TCRbeta Sequences Research Articles

T-cell tolerant fraction as a predictor of immune-related adverse events

BackgroundImmune checkpoint inhibitor (ICI) therapies may cause unpredictable and potentially severe autoimmune toxicities termed immune-related adverse events (irAEs). Because T cells mediate ICI effects, T cell profiling may provide insight...

CTIM-31. IMMUNOLOGIC MONITORING AFTER ADOPTIVE CELL THERAPY IN PEDIATRIC PATIENTS WITH RECURRENT MEDULLOBLASTOMA USING IMMUNOGENOMICS APPROACH

Abstract BACKGROUND Adoptive cellular therapy (ACT) using transfer of tumor-specific lymphocytes has emerged as a potent strategy for treatment of advanced and refractory malignancies. We have employed the use of total tumor RNA (TTRNA)-pulsed DCs in the ex vivo expansion of tumor-specific lymphocytes for use in adoptive cellular therapy targeting pediatric malignant brain tumors. METHODS Total RNA was isolated from patient PBMC samples collected prior to adoptive cellular therapy and after 2-weeks and 4-weeks following immunotherapy treatment from 18 patients (Re-MATCH protocol, FDA IND BB-14058). We applied bulk RNA-Seq and high-throughput T cell receptor sequencing to monitor changes in blood draws after ACT. RESULTS We observed an increase of expression level of genes responsible for T cell activation in PBMC after adoptive cell therapy. We found that these genes were involved in T cell signaling pathways, proliferative signals, and cytokine production. The data revealed that certain T-cells were clonally expanded after ACT, with an increasing number of “hyper-expanded” TCR clones (comprising > 1% of all TCR beta or alpha sequences) after adoptive cellular therapy. These TCR clones established a new balance in the peripheral blood that was stable over the month following the completion of cellular immunotherapy. Hyper-expanded TCR clones and increased TCR diversity following ACT was associated with radiographic response to ACT and prolonged progression-free and overall survival. Limited TCR expansion and diversity following ACT was observed in patients with short overall and progression-free survival. CONCLUSIONS These results emphasize the importance of gene expression profiling to achieve higher resolution in monitoring immune responses in patients receiving immunotherapeutic treatment. Also, these findings support further study of the use of TCR sequencing to monitor responses to adoptive cellular therapy and suggest that TCR clonal expansion and increasing TCR diversity following treatment may be associated with positive clinical responses.

T cell receptor dynamic and transcriptional determinants of T cell expansion in glioma-infiltrating T cells.

BackgroundGlioblastoma (GBM) is characterized by low numbers of glioma-infiltrating lymphocytes (GIL) with a dysfunctional phenotype. Whether this dysfunctional phenotype is fixed or can be reversed upon ex vivo culturing is poorly understood. The aim of this study was to assess T cell receptor (TCR)-dynamics and -specificities as well as determinants of in vitro GIL expansion by sequencing-based technologies and functional assays to explore the use of GIL for cell therapy.MethodsBy means of flow cytometry, T cell functionality in GIL cultures was assessed from 9 GBM patients. TCR beta sequencing (TCRB-seq) was used for TCR repertoire profiling before and after in vitro expansion. Microarrays or RNA sequencing (RNA-seq) were performed from 6 micro-dissected GBM tissues and healthy brain RNA to assess the individual expression of GBM-associated antigens (GAA). GIL reactivity against in silico predicted tumor-associated antigens (TAA) and patient-individual GAA was assessed by ELISpot assay. Combined ex vivo single cell (sc)TCR-/RNA-seq and post-expansion TCRB-seq were used to evaluate transcriptional signatures that determine GIL expansion.ResultsHuman GIL regains cellular fitness upon in vitro expansion. Profound TCR dynamics were observed during in vitro expansion and only in one of six GIL cultures, reactivity against GAA was observed. Paired ex vivo scTCR/RNA-seq and TCRB-seq revealed predictive transcriptional signatures that determine GIL expansion.ConclusionsProfound TCR repertoire dynamics occur during GIL expansion. Ex vivo transcriptional T cell states determine expansion capacity in gliomas. Our observation has important implications for the use of GIL for cell therapy including genetic manipulation to maintain both antigen specificity and expansion capacity.

Abstract PR-007: Lung-tropic, liver-averse, primary PDAC tumors are associated with greater peripheral T cell diversity and have a unique, subtype-independent, gene-expression signature that significantly correlates with longer survival

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related death in the United States, and most patients who present with metastatic PDAC die within a year. However, we and others have found that patients with lung metastases in the absence of liver metastases survive significantly longer than patients who present with liver metastases. We analyzed an unpublished RNASeq dataset from ~300 tumor-enriched samples from primary and metastatic PDAC specimens. Consistent with many previous publications, we found that patients with basal/squamoid-subtype tumors had significantly worse outcomes than patients with classical/ductal-subtype tumors. Additionally, we identified that most primary tumors from patients who develop lung – but not liver – metastases are classical subtype. However, this association did not wholly account for the pro-survival effect of lung-tropic, liver-averse metastatic disease because patients with lung-tropic, liver-averse, classical-subtype, primary tumors had significantly better outcomes than patients with liver-tropic, classical-subtype tumors. To identify and parse metastatic organotropism from subtype, we used organotropism-independent and subtype-independent, primary-tumor training cohorts to generate two non-overlapping gene sets that were significantly enriched in test cohorts of either primary, basal-subtype or liver-tropic tumors over primary tumors that were classical-subtype or lung-tropic and liver-averse, respectively. When applied to all primary tumors in our dataset, both the subtype-specific and organotropism-specific gene sets significantly correlated with patient outcome. From an unpublished analysis of TCRbeta CDR3 sequences from ~250 paired blood and primary tumor samples, we identified significantly greater TCRbeta diversity in blood and primary tumors from patients with lung-tropic, liver-averse disease. Additionally, we found evidence that TCRbeta rearrangements from liver-tropic primary tumors were more likely to be found in autologous peripheral blood samples than TCRbeta rearrangements from lung-tropic, liver-averse primary tumors. We also found that TCRbeta sequences were often shared between samples from patients with liver-tropic disease but never shared between samples from patients with lung-tropic, liver-averse disease. Overall, our results point to a lung-tropic, liver-averse form of PDAC that – independent of tumor subtype – leads to positive outcomes, and that T cell diversity may have a causal relationship and/or may serve as a biomarker of long-term survival with lung-tropic, liver-averse disease. Citation Format: Jason M. Link, Patrick J. Worth, Dove Keith, Sydney Owen, Alison Grossblatt-Wait, Carl Pelz, Hannah Holly, Motoyuki Tsuda, Kevin MacPherson, Jonathan Brody, Charles Lopez, Brett C. Sheppard, Rosalie C. Sears. Lung-tropic, liver-averse, primary PDAC tumors are associated with greater peripheral T cell diversity and have a unique, subtype-independent, gene-expression signature that significantly correlates with longer survival [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-007.

T cell receptor beta locus sequencing early post-allogeneic stem cell transplant identifies patients at risk of initial and recurrent cytomegalovirus infection.

Identification of patients at risk of initial & recurrent cytomegalovirus (CMV) reactivation following allogeneic stem cell transplant (alloSCT) may help guide prophylactic strategies. T-cell receptor beta (TRB) deep sequencing was used to identify and enumerate the T-cell repertoire harbouring TRB sequences with annotated specificity to CMV (pubCMVrep), as well as the overall T-cell receptor (TCR) repertoire diversity at day +30 & day +60 post-alloSCT for 65 patients. T-cells harbouring TRB sequences with annotated specificity for CMV were identifiable in all patients. 56% of patients required CMV treatment and 23% of the cohort developed recurrent CMV. PubCMVrep size at day +30 was not associated with reactivation, however amongst patients with antecedent CMV viremia a low day +60 pubCMVrep was associated with a greater incidence of recurrent CMV (75% vs. 21%, HR 6.16, 95% CI 1.29-29.40, P = 0.0008). Moreover, patients with high pubCMVrep only developed recurrent CMV in the setting of GVHD. Low TCR diversity at day +30 was associated with a greater incidence of initial CMV reactivation (71% vs. 22%, HR 5.39, 95% CI 1.70-17.09, p = 0.0002). pubCMVrep and TCR diversity are promising biomarkers to identify patients at risk of initial & recurrent CMV who may benefit from novel prophylactic strategies.

Somatic mutations in lymphocytes in patients with immune-mediated aplastic anemia

The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients’ CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.

The T cell Repertoire as a biomarker for breast cancer detection

To study the T-cell repertoire during tumor progression, we followed 10 female mice of a transgenic mouse strain that expresses the un-activated rat neu (Erbb2) oncogene, along with 5 control mice. These mice develop mammary tumors spontaneously over 5-8 months. To quantify the peripheral T cell repertoire, we extracted T cells from blood, every month, over the period of 9 months. Cells from these samples were sorted and later processed through a cDNA TCR С and С library preparation protocol using single-molecule barcoding and then NGS sequenced. We were able to use the repertoire to classify tumor and non-tumor mice, using their immunological repertoire. Using feature selection algorithms, we were able to provide superior classification using a small subset (3 to 6 clones) of the T cell repertoire. Thus, machine learning and feature selection allowed us to reduce the hundreds of thousands of TCR alpha and beta sequences obtained during repertoire sequencing, to a set of six clones, with which we can identify the source of a blood sample as tumor or control. We can further stratify older transgenic mice (older than 5 months) and those of older control mice, using the same small T cell clones’ subset. This latter classification has been obtained with as little as three T cell clones.

Serial T-cell Receptor (TCR) Repertoire Sequencing to Predict Outcomes in Gastrointestinal (GI) Malignancies

Severe treatment-induced lymphopenia is associated with worse prognosis in multiple solid malignancies. Emerging data suggests that increased TCR repertoire diversity (RD) is associated with improved survival. We hypothesized that increased TCR RD following severe treatment-induced lymphopenia would predict for improved survival. Patients within a prospective cohort were retrospectively analyzed for severe (grade 4) lymphopenia during radiotherapy (RT) at a single institution between 2007 and 2016. Peripheral blood samples were collected weekly. TCR repertoire at 3 time points were evaluated: pre-RT, during or immediately post-RT at absolute lymphocyte count (ALC) nadir, and ∼2 months post-RT. Peripheral blood was analyzed via multiparametric flow cytometry. TCR beta sequencing was performed on a commercially available assay. A Gini diversity index reflecting TCR RD was calculated as well as percentage change between ALC nadir and ALC 2 months post-RT. Primary endpoint was overall survival (OS). Cox modeling and Kaplan Meier estimates were used for survival analyses. 109 patients were enrolled. 20 patients were evaluable with complete data. Median follow up was 39.9 months. Median age was 68. 55% were male. 80% had ECOG 0 and 20% had ECOG 1. Primary malignancy was pancreatic adenocarcinoma in 40%, gastric adenocarcinoma in 30%, and cholangiocarcinoma in 30%. Clinical stage was I-II in 29%, III in 50%, and IV in 21%. 80% underwent chemotherapy (CTX) pre-RT and none received CTX post-RT. 80% had concurrent CTX during RT. Median RT dose was 56 Gy. Among those with pathology available (n = 12), 17% had a pathologic complete response (pCR) and 83% had R0 resection. Median baseline white blood cell (WBC) was 7 k/ul. Median ALC at each of the three study timepoints was 1.26 k/ul, 0.32 k/ul, and 1.02 k/ul, respectively. 4 (20%) patients had increased TCR RD as defined by the change in Gini diversity index while 16 (80%) had decreased TCR RD post-RT. There were no significant differences in baseline characteristics between the two cohorts. On univariable analysis of OS, increased TCR RD from ALC nadir to 2 months post-RT significantly correlated with better OS (HR = 0.20 [95% CI 0.04-0.93]; p = 0.041). Other variables including ECOG, stage, RT dose, CTX use, pCR, R0 resection, baseline WBC, and ALC at three study time points did not correlate with OS. Patients with increased vs decreased TCR RD post-RT had a 3-year OS of 75% vs 22%, respectively (log rank p = 0.026). Increased TCR RD predicted for better OS in our cohort of GI malignancies. Our findings provide biologic insight and refine the association between ALC and cancer outcomes. TCR RD warrants further study as a predictive and prognostic biomarker in GI cancers. Therapeutic approaches to limit severe lymphopenia are ongoing and should include TCR assessment.

Deep Immunoprofiling By Flow Cytometry and NGS Reveals Distinct T Cell Profile in Pediatric Hepatitis-Associated Aplastic Anemia

Hepatitis-associated aplastic anemia (HAAA) is a rare variant of bone marrow (BM) failure that is typically diagnosed 2-3 months after an acute attack of hepatitis in patients that are seronegative for known hepatitis viruses. Although very little is known about its etiology, an autoimmune mechanism is presumed based on clinical response to immunosuppressive therapy (IST) and reports of oligoclonal T-lymphocyte expansion and skewing of CDR3 region length. However due to the rareness of this disease, the published evidence is still limited. We analyzed 18 pediatric patients consecutively diagnosed with HAAA in the Czech Republic between 2004-2019. Based on BM biopsy examination, 14 patients had aplastic anemia (AA) and interestingly 4 patients were histologically consistent with refractory cytopenia of childhood (RCC). Median age of these patients was 6.6 years (range 1-18) with a male to female ratio of 2:1. The prevalence of HAAA in our cohort of pediatric patients with BM failure (n=107; excluding patients with known genetic cause) was 17%. The presence of a paroxysmal nocturnal hemoglobinuria clone was excluded and cytogenetic evaluation showed no abnormalities. We examined both the diagnostic BM and peripheral blood by flow cytometry in all patients (with the exception of one peripheral blood sample) and performed deep immunophenotyping of T cells in 7 of them. Ten patients were selected for T cell receptor beta (TRB) repertoire and whole-exome sequencing (WES) based on the availability of DNA samples isolated from the BM before the start of the treatment. TRB sequencing was performed following the SOP developed by the EuroClonality-NGS working group with DNA input normalized to the equivalent of 20 000 CD3+ cells per sample based on flow cytometry data. Bioinformatic analysis was performed with ARResT/Interrogate. WES was performed on Illumina NextSeq 500 and libraries prepared using the Agilent SureSelectXT Human All Exon V6+UTRs kit. Vertebrate virus detection was performed using the VirCapSeq-VERT probe capture enrichment set and massive parallel sequencing from 8 BM DNA samples taken at the time of diagnosis. We found significant activation of CD8 positive T cells based on the expression of HLA-DR compared to non-HAAA RCC and AA patients (n=20 and n=21 respectively, Mann-Whitney p<0.0001). Three out of 7 patients with deep T cell immunophenotype available showed a decrease of naive CD27pos45RAposCD8pos T cells. From TRB repertoire analysis we were able to identify a total of 5 private immunodominant clones in 3 patients with relative abundance of up to 11% from all productive TRB sequences. These clones were not found in any previous publications or clonotype databases based on their CDR3 sequence. We detected 5 clones shared by 3 or more patients; these were previously published in other cohorts and are not specific for HAAA. WES analysis did not reveal any previously published variant in genes associated with immune dysregulation or immunodeficiency, or any candidate variant for functional validation. No relevant vertebrate virus signal was detected in the 8 samples. Out of all 18 patients, 3 patients underwent MSD-HSCT without preceding immunotherapy. Fifteen patients were treated according to standard European Working Group on MDS/SAA protocols with anti-thymocyte globulin (horse n=6; rabbit n=9), corticosteroids and cyclosporin A. In total, 7 patients reached complete remission on IST by day 120 and 7 patients with insufficient response underwent MUD-HSCT. One patient died shortly after MUD-HSCT because of toxicity. We did not observe any correlation of T cell populations at diagnosis or day 120 of IST (activated and exhausted T cells) and clinical response. Similarly, the presence of an immunodominant clone or oligoclonal TRB repertoire with lower diversity at diagnosis had no significant impact on the outcome of these patients in our cohort. In conclusion, we present the biggest cohort of pediatric patients with HAAA analyzed by next-generation sequencing and flow cytometry so far. Even though our results show significantly increased activation of CD8 positive T-cells and presence of expanded clones, we did not confirm that these factors would be useful and significant for the prediction of treatment outcome. Large number of our patients did not fully respond to IST, requiring HSCT. Supported by AZV 18-07-00430, 16-32568A and PRIMUS/17/MED/11. Disclosures No relevant conflicts of interest to declare.

Haplotype Analysis of the T-Cell Receptor Beta (TCRB) Locus by Long-amplicon TCRB Repertoire Sequencing

Abstract Background: Polymorphism within the human T-cell receptor beta variable (TRBV) gene has been proposed as a risk factor for autoimmune disease and immune-related adverse events (IRAEs) during immunotherapy. Previous efforts to evaluate TRBV polymorphism by whole genome sequencing have been hampered by the repetitive nature of the T-cell receptor beta (TCRB) locus. We present a novel long-amplicon TCRB repertoire sequencing approach to enable TRBV haplotype analysis from peripheral blood. Methods: Peripheral blood leukocyte total RNA from 81 Caucasians was used for sequencing of TCRB chains via the Oncomine TCRB-LR assay (amplicon spanning CDR1, 2 and 3) and the Ion Gene Studio S5. VDJ rearrangements were annotated by comparison to the IMGT database, then mined to construct TRBV allele profiles for each individual including, where detected, novel alleles not present in the ImMunoGeneTics (IMGT) database. Finally, TRBV allele profiles were subjected to principal component analysis and k-means clustering to identify TRBV allele haplotypes. Results: Clustering analysis revealed the presence of six major sets of coincident TRBV alleles, which we term haplotype groups. Allelic diversity varied markedly across haplotype groups, with approximately one third of the cohort showing limited TRBV allelic diversity and few uncommon alleles compared to members of other groups. Analysis revealed 37 putatively novel TRBV alleles that are absent from the IMGT database. Conclusion: We demonstrate a straightforward and cost-efficient method for TRBV haplotype analysis from long-amplicon TCRB sequencing data.

Abstract PR14: Identification of specificity TCR groups of tumor antigen specific T-cells

Abstract A major breakthrough in the treatment of advanced melanoma has been the development and FDA approval of immune checkpoint inhibitors. Approximately 20% of the patients who received anti-CTLA-4 or anti-PD-1 therapy have long-term remissions. At the core of the clinical success lies the fact that cancer patients bear T lymphocytes that recognize tumor-specific antigens. It has been proposed that efficacy of immune checkpoint inhibitors is attributable, at least in part, to diversification of T-cell receptor (TCR) repertoire in peripheral circulation. However, current TCR repertoire analyses mostly rely on counting unique TCR sequences, which does not consider the degenerate nature of TCR recognition, that is, many different TCRs can recognize the same antigen peptide and the same TCR can recognize different antigen peptides. This will create roadblocks to identify recurrent TCR clones that likely recognize the same clinically relevant tumor antigen. To understand how TCR sequences encode its specificity, our lab sequenced several thousands of TCRs from T-cells enriched by peptide-major histocompatibility complex multimer (pMHC)-guided cell sorting. We established a TCR repertoire database that contains our own sequences and published TCRs with known specificity. We used this database as the training dataset for a machine learning algorithm that classified TCRs (mainly TCR beta) based on their similarity and specificity. Although the orginal algorithm—termed Grouping of lymphocyte interactions by paratope hotspots(GLIPH)—was proficient in identifying TCR clusters in a small dataset such as those generated by single-cell TCR sequences, it failed to perform reliably when applied to larger data set generated by high-throughput bulk TCR beta sequences. Most importantly, the reference sequences used by GLIPH 1.0 are insufficient when the targeted TCR dataset is much larger. The new version of GLIPH (GLIPH 2.0) was significantly improved in performance and reliability. As a proof of concept, we applied GLIPH 2.0 to TCR-sequences generated from peripheral T-cells of a cohort of melanoma patients. Each of these patients received one infusion of poly-clonal CTLs specific for HLA-A2/MART1. These autologous MART-specific CTLs were generated by priming with MART1 peptide-pulsed dendritic cells and enriched by pMHC-tetramer-guided cell sorting. MART1 peptide stimulation promotes expansion of CTLs based on TCR clonality. However, the overlap of sequences among these polyclonal CTLs is marginal. These TCR sequences were then analyzed by GLIPH and we found that MART1-specific CTLs showed significant enrichment of CDR3 motifs. As a control, we analyzed peripheral TCR repertoire from a group HLA-A2+ healthy donors and found no significant convergence in TCR sequences. This proves that GLIPH 2.0 can reliably identify TCR convergence and antigen specificity CD3 motifs from large-scale TCR beta repertoires. This algorithm can further facilitate the identification of recurrent tumor antigens in melanoma patients receiving T-cell-based immunotherapy. Citation Format: Liang Chen, Chunlin Wang, Mark Davis. Identification of specificity TCR groups of tumor antigen specific T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR14.

Adoptive Transfer of CMV- and EBV- Specific Peptide-Stimulated T Cells after Allogeneic Stem Cell Transplantation: First Results of a Phase I/IIa Clinical Trial [Multivir-01

Background: Reactivation of CMV and EBV negatively impacts on outcome after allogeneic stem cell transplantation (aSCT). Specific antiviral therapy is only available for CMV. With the exception of ganciclovir all drugs are being used off-label. 40-50% of patients reactivate CMV following aSCT. For the 20-30% of patients reactivating EBV, only rituximab is available to control EBV. Rituximab leads to long term B-cell depletion requiring frequent administration of immunoglobulins. To cover the unmet medical need of CMV and EBV control after aSCT, we investigate a somatic cell therapy approach by means of CMV- and EBV-specific peptide-stimulated T cells. We have set up a prospective randomized controlled phase I/IIa multi-center clinical trial to evaluate the preventive and preemptive adoptive transfer of this ATMP in patients after aSCT (EudraCT number 2012-004240-30). The multi-center trial is currently recruiting.Methods: For manufacturing of the cell product two peptide pools (CMV and EBV) each covering 17 well-defined HLA class I and class II epitopes for stimulation of donor derived PBMC are used. PBMC collected by leukapheresis of mobilized or non-mobilized donors can be used as starting material. To avoid a second leukapheresis of the donor, CMV- and EBV-specific T cells are preferentially expanded from a small fraction of the stem cell graft. A strong expansion of virus-specific T cells could be observed in the cell products as determined by HLA class I multimer analysis. Reconstitution and cell counts of leukocytes after aSCT are monitored for both treatment and control group. To obtain further insights in the expansion of transferred T cells, the TCR beta (TCRb) repertoire of the T-cell product before and after adoptive transfer in the patient is monitored by high throughput sequencing. Specificities of TCRb sequences can be assigned by determining the repertoire of HLA/peptide-multimer-sorted CD8+ T cells. New virus-specific TCRb sequences can be identified thereby. Furthermore, TCR sequences within the T-cell product can be tracked in the patient.Study design: After recruitment patients are randomized in intervention or control group. Patients of the intervention group receive three applications of virus specific T cells (5x10e4/kg bodyweight) starting the first adoptive transfer 30 days after allogeneic stem cell transplantation. Cells are transferred as preventive, preemptive, or also as therapeutic treatment. Patients are monitored for occurrence of GvHD, for viral load, as well as for immune reconstitution, especially of virus-specific T cells.Results: So far, 10 patients have been randomized. The reconstitution of virus-specific T cells of treated patients looks encouraging after transfer so far. The immunomonitoring of six included patients is completed. New CMV- and EBV-specific TCRb sequences could be identified and tracked.Conclusion: Our first observations show promising results regarding feasibility and efficacy of our approach under clinical trial conditions. DisclosuresHennig:HS Diagnomics GmbH: Employment, Equity Ownership.

PDCT-11. QUANTITATIVE TRACKING OF T-CELL CLONES AFTER ADOPTIVE CELLULAR THERAPY TARGETING RECURRENT MEDULLOBLASTOMA USING MASSIVELY PARALLEL RNA SEQUENCING TECHNOLOGY

Adoptive cellular therapy (ACT) using transfer of tumor-specific lymphocytes has emerged as a potent strategy for treatment of advanced and refractory malignancies. We have established a novel next-generation sequencing approach based on comprehensive characterization of the T-cell receptor (TCR) repertoire changes in patients with recurrent medulloblastoma and PNETs undergoing adoptive cellular therapy at our center (Re-MATCH protocol, FDA IND BB-14058). Total RNA was isolated from patient PBMC samples collected prior to adoptive cellular therapy and weekly for one month and then 5 months following immunotherapy treatment. cDNA was generated with addition of a common adapter at 5’ end of cDNA using SMART technology. PCR was performed to amplify TCR alpha and beta chains and included adapter and index sequences compatible with the Illumina sequencing platform. Libraries were sequenced on Illumina MiSeq using 600 cycle reagent kit with paired end, 300 + 300 base pair reads. T-cell sequences were analyzed using MiXCR software package, hosted on BaseSpace, Illumina’s cloud computing environment. The observed sequence reads in the peripheral blood allowed us to identify 54 TCRAV and 64 TCRBV exons which indicated a good coverage of TCR genes by our cDNA sequencing approach. The data revealed that certain T-cells were clonally expanded after ACT, with an increasing number of “hyper-expanded” TCR clones (comprising >1% of all TCR beta or alpha sequences) after adoptive cellular therapy. These TCR clones established a new balance in the peripheral blood that was stable over the next 5 months following the completion of cellular immunotherapy. This new method is useful for routine monitoring of clonotypic TCR expansion in the peripheral blood of patients undergoing immunotherapy, requires low blood sample volume, and may significantly enhance capacity to monitor the response to immunotherapy in patients with brain tumors.

Immune DNA signature of T-cell infiltration in breast tumor exomes.

Tumor infiltrating lymphocytes (TILs) have been associated with favorable prognosis in multiple tumor types. The Cancer Genome Atlas (TCGA) represents the largest collection of cancer molecular data, but lacks detailed information about the immune environment. Here, we show that exome reads mapping to the complementarity-determining-region 3 (CDR3) of mature T-cell receptor beta (TCRB) can be used as an immune DNA (iDNA) signature. Specifically, we propose a method to identify CDR3 reads in a breast tumor exome and validate it using deep TCRB sequencing. In 1,078 TCGA breast cancer exomes, the fraction of CDR3 reads was associated with TILs fraction, tumor purity, adaptive immunity gene expression signatures and improved survival in Her2+ patients. Only 2/839 TCRB clonotypes were shared between patients and none associated with a specific HLA allele or somatic driver mutations. The iDNA biomarker enriches the comprehensive dataset collected through TCGA, revealing associations with other molecular features and clinical outcomes.

T-Prolymphocytic Leukemia Is Sensitive to Polyclonal T Cell-Derived Graft-Versus-Leukemia Effects: Evidence from Minimal Residual Disease Kinetics and TCR Repertoire Diversity Analyses

▪ INTRODUCTIONT-prolymphocytic leukemia (T-PLL) is a rare T cell malignancy with an aggressive clinical course. Preliminary clinical data suggest that allogeneic stem cell transplantation (alloSCT) may provide long-term disease control in a proportion of patients. However, direct evidence that graft-versus-leukemia (GVL) activity is indeed effective in T-PLL is lacking. We sought to investigate GVL in T-PLL by correlating minimal residual disease (MRD) kinetics with immune modulatory interventions (immunosuppression tapering, donor lymphocyte infusions (DLI), chronic graft-versus-host disease (cGvHD)), and T cell receptor (TCR) repertoire diversity alterations after alloSCT. METHODSThe study sample consisted of 10 consecutive patients who received alloSCT for T-PLL at the University of Heidelberg between 2007 and 2015. Quantitative MRD monitoring was performed using clone-specific real-time quantitative PCR (RQ-PCR) of clonal TCR beta (TRB) and/or gamma (TRG) gene rearrangements. Data interpretation followed EuroMRD guidelines. In selected patients, TCR repertoire diversity was analyzed longitudinally by next-generation sequencing (NGS). TRBV-TRBD-TRBJ gene rearrangements were amplified according to BIOMED2 protocol on genomic DNA. PCR products were sequenced on Illumina's MiSeq platform. NGS data were analyzed through a purpose-built bioinformatics immunoprofiler (EuroClonality-NGS consortium). Rearrangements with very similar junctional amino acid sequences and identical TRBV and TRBJ gene usage were defined as clonotypes. RESULTSPatients underwent alloSCT in remission after first-line (8) or salvage (2) alemtuzumab-based therapy. 5 patients were allografted with an unrelated donor, 4 with a related donor, and one received haploidentical alloSCT. Conditioning was fludarabine with cyclophosphamide and/or total body irradiation-based. All patients had a cytological complete response (CR) after alloSCT. 2 patients died early because of acute GvHD, and one had no MRD marker, leaving 7 patients for MRD monitoring. Of these, 3 were MRD- at alloSCT, whereas 5 patients remained or became MRD+ early after alloSCT. In all of these 5 patients, immunosuppression tapering (3) or DLI (2) resulted in significant reduction of MRD levels (range 1-3 log) and was accompanied by cGvHD in 3 patients. However, durable MRD- was obtained in only 2 patients (alive 81+ and 12+ months post transplant), whilst MRD re-increased in 3 patients after 5-28 months despite ongoing cGvHD in one of them. NGS of the TCR repertoire was performed longitudinally in 3 patients with the longest follow-up. A total of 104 samples (blood (n=91) and BM (n=10) plus 3 donor blood samples) were sequenced. The sample at diagnosis showed one or two major clonotypes in all 3 sequenced T-PLLs, predominantly reflecting a mono- or biallelic leukemic TRB gene rearrangement. Kinetics of this leukemic clonotype followed kinetics of RQ-PCR MRD measurement, demonstrating that NGS can be used to quantify MRD in T cell lymphoma. Immediately after transplantation, the TRB repertoire was heavily skewed in all 3 patients, but recovered over time. Also in all 3 patients, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. This corresponded to the donor clonotype repertoire and disappeared with increasing MRD levels. In each of the samples after alloSCT, several expanded non-leukemic clonotypes were observed at a maximum frequency from 3% to 54% of total TRB sequences. However, during MRD response, novel dominant clonotypes that could explain a clonal GVL effect did not emerge. Figure 1 shows an example of a patient repeatedly showing MRD response to immune interventions. Currently, 5 patients are alive and in cytological CR (4-81 months after alloSCT), translating into a median relapse-free survival of 40 months. CONCLUSIONSThe MRD responses to immune interventions observed here provide the first direct evidence for the efficacy of GVL in T-PLL. However, the GVL effect in T-PLL appears to be often only limited or transient. It also does not seem to be caused by the emergence of novel dominant T cell clones but is rather relying on a poly-/oligoclonal T cell response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High-throughput pairing of T cell receptor alpha and beta sequences (TECH2P.930)

Abstract Conventional high-throughput immunosequencing methods profile TCR alpha (TRA) and/or TCR beta (TRB) repertoires, but do not enable pairing of the cognate subunits comprising functional TCRs. We developed a method called pairSEQ that can pair hundreds of thousands of TRA and TRB sequences in a single experiment. Our approach works by allocating random samples of T cells to a micro-titer plate, sequencing amplified cDNA from TRA and TRB genes, and DNA bar-coding the wells. Most TCR sequences uniquely identify clonal cell populations, so pairs can be assigned based on TRA and TRB sequences that occupy the same wells. To validate our method we identified cognate pairs from a sample of cells mixed from two people. The cross-sample pairs directly measure false pairing rates, which closely matched predictions from our statistical model. To show our method can be targeted to any clonal frequency range of interest, we designed a 3-plate experiment to capture pairs with repertoire frequencies from 0.5% to less than 1 in 10 million. These experiments yielded over 300,000 confident TCR pairs with one 96-well plate alone providing over 200,000 pairs. This technology is not restricted by material; in a single experiment we used this method to identify 6,349 TCR pairs from tumor infiltrating lymphocytes in 9 tumor samples. Our TCR pairing method is highly scalable, and should be broadly applicable to all somatically rearranged immune receptor loci, including immunoglobulins.

Abstract 3581: Characterization of T cell repertoire in transplanted patients with graft versus host disease using next generation DNA sequencer

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective therapy in treating patients for a range of high-risk hematologic malignancies. However, the success of allogeneic HSCT is limited due to graft versus host disease (GVHD). T cell receptor (TCR), a heterodimer of alpha and beta chains, is expressed on the surface of T cells, recognizes some antigens on the HLA molecule on host cells, enhances T cell proliferation, and release cytotoxic agents that cause the damage on host cells. The TCR in each T cell is unique due to the recombination of VJ (alpha) or VDJ (beta) segments that generates a diverse repertoire of T cells and enables T cells to recognize a vast number of different antigens. The development of next generation sequencing (NGS) has enabled us to obtain massive profiling of TCR to unravel the complexity of T cell diversity. In this study, we attempted to identify expanded T cell populations which are induced during the course of GVHD development after HSCT. To characterize millions of the TCR alpha and beta chains from a single individual, we performed very deep sequencing using cDNA derived from mRNA isolated from peripheral blood mononuclear cells of GVHD patients at different time-points (before and after HSCT) with Ion Personal Genome Machine (PGM™) Sequencer and a 400-bp reading kit. This sequencing technology has allowed us to obtain several million TCR sequences in a single experiment. A newly-developed algorithm was then applied for characterization of TCRs at each time-point. This approach has identified expansion of oligoclonal TCR alpha and beta chains, with unique V(D)J junction sequences around the rearranged regions, in T cells that are likely to be associated with development of GVHD. In the present study, a total of nine GVHD patients with different time-points (before and after HSCT) were examined. We obtained an average of 3,560,634 TCR alpha sequences with VJ combinations and 2,161,990 TCR beta sequences with VDJ combinations. Diversified TCR repertoire is observed in these samples. Further analysis of TCR alpha and beta chains for various time-points has suggested that a specific type(s) of T cells might be activated in the GVHD patients. Characterization of TCR may help delineate the molecular mechanisms of GVHD more precisely. Furthermore, it may aid in characterizing a personalized GVHD phenotype and predicting and/or monitoring the immunological responses after HSCT. Citation Format: Poh Yin Yew, Rui Yamaguchi, Michael Bishop, Amittha Wickrema, Andrew Artz, Satoru Miyano, Yusuke Nakamura. Characterization of T cell repertoire in transplanted patients with graft versus host disease using next generation DNA sequencer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3581. doi:10.1158/1538-7445.AM2014-3581

Nonsense Codons Trigger an RNA Partitioning Shift

T-cell receptor-beta (TCRbeta) genes naturally acquire premature termination codons (PTCs) as a result of programmed gene rearrangements. PTC-bearing TCRbeta transcripts are dramatically down-regulated to protect T-cells from the deleterious effects of the truncated proteins that would otherwise be produced. Here we provide evidence that two responses collaborate to elicit this dramatic down-regulation. One is rapid mRNA decay triggered by the nonsense-mediated decay (NMD) RNA surveillance pathway. We demonstrate that this occurs in highly purified nuclei lacking detectable levels of three different cytoplasmic markers, but containing an outer nuclear membrane marker, suggesting that decay occurs either in the nucleoplasm or at the outer nuclear membrane. The second response is a dramatic partitioning shift in the nuclear fraction-to-cytoplasmic fraction mRNA ratio that results in few TCRbeta transcripts escaping to the cytoplasmic fraction of cells. Analysis of TCRbeta mRNA kinetics after either transcriptional repression or induction suggested that this nonsense codon-induced partitioning shift (NIPS) response is not the result of cytoplasmic NMD but instead reflects retention of PTC(+) TCRbeta mRNA in the nuclear fraction of cells. We identified TCRbeta sequences crucial for NIPS but found that NIPS is not exclusively a property of TCRbeta transcripts, and we identified non-TCRbeta sequences that elicit NIPS. RNA interference experiments indicated that NIPS depends on the NMD factors UPF1 and eIF4AIII but not the NMD factor UPF3B. We propose that NIPS collaborates with NMD to retain and degrade a subset of PTC(+) transcripts at the outer nuclear membrane and/or within the nucleoplasm.

TCR beta-chain sharing in human CD8+ T cell responses to cytomegalovirus and EBV.

The CD8(+) TCR repertoires specific for many immunogenic epitopes of CMV and EBV are dominated by a few TCR clonotypes and involve public TCRs that are shared between many MHC-matched individuals. In previous studies, we demonstrated that the observed sharing of epitope-specific TCRbeta chains between individuals is strongly associated with TCRbeta production frequency, and that a process of convergent recombination facilitates the more efficient production of some TCRbeta sequences. In this study, we analyzed a total of 2836 TCRbeta sequences from 23 CMV-infected and 10 EBV-infected individuals to investigate the factors that influence the sharing of TCRbeta sequences in the CD8(+) T cell responses to two immunodominant HLA-A*0201-restricted epitopes from these viruses. The most shared TCRbeta amino acid sequences were found to have two features that indicate efficient TCRbeta production, as follows: 1) they required fewer nucleotide additions, and 2) they were encoded by a greater variety of nucleotide sequences. We used simulations of random V(D)J recombination to demonstrate that the in silico TCRbeta production frequency was predictive of the extent to which both TCRbeta nucleotide and amino acid sequences were shared in vivo. These results suggest that TCRbeta production frequency plays an important role in the interindividual sharing of TCRbeta sequences within CD8(+) T cell responses specific for CMV and EBV.

Selecting highly affine and well-expressed TCRs for gene therapy of melanoma

AbstractA recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumor-reactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well- and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.