Intratumor expanded T cell clones can be non-sentinel lymph node derived in breast cancer revealed by single-cell immune profiling

BackgroundSentinel lymph nodes (LNs) are regarded as key immune surveillance sites in cancer wherein mature dendritic cells present tumor-derived antigens to prime and activate T cells, which then migrate to the tumor site. However, it is unclear whether the tumor-specific T cells can be elicited within the tumor independent of the sentinel LNs.MethodsWe performed an integrative analysis of gene expression profiles of 65,285 cells and T cell receptor sequences of 15,831 T cells from 5 paired primary breast tumors and sentinel LNs to identify where clonal T cells come from and the characteristics of those clonal T cells.ResultsThe proportion of clonal T cells was higher in the primary tumors compared with the sentinel LNs, whereas all expanded clones identified in the sentinel LN were also present in the primary tumors. In contrast, 10.91% of the expanded clones in the primary tumors were not found in the sentinel LNs. These novel intratumoral T cell clones were characterized by high tissues retention capacity (CXCR6 +ITGAE+) and a distinct coinhibitory pattern (CD39 +NKG2A+) compared with the expanded T cell clones common to both sites. Furthermore, multiplex immunofluorescence imaging showed the presence of tertiary lymphoid structures (TLS) in the primary breast tumors wherein the activated cytolytic T cells were concentrated, indicating its possible role in eliciting non-sentinel LN-derived T cell clones.ConclusionsOur study revealed expanded intratumor non-sentinel LN derived T cell clones located in the TLS, which points to the need for exploring the role of TLS in antitumor immunity.

Divergent clonal differentiation trajectories of T cell exhaustion

T cells activated by chronic antigen exposure in the setting of viral infections or cancer can adopt an exhausted T cell (Tex) state, characterized by reduced effector function and proliferative capacity, and the upregulation of inhibitory receptors. However, whether all antigen-specific T cell clones follow the same molecular and cellular Tex differentiation trajectory remains unclear. Here, we generate a single-cell multi-omic atlas of T cell exhaustion that redefines the phenotypic diversity and molecular regulation of Tex phenotypes. Longitudinal analysis during chronic viral infection identifies an early effector phenotype that is epigenetically primed for Tex differentiation and two late-stage Tex cell states with either a terminal exhaustion or a killer cell lectin-like receptor (KLR)-expressing cytotoxic gene signature. We define clonal trajectories of antigen-specific T cells using paired single-cell RNA and T cell receptor sequencing and reveal distinct differentiation trajectories resulting in terminal Tex-biased, KLR Tex-biased, or divergent clones that differentiate into both phenotypes. Comparison of Tex phenotypes among shared T cell clones that traffic to multiple organs reveals that clonal differentiation trajectories are maintained across tissues. Finally, we show that differences in clonal differentiation trajectory are driven by TCR signal strength, whereby high-affinity T cell clones preferentially adopt a terminal Tex fate, while low-affinity clones adopt an effector-like KLR Tex fate that is detectable long-term but depleted in high antigen settings. These findings reveal clonal heterogeneity in the T cell response to chronic antigen and genomic programs that underlie Tex fates and persistence.

Chronic Graft-Versus-Host Disease Immunoprofiling Reveals T Cell Clonal Dynamics That Correlate with Disease Activity: A Novel Molecular "Biomarker"?

Chronic graft-versus-host disease (cGVHD) is the main cause of morbidity and transplant-related mortality following allogeneic hematopoietic stem cell transplantation (alloHSCT), however effective treatment options are limited. Lack of objective surrogates (biomarkers) for treatment response has hindered progress in this respect. T cells are considered the major effectors of cGVHD, yet the respective repertoires are insufficiently charted. Here, we investigated the dynamic architecture of T-cell repertoires in cGVHD by exploiting next-generation sequencing (NGS), aiming to uncover immunogenetic signatures linked with cGHVD occurrence and response to treatment. We analyzed 53 blood samples from 15 patients with hematological malignancies who underwent alloHSCT, with an intended bias towards patients who developed cGVHD (n=12). The remaining 3 patients had no cGVHD (control group). Patients with cGVHD were analyzed at cGVHD onset and/or prior to a new line of treatment (n=17 samples) as well as at clinically relevant timepoints following treatment: (i) partial response (PR, n=8), (ii) complete response (CR, n=3), (iii) stable disease (SD, n=18), (iv) progression (PD, n=1). Treatment modalities involved corticosteroids, mycophenolate myfetil, extracorporeal phototherapy, ruxolitinib and ibrutinib. Patients with no cGVHD were analyzed at 3 months (+3mo) and 6 months (+6mo) post-alloHSCT (n=6 samples). Starting material was PB mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS and detailed bioinformatics analysis. Only productive TRBV-TRBD-TRBJ rearrangements were evaluated (n= 13,059,730, 89.9% of filtered-in sequences, median 219,063/sample). For repertoire characterization, clonotypes (i.e., TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence) were considered (median 9,725 distinct clonotypes/sample). The 10 most frequent clonotypes/sample were defined as "major". To purge T cell clones expanded secondary to viruses common in the alloHSCT setting, we compared all major clonotypes against the GenBank TRB sequence database (n=18,402), as well as an extensive TR repertoire dataset from a previous study by our group, profiling tri-virus-specific (CMV, EBV, BK) T cell products generated from immunocompetent donors (n=947,298). Overall, we identified 289 unique major clonotypes; 38 were excluded due to match within the tri-VST database (no match within GenBank). All cases with cGVHD displayed significant clonal T cell expansions both pre- and post-treatment (overall median cumulative frequency of the 6 most expanded T cell clonotypes/sample 32.4%). However, clonality tended to decrease in PR samples compared to pre-treatment (19.8% vs 33.9%, respectively), although not reaching statistical significance possibly due to small sample size (p=0.06). Patients with no cGVHD, on the other hand, consistently displayed a clonality decrease overtime (31.8% at +3mo vs 18.4% at +6mo, p=0.02). Importantly, patients with no GVHD displayed TR repertoire reconstitution with few major T cell clones of the +3mo timepoint persisting at +6mo (median 20%). In clear contrast, cGVHD T cell repertoires were dominated by clones which persisted overtime (median 60%). In fact, repertoire persistence was most evident in SD (median 66.7%) and significantly lower in PR and CR (33.3% and 10.0%, respectively, p<0.05), suggesting that the persisting T cell clones are implicated in cGVHD pathogenesis. Notably, repertoire comparisons across patients in our cohort revealed 6 "public" clonotypes [5 clonotypes shared among a single pair of patients (Pt1 and Pt2) and 1 clonotype shared among the same pair plus an additional patient, Pt3], suggesting the existence of a common antigenic trigger. In conclusion, NGS immunoprofiling in cGVHD reveals expanded T cell clones with clonal dynamics that correlate with clinical response, indicating a causal relationship to cGVHD pathogenesis. Identification and longitudinal tracking of T cell clones associated with cGHVD could serve as a molecular surrogate marker for disease activity, with evident benefits for cGVHD monitoring and evaluation of response to various treatments. Disclosures Anagnostopoulos: Abbvie: Other: clinical trials; Sanofi: Other: clinical trials ; Ocopeptides: Other: clinical trials ; GSK: Other: clinical trials; Incyte: Other: clinical trials ; Takeda: Other: clinical trials ; Amgen: Other: clinical trials ; Janssen: Other: clinical trials; novartis: Other: clinical trials; Celgene: Other: clinical trials; Roche: Other: clinical trials; Astellas: Other: clinical trials . Chatzidimitriou: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.

Mapping the Multiple Myeloma T Cell Landscape By Immunotherapeutic Perturbation Reveals Mechanism and Determinants of Response to Bispecific T Cell Engagers

Immunotherapies have transformed the clinical care of patients with cancer. Bispecific T cell engagers (TCEs) have recently entered early-phase clinical trials of multiple myeloma (MM) and shown remarkable response rates even in heavily pretreated patients. However, T cells are heterogeneous with respect to phenotype, function and specificity for tumor antigens and currently we have limited understanding how to identify and monitor tumor specific T cells in hematological malignancies. It is furthermore unclear why individual patients fail to elicit an antitumor immune response upon treatment with TCEs and whether a persistent T cell response to TCEs relies on reinvigoration of pre-existing tumor-infiltrating lymphocytes or on recruitment of novel T cells. Here we performed longitudinal paired single-cell RNA and T cell receptor (TCR) sequencing on >100,000 immune cells from patients with MM before, during and after TCE therapy. We defined transcriptional gradients of MM-infiltrating immune cells between n=5 healthy bone marrow donors, n=10 newly diagnosed MM patients and n=11 refractory MM patients undergoing immunotherapy with bispecific BCMA-targeting antibodies. By tracking T cell clones over time using their TCR as individual barcode, we further integrated these longitudinal in vivo data with protein-level analysis and functional validation in MM bone-marrow cultures exposed to TCEs. Refractory MM patients exhibited a highly individual bone-marrow immune composition, that was significantly perturbed compared to healthy or diseased, but therapy-naïve bone marrow. We observed that the inter-patient heterogeneity in the T cell landscape composition is superimposed by conserved TCR repertoire dynamics forming a trajectory between early anti-tumor effector states and exhaustion. In all patients, we observed a dichotomy of TCE-responsive versus TCE-refractory T cell clones. Longitudinal tracking of TCE-responsive T cell clones and their transcriptional phenotypes revealed coupling of tumor recognition, clonal expansion and T cell dysfunction marked by expression of cytotoxicity (GZMB, GNLY) and terminal exhaustion markers, such as TOX and CD39. Significant clonal replacement of T cells was evident in n=5 clinically responding patients with MM throughout continued TCE therapy and driven by a subset of non-exhausted, naïve-like CD8 + T cells. The top 1% TCE-responsive clones were fate-determined and either followed a memory-exhaustion or cytotoxicity trajectory. Patients who did not respond to TCE therapy exhibited a dysfunctional T cell landscape before therapy that limited clonal expansion and TCR persistence. As proof-of-concept, we matched single-cell profiling data of n=10 individual patients with protein-level analysis and functional validation of TCE-driven T cell expansion in vitro, providing the first signals of preferential expansion of specific fate- and avidity-determined clones upon TCE-mediated stimulation. We propose the mode of action of TCE therapy in MM to be driven by pre-existing T cell fate commitments that determine clonotype diversification and persistence, and ultimately, clinical response. Our results further demonstrate that clinical TCE response derives from a distinct repertoire of pre-existing T cell clones, whereas other clonotypes are functionally excluded from the repertoire and subsequently lost during therapy. We define the determinants of response to TCE treatment to be inherent to the individual's T cell repertoire before therapy. Our results provide the rationale for response prediction and monitoring of future immunotherapy approaches in MM patients beyond TCE therapy. Figure 1 Figure 1. Disclosures Neri: BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Goldschmidt: Amgen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Adaptive Biotechnology: Consultancy; Celgene: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; BMS: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Chugai: Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; GSK: Honoraria; Incyte: Research Funding; Janssen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Johns Hopkins University: Other: Grant; Molecular Partners: Research Funding; MSD: Research Funding; Mundipharma: Research Funding; Novartis: Honoraria, Research Funding; Dietmar-Hopp-Foundation: Other: Grant; Sanofi: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Takeda: Consultancy, Research Funding. Weinhold: Sanofi: Honoraria. Raab: Abbvie: Consultancy, Honoraria; Roche: Consultancy; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bahlis: Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genentech: Consultancy; Janssen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Single-Cell Clonal Tracking in Allogeneic Hematopoietic Stem Cell Transplantation Reveals Time Dependent and Distinct Functional Patterns in Traceable Donor T Cell Clones

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only curative treatment option for various malignant hematological diseases. The therapeutic effect of alloHSCT is a long-lasting graft-versus-leukemia (GvL) effect of the transferred graft. T cells are important mediators of GvL and the longitudinal tracking of T-cell clones from donor to recipient is of particular interest in the setting of alloHSCT as this might provide further insight into mechanisms leading to survival and expansion of particular clones. In a broader sense, we used the unique setting of alloHSCT to study survival and expansion of mature T-cell clones after transfer into an immune cell depleted and allogeneic patient. We used single-cell RNA sequencing (scRNAseq) to integrate immune subset delineation, clone identification and transcriptome information of about 35500 single T cells in peripheral blood of 14 paired donor-recipient samples in four alloHSCT pairs. Donor samples were collected before and after treatment with Granulocyte-Colony Stimulating Factor (GCSF), and recipient samples were collected on days +90 and +180 post-transplant. Looking at common diversity scores of pooled donor versus pooled recipient time points we observe an expected decrease of TCR diversity after transplantation (median inv. Simpson's 379 in donor vs. 20 in recipient samples, p=0.011, Figure 1A). On single cell level, we observe a substantial decrease of unique T-cell clones after transplantation compared to donor samples, which in return means that certain TCR clones markedly expand, contributing to a skewing of the TCR repertoire in the post-transplant course. The majority of these cells represent CD8 effector memory T cells. Our main interest was a better understanding of traceable and persisting T-cell clones. In a first step, we looked at the overall clonal overlap between time points of the different donor-recipient pairs, using only combined TCR alpha and beta chain information to define specific T-cell clones. We find the highest overlaps of T-cell clones between time points within individuals (e.g., Morisita score 0.91 between preGCSF and postGCSF of donor 16 and Morisita score 0.65 between days +90 and +180 of recipient 16, Figure 1B). Additionally, we demonstrate an inter-individual overlap between donors and their respective recipients in all pairs on single cell level (Figure 1C). Next, we compared the differential gene expression of traceable and non-traceable T cell clones and found that the traceable T cell clones exhibit a distinct transcriptional program, characterized by upregulation of genes related to T cell proliferation and chemotaxis as well as antigen presentation, while housekeeping functions such as translation are downregulated. In order to examine the dynamic changes of the T-cell transcriptome, we looked at the differential gene expression at the consecutive time points of pooled traceable clones in all pairs. This shows an induction of an activation pattern during the donor-recipient transfer and post-transplant phase involving genes related to the cell cycle and graft-versus-host disease (Figure 1D). Phenotype analysis via antibody-derived tags accordingly revealed an upregulation of activation markers in the recipients. To our knowledge, this is the first time that longitudinal inter-individual (donor-to-recipient) overlap of single-cell TCR alpha/beta clones is demonstrated in the setting of alloHSCT revealing time point-dependent and distinct functional patterns in traceable donor T cell clones. Figure 1 Figure 1. Disclosures Penack: Neovii: Honoraria; MSD: Honoraria; Incyte: Research Funding; Priothera: Consultancy; Therakos: Honoraria; Gilead: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Takeda: Research Funding; Astellas: Honoraria; Jazz: Honoraria; Omeros: Consultancy; Shionogi: Consultancy. Bullinger: Amgen: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Hexal: Consultancy; Abbvie: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Astellas: Honoraria; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bayer: Research Funding; Seattle Genetics: Honoraria; Novartis: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Menarini: Consultancy; Gilead: Consultancy; Celgene: Consultancy, Honoraria; Sanofi: Honoraria. Na: Bristol Myers Squibb: Research Funding; Shire/Takeda: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding.

691 Identification of shared tumor epitopes from endogenous retroviruses inducing high avidity cytotoxic T cells for cancer immunotherapy

BackgroundHuman endogenous retroviruses (HERVs) are aberrantly expressed by tumor cells and may represent a source of T cell epitopesMethodsUsing TCGA pancancer RNAseq data (n=8,893 samples), we developed a bioinformatics-based method to select cancer-specific HERVs associated with a cytotoxic T cell response (“cyt-HERVs”) and identify shared T cell epitope candidates. T cells were primed with selected short and long peptide candidates from HLA-A2+ healthy donors. Peptide-specific dextramers were used to sort and expand specific CD8+ T cell clones and determine their TCR sequences and avidity. Cytotoxicity was assessed against HERV-expressing tumor cell lines and patient-derived organoids using Incucyte and Nanolive technologies (Flowchart, figure 1).ResultsIn a pancancer analysis, we identified 57 HML-2/HERV-K HLA-A*0201 epitope candidates from 27 distinct open reading frames. Six shared HLA-A2 strong binders 9-mer peptides, present on multiple HERVs located on different chromosomes, and with translational evidence found in mass spectrometry public datasets, were selected and synthetized. In vitro HLA binding assay confirmed peptide-HLA affinity. Priming assays showed the presence of specific CD8+ T cells leading to polyfunctional IFN-γ+ TNF-α+ T cell responses with upregulation of the degranulation marker CD107A upon co-culture with peptide-pulsed T2 cells. Synthetic long peptides containing the epitopes were used to confirm the correct processing by antigen-presenting cells. The functionality of the sorted T cell clones was confirmed using an Elispot assay (GrzB+ IFN-γ+). Their sequenced TCRs were predicted to stably interact with their respective MHC-peptide complexes in a 3D model. This was confirmed by measurement of the functional avidity, which was in the same order as CMV-specific T cell clones. HERV-specific CD8+ T cells induced specific cell death of HLA-A2+ cancer cell lines, associated with IFN-g production, in a HLA-A2 restricted manner. Finally, pre-existing HERV-specific CD8+ T cells were identified using dextramers among tumor infiltrating lymphocytes (TILs) from cancer patients. HERV-specific T cells co-cultured with patient derived organoids showed signs of activation with lysis of the organoid.ConclusionsOur bioinformatics-based approach allowed us to identify shared HERV-derived CD8+ T cell epitopes specifically expressed by tumor cells and inducing high avidity T cell clones able to kill tumor cells in a class I-restricted manner. The detection of TILs recognizing HERV peptides suggests natural presentation of these epitopes in the tumors. These HERV-derived epitopes may thus represent relevant targets for the development of new cancer vaccines or T cell-based therapies, especially in tumors with low mutational burden.Abstract 691 Figure 1Graphical flowchart of HERV antigen validation. Graphical representation of the flowchart used to identify and validate specific CD8+ T cells for shared tumor epitopes from endogenous retroviruses http://dx.doi.org/10.1136/jitc-2021-SITC2021.691

453 Personalized DNA neoantigen vaccine (GNOS-PV02) in combination with plasmid IL-12 and pembrolizumab for the treatment of patients with advanced hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death. Immune checkpoint inhibitors targeting PD-1 have limited activity in HCC as monotherapy, with response rates ranging from 14–17%. Tumor neoantigens derived from tumor-specific mutations can be incorporated into personalized therapeutic cancer vaccines to generate tumor-specific T cell immunity, potentially priming the immune system for anti-PD1 therapy. DNA vaccines have been shown to elicit strong CD8 and CD4 T cell responses in preclinical and clinical trials. GNOS-PV02 is a personalized DNA vaccine, encoding up to 40 patient-specific neoantigens. In the GT-30 trial, it is used in combination with INO-9012 (plasmid-encoded IL-12) and pembrolizumab for the treatment of advanced HCC.MethodsGT-30 is a single-arm phase I/II clinical trial to assess the safety, immunogenicity, and preliminary efficacy of GNOS-PV02 in combination with INO-9012 and pembrolizumab in patients with advanced HCC. Twenty-four patients are anticipated to be enrolled. Patients are recruited upon diagnosis or during first-line treatment with tyrosine kinase inhibitors (TKI). Tumors are biopsied for exome and transcriptome sequencing, and peripheral blood collected for germline sequencing and histogenetics. The tumor specific vaccine is designed, optimized and manufactured during first-line therapy. Each vaccine encodes up to 40 neoantigens. After progression or intolerance with first-line therapy, patients commence concurrent personalized vaccine and pembrolizumab. GNOS-PV02 + INO-9012 are administered Q3w for the first 4 doses and Q9w thereafter. Pembrolizumab is delivered Q3w.ResultsWe performed a data cut-off on the first 12 patients. The median age was 66 years (range 55–75 years). GNOS-PV02 + INO-9012 with pembrolizumab has had no reported DLTs or drug related SAEs. The most common treatment-related AE were grade 1 fatigue (25%) and grade 1 injection site reactions (17%). By including up to 40 epitopes in the vaccine we were able to target all neoantigens present in 83% of the patients. The objective response rate was 25% (3/12 partial response, 5/12 stable disease, 4/12 progressive disease). Analysis of the TCR repertoire in peripheral blood and tumor tissue identified novel and significantly expanded T cell clones post-vaccination in all patients analyzed. Many of the novel peripheral T cell clones were also identified to have trafficked to the TME at week 9, potentially mediating the observed tumor regressions.ConclusionsThese data demonstrate the potential of GNOS-PV02 + INO-9012 with pembrolizumab to target multiple neoepitopes, and provide initial support for the safety and efficacy of this regimen in patients with advanced HCC.Trial RegistrationNCT04251117Ethics ApprovalThe study obtained IRB approval (IRB) and all patients signed informed consent prior to taking part in the clinical trial. NZCR EC: 20/NTA/6; JHU: IRB00227771; Mount Sinai: HS#: 20–00076

Cross-Reactivity to Mutated Viral Immune Targets Can Influence CD8+ T Cell Functionality: An Alternative Viral Adaptation Strategy

Loss of T cell immunogenicity due to mutations in virally encoded epitopes is a well-described adaptation strategy to limit host anti-viral immunity. Another described, but less understood, adaptation strategy involves the selection of mutations within epitopes that retain immune recognition, suggesting a benefit for the virus despite continued immune pressure (termed non-classical adaptation). To understand this adaptation strategy, we utilized a single cell transcriptomic approach to identify features of the HIV-specific CD8+ T cell responses targeting non-adapted (NAE) and adapted (AE) forms of epitopes containing a non-classical adaptation. T cell receptor (TCR) repertoire and transcriptome were obtained from antigen-specific CD8+ T cells of chronic (n=7) and acute (n=4) HIV-infected subjects identified by either HLA class I tetramers or upregulation of activation markers following peptide stimulation. CD8+ T cells were predominantly dual tetramer+, confirming a large proportion of cross-reactive TCR clonotypes capable of recognizing the NAE and AE form. However, single-reactive CD8+ T cells were identified in acute HIV-infected subjects only, providing the potential for the selection of T cell clones over time. The transcriptomic profile of CD8+ T cells was dependent on the autologous virus: subjects whose virus encoded the NAE form of the epitope (and who transitioned to the AE form at a later timepoint) exhibited an ‘effective’ immune response, as indicated by expression of transcripts associated with polyfunctionality, cytotoxicity and apoptosis (largely driven by the genes GZMB, IFNɣ, CCL3, CCL4 and CCL5). These data suggest that viral adaptation at a single amino acid residue can provide an alternative strategy for viral survival by modulating the transcriptome of CD8+ T cells and potentially selecting for less effective T cell clones from the acute to chronic phase.

Prospective Tracking of Donor-Reactive T-Cell Clones in the Circulation and Rejecting Human Kidney Allografts

BackgroundAntigen recognition of allo-peptides and HLA molecules leads to the activation of donor-reactive T-cells following transplantation, potentially causing T-cell-mediated rejection (TCMR). Sequencing of the T-cell receptor (TCR) repertoire can be used to track the donor-reactive repertoire in blood and tissue of patients after kidney transplantation.Methods/DesignIn this prospective cohort study, 117 non-sensitized kidney transplant recipients with anti-CD25 induction were included. Peripheral mononuclear cells (PBMCs) were sampled pre-transplant and at the time of protocol or indication biopsies together with graft tissue. Next-generation sequencing (NGS) of the CDR3 region of the TCRbeta chain was performed after donor stimulation in mixed lymphocyte reactions to define the donor-reactive TCR repertoire. Blood and tissue of six patients experiencing a TCMR and six patients without rejection on protocol biopsies were interrogated for these TCRs. To elucidate common features of T-cell clonotypes, a network analysis of the TCR repertoires was performed.ResultsAfter transplantation, the frequency of circulating donor-reactive CD4 T-cells increased significantly from 0.86 ± 0.40% to 2.06 ± 0.40% of all CD4 cells (p < 0.001, mean dif.: -1.197, CI: -1.802, -0.593). The number of circulating donor-reactive CD4 clonotypes increased from 0.72 ± 0.33% to 1.89 ± 0.33% (p < 0.001, mean dif.: -1.168, CI: -1.724, -0.612). No difference in the percentage of donor-reactive T-cells in the circulation at transplant biopsy was found between subjects experiencing a TCMR and the control group [p = 0.64 (CD4+), p = 0.52 (CD8+)]. Graft-infiltrating T-cells showed an up to six-fold increase of donor-reactive T-cell clonotypes compared to the blood at the same time (3.7 vs. 0.6% and 2.4 vs. 1.5%), but the infiltrating TCR repertoire was not reflected by the composition of the circulating TCR repertoire despite some overlap. Network analysis showed a distinct segregation of the donor-reactive repertoire with higher modularity than the overall TCR repertoire in the blood. These findings indicate an unchoreographed process of diverse T-cell clones directed against numerous non-self antigens found in the allograft.ConclusionDonor-reactive T-cells are enriched in the kidney allograft during a TCMR episode, and dominant tissue clones are also found in the blood.Trial RegistrationClinicaltrials.gov: NCT: 03422224 (https://clinicaltrials.gov/ct2/show/NCT03422224).