cell compartment (data not shown). This suggests that infused Tregs are not reverting to Tcons, an important control and promising result. We apply the same type of analysis to track clonotypes that were shared between the infused product and the patient prior to infusion, in addition to those clonotypes that were unique to the patient before infusion and not present in the infused product.ConclusionBy applying our robust and sensitive TCRβ sequencing platform to samples from cGvHD patients infused with donor Treg, we are able to detect unique clonotypes of the infused product up to 1 year post-infusion. It remains to be be determined if there is any correlation between the level of detection of the infused clonotypes and the clinical responses of cGVHD to donor Treg therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clonotypic T-cell responses in metastatic castration-resistant prostate cancer (mCRPC) patients (pts) treated with standard sipuleucel-T (sip-T) compared with patients receiving a booster treatment.

107 Background: Sip-T is an FDA-approved autologous cellular immunotherapy for asymptomatic or minimally symptomatic mCRPC. Neoadjuvant sip-T induced activated T-cell infiltration to the prostate tissue (Fong 2014) and broadened the T cell receptor (TCR) repertoire vs control (Sheikh 2016). To test if sip-T induces memory T-cell responses, we compared treatment-induced changes in TCR repertoire of treatment-naïve pts (STRIDE) with those retreated with sip-T (P10-1). Methods: In STRIDE (N=52), pts received sip-T with concurrent or sequential enzalutamide (Petrylak 2015). In P10-1 (N=8), pts previously treated with sip-T in an androgen-dependent setting were retreated with a booster course after a median of 8.6 years (Beer 2013). PBMCs were collected at baseline and during/post–sip-T. Deep sequencing was performed using the ImmunoSEQ assay (Adaptive Biotechnologies). TCR diversity was assessed by Shannon diversity index and clonality. TCR dynamics was evaluated by Morisita’s distance (Zhang 2017). Results: Baseline TCR diversity was similar between the two trials (p=0.590, Shannon diversity index; p=0.700, clonality). Significant decreases in clonality were observed from baseline to wk 4 (p&lt;0.001) and wk 6 (p=0.030) in STRIDE, but not in P10-1, indicating more expansion of T cell clones in STRIDE. Morisita’s distance was significantly higher at wk 2 (p=0.040) and 26 (p=0.013) in STRIDE vs P10-1, indicating a more consistent TCR repertoire in STRIDE across timepoints. The proportion of the clones enriched after sip-T treatment was significantly higher in P10-1 vs STRIDE (wk 6: p=0.007, wk 26: p=0.015, wk 52: p=0.026). The extent of change within the top 100 most abundant TCR sequences (clonal shuffling) was greater and initiated earlier in P10-1 vs STRIDE. Conclusions: Initial sip-T treatment of naïve mCRPC pts programs the TCR repertoire, which is maintained over time. Consistent with immunologic memory, retreatment with sip-T rapidly expands the number of enriched T-cell clones persisting up to 1 year after retreatment, which is characteristic of immunological boosting following successful treatment.

Identification of flu-specific ICOS+CD38+ cTfh after influenza vaccination

Abstract The foundation of successful vaccines lies in the ability to prime the immune system to target a specific pathogen. The interaction between follicular helper T cells (Tfh) and B cells within germinal centers is necessary for the production of class-switched, affinity-matured antibodies. However, the size, diversity, and specificity of the T cell receptor (TCR) repertoire of the Tfh during an immune response remains unknown. We identified a subset of circulating Tfh (cTfh) coexpressing ICOS and CD38 that expresses markers of recent activation, such as the proliferation marker Ki67 and the transcription factor Helios. This activated cTfh subset increases following influenza vaccination. To begin to interrogate how the TCR repertoire changes in human cTfh cells responding to a vaccine, we performed T-cell receptor beta chain sequencing on cTfh from individuals before and after influenza vaccination. Although the ICOS−CD38− cTfh maintained a stable TCR repertoire pre- and 7 days post vaccination, the ICOS+CD38+ cTfh exhibited increased clonality indicative of antigen-driven activation and expansion. Using HLA class II HA306- and HA40-specific tetramer staining on peripheral blood from HLA-DR0401 individuals, we then further assessed the specificity of the responding cTfh at day 7 after vaccination. We found many tetramer-positive cells that expressed ICOS and CD38 in the cTfh subset, confirming that the ICOS+CD38+ response after vaccination contains influenza-specific cells. Ultimately, rational vaccine design will depend on better understanding of the clonotypic response elicited in response to various pathogens and their relevance to the overall immune response.

Quantifying Distribution of Flow Cytometric TCR-Vβ Usage with Economic Statistics.

Measuring changes of the T cell receptor (TCR) repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vβ usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vβ usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vβ analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vβ distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vβ analysis will greatly help to gain better understanding of the TCR repertoire in health and disease.

Reconstitution of T-cell receptor repertoire diversity following T-cell depleted allogeneic bone marrow transplantation is related to hematopoietic chimerism

CDR3 spectratyping was used to analyze the complexity of the T-cell repertoire and to define the mechanisms and kinetics of the reconstitution of T-cell immunity after allogeneic bone marrow transplantation (BMT). This method, which is based on polymerase chain reaction amplification of all CDR3 regions using the T-cell receptor (TCR) Vβ genes, was used to examine serial samples of peripheral blood lymphocytes from 11 adult patients with chronic myelogenous leukemia (CML) who underwent T-cell–depleted allogeneic BMT. In contrast to 10 normal donors who display highly diverse and polyclonal spectratypes, patient samples before and early after BMT revealed markedly skewed repertoires, consisting of absent, monoclonal, or oligoclonal profiles for the majority of Vβ subfamilies. To quantify changes in TCR repertoire over time, we established an 8-point scoring system for each Vβ subfamily. The mean complexity score for patient samples before transplant (130.8) was significantly lower than that for normal donors (183; P = 0.0007). TCR repertoire complexity was abnormal in all patients at 3 months after BMT (mean score = 87). Normalization of repertoire began in 4 patients at 6 months after BMT, but the majority of patients continued to display abnormal repertoires for up to 3 years after BMT. To determine whether the reconstituted T-cell repertoire was derived from the donor or recipient, unique microsatellite loci were examined to establish chimeric status. At 3 months after BMT, 7 patients demonstrated mixed chimerism; 4 had complete donor hematopoiesis (CDH). CDH strongly correlated with likelihood of restoration of T-cell repertoire complexity (P = 0.003). In contrast, patients who demonstrated persistence of recipient hematopoiesis failed to reconstitute a diverse TCR repertoire. These findings suggest that the reconstitution of a normal T-cell repertoire from T-cell progenitors in adults is influenced by interactions between recipient and donor hematopoietic cells. (Blood. 2000;95: 352-359)

Reconstitution of T-cell receptor repertoire diversity following T-cell depleted allogeneic bone marrow transplantation is related to hematopoietic chimerism

AbstractCDR3 spectratyping was used to analyze the complexity of the T-cell repertoire and to define the mechanisms and kinetics of the reconstitution of T-cell immunity after allogeneic bone marrow transplantation (BMT). This method, which is based on polymerase chain reaction amplification of all CDR3 regions using the T-cell receptor (TCR) Vβ genes, was used to examine serial samples of peripheral blood lymphocytes from 11 adult patients with chronic myelogenous leukemia (CML) who underwent T-cell–depleted allogeneic BMT. In contrast to 10 normal donors who display highly diverse and polyclonal spectratypes, patient samples before and early after BMT revealed markedly skewed repertoires, consisting of absent, monoclonal, or oligoclonal profiles for the majority of Vβ subfamilies. To quantify changes in TCR repertoire over time, we established an 8-point scoring system for each Vβ subfamily. The mean complexity score for patient samples before transplant (130.8) was significantly lower than that for normal donors (183; P = 0.0007). TCR repertoire complexity was abnormal in all patients at 3 months after BMT (mean score = 87). Normalization of repertoire began in 4 patients at 6 months after BMT, but the majority of patients continued to display abnormal repertoires for up to 3 years after BMT. To determine whether the reconstituted T-cell repertoire was derived from the donor or recipient, unique microsatellite loci were examined to establish chimeric status. At 3 months after BMT, 7 patients demonstrated mixed chimerism; 4 had complete donor hematopoiesis (CDH). CDH strongly correlated with likelihood of restoration of T-cell repertoire complexity (P = 0.003). In contrast, patients who demonstrated persistence of recipient hematopoiesis failed to reconstitute a diverse TCR repertoire. These findings suggest that the reconstitution of a normal T-cell repertoire from T-cell progenitors in adults is influenced by interactions between recipient and donor hematopoietic cells. (Blood. 2000;95: 352-359)