TCR Repertoire Research Articles

Clinical efficacy of T-cell therapy after short-term BRAF-inhibitor priming in patients with checkpoint inhibitor-resistant metastatic melanoma

PurposeDespite impressive response rates following adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, improvement is needed to increase the efficacy and broaden the applicability of this...

Abstract LB150: Clinical Feasibility and Treatment Outcomes with Unselected Autologous Tumor Infiltrating Lymphocyte Therapy in Patients with Advanced Cutaneous Melanoma

Abstract Introduction: A minority of patients (pts) with advanced melanoma achieve long-term survival with immunotherapy and those who relapse following immune checkpoint inhibition (ICI) or BRAFi have limited treatment options. The intrinsic anti-tumor activity and broad neoantigen-specific TCR repertoire of unselected autologous TIL may provide advantages over other treatments in solid tumors, including ICI-refractory melanoma. Methods: This is a retrospective analysis of a single-center experience of TIL for treatment of advanced cutaneous melanoma. Unselected autologous young TIL derived from digested tumor tissue were manufactured under a MHRA Manufacturing Specials license. Pts with advanced cutaneous melanoma and no other treatment options received non-myeloablative lymphodepleting chemotherapy (cyclophosphamide 60 mg/kg/d x 2d, fludarabine 25 mg/m2/d x 5 d [Cy/Flu]), followed by TIL infusion and post-TIL high-dose (HD) IL-2 (600,000-720,000 IU/Kg) on a compassionate use basis. Efficacy for 15 imaging-evaluable pts was reported by investigator assessment of CT/MRI per RECIST 1.1; 6 additional pts were followed using non-RECIST 1.1 imaging (PET) and clinical monitoring. Clinically significant adverse events (AEs) with onset post-TIL infusion were reported for all treated pts. Data cutoff date: 31-DEC-2019. Results: Between OCT-2011 and AUG-2019, 21 pts with advanced cutaneous melanoma were treated with Cy/Flu, TIL (median 31.9 x109 cells infused), and HD IL-2 (median 8 doses). All had high-risk metastatic disease (median 4 sites, stage IV M1d [38%]), an average of 3 prior therapies (any ICI [91%], PD-1 [57%], BRAFi [52%], and MEKi [24%]). With a median follow up of 52.2 mos, the response rate in imaging-evaluable pts (n=15) was 53% with a CR rate of 13%; DCR was 73% including 3 (20%) pts with stable disease. Additional durable responses were observed in the 6 pts followed by PET and clinical monitoring. Responses were generally consistent across subgroups including age, number of disease sites, tumor burden, brain metastases, number of prior lines of therapies, prior PD-1 blockade, prior BRAFi, and prior MEKi. For all treated pts, the median survival was 21.3 mos. Toxicity was generally self-limited and consistent with Cy/Flu and HD IL-2. Common AEs (≥20%, any grade) were thrombocytopenia (62%), pyrexia (57%), rigors (43%), neutropenia (29%), tachycardia (29%), pulmonary edema (24%), and vascular leak (24%); no treatment-related deaths were observed. Conclusion: The high response rate observed in this series exceeds the 41% ORR estimated for TIL in advanced cutaneous melanoma (meta-analysis by Dafni et al. Ann Oncol., 2019) and highlights the successful bench-to-bedside application of unselected autologous TIL to address unmet medical need in advanced melanoma. Use of tumor digests as starting material for manufacturing of TIL demonstrates feasibility of this approach. A multicenter Phase 2 trial of this therapy in advanced melanoma is planned for 2021. Citation Format: Robert E. Hawkins, Yizhou Jiang, Paul C. Lorigan, Fiona C. Thistlethwaite, Manon Pillai, Martine Thomas, Natalia Kirilova, John S. Bridgeman, Gray Kueberuwa, Ryan S. Guest, Zachary J. Roberts. Clinical Feasibility and Treatment Outcomes with Unselected Autologous Tumor Infiltrating Lymphocyte Therapy in Patients with Advanced Cutaneous Melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB150.

Abstract 73: A personal neoantigen vaccine NEO-PV-01 in combination with chemotherapy and pembrolizumab induces broad de novo immune responses in first-line non-squamous NSCLC: Associations with clinical outcomes

Abstract Background: Neoantigens arising from mutations in cancer cell DNA are important targets for T cell-mediated anti-tumor immunity. NEO-PV-01 is a personal neoantigen vaccine of up to 20 peptides (14-35 amino acids) based on a patient's HLA profile and bioinformatic analysis of tumor neoantigens. We report here relationships between baseline tumor characteristics, immune response, and clinical outcomes from NT-002, a Phase 1b study of NEO-PV-01 with pemetrexed, carboplatin, and pembrolizumab as first-line therapy for advanced non-squamous NSCLC (NCT03380871). The primary objective of this study was to evaluate the safety of the combination. Materials: Serial blood and tumor biopsies were collected at: i) prior to treatment, ii) after 12 weeks of chemotherapy-pembrolizumab treatment, and iii) after completion of NEO-PV-01 vaccination. Tumor biopsies were characterized by immunohistochemistry for immune and tumor markers, gene expression, whole-exome and TCR sequencing, and single-cell analysis. Antigen-specific responses were measured in blood samples by IFNγ ELISpot, intracellular cytokine staining and functional phenotyping by FACS. Results: A total of 38 patients initiated study treatment (ITT); 21 patients received at least 1 dose of NEO-PV-01 (VAX). The regimen was well-tolerated and consistent with the pembrolizumab plus pemetrexed/carboplatin safety profile. The overall response rate (ORR)/clinical benefit rate (CBR) for the ITT and VAX were 37%/69% and 57%/95%, respectively. Median PFS was 7.2 months (95% CI: 5.6,16.8) for both the ITT and VAX, and median OS 16.8 months (95% CI: 11.6, NR) for both groups. Immune analysis on 12 patients with available samples revealed neoantigen-specific CD4+ and CD8+ T cell responses in all patients tested with an average of 55% of vaccine peptides generating an immune response post-vaccination. Vaccine-induced immune responses were mutant-specific and durable at 52-week treatment timepoint. T cell responses were polyfunctional, as evident by secretion of multiple cytokines (TNFα, IL2, IFNγ), and were activated memory cells with a cytotoxic phenotype. Epitope spread was observed in 7 of 11 patients analyzed thus far. Comprehensive analysis by gene expression, ctDNA and TCR repertoire analysis demonstrated correlations to extended PFS. Additional data on single-cell sequencing of neoantigen-specific T cells and tumor biopsies and correlates to clinical outcomes will be presented. Conclusions: NEO-PV-01 in combination with pembrolizumab and carboplatin/pemetrexed has a good safety profile and induces de novo immune responses in first-line non-squamous NSCLC. The association of baseline disease characteristics to prolonged PFS suggests future patient enrichment strategies for evaluation of this novel regimen in a phase 2 trial. Citation Format: Mark M. Awad, Ramaswamy Govindan, David R. Spigel, Edward B. Garon, Victoria Kohler, Rohit Vyasamneni, Suchitra Ramesh, Tracey E. Sciuto, Melissa A. Moles, Jennifer Tepper, Amy Wanamaker, Zakaria S. Khondkar, John Srouji, Jesse Z. Dong, Kristen N. Balogh, Asaf Poran, Meghan E. Bushway, Mark DeMario, Richard B. Gaynor, Lakshmi Srinivasan. A personal neoantigen vaccine NEO-PV-01 in combination with chemotherapy and pembrolizumab induces broad de novo immune responses in first-line non-squamous NSCLC: Associations with clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 73.

Follicular Lymphoma Regulatory T Cell Function and Origin

Follicular Lymphoma (FL) is the most frequent indolent lymphoma and results from the malignant transformation of germinal center (GC) B cells. Among FL recurrent genetic alterations, some are not only oncogenic per se but favor the interactions of FL B cells with their microenvironment including in particular surrounding follicular helper T cells (Tfh). During normal GC reaction Tfh support early GC B-cell selection and differentiation whereas follicular regulatory T cells (Tfr) expand later and inhibit both Tfh activity and B-cell antibody secretion. Whereas the role of Tfh in human diseases is an intense matter of interest, very few data are available concerning the origin and function of human Tfr. We previously demonstrated that Tfh and Tfr proportions are increased within FL lymph nodes and FL-Tfh contribute, through a specific polarization profile, to FL B cell survival and drug resistance. Interestingly FL-Tfr expansion is correlated to the one of FL-Tfh but not FL-Treg. The aim of this work was to characterize human Tfr in non-malignant (chronically inflamed tonsils) and malignant (FL) settings through transcriptomic, DNA methylation, TCR repertoire, and functional studies. We demonstrated for the first time that human Tfr purified from FL samples and tonsils directly inhibit autologous Tfh proliferation and trigger a downregulation of the co-stimulatory markers CD86 and HLA-DR on autologous normal and malignant GC B cells. In agreement FL-Tfh proliferated less than tonsil-Tfh in situ and after in vitro restimulation. Moreover, we found that Tfr clustered with Treg at both transcriptomic and methylome levels including for the high demethylation level of the FOXP3 TSDR, suggesting a stable regulatory phenotype. However, TCR sequencing demonstrated that tonsil-Tfr shared a common repertoire only with tonsil-Tfh unlike tonsil-Treg, arguing for their peripheral unlike thymic origin. Conversely in FL, even if the majority of common clones were found between Tfr and Tfh, a small proportion of TCR repertoire was shared between Tfr and Treg. Altogether, these results are the first detailed characterization of human Tfr and challenge our current understanding of Tfr commitment pathway. FL-Tfr retain their capacity to inhibit Tfh thus questioning their role in FL and their potential as therapeutic target in this fatal malignancy. DisclosuresTarte:Roche: Consultancy; Novimmune: Research Funding; Celgene: Consultancy, Research Funding. Lamy:Roche: Consultancy, Honoraria.

Surveillance of the Immune Repertoire of Aplastic Anemia Patients Using Deep Sequencing

Cytotoxic T cells (CTL) responses have been implicated in the pathogenesis of idiopathic aplastic anemia (AA). In the past, CDR3 region of variable portion of B-chain (VB) of TCR has been analyzed by capillary electrophoresis and later by sequencing to identify clonal expansions or oligoclonal skewing of otherwise polyclonal TCR VB repertoire. CTL clonal expansions, most likely, a result of aberrant antigenic drive, lead to increased numbers of identical CDR3 sequences which can serve as clonal barcodes for individually expanded T cell clones. In AA oligoclonal skewing of VB TCR has been detected at presentation using manual clonotyping and interpreted as an overheated cellular response to putative autoimmune targets located within hematopoietic stem cell compartment. With the advent of NGS, the process of analysis of TCR repertoire has been improved: while manual sequencing allowed for characterization of only hundreds of VB CDR3 clonotypes, deep TCR sequencing affords incredible sequencing depth and therefore precision and resolution. We hypothesized that deep TCR sequencing (NGS clonotyping) can be used to identify changes in the T cell repertoire, including clonal expansions which would either qualitatively or quantitatively enable to us to predict subsequent responsiveness to immunosuppression (IST), propensity of relapse or potentially monitor the IST intensity.We have identified 26 AA patients (56% severe AA) from whom samples were obtained at presentation and following IST with ATG/CsA including at recovery and relapse. In 10 patients serial samples were analyzed to allow for comparisons between clonal repertoire at presentation and in intervals following IST including when hematologic response was achieved. A hematologic response was seen in 88% of patients. PNH clone was present in 15/23 AA (AA/PNH).Genomic DNA was sequenced according to the ImmunoSeq NGS protocols. A group of 587 controls with an average sequencing depth of 30± 11 x 104 reads was used as reference. The average sequencing depth in AA patients was 74± 53 x103 productive reads. The healthy individuals showed a Simpson's Diversity value (SDV) of .998 (i.e ., number of unique clonotypic reads/number of total clonotypic reads). A total of 341/587 (58%) healthy controls had >1 rearrangement >1% of total repertoire with a mean productive frequency of 2.4%± 2.1%.The average SDV for AA was 0.993. Analyzing the frequency of productive rearrangements in patients at diagnosis, 22/26 patients had >1 rearrangement >1% of total repertoire with a mean productive frequency of 2.9%± 2.1%.We then analyzed the homology of the amino acid sequence of each expanded TCRB clone >1%. If the corresponding CDR3 amino acid sequence was absent in any other individuals (AA patients or controls), we referred to it as a “private” clone. In contrast, “public” clones had CDR3 sequences, which were also present in other individuals; to that end, a clone was defined as “public expanded” if the corresponding productive frequency was >1% in any other control or patient. We estimated that of all the immunodominant clones found in AA patients, 41% were private, likely recognizing patient specific antigens, while 33% were shared only between AA patients (42% in 2, 32% between 3 and 5, 26%>5 patients, N=22) and thus were AA-related. Public specificities constituted 59% of expanded clonotypes. The public clonotypes likely recognize ubiquitous antigens, which also exist in a tolerant form in healthy individuals. Any expanded clonotypes present in controls even if also found in patients (at any frequency) are the least likely to be pathogenic.We subsequently analyzed serial samples to assess the changes in size of the individual clones and establish a correlation between response and contraction of pathogenic clones. Average clonal contraction by 10.9 fold was observed in 50% of responders, and thus the remaining stable or expanding clones are unlikely pathogenic. In contrast, in non-responders only 33% of clones contracted by an average of 2.4 fold. Reappearance of originally expanded clones as well as new expanded clonotypes was observed upon relapse.In sum, our studies indicate that deep TCR sequencing will provide intricate insights into the dynamics of autoimmune process in AA and in the future may be used for monitoring of the efficiency of IST in the clinic and for the study of potentially pathogenic clones in the laboratory. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.

Conversion of Adult Human Endothelium to Lymphoid Competent Haematopoietic Stem Cells

We devised a tractable approach to fully reprogram adult human endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors.rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to adult haematopoietic stem cells, and can be used for clonal engraftment with serial primary and secondary multi-lineage reconstitution. rEC-HSCs give rise to fully mature lymphoid CD4+ and CD8+ T cells. These T cells exhibit a diverse TCR repertoire and are endowed with antigen-dependent adaptive immune function.These T cells are capable of anti-tumor response, and are amenable to chimeric antigen receptor (CAR) gene editing without losing their T lymphoid properties.Our combined approach of transcription factor and niche-mediated cell fate conversion produces haematopoietic stem cells from adult human endothelium and holds promise for modelling hematologic disease in humanized mice and for immune-therapeutic strategies in malignant blood disorders. DisclosuresNo relevant conflicts of interest to declare.

The Orally Available Selective Axl Inhibitor BGB324 Induces Diversification of the Immune Repertoire and Specific Changes in Plasma Biomarker Profiles

Introduction: The RTK Axl represents a novel therapeutic target in AML promoting proliferation and therapy resistance. In addition, Axl suppresses immune responses by decreasing antigen presentation and inhibiting NK cells. BGB324 is a first-in-class, orally available, selective Axl kinase inhibitor which is being explored as a therapy for solid tumours and myeloid malignancies. Selective blockade of Axl by BGB324 in AML is currently being explored in the Phase 1/2 trial BGBC003 (NCT02488408). Until now, n=25 patients have been treated in a classical 3+3 dose escalation design and treatment was generally well-tolerated (n=22 with AML and n=3 with MDS). Steady-state levels of BGB324 were achieved between three and six days after initiation of treatment. One AML patient achieved a CRi of five months, a second achieved a PR and another achieved clearance of circulating blasts accompanied by peripheral blood count recovery > three months (ORR 10%). Four additional AML patients (25%) experienced disease stabilisation for more than four months. Two MDS patients experienced a PR.Here, we report the spectrum of plasma proteins modulated by BGB324 in patients with AML/MDS and their relationship to treatment outcome and compound exposure. Furthermore, we analysed the B- and T- cell repertoire in order to assess potential effects of BGB324 on the immune response in AML patients.Methods: We used the DiscoveryMap panel (Myriad RBM) to measure plasma protein biomarker levels in a selection of patients pre-dose and at C2D1 and performed a cross-comparison with samples from an ongoing NSCLC study with BGB324 (BGBC004, NCT02424617) to identify candidates specifically modulated in AML. The TCRß repertoire was quantified by Next Generation Sequencing of DNA isolated from peripheral blood MNCs using an Illumina MiSeq sequencer. TCRß gene containing the entire Vß, Dß and Jß segments were amplified with BIOMED2-TCRß-A and -B primer pools. The IGH repertoire was analysed using BIOMED2-FR1/-FR3 primer pools. Using genomic DNA as template, the amplicons were tagged with Illumina adapters and indices in two consecutive PCR reactions. Demultiplexing and FastQ formated data output was generated by the MiSeq reporter. Analysis of TCRß and IGH data was performed on a Microsoft Cloud using our in-house analysis pipeline Pippa, which relies on MiXCR analysis tools.Results: Treatment with BGB324 significantly modulated Gas6, soluble Axl (sAxl), Angpt2, CXCL1 (GROalpha), SCGB1A1 (uteroglobin, club cell 10kDa protein) and PLAU (uPAR) levels in patient plasma. Of these, Gas6 was modulated after treatment with BGB324 in AML but not in lung cancer patients while CXCL1 and SCGB1A1 were only influenced in lung cancer patients. sAxl levels correlated with BGB324 exposure indicating that the shed Axl receptor represents a novel biomarker of Axl kinase inhibition.Furthermore, we studied the effects of BGB324 on the T- and B- cell receptor repertoire in n=5 available matched PB samples. We could demonstrate diversification of the TCR repertoire in PB by higher abundance of small T-cell clones at C2D1 with BGB324 compared to pre-treatment levels in three out of five analysed patients.The analyses of the IGH repertoire in the PB indicates higher diversity at C2D1 in two out of five available patients.Thus, treatment with BGB324 can increase the diversity of the IGH and TCRb repertoire in the PB of AML patients.Conclusion: BGB324 is well tolerated in AML patients, exhibits anti-leukemic activity and induces exposure-related modulation of biomarker candidates. Furthermore, BGB324 can induce a diversification of the TCR repertoire in AML patients and might hold potential as an immune-activating drug. DisclosuresLoges:BerGenBio ASA: Research Funding. Ben-Batalla:BerGenBio ASA: Research Funding. Micklem:BerGenBio ASA: Employment, Equity Ownership, Patents & Royalties. Brown:BerGenBIo ASA: Employment. Lorens:BerGenBIo ASA: Employment, Equity Ownership, Patents & Royalties, Research Funding. Yule:BerGenBIo ASA: Employment. Fiedler:Amgen: Patents & Royalties; Amgen, ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen, Pfizer: Research Funding; Amgen, Gilead, GSO, Teva, Jazz Pharmaceuticals: Other: Support for meeting attendance. Cortes:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Teva: Research Funding.

Modelling the interplay between the CD4^{+}/CD8^{+} T-cell ratio and the expression of MHC-I in tumours

Describing the anti-tumour immune response as a series of cellular kinetic reactions from known immunological mechanisms, we create a mathematical model that shows the CD4^{+}/CD8^{+} T-cell ratio, T-cell infiltration and the expression of MHC-I to be interacting factors in tumour elimination. Methods from dynamical systems theory and non-equilibrium statistical mechanics are used to model the T-cell dependent anti-tumour immune response. Our model predicts a critical level of MHC-I expression which determines whether or not the tumour escapes the immune response. This critical level of MHC-I depends on the helper/cytotoxic T-cell ratio. However, our model also suggests that the immune system is robust against small changes in this ratio. We also find that T-cell infiltration and the specificity of the intra-tumour TCR repertoire will affect the critical MHC-I expression. Our work suggests that the functional form of the time evolution of MHC-I expression may explain the qualitative behaviour of tumour growth seen in patients.

Back cover story: Eur. J. Immunol. 6'21

European Journal of ImmunologyVolume 51, Issue 6 Back CoverFree Access Back cover story: Eur. J. Immunol. 6'21 First published: 07 June 2021 https://doi.org/10.1002/eji.202170069AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Our back cover features TRAV and TRBV gene pairing of intraepithelial CD8+ αβ lymphocytes obtained from duodenal biopsies of untreated celiac disease patients. The TRAV/TRBV gene pairing is here depicted using Circos plots. The image is taken from Eggesbø et al. (pp. 1542–1545) where the authors by single-cell TCR sequencing demonstrate that TCR repertoires of celiac disease patients do not show any obvious V gene pairing preference. Furthermore, untreated celiac disease patients display a more polyclonal and diverse TCR repertoire compared to treated patients and healthy subjects. Volume51, Issue6June 2021 RelatedInformation

C‐Rel employs multiple mechanisms to promote the thymic development and peripheral function of regulatory T cells in mice

The NF-κB transcription factor c-Rel is a critical regulator of Treg ontogeny, controlling multiple points of the stepwise developmental pathway. Here, we found that the thymic Treg defect in c-Rel-deficient (cRel-/- ) mice is quantitative, not qualitative, based on analyses of TCR repertoire and TCR signaling strength. However, these parameters are altered in the thymic Treg-precursor population, which is also markedly diminished in cRel-/- mice. Moreover, c-Rel governs the transcriptional programme of both thymic and peripheral Tregs, controlling a core of genes involved with immune signaling, and separately in the periphery, cell cycle progression. Last, the immune suppressive function of peripheral cRel-/- tTregs is diminished in a lymphopenic model of T cell proliferation and is associated with decreased stability of Foxp3 expression. Collectively, we show that c-Rel is a transcriptional regulator that controls multiple aspects of Treg development, differentiation, and function via distinct mechanisms.

Integrating the genomic and TCR repertoire signatures predicts prognosis in primary epithelial ovarian cancer.

e17547 Background: Epithelial ovarian cancer is one of the common causes of gynecologic cancer death around the world. Histologically, EOC is classified into 5 major sub-types: high-grade serous (HGSOC), low grade serous (LGSOC), clear cell (CCOC), endometrioid (ENOC) and mucinous (MOC). Baseline genomic and immunomicroenvironmental characteristics of treatment-naive patients may offer an insight into recurrent risks. Methods: 101 patients with treatment-naive EOC were enrolled, including 60 HGSOC, 14 LGSOC, 8 ENOC, 14 CCOC, 5 MOC. Tumor tissues and paired peripheral blood samples were collected and performed with target sequencing of 1021 genes and TCR repertoire sequencing. Patients were intensively followed up for at least one year. To explored the molecular characteristic among 5 sub-types, genomic features and TCR repertoire signature were evaluated. Results: The mutation rate of PTEN loss was comparable among 5 sub-types for 53.3% (32/60) in HGSOC, 57.1% (8/14) in LGSOC, 42.9% (6/14) in CCOC, 50% (4/8) in ENOC, 40% (2/5) in MOC. Patients with PTEN loss tended to have lower tumor mutation burden (p = 0.077, Wilcoxon test), especially in HGSOC (p = 0.048, Wilcoxon test). Patients with CCOC and ENOC had significantly higher clonality, which indicates T cell expansion, than SOC tumor (HGSOC, LGSOC) (p = 0.036, Wilcoxon test). Moreover, the proportion of top 10, top 50 and top 100 T cell clones were all significantly higher in CCOC and ENOC than that in SOC(p = 0.0072, 0.0041, 0.0058, Wilcoxon test).In addition, richness and Shannon-index, two metrics for T cell diversity, were significantly higher in SOC comparing with CCOC and ENOC(p = 0.032, 0.0018, Wilcoxon test). After integrated the genomic and TCR features, more favorite disease-free survival (DFS) was found in patients with PTEN loss wild type, higher TMB, higher clonality and lower Shannon-index (p = 0.041, HR = 0.24, Mantel–Cox test). Conclusions: Our results highlight the traits of genomic and TCR repertoire in different subtypes of epithelial ovarian cancer and their prognostic significance. Interestingly, higher T cell clonality in CCOC and ENOC patients were found, which suggested the anti-cancer immune response was better activated. However, our study gets a preliminary result, and further verification work is needed.

The correlation between tumor microenvironment and the clonality of the T-cell repertoire in lung cancer and colorectal cancer.

e14524 Background: The tumor microenvironment has been shown to affect the responsiveness of immunotherapy. Effective anti-tumor immune response requires the activation and expansion of specific antigen-reactive T cell clones. It was reported that increased T cell clonality was associated with improved response to immunotherapy. However, what type of tumor microenvironment facilitates T cell clonal expansion remained controversial. The study aims to investigate the correlation between tumor microenvironment and the clonality of the T cell repertoire in lung cancer and colorectal cancer. Methods: 4 lung cancer patients and 4 colorectal cancer patients were enrolled in this study. Tumor tissues and peripheral blood samples were collected for RNA sequencing and T cell receptor CDR3 sequencing. The infiltration levels of 28 immune cells were estimated based on the mRNA expression of the genetic markers. The T cell clonality was defined as 1-Peilou’s evenness. Data were presented as mean± S.E.M. Results: The mean T cell clone counts in the blood samples of the 8 patients were 25676±4782 (ranging from 10259 to 45016), and the mean clonality of the TCR repertoires was 0.20±0.02 (ranging from 0.11 to 0.27). The clonality of T cells in colorectal cancer patients was similar to that of the lung cancer patients (0.22±0.02 versus 0.18±0.03, p = 0.31), showing comparable potentials of antigenic responses. The tumor infiltration of regulatory T cells, type 17 T helper cells, CD56bright natural killer cells, and natural killer cells varied greatly among patients, the coefficient of variation of those cells were 54.61%, 54.61%, 54.43%, and 55.62% respectively. In contrast, the coefficient of variation of monocytes was 23.34%, displaying a relatively even distribution among patients. The Pearson’s correlation coefficient was calculated to show the correlation between T cell clonality and the infiltration level of all 28 types of immune cells. Notably, only the infiltration of type 17 T helper cells significantly associated with T cell clonality, the positive correlation gave an R square value of 0.68 (r = 0.82, 95% confidence interval of 0.04-0.98, p = 0.04). The infiltration levels of CD4+ T cells, CD8+ T cells, regulatory T cells, type 1 and type 2 T cells, and gamma delta T cells were not affected by T cell clonal expansion. The expression of B cells, dendritic cells, macrophages, natural killer cells, and monocytes did not correlate with T cell clonal expansion. However, the abundance of neutrophils appears to positively correlate with T cell clonality (p = 0.09). Conclusions: The clonal expansion was significantly associated with the infiltration of type 17 T helper cells but not other subtypes of T cells, showing that the type 17 T helper cells are crucial to the antigenic responses in lung cancer and colorectal cancer. A neutrophil enriched tumor microenvironment may facilitate T cell clonal expansion.

Robust prediction of response to immunotherapy in a mixed cohort of previously treated and immunotherapy-naive melanoma patients.

e21548 Background: While immune checkpoint blockade (ICB) has become a pillar of cancer treatment, biomarkers that consistently predict patient response remain elusive due to the complex mechanisms driving immune response to tumors. We hypothesized that a multi-dimensional approach modeling both tumor and immune-related molecular mechanisms would better predict ICB response than simpler mutation-focused biomarkers. Methods: Pre-treatment samples from 51 unresectable, stage III/IV melanoma patients who underwent anti-PD-1 therapy were characterized to assess factors influencing response. RECIST criteria were used to evaluate tumor response to therapy, with a median follow-up of 24 months. For each patient, a single paired FFPE tumor and normal blood sample was collected and profiled using Personalis’ ImmunoID NeXT Platform; an augmented exome/transcriptome platform and analysis pipeline which produces comprehensive tumor mutation information, gene expression profiling, neoantigen characterization, HLA typing and LOH, TCR repertoire profiling, and tumor microenvironment profiling. These data were then integrated to form a composite neoantigen presentation score (NEOPS) for each patient. Results: We demonstrate increasing predictive power with deeper modeling of neoantigens and immune-related resistance mechanisms to ICB. Neoantigen burden score (NBS) was derived from SHERPA, our exome and transcriptome integrative neoantigen prediction model. We found that NBS more significantly stratified responders and non-responders (P = 0.016) than TMB alone (P = 0.049). A broader score, NEOPS, incorporated DASH, to capture HLA allele-specific loss of heterozygosity and additional antigen presenting machinery resistance mechanisms. NEOPS consequently demonstrated a stronger association with response (p = 0.002). We used area under the curve (AUC) to benchmark NEOPS performance against established mutational and transcriptomic predictive models, demonstrating the model was more predictive of response in both the treatment naive setting, as well as in a mixed cohort of treatment-naive and experienced patients. These findings were confirmed in an independent cohort of patients (n = 110), suggesting that NEOPS is a robust, novel biomarker of ICB response in melanoma. Conclusions: Given the complex nature of resistance to immunotherapy, as well as potential toxicities associated with treatment, there is a need for biomarkers that can more accurately predict response. Here we demonstrate that NEOPS can significantly improve stratification of patient response. We also demonstrate that data intensive biomarkers like NEOPS can be clinically practical, with comprehensive tumor profiling in our clinical cohort achieved using very limited tumor tissue.

Multiomics analysis reveals high-immune infiltration in tumor-adjacent lung tissues affects the prognosis of stage I NSCLC.

8540 Background: Along with the wide application of low-dose spiral CT in lung cancer screening, a large number of resectable patients with lung cancer are identified, especially stage I Non-Small Cell Lung Cancer (NSCLC). However, their prognosis varies greatly and 5-year recurrence rate of stage I NSCLC is around 30%. Therefore, it is crucial to explore the potential mechanism of recurrence of early NSCLC. Methods: Eighty-seven patients with stage I NSCLC were enrolled from April 2008 to July 2015, including 79 squamous carcinoma and 28 adenocarcinoma. Frozen tumor tissues and paired tumor-adjacent lung tissues were collected to employ targeted panel sequencing, RNA sequencing and TCR repertoire sequencing. Results: Ninety-five non-silent mutations were detected in tumor-adjacent lung tissues with a median tumor mutation burden of 1.5/Mb, significantly lower than that in tumor tissues (11/Mb), p < 0.05. 42 mutations were specifically detected in the adjacent normal tissues, enriching in the immune response pathways. Comparing with paired tumors, tumor-adjacent lung tissues were found more favorable immune infiltration including higher immune cell activity like CD8+ T cell (0.50 vs 0.43, p < 0.0001), up-regulated immune-associated pathways, higher TCR clonality (0.19 vs 0.18, p < 0.05), less loss of heterozygosity (LOH) of human leukocyte antigen (HLA) (6.25% vs 50%, p < 0.0001) and observed predicted neoantigens expression (1 vs 128, p < 0.01). However, more shared viral-associated TCRs in tumor and tumor-adjacent tissues were found using the GLIPH algorithm. Prognostic analysis showed patients with higher overlap of TCR in tumors and tumor-adjacent lung tissues were prone to recur in five years. Furthermore, patients with higher immune infiltration in tumor-adjacent lung tissues had favorable outcomes than those with lower immune infiltration (HR: 0.37 for DFS, p < 0.05, HR: 0.31 for OS, p < 0.05), irrelevant of the infiltration level in tumor tissues. Conclusions: Immune microenvironment in tumor-adjacent lung tissues plays important roles in progress of stage I NSCLC.

Weighting tumor-specific TCR repertoires as a classifier to stratify the immunotherapy delivery in non-small cell lung cancers.

Analysis of T cell receptor (TCR) repertoires may contribute to better understanding of the response to immunotherapy. By deep sequencing of the TCR β chain complementarity-determining regions in the paired biopsies and peripheral blood specimens of 31 patients with non-small cell lung cancer (NSCLC) treated with anti-programmed death 1 (PD-1) or PD-ligand 1 (PD-L1) therapy, we developed a previously unidentified index, the TCR-based immunotherapy response index (TIR index), that estimated the degree of overlap of the TCR repertoire between tumor-infiltrating lymphocytes and circulating PD-1+CD8+T cells (shared TCR clones). This index correlated with response and survival outcomes of anti-PD-(L)1 treatment. All the TCR sequences of neoantigen-stimulated T cells were included in the shared TCR clones, indicating that TCR clones involved in TIR index estimation represented tumor-specific T cells. Therefore, the TIR index is a feasible approach for assessing tumor-specific TCR and stratifying patients with NSCLC for anti-PD-(L)1 therapy.

HLA class I-associated expansion of TRBV11-2 T cells in multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. Superantigen specificity for different Vβ chains results in Vβ skewing, whereby T cells with specific Vβ chains and diverse antigen specificity are overrepresented in the T cell receptor (TCR) repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCRβ variable gene 11-2 (TRBV11-2), with up to 24% of clonal T cell space occupied by TRBV11-2 T cells, which correlated with MIS-C severity and serum cytokine levels. Analysis of TRBJ gene usage and complementarity-determining region 3 (CDR3) length distribution of MIS-C expanded TRBV11-2 clones revealed extensive junctional diversity. Patients with TRBV11-2 expansion shared HLA class I alleles A02, B35, and C04, indicating what we believe is a novel mechanism for CDR3-independent T cell expansion. In silico modeling indicated that polyacidic residues in the Vβ chain encoded by TRBV11-2 (Vβ21.3) strongly interact with the superantigen-like motif of SARS-CoV-2 spike glycoprotein, suggesting that unprocessed SARS-CoV-2 spike may directly mediate TRBV11-2 expansion. Overall, our data indicate that a CDR3-independent interaction between SARS-CoV-2 spike and TCR leads to T cell expansion and possibly activation, which may account for the clinical presentation of MIS-C.

Hallmarks of T cell aging.

The aged adaptive immune system is characterized by progressive dysfunction as well as increased autoimmunity. This decline is responsible for elevated susceptibility to infection and cancer, as well as decreased vaccination efficacy. Recent evidence indicates that CD4+ T cell-intrinsic alteratins contribute to chronic inflammation and are sufficient to accelerate an organism-wide aging phenotype, supporting the idea that T cell aging plays a major role in body-wide deterioration. In this Review, we propose ten molecular hallmarks to represent common denominators of T cell aging. These hallmarks are grouped into four primary hallmarks (thymic involution, mitochondrial dysfunction, genetic and epigenetic alterations, and loss of proteostasis) and four secondary hallmarks (reduction of the TCR repertoire, naive-memory imbalance, T cell senescence, and lack of effector plasticity), and together they explain the manifestation of the two integrative hallmarks (immunodeficiency and inflammaging). A major challenge now is weighing the relative impact of these hallmarks on T cell aging and understanding their interconnections, with the final goal of defining molecular targets for interventions in the aging process.

Identification of a unique TCR repertoire, consistent with a superantigen selection process in Children with Multi-system Inflammatory Syndrome

Abstract Multisystem Inflammatory Syndrome in Children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares many clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. The superantigen specificity for binding different Vβ-chains results in Vβ-skewing, whereby T cells with specific Vβ-chains and diverse antigen specificity are overrepresented in the TCR repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCR Beta Variable gene (TRBV)11-2. Furthermore, TRBV11-2 skewing was remarkably correlated with MIS-C severity and serum cytokine levels. Further analysis of TRBJ gene usage and CDR3 length distribution of MIS-C expanding TRBV11-2 clones revealed extensive junctional diversity, indicating a superantigen-mediated selection process for TRBV expansion. In silico modelling indicates that polyacidic residues in TCR Vβ11-2 engage in strong interactions with the superantigen-like motif of SARS-CoV-2 spike glycoprotein. Overall, our data indicate that the immune response in MIS-C is consistent with superantigenic activation.

Single-cell proteogenomics reveals that SLAM/SAP signaling regulates the development of innate-like γδT cell subsets with distinct TCR repertoires

Abstract Innate-like T cells are unusual T cells that are enriched in mucosal tissues, and which constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. While it is known that SLAM/SAP signaling is required for the development of innate-like iNKT and MAIT αβ T cells, far less is known about the role of this pathway in the development and function of γδ T cells. Here, we utilized a single-cell proteogenomics approach coupled with γδ V(D)J profiling to define the transcriptional landscape and developmental checkpoints of SAP-dependent γδ T cells. We found that SAP-dependent γδNKT TCRs utilized TRGV1 paired with TRAV15N-1 or TRAV15-1/DV6-1, and we identified two distinct developmental γδNKT stages in the neonatal thymus that were distinguished by SLAMF6 and SLAMF7 expression. Moreover, we found that a significant fraction of SLAMf1+innate-like γδT17 cells utilized TRGV4 and TRGV6, γ-chains paired with TRDV2 or TRDV5 δ-chains of limited diversity. Examination of SAP-deficient neonatal thymus revealed decreased numbers of mature SLAMF1+ γδT17 cells, which was associated with lower numbers of an invariant germline-encoded TRGV4/TRDV5 clonotype. Accordingly, we observed significant alterations in the SLAMF1+ γδT17 TCR repertoire in adult lung. We found that lung γδTIFN were CD44+CD45RB+CD27+ cells that co-expressed SLAMF6and SLAMF7, and that these cells predominantly utilized a diverse TRDV7 paired with TRGV4. Interestingly, we observed a specific decrease of these TRDV7+ γδTIFN cells in SAP-deficient mice, which we confirmed using qPCR. Altogether, these data indicate a crucial link between SLAM/SAP signaling and the development of functionally distinct innate-like γδ TCR clonotypes.

The Role of Aire in the selection of Regulatory T cells in Type 1 Diabetes

Abstract In autoimmune diseases like T1D, it is proposed that failure in central tolerance mechanisms towards pancreatic islet self-antigens leads to the escape of pathogenic clones and loss of critical immunosuppressive antigen-specific Tregs. The importance of Aire in maintaining central tolerance against self-reactive antigens is evidenced by the multiorgan autoimmune disease seen in individuals with mutation the Aire gene. When Aire-expression is specifically ablated in mouse mTECs, there is a dramatic increase in the number of self-reactive T cells that escape negative selection, in addition, a dramatic loss of Tregs. Utilizing novel high-throughput platforms and single-cell DNA barcoding technology has allowed us to assess several thousand CD4+ T-cell clones. The use of FoxP3-GFP reporter mice permits us to identify individual FoxP3+ Tregs that have undergone Aire-dependent selection in the thymus and compare the TCR repertoire to mice that have had Aire specifically ablated from the mTECs. Identification of unique clones and gene signatures within our large datasets has allowed us to start to qualitatively assess the functional role of the identified TCRs within the model of T1D. GLIPH2 clustering using our extensive insulin-specific TCRs have identified a number of motifs shared within our TCR dataset, as well as a unique V-gene usage that may predict TCR affinity. Our proposed study aims to expand our knowledge on TCR repertoire of Aire-dependent FoxP3+ Tregs and the antigen-specificity in T1D, improving our understanding of the respective function of thymic-derived antigen-specific Treg suppression in peripheral tissues associated with autoimmune T1D.