The signature of a T-cell response to KSHV persists across space and time in individuals with epidemic and endemic KS from Uganda.

Inadequate T-cell control of Kaposi sarcoma-associated herpesvirus (KSHV) infection predisposes to development of Kaposi sarcoma (KS), but little is known about the T-cell response to KSHV. Postulating that KS tumors contain abundant KSHV-specific T-cells, we performed transcriptional profiling and T-cell receptor (TCR) repertoire analysis of tumor biopsies from 144 Ugandan adults with KS. We show that CD8+ T-cells and M2-polarized macrophages dominate the tumor micro-environment (TME). The TCR repertoire of KS tumor infiltrating lymphocytes (TIL) is shared across non-contiguous tumors and persists across time. Clusters of T-cells with predicted shared specificity for uncharacterized antigens, potentially encoded by KSHV, comprise ~25% of KS TIL, and are shared across tumors from different time points and individuals. Single-cell RNA-sequencing of blood identifies a non-proliferating effector memory phenotype and captured the TCRs in 14,698 putative KSHV-specific T-cells. These results suggest that a polyspecific KSHV-specific T-cell response inhibited by M2 macrophages exists within the KS TME, and provide a foundation for studies to define its specificity at a large scale.

Class II HLA-DRB4 is a predictive biomarker for survival following immunotherapy in metastatic non-small cell lung cancer.

Immune checkpoint inhibitors (ICI) are important treatment options for metastatic non-small cell lung cancer (mNSCLC). However, not all patients benefit from ICIs and can experience immune-related adverse events (irAEs). Limited understanding exists for germline determinants of ICI efficacy and toxicity, but Human Leukocyte Antigen (HLA) genes have emerged as a potential predictive biomarker. We performed HLA typing on 85 patients with mNSCLC, on ICI therapy and analyzed the impact of HLA Class II genotype on progression free survival (PFS), overall survival (OS), and irAEs. Most patients received pembrolizumab (83.5%). HLA-DRB4 genotype was seen in 34/85 (40%) and its presence correlated with improved OS in both univariate (p = 0.022; 26.3 months vs 10.2 months) and multivariate analysis (p = 0.011, HR 0.49, 95% CI [0.29, 0.85]). PFS did not reach significance (univariate, p = 0.12, 8.2 months vs 5.1 months). Eleven patients developed endocrine irAEs. HLA-DRB4 was the predominant genotype among these patients (9/11, 81.8%). Cumulative incidence of endocrine irAEs was higher in patients with HLA-DRB4 (p = 0.0139). Our study is the first to suggest that patients with metastatic NSCLC patients on ICI therapy with HLA-DRB4 genotype experience improved survival outcomes. Patients with HLA-DRB4 had the longest median OS (26.3 months). Additionally, we found a correlation between HLA-DRB4 and the occurrence of endocrine irAEs.

Integrated TCR repertoire analysis and single-cell transcriptomic profiling of tumor-infiltrating T cells in renal cell carcinoma identifies shared and tumor-restricted expanded clones with unique phenotypes.

Objective responses of metastatic renal cell carcinoma (RCC) associated with systemic immunotherapies suggest the potential for T-cell-mediated tumor clearance. Recent analyses associate clonally expanded T cells present in the tumor at diagnosis with responses to immune checkpoint inhibitors (ICIs). To identify and further characterize tumor-associated, clonally expanded T cells, we characterized the density, spatial distribution, T-cell receptor (TCR) repertoire, and transcriptome of tumor-infiltrating T cells from 14 renal tumors at the time of resection and compared them with T cells in peripheral blood and normal adjacent kidney. Multiplex immunohistochemistry revealed that T-cell density was higher in clear cell RCC (ccRCC) than in other renal tumor histologies with spatially nonuniform T-cell hotspots and exclusion zones. TCR repertoire analysis also revealed increased clonal expansion in ccRCC tumors compared with non-clear cell histologies or normal tissues. Expanded T-cell clones were most frequently CD8+ with some detectable in peripheral blood or normal kidney and others found exclusively within the tumor. Divergent expression profiles for chemokine receptors and ligands and the Ki67 proliferation marker distinguished tumor-restricted T-cell clones from those also present in blood suggesting a distinct phenotype for subsets of clonally expanded T cells that also differed for upregulated markers of T-cell activation and exhaustion. Thus, our single-cell level stratification of clonally expanded tumor infiltrating T-cell subpopulations provides a framework for further analysis. Future studies will address the spatial orientation of these clonal subsets within tumors and their association with treatment outcomes for ICIs or other therapeutic modalities.

Case report: A persistently expanded T cell response in an exceptional responder to radiation and atezolizumab for metastatic non-small cell lung cancer.

Most patients with advanced non-small cell lung cancer (NSCLC) do not achieve a durable remission after treatment with immune checkpoint inhibitors. Here we report the clinical history of an exceptional responder to radiation and anti-program death-ligand 1 (PD-L1) monoclonal antibody, atezolizumab, for metastatic NSCLC who remains in a complete remission more than 8 years after treatment. Sequencing of the patient's T cell repertoire from a metastatic lesion and the blood before and after anti-PD-L1 treatment revealed oligoclonal T cell expansion. Characterization of the dominant T cell clone, which comprised 10% of all clones and increased 10-fold in the blood post-treatment, revealed an activated CD8+ phenotype and reactivity against 4 HLA-A2 restricted neopeptides but not viral or wild-type human peptides, suggesting tumor reactivity. We hypothesize that the patient's exceptional response to anti-PD-L1 therapy may have been achieved by increased tumor immunogenicity promoted by pre-treatment radiation therapy as well as long-term persistence of oligoclonal expanded circulating T cells.

Serial Analysis of the T-Cell Receptor ß-Chain Repertoire in People Living With HIV Reveals Incomplete Recovery After Long-Term Antiretroviral Therapy.

Long-term antiretroviral therapy (ART) in people living with HIV (PLHIV) is associated with sustained increases in CD4+ T-cell count, but its effect on the peripheral blood T-cell repertoire has not been comprehensively evaluated. In this study, we performed serial profiling of the composition and diversity of the T-cell receptor ß-chain (TRB) repertoire in 30 adults with HIV infection before and after the initiation of ART to define its long-term impact on the TRB repertoire. Serially acquired blood samples from 30 adults with HIV infection collected over a mean of 6 years (range, 1-12) years, with 1-4 samples collected before and 2-8 samples collected after the initiation of ART, were available for analysis. TRB repertoires were characterized via high-throughput sequencing of the TRB variable region performed on genomic DNA extracted from unsorted peripheral blood mononuclear cells. Additional laboratory and clinical metadata including serial measurements of HIV viral load and CD4 + T-cell count were available for all individuals in the cohort. A previously published control group of 189 TRB repertoires from peripheral blood samples of adult bone marrow transplant donors was evaluated for comparison. ART initiation in PLHIV was associated with a sustained reduction in viral load and a significant increase in TRB repertoire diversity. However, repertoire diversity in PLHIV remained significantly lower than in the control group even after long-term ART. The composition of TRB repertoires of PLHIV after ART also remained perturbed compared to the control cohort, as evidenced by large persistent private clonal expansions, reduced efficiency in the generation of TRB CDR3 amino acid sequences, and a narrower range of CDR3 lengths. Network analysis revealed an antigen-experienced structure in the TRB repertoire of PLHIV both before and after ART initiation that was quite distinct from the structure of control repertoires, with a slight shift toward a more naïve structure observed after ART initiation. Though we observe significant improvement in TRB repertoire diversity with durable viral suppression in PLHIV on long-term ART, the composition and structure of these repertoires remain significantly perturbed compared to the control cohort of adult bone marrow transplant donors.

A Pilot Study of Atezolizumab Plus Hypofractionated Image Guided Radiation Therapy for the Treatment of Advanced Non-Small Cell Lung Cancer.

PURPOSE: Preclinical data and subset analyses from immunotherapy clinical trials indicate that prior radiation therapy was associated with better progression-free survival and overall survival when combined with immune checkpoint inhibitors in patients with non-small cell lung cancer. We present a prospective study of hypofractionated image guided radiation therapy (HIGRT) to a single site of metastatic disease concurrently with atezolizumab in patients with metastatic non-small cell lung cancer. METHODS AND MATERIALS: Patients meeting eligibility criteria received 1200 mg of atezolizumab intravenously every 3 weeks with concurrent 3- or 5-fraction HIGRT starting no later than the second cycle. The 3-fraction regimen employed a minimum of 8 Gy per fraction compared with 6 Gy for the 5-fraction regimen. Imaging was obtained every 12 weeks to assess response. RESULTS: From October 2015 to February 2017, 12 patients were enrolled in the study (median age 64; range, 55-77 years). The best response by the Response Evaluation in Solid Tumors criteria was partial response in 3 and stable disease in 3, for a disease control rate of 50%. Five patients had a grade 3 immune-related adverse event, including choreoretinitis (n = 1), pneumonitis (n = 1), transaminitis (n = 1), fatigue (n = 1), and peripheral neuropathy (n = 1). The median progression-free survival was 2.3 months, and the median overall survival was 6.9 months (range, 0.4-not reached). There was no clear association between peripheral blood T cell repertoire characteristics at baseline, PD-L1, or tumor mutations and response or outcome. One long-term survivor exhibited oligoclonal T cell populations in a baseline tumor biopsy that were consistently detected in peripheral blood over the entire course of the study. CONCLUSIONS: HIGRT plus atezolizumab resulted in an overall response rate of 25% and disease control rate of 50% in this pilot study. The incidence of grade 3 adverse events was similar to that of atezolizumab alone. Alhough it was a pilot study with limited sample size, the results generated hypotheses worthy of further investigation.

Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 tumor antigen on renal cell carcinoma.

5T4 (trophoblast glycoprotein, TPBG) is a transmembrane tumor antigen expressed on more than 90% of primary renal cell carcinomas (RCC) and a wide range of human carcinomas but not on most somatic adult tissues. The favorable expression pattern has encouraged the development and clinical testing of 5T4-targeted antibody and vaccine therapies. 5T4 also represents a compelling and unexplored target for T-cell receptor (TCR)-engineered T-cell therapy. Our group has previously isolated high-avidity CD8+ T-cell clones specific for an HLA-A2-restricted 5T4 epitope (residues 17-25; 5T4p17). In this report, targeted single-cell RNA sequencing was performed on 5T4p17-specific T-cell clones to sequence the highly variable complementarity-determining region 3 (CDR3) of T-cell receptor α chain (TRA) and ß chain (TRB) genes. Full-length TRA and TRB sequences were cloned into lentiviral vectors and transduced into CD8+ T-cells from healthy donors. Redirected effector T-cell function against 5T4p17 was measured by cytotoxicity and cytokine release assays. Seven unique TRA-TRB pairs were identified. All seven TCRs exhibited high expression on CD8+ T-cells with transduction efficiencies from 59 to 89%. TCR-transduced CD8+ T-cells demonstrated redirected cytotoxicity and cytokine release in response to 5T4p17 on target-cells and killed 5T4+/HLA-A2+ kidney-, breast-, and colorectal-tumor cell lines as well as primary RCC tumor cells in vitro. TCR-transduced CD8+ T-cells also detected presentation of 5T4p17 in TAP1/2-deficient T2 target-cells. TCR-transduced T-cells redirected to recognize the 5T4p17 epitope from a broadly shared tumor antigen are of interest for future testing as a cellular immunotherapy strategy for HLA-A2+ subjects with 5T4+ tumors.

A Multi-center Phase I Trial of Ipilimumab in Patients with Myelodysplastic Syndromes following Hypomethylating Agent Failure.

Purpose: After failure of hypomethylating agents (HMA), patients with myelodysplastic syndromes (MDS) have dismal survival and no approved treatment options.Patients and Methods: We conducted a phase 1b investigator-initiated trial of ipilimumab in patients with higher risk MDS who have failed HMAs. Patients received monotherapy at two dose levels (DL; 3 and 10 mg/kg) with an induction followed by a maintenance phase. Toxicities and responses were evaluated with CTCAE.4 and IWG-2006 criteria, respectively. We also performed immunologic assays and T-cell receptor sequencing on serial samples.Results: Twenty-nine patients from 7 centers were enrolled. In the initial DL1 (3 mg), 3 of 6 patients experienced grade 2-4 immune-related adverse events (IRAE) that were reversible with drug discontinuation and/or systemic steroids. In DL2, 4 of 5 patients experienced grade 2 or higher IRAE; thus, DL1 (3 mg/kg) was expanded with no grade 2-4 IRAEs reported in 18 additional patients. Best responses included marrow complete response (mCR) in one patient (3.4%). Prolonged stable disease (PSD) for ≥46 weeks occurred in 7 patients (24% of entire cohort and 29% of those treated with 3 mg/kg dose), including 3 patients with more than a year of SD. Five patients underwent allografting without excessive toxicity. Median survival for the group was 294 days (95% CI, 240-671+). Patients who achieved PSD or mCR had significantly higher frequency of T cells expressing ICOS (inducible T-cell co-stimulator).Conclusions: Our findings suggest that ipilimumab dosed at 3 mg/kg in patients with MDS after HMA failure is safe but has limited efficacy as a monotherapy. Increased frequency of ICOS-expressing T cells might predict clinical benefit. Clin Cancer Res; 24(15); 3519-27. ©2018 AACR.

Plasma-derived proteomic biomarkers in human leukocyte antigen-haploidentical or human leukocyte antigen-matched bone marrow transplantation using post-transplantation cyclophosphamide.

Recent studies have suggested that plasma-derived proteins may be potential biomarkers relevant for graft-versus-host disease and/or non-relapse mortality occurring after allogeneic blood or marrow transplantation. However, none of these putative biomarkers have been assessed in patients treated either with human leukocyte antigen-haploidentical blood or marrow transplantation or with post-transplantation cyclophosphamide, which has been repeatedly associated with low rates of severe acute graft-versus-host disease, chronic graft-versus-host disease, and non-relapse mortality. We explored whether seven of these plasma-derived proteins, as measured by enzyme-linked immunosorbent assays, were predictive of clinical outcomes in post-transplantation cyclophosphamide-treated patients using plasma samples collected at serial predetermined timepoints from patients treated on prospective clinical studies of human leukocyte antigen-haploidentical (n=58; clinicaltrials.gov Identifier: 00796562) or human leukocyte antigen-matched-related or -unrelated (n=100; clinicaltrials.gov Identifiers: 00134017 and 00809276) T-cell-replete bone marrow transplantation. Day 30 levels of interleukin-2 receptor α, tumor necrosis factor receptor 1, serum STimulation-2 (IL1RL1 gene product), and regenerating islet-derived 3-α all had high areas under the curve of 0.74-0.97 for predicting non-relapse mortality occurrence by 3 months post-transplant in both the human leukocyte antigen-matched and human leukocyte antigen-haploidentical cohorts. In both cohorts, all four of these proteins were also predictive of subsequent non-relapse mortality occurring by 6, 9, or 12 months post-transplant and were significantly associated with non-relapse mortality in univariable analyses. Furthermore, day 30 elevations of interleukin-2 receptor α were associated with grade II-IV and III-IV acute graft-versus-host disease occurring after day 30 in both cohorts. These data confirm that plasma-derived proteins previously assessed in other transplantation platforms appear to retain prognostic and predictive utility in patients treated with post-transplantation cyclophosphamide.

High-throughput sequencing of the B-cell receptor in African Burkitt lymphoma reveals clues to pathogenesis.

Burkitt lymphoma (BL), the most common pediatric cancer in sub-Saharan Africa, is a malignancy of antigen-experienced B lymphocytes. High-throughput sequencing (HTS) of the immunoglobulin heavy (IGH) and light chain (IGK/IGL) loci was performed on genomic DNA from 51 primary BL tumors: 19 from Uganda and 32 from Ghana. Reverse transcription polymerase chain reaction analysis and tumor RNA sequencing (RNAseq) was performed on the Ugandan tumors to confirm and extend the findings from the HTS of tumor DNA. Clonal IGH and IGK/IGL rearrangements were identified in 41 and 46 tumors, respectively. Evidence for rearrangement of the second IGH allele was observed in only 6 of 41 tumor samples with a clonal IGH rearrangement, suggesting that the normal process of biallelic IGHD to IGHJ diversity-joining (DJ) rearrangement is often disrupted in BL progenitor cells. Most tumors, including those with a sole dominant, nonexpressed DJ rearrangement, contained many IGH and IGK/IGL sequences that differed from the dominant rearrangement by < 10 nucleotides, suggesting that the target of ongoing mutagenesis of these loci in BL tumor cells is not limited to expressed alleles. IGHV usage in both BL tumor cohorts revealed enrichment for IGHV genes that are infrequently used in memory B cells from healthy subjects. Analysis of publicly available DNA sequencing and RNAseq data revealed that these same IGHV genes were overrepresented in dominant tumor-associated IGH rearrangements in several independent BL tumor cohorts. These data suggest that BL derives from an abnormal B-cell progenitor and that aberrant mutational processes are active on the immunoglobulin loci in BL cells.

Origin and evolution of the T cell repertoire after posttransplantation cyclophosphamide.

Posttransplantation cyclophosphamide (PTCy) effectively prevents graft-versus-host disease (GVHD), but its immunologic impact is poorly understood. We assessed lymphocyte reconstitution via flow cytometry (n = 74) and antigen receptor sequencing (n = 35) in recipients of myeloablative, HLA-matched allogeneic BM transplantation using PTCy. Recovering T cells were primarily phenotypically effector memory with lower T cell receptor ß (TRB) repertoire diversity than input donor repertoires. Recovering B cells were predominantly naive with immunoglobulin heavy chain locus (IGH) repertoire diversity similar to donors. Numerical T cell reconstitution and TRB diversity were strongly associated with recipient cytomegalovirus seropositivity. Global similarity between input donor and recipient posttransplant repertoires was uniformly low at 1-2 months after transplant but increased over the balance of the first posttransplant year. Blood TRB repertoires at ≥3 months after transplant were often dominated by clones present in the donor blood/marrow memory CD8+ compartment. Limited overlap was observed between the TRB repertoires of T cells infiltrating the skin or gastrointestinal tract versus the blood. Although public TRB sequences associated with herpesvirus- or alloantigen-specific CD8+ T cells were detected in some patients, posttransplant TRB and IGH repertoires were unique to each individual. These data define the immune dynamics occurring after PTCy and establish a benchmark against which immune recovery after other transplantation approaches can be compared.

Phenotypic and transcriptional fidelity of patient-derived colon cancer xenografts in immune-deficient mice.

Xenografts of human colorectal cancer (CRC) in immune-deficient mice have great potential for accelerating the study of tumor biology and therapy. We evaluated xenografts established in NOD/scid/IL2Rγ-null mice from the primary or metastatic tumors of 27 patients with CRC to estimate their capacity for expanding tumor cells for in vitro studies and to assess how faithfully they recapitulated the transcriptional profile of their parental tumors. RNA-seq analysis of parental human CRC tumors and their derivative xenografts demonstrated that reproducible transcriptional changes characterize the human tumor to murine xenograft transition. In most but not all cases, the human stroma, vasculature, and hematopoietic elements were systematically replaced by murine analogues while the carcinoma component persisted. Once established as xenografts, human CRC cells that could be propagated by serial transplantation remained transcriptionally stable. Three histologically atypical xenografts, established from patients with peritoneal metastases, contained abundant human stromal elements and blood vessels in addition to human tumor cells. The transcriptomes of these mixed tumor/stromal xenografts did not closely resemble those of their parental tumors, and attempts to propagate such xenografts by serial transplantation were unsuccessful. Stable expression of numerous genes previously identified as high priority targets for immunotherapy was observed in most xenograft lineages. Aberrant expression in CRC cells of human genes that are normally only expressed in hematopoietic cells was also observed. Our results suggest that human CRC cells expanded in murine xenografts have great utility for studies of tumor immunobiology and targeted therapies such as immunotherapy but also identify potential limitations.

Epigenetic modulation to enable antigen-specific T-cell therapy of colorectal cancer.

Development of specific immunotherapy for colorectal cancer (CRC) will require identification of antigens selectively or exclusively expressed on CRC cells and strategies to induce and enhance immune responses against these antigenic targets. Cancer-testis (C-T) antigens are proving to be excellent targets for immunotherapy of solid tumors such as melanoma, but their clinical utility for treatment of CRC has to date been limited by their infrequent expression in CRC cells. Here we report that the hypomethylating agent 5-aza-2'-deoxycytidine (DAC) induces expression of NY-ESO-1 and other C-T genes in CRC cells both in vitro and in vivo in a dose-dependent manner but has negligible effects on the expression of C-T genes in normal nontransformed cells such as fibroblasts. The induction by DAC of NY-ESO-1 expression in CRC cells persists over 100 days after DAC exposure and is associated with increased levels of NY-ESO-1 protein. CRC cells exposed to DAC at concentrations that can be readily achieved in vivo are rendered susceptible to major histocompatibility complex-restricted recognition by CD8 NY-ESO-1-specific T cells. We also demonstrate that retroviral transduction of polyclonal peripheral blood T cells from a metastatic CRC patient with the T-cell receptor α-chain and ß-chain genes encoding a human leukocyte antigen-A2-restricted, NY-ESO-1157-165-specific T-cell receptor can be used to generate both CD8 and CD4 NY-ESO-1157-165-specific T cells that selectively recognize DAC-treated CRC but not nontransformed cells. Collectively, these results suggest that the combination of epigenetic modulation and adoptive transfer of genetically engineered T lymphocytes may enable specific immunotherapy for CRC.