T-cell Clonal Expansion Research Articles

Intra-Tumoral CD8+ T-Cells in Follicular Lymphoma Contain Large Clonal Expansions That Are Amenable to Dual-Checkpoint Blockade

Introduction. Intra-tumoral T-cell infiltration is associated with R-CHOP responsiveness in aggressive B-cell lymphoma (Keane, Lancet Haem 2015). These patients also have a broad (i.e. diverse) intra-tumoral T-cell receptor (TCR) repertoire with a ~20% superior survival compared to those with a narrow (i.e. clonal) repertoire after R-CHOP therapy. Here, the major contributor to the TCR clonal expansion were CD8+ T cells (Keane, CCR 2017). Paradoxically, our recent results in Follicular Lymphoma (FL) (Tobin, JCO in press) found that clonal T-cell expansions were markedly enriched in those patients that experienced progression of disease within 24 months (POD24). Given that FL is a histological subtype associated with a tumor microenvironment distinct from DLBCL including numerous CD4+ T-follicular helper cells (TFH), we now expand upon these findings by comparing TCR repertoires across histological subtypes. We then established whether the TCR repertoire in FL is related to differential TCR clonal expansions between different T-cell subsets and immune checkpoints. Finally, the overlap between tissue and blood TCR repertoires was investigated. Methods. Firstly, unbiased, high-throughput TCRβ sequencing (ImmunoSEQ, Adaptive Biotechnologies) was compared in 164 FFPE tissues (12 healthy nodes, 40 FL, 88 DLBCL, and as a comparator tumor known to be sensitive to checkpoint blockade and to have a high neoantigen burden, 24 melanoma tissues). Next, to determine the contribution of individual T-cell subsets to overall clonality, a further 21 fresh de-aggregated/cryopreserved FL tumor samples were FACS sorted into four T-cell groupings: CD8+ cytotoxic T-lymphocytes (CTLs), CD4+ TFH, CD4+ regulatory T-cells (TREGs) and 'other' (non-TFH/TREG) CD4+ T-cells. Flow cytometry quantified the expression of the checkpoints LAG3, TIM3 and PD1. Then, 5 FL paired tissue/blood samples were tested for shared TCR clones. Results. FL exhibited strikingly reduced TCR repertoire clonality (higher diversity) compared to DLBCL, melanoma and healthy lymph nodes (Fig 1A). Analysis of de-aggregated sorted nodal T-cells revealed a more complex TCR repertoire. The outcome measure was median clonality index (CIx ranging from '0' or minimal, to '1' or maximal clonality). Large T-cell clones in FL (CIx=0.12) predominantly resided within the CTL subset (34% all T-cells). By contrast, there was marked T-cell diversity in TFH (CIx=0.04; 27% all T-cells), TREG (CIx=0.02; 7% all T-cells) and 'other' CD4+ T-cells (CIx=0.02; 32% all T-cells) (Fig 1B). The CTL population had a bimodal expression for PD1 (+51%/-49%), a marker in FL that has been shown to remain functionally active unless co-expressed with LAG3 and/or TIM3 (Yang, Oncotarget 2017). These dual-checkpoint expressing CTLs have reduced capacity to produce cytokines or lytic granules (i.e. they are 'exhausted'). Notably, 54% of the PD1+ CTLs co-expressed either LAG3 or TIM3. Put together, these results are consistent with expanded CTL clones that are frequently functionally exhausted. In contrast, TFH, TREG and 'other' CD4+ T-cells had a low expression of LAG3 and TIM3, although PD1 was frequently found (as expected, particularly in the TFH cells). Finally, in paired tissue/blood samples, there was weak overlap between the circulating and intra-tumoral TCR repertoire in CTLs and TFH T-cells. Conclusion. Although FL has a markedly less clonal TCR repertoire compared to DLBCL, melanoma and even healthy nodes, this result is misleading. Detailed analysis on sorted intra-tumoral T-cell subsets in FL revealed large clonal expansions in CTLs, with approximately half of these classified as functionally exhausted (dual-positive for PD1 and LAG3 and/or TIM3), a state potentially amenable to reversal by dual-checkpoint blockade. The explanation for TCR repertoire diversity lies in CD4+ T-cells (representing approximately two-thirds of T-cells, including the large TFH subset). T-cells in blood did not reflect FL tissue T-cell clones, further highlighting the need for sorted intra-tumoral nodal tissues to accurately assess TCR repertoires in FL. Further characterization of the neo-antigenic targets that CTL clones potentially recognize is required. These results have implications for therapeutic vaccine design and selective recruitment of patients for immune checkpoint blockade. Disclosures Keane: MSD: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Roche: Consultancy, Other: Travel Grant; BMS: Research Funding. Gandhi:Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding.

T-Cell Clonal Expansion and Activation Associated with Durable Clinical Response to Dual BTK and CDK4/6 Inhibitor Therapy in Mantle Cell Lymphoma

T-Cell Clonal Expansion and Activation Associated with Durable Clinical Response to Dual BTK and CDK4/6 Inhibitor Therapy in Mantle Cell Lymphoma

Microrna-191 Regulates T-Cell Clonal Expansion during Graft-Versus-Host Disease

Allogeneic hematopoietic cell transplantation is a potentially curative treatment choice for a wide variety of hematological malignancies. However, graft-versus-host disease (GVHD), which is mediated by donor alloreactive T cells, limits the success of this procedure. Previous studies have demonstrated that several microRNAs (miRs) modulate graft-versus-host disease. miR-191 was previously reported to be able to support T cell survival after TCR stimulation. We hypothesize that miR191 regulates T cell response during GVHD. To test this hypothesis, we first studied miR-191 expression in alloreactive T cells. The result demonstrated that miR-191 was up-regulated in donor T cells isolated from murine GVHD recipients, suggesting that miR-191 may play a role in GVHD induction. We further studied the role of miR-191in GVHD using miR-191 deficient T cells (KO). Lethally irradiated (8.5 Gy) BALB/c mice were injected intravenously with 1×107 T cell-depleted bone marrow (TCDBM) cells along with 1×106 purified T cells from wild-type (WT) or KO mice, which are in C57BL/6 background. Interestingly, all recipients in the WT group died within 35 days after transplantation, while only one out of ten animals died in the KO group during an observation period of 56 days. Body weights and clinical scores were also improved in KO T cell recipients when compared with the WT controls. Similar results were also observed in a second GVHD model (C57BL/6→C3H/HeJ). To understand the mechanism by which miR-191 KO T cells have decreased ability to mediate GVHD, we first measured the ability of KO T cells to respond to alloantigens in vitro in a mixed lymphocytes reaction assay. Dramatically decreased alloresponse was observed with KO T cells as compared with WT T cells. Similarly, decreased clonal expansion was observed in KO T cells in vivo upon challenge with alloantigens as measured by bioluminescent imaging (Figure 1A). These results were further supported by data from a co-transfer experiment, in which equal numbers of WT and KO T cells were transplanted into the same GVHD recipient. At day7 after transplantation, KO T cells showed significantly reduced expansion in the spleen and liver compared with WT T cells. Reduced alloresponses mediated by KO T cells may not due to decreased proliferative capability directly as an in vivo carboxyfluorescein succinimidyl ester (CFSE) assay showed a comparable cell division between WT and KO T cells upon challenge with alloantigens. Rather, increased cell death is responsible for decreased alloresponse observed in KO T cells because dramatically increased number of dead cells was observed in KO group compared with WT group upon response to alloantigens in vitro and vivo. To determine the genes that are regulated by miR-191, we did a screening based on the prediction. Humans and mice share more than 100 predicted targets for miR-191. We chose top 20 of these targets for RT-qPCR screening. The result demonstrated that Taf5 was a target gene of miR-191. Expression of TAF5 protein was down-regulated in activated KO T cells when compared with the WT T cells. Finally, we investigated whether miR-191 KO T cells preserve graft-versus-leukemia effects. 1×106 T cells from WT or KO mice were transplanted into lethally irradiated BALB/c mice along with 1×107 TCDBM cells and 1×105 host-type BCL-1 cells. While all recipients that received only TCDBM and tumor cells developed lethal leukemia/lymphoma, none of WT and KO T cells recipients developed tumor. In conclusion, our findings reveal a critical role of miR-191 during GVHD process and demonstrate that miR-191 is a novel therapeutic target for GVHD. Figure 1 Disclosures No relevant conflicts of interest to declare.

Histone deacetylase inhibition promotes intratumoral CD8+ T-cell responses, sensitizing murine breast tumors to anti-PD1

Histone deacetylase (HDAC) inhibitors impair tumor cell proliferation and alter gene expression. However, the impact of these changes on anti-tumor immunity is poorly understood. Here, we showed that the class I HDAC inhibitor, entinostat (ENT), promoted the expression of immune-modulatory molecules, including MHCII, costimulatory ligands, and chemokines on murine breast tumor cells in vitro and in vivo. ENT also impaired tumor growth in vivo-an effect that was dependent on both CD8+ T cells and IFNγ. Moreover, ENT promoted intratumoral T-cell clonal expansion and enhanced their functional activity. Importantly, ENT sensitized normally unresponsive tumors to the effects of PD1 blockade, predominantly through increases in T-cell proliferation. Our findings suggest that class I HDAC inhibitors impair tumor growth by enhancing the proliferative and functional capacity of CD8+ T cells and by sensitizing tumor cells to T-cell recognition.

Differences in Tumor Microenvironment Dictate T Helper Lineage Polarization and Response to Immune Checkpoint Therapy

Immune checkpoint therapy (ICT) shows encouraging results in a subset of patients with metastatic castration-resistant prostate cancer (mCRPC) but still elicits a sub-optimal response among those with bone metastases. Analysis of patients' bone marrow samples revealed increased Th17 instead ofTh1 subsets after ICT. To further evaluate the different tumor microenvironments, we injected mice with prostate tumor cells either subcutaneously or intraosseously. ICT in the subcutaneous CRPC model significantly increases intra-tumoral Th1 subsets and improves survival. However, ICT fails to elicit an anti-tumor response in the bone CRPC model despite an increase in the intra-tumoral CD4 Tcells, which are polarized to Th17 rather than Th1 lineage. Mechanistically, tumors in the bone promote osteoclast-mediated bone resorption that releases TGF-β, which restrains Th1 lineage development. Blocking TGF-β along with ICT increases Th1 subsets and promotes clonal expansion of CD8 Tcells and subsequent regression of bone CRPC and improves survival.

Immune adaptor SKAP1 acts a scaffold for Polo-like kinase 1 (PLK1) for the optimal cell cycling of T-cells

While the immune cell adaptor protein SKAP1 mediates LFA-1 activation induced by antigen-receptor (TCR/CD3) ligation on T-cells, it is unclear whether the adaptor interacts with other mediators of T-cell function. In this context, the serine/threonine kinase, polo-like kinase (PLK1) regulates multiple steps in the mitotic and cell cycle progression of mammalian cells. Here, we show that SKAP1 is phosphorylated by and binds to PLK1 for the optimal cycling of T-cells. PLK1 binds to the N-terminal residue serine 31 (S31) of SKAP1 and the interaction is needed for optimal PLK1 kinase activity. Further, siRNA knock-down of SKAP1 reduced the rate of T-cell division concurrent with a delay in the expression of PLK1, Cyclin A and pH3. Reconstitution of these KD cells with WT SKAP1, but not the SKAP1 S31 mutant, restored normal cell division. SKAP1-PLK1 binding is dynamically regulated during the cell cycle of T-cells. Our findings identify a novel role for SKAP1 in the regulation of PLK1 and optimal cell cycling needed for T-cell clonal expansion in response to antigenic activation.

Abstract CT010: A phase I study of the oncolytic peptide LTX-315 generatesde novoT-cell responses and clinical benefit in patients with advanced sarcoma

Abstract Background: LTX-315 is a first-in class oncolytic peptide that has the ability to kill cancer cells and induce specific anticancer immune response when injected locally into experimental tumors established in immunocompetent mice. The underlying mechanism of action include induction of immunogenic cell death with subsequent infiltration of T-cells into the tumor microenvironment. A multicenter Phase I study was conducted to evaluate the safety and tolerability of LTX-315 in patients with solid tumors who had exhausted standard treatment options. Methods: The primary objective of the trial was to assess the safety and tolerability of multiple, intratumoral doses of LTX-315 as a monotherapy. The secondary objective was to evaluate clinical activity (assessed using immune-related response criteria (irRC)) and the ability of LTX-315 to evoke local and systemic immune responses. Paired pre and post treatment tumor samples were obtained throughout the study. Immunohistochemistry and gene expression analysis were performed on these paired tumor biopsies. T-cell receptor (TCR) repertoire in peripheral blood and tumor biopsies was assessed by TCRβ-gene sequencing. Results: Twenty-seven patients were treated with LTX-315 monotherapy and had at least one post baseline efficacy assessment, of which 5 patients had the diagnosis of sarcoma. Best overall response seen in the sarcoma patients was SD in 4 out of 5 patients (80%). In one patient, a marked tumor regression was observed in two distant non-injected lesions (39% and 66% reduction). All patients experienced a treatment emergent adverse event (TEAE) and all patients experienced at least one episode of LTX-315 related adverse event, most commonly an acute reaction to injection of LTX-315 such as hypotension, rash, flushing or itching. The majority (94%) of these injection-related events were grade 1. There were 2 episodes (6%) of grade 3 AE, no serious adverse events were reported. Enhanced tumor infiltration of CD3+ and CD8+T cells, assessed by immunoscore®, was observed in 100% and 75% of the responding sarcoma patients, respectively. Gene expression profiling by Immunosign ®21 in one injected lesion showed a distinct “cold to hot’’ signature change.TCR sequencing of the patient`s T cells in peripheral blood revealed significant clonal expansion of T-cells, 39% of these T cell clones were also detected in post-treatment biopsied tumors. Conclusions: Intratumoral LTX-315 is generally safe and tolerable. LTX-315 monotherapy enhances TIL population and induces polyclonal T-cell responses Based on the data from the Phase I study, the dosing regimen of LTX-315 will be assessed and optimized to position LTX-315 as a therapeutic agent in combination with other targeted immune therapies such as checkpoint inhibitors to address an unmet need in a select group of indications. Registration number; NCT01986426 Citation Format: Jean-Francois Baaurain, Christiane Jungels, Rebecca S. Kristeleit, Dag E. Jossang, Nina L. Jebsen, Øystein Rekdal, Baldur Sveinbjornsson, Vibeke S. Gjerstad, Paal Brunsvig, Jerome Galon, Fabienne Hermitte, Hamina Patel. A phase I study of the oncolytic peptide LTX-315 generatesde novoT-cell responses and clinical benefit in patients with advanced sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT010.

Clonal expansion of tumor infiltrating lymphocytes (TILs) in the peripheral blood of metastatic melanoma patients is significantly associated with response to CTLA4 blockade-based immunotherapy.

2541 Background: Patients with metastatic melanoma were treated on a clinical trial with tremelimumab and High Dose Interferon-Alfa (HDI) (Tarhini. J Clin Oncol. 2012). We previously reported that patients who achieved disease control and clinical response had significantly greater T-cell clonality (p = 0.0008) and T-cell fraction (p = 0.044) respectively in their pretreatment tumor biopsy samples (Tarhini. J Clin Oncol. 2017). In this study, we further characterize T-cell repertoire clonality and clonal expansion in the peripheral blood at different time points to evaluate the association between repertoire features and clinical response. Methods: Patients received tremelimumab 15 mg/kg I.V. every 12 weeks and HDI was given concurrently. Responses were assessed by RECIST as complete (CR) or partial (PR), stable disease (SD) or progression (PD). Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 33) were obtained at baseline, day 29, and day 85 (following tremelimumab-HDI treatment); tumor samples at baseline were also obtained (N = 18). The T-cell receptor beta chain (TCRB) repertoire of PBMCs and tumor samples was immunosequenced using the immunoSEQ assay (Adaptive Biotechnologies), and repertoire clonality was assessed at baseline, day 29, and day 85. Differential abundance analysis was used to detect and quantify peripheral clonal expansion pre- versus post-treatment and identify the subset of peripheral clones also detected in the tumor repertoire. The Morisita Index of repertoire similarity was also calculated to compare global repertoire changes between pre- and post-treatment PBMC samples. Results: T-cell repertoire turnover, as measured by the Morisita Index, showed a trend towards responders (CR/PR) having greater turnover (lower Morisita Index) post-treatment than non-responders (SD/PD). Similarly, the total number of clones expanding in the peripheral repertoire varied over time within an individual (p = 0.034) but was not significantly affected by response to therapy (p = 0.275) or by on-treatment time point (p = 0.768). When the analysis was restricted to peripherally expanded clones that were also found in the tumor repertoire, responders had significantly more TILs expanded in the periphery at day 29 than non-responders (p = 0.036). Conclusions: Our analysis of the peripheral T-cell repertoire following treatment showed that detection of TILs in early peripheral clonal expansion correlates with response to therapy.

Neoadjuvant nivolumab in resectable non-small cell lung cancer: Extended follow-up and molecular markers of response.

8524 Background: Improved therapy is needed for patients (pts) with early-stage non-small cell lung cancer (NSCLC), as the majority relapse after curative resection. Our group reported the first trial of neoadjuvant PD-1 blockade in resectable NSCLC, finding therapy to be safe and feasible. Here we report extended clinical follow-up and long-term molecular response data from this trial. Methods: IV nivolumab 3 mg/kg was given every 2 weeks for 2 doses prior to surgery in 20 pts with resectable NSCLC at Johns Hopkins and MSKCC. Blood for correlative studies was taken prior to each dose of nivolumab, prior to surgery, 2-4 weeks post-surgery, and during long-term follow up. In a subgroup of pts, longitudinal molecular data was assessed in peripheral blood for circulating tumor DNA (ctDNA) and dynamics of tumor-infiltrating T-cell clonotypes. Results: At median follow up of 30 months (m), 15 of 20 pts are disease-free and alive. Two pts have died (one from relapsed disease). Median recurrence free survival (RFS) has not been reached. The 24m RFS rate is 69% (95% CI: 51-93). Thus far, presence of ctDNA at diagnosis and major pathologic response (MPR - ≤10% viable tumor in resected specimen) do not associate with RFS. One long-term immune-related adverse event has occurred (skin, G3). All pts who on pathologic review had ≥30% reduction in viable tumor in response to nivolumab demonstrated clearance of detectable ctDNA from blood prior to surgery. Pts with MPR experienced expansion of neoantigen-specific T-cells in peripheral blood. In one patient with ongoing disease free status, expansion of tumor-associated T-cells has persisted in peripheral blood beyond 15m from surgery. By contrast, in a patient who had detectable peri-operative ctDNA and 75% residual disease at surgery, minimal T-cell expansion was observed in peripheral blood, with a decreasing frequency of expanded T-cell clones over time that correlated with eventual cancer relapse. Conclusions: Long-term follow up reinforces the safety of neoadjuvant nivolumab in resectable NSCLC. Analysis of ctDNA and peripheral T-cell expansion in responders compared with non-responders suggests potential biomarkers for response and surveillance. While RFS data is encouraging, phase 3 trials are ongoing to evaluate efficacy of PD-(L)1 blockade in early-stage NSCLC. Clinical trial information: NCT02259621.

Diacylglycerol Lipase-β Is Required for TNF-α Response but Not CD8+ T Cell Priming Capacity of Dendritic Cells

Diacylglycerol lipase-β (DAGLβ) hydrolyzes arachidonic acid (AA)-esterified diacylglycerols to produce 2-arachidonoylglycerol (2-AG) and downstream prostanoids that mediate inflammatory responses of macrophages. Here, we utilized DAGL-tailored activity-based protein profiling and genetic disruption models to discover that DAGLβ regulates inflammatory lipid and protein signaling pathways in primary dendritic cells (DCs). DCs serve as an important link between innate and adaptive immune pathways by relaying innate signals and antigen to drive Tcell clonal expansion and prime antigen-specific immunity. We discovered that disruption of DAGLβ in DCs lowers cellular 2-AG and AA that is accompanied by reductions in lipopolysaccharide (LPS) stimulated tumor necrosis factor α secretion. Cell-based vaccination studies revealed that DC maturation exvivo and immunogenicity invivo was surprisingly unaffected by DAGLβ inactivation. Collectively, we identify DAGLβ pathways as a means for attenuatingDC inflammatory signaling while sparing criticaladaptive immune functions and further expand theutility of targeting lipid pathways for immunomodulation.

T-Cell Compartmentalization and Functional Adaptation in Autoimmune Inflammation: Lessons From Pediatric Rheumatic Diseases.

Chronic inflammatory diseases are characterized by a disturbed immune balance leading to recurring episodes of inflammation in specific target tissues, such as the joints in juvenile idiopathic arthritis. The tissue becomes infiltrated by multiple types of immune cell, including high numbers of CD4 and CD8 T-cells, which are mostly effector memory cells. Locally, these T-cells display an environment-adapted phenotype, induced by inflammation- and tissue-specific instructions. Some of the infiltrated T-cells may become tissue resident and play a role in relapses of inflammation. Adaptation to the environment may lead to functional (re)programming of cells and altered cellular interactions and responses. For example, specifically at the site of inflammation both CD4 and CD8 T-cells can become resistant to regulatory T-cell-mediated regulation. In addition, CD8 and CD4 T-cells show a unique profile with pro- and anti-inflammatory features coexisting in the same compartment. Also regulatory T-cells are neither homogeneous nor static in nature and show features of functional differentiation, and plasticity in inflammatory environments. Here we will discuss the recent insights in T-cell functional specialization, regulation, and clonal expansion in local (tissue) inflammation.

Model to improve specificity for identification of clinically-relevant expanded T cells in peripheral blood.

Current methods to quantify T-cell clonal expansion only account for variance due to random sampling from a highly diverse repertoire space. We propose a beta-binomial model to incorporate time-dependent variance into the assessment of differentially abundant T-cell clones, identified by unique T Cell Receptor (TCR) β-chain rearrangements, and show that this model improves specificity for detecting clinically relevant clonal expansion. Using blood samples from ten healthy donors, we modeled the variance of T-cell clones within each subject over time and calibrated the dispersion parameters of the beta distribution to fit this variance. As a validation, we compared pre- versus post-treatment blood samples from urothelial cancer patients treated with atezolizumab, where clonal expansion (quantified by the earlier binomial model) was previously reported to correlate with benefit. The beta-binomial model significantly reduced the false-positive rate for detecting differentially abundant clones over time compared to the earlier binomial method. In the urothelial cancer cohort, the beta-binomial model enriched for tumor infiltrating lymphocytes among the clones detected as expanding in the peripheral blood in response to therapy compared to the binomial model and improved the overall correlation with clinical benefit. Incorporating time-dependent variance into the statistical framework for measuring differentially abundant T-cell clones improves the model's specificity for T-cells that correlate more strongly with the disease and treatment setting of-interest. Reducing background-level clonal expansion, therefore, improves the quality of clonal expansion as a biomarker for assessing the T cell immune response and correlations with clinical measures.

Determination of the complexity and diversity of the TCR β-chain CDR3 repertoire in bladder cancer using high-throughput sequencing.

The present study aimed to investigate the complexity and diversity of the T lymphocyte immune repertoire in patients with bladder cancer. To do so, the immune state of patients was assessed. The study also aimed to elucidate the aetiology and pathogenesis of bladder cancer to provide a novel theoretical basis for disease prevention, diagnosis, treatment and prognosis monitoring. Cancerous and paracancerous (control) tissue samples were collected from five patients diagnosed with muscle-invasive bladder cancer. Multiplex polymerase chain reaction and Illumina high-throughput sequencing were used to determine the characteristics and clonal diversity of the T-cell receptor (TCR) β-chain complementarity-determining region 3 (CDR3) gene in the cancerous and paracancerous tissues of patients with bladder cancer. The degree of clonal expansion in malignant samples was significantly higher than in adjacent samples. Furthermore, ΤCRβ variable (TRBV), ΤCRβ diversity (TRBD) and ΤCRβ joining (TRBJ) repertoires were significantly different in cancerous samples compared with adjacent samples. In addition, 13 identified V-J pairs were highly expressed in cancerous samples whereas they had low expression in control samples. In conclusion, the degree of T-cell clonal expansion in bladder cancerous tissue was higher than in paracancerous tissue, whereas the immune diversity of the tissues of patients with bladder cancer was significantly lower. The DNA sequence and amino acid sequences, and V-J combination level may be used to comprehensively understand the diversity and characteristics of TCR CDR3 in bladder cancer and paracancerous tissues, and to evaluate the immune status of bladder cancer to develop therapeutic targets and biomarkers for prognosis monitoring.

Abstract A016: PEGylated IL-10 (pegilodecakin) induces systemic immune activation, CD8+ T-cell invigoration and polyclonal T-cell expansion in cancer patients

Abstract Background: Immune therapies rely on successful activation of systemic immunity as well as expansion of the T-cell clones for tumor regression and successful therapeutic outcomes. PEGylated IL-10 (pegilodecakin or AM0010) monotherapy has been reported to achieve 25% objective tumor responses (ORR) in intermediate to poor risk renal cell cancer (RCC) in median 4th line of treatment (LOT) (range 1-8) (Naing A et al, JCO 2016). Here we report the immunological underpinnings of pegilodecakin induced tumor responses alone and in combination with anti-PD-1. Methods: Samples were collected post written consent from patients enrolled in a multi-basket trial (NCT0200944) and were analyzed in accordance with the IRB. Patients on AM0010 alone administered daily self-injection of pegilodecakin at 20 µg/kg, SC. Patients in the pembrolizumab + pegilodecakin received in addition pembrolizumab at 2mg/kg IV, Q3W. Normal healthy volunteers (NCT03267732) received a single (day 1) and multiple doses (days 4-9) of 5 µg/kg or 10 µg/kg AM0010 SC. Systemic and cellular antitumor immune responses were assessed by serum cytokine analysis (luminex) and by PBMC flow cytometry respectively. Additionally, immune fluorescence (IF) or immunohistochemistry was performed on formaldehyde fixed archival, pretreatment biopsies and on treatment biopsies CD8, granzyme B, phospho-STAT-3 or LAG-3, T-bet/CD3 and HLA-A. T-cell clones were quantified by TCR deep sequencing (Adaptive biotechnology) from DNA isolated using EDTA blood samples. Results: Pegilodecakin treatment induced a systemic anti-tumor immune cytokine response biased towards Th1 & Th2 cytokines (IFNg, IL-18, TNFa, IL-3, IL-4) along with IL-7. Tuppressive (TGFb) and Th17 cytokines (IL-23, IL-17) were reduced. Cytotoxic effector molecules (Granzyme B, FasL, lymphotoxinB) were increased in the serum. PBMC analysis by flow cytometry in pre- and post-treatment samples show invigoration of the exhausted, T-cells, with increased proliferative index among CD8+ T-cells expressing LAG3 and PD1 throughout pegilodecakin treatment. The increase of PD-1+ Lag-3+ Ki-67+ CD8+ T-cells correlates with objective response to AM0010. On-treatment biopsies showed that pegilodecakin increased GzmB+, Phospho-Stat3+, Lag-3+ CD8+ T-cells in the tumor. T-cell clonal analysis by TCR sequencing on PBMCs from patients during pegilodecakin treatment showed expansion of several hundred previously undetected T-cell clones per patient. Expansion of these T-cell clones in the blood correlated with tumor response, with patients with objective response showing increased number of novel clones as compared to patients with progressive disease. Conclusion: We report that pegilodecakin treatment induced a systemic Th1 immune activation with reduction of Th17 related cytokines. We further report the hallmarks of CD8+ T-cell immunity in these cancer patients, including the systemic elevation of IFNg and GranzymeB levels, expansion and activation of CD8+ TILs, and the proliferation and invigoration and expansion of PD-1+/Lag-3+ CD8+ T-cell sub-set. In addition, pegilodecakin treatment led to the expansion of T-cell clones that were undetectable pretreatment. Clinically, expansion of these novel T-cell clones during pegilodecakin treatment correlated with achievement of objective response. Citation Format: Martin Oft, Aung Naing, Jeffrey R. Infante, Kyriakos P. Papadopoulos, Ivan H. Chan, Cong Shen, Navneet P. Ratti, Karen A. Autio, Deborah J. Wong, Manish R. Patel, Patrick A. Ott, Gerald S. Falchook, Shubham Pant, Annie Hung, John B. Mumm, Matthew Adamow, Scott McCauley, Rakesh Verma, Phillip Wong, Peter VanVlasselaer, Joseph Leveque, Nizar M. Tannir. PEGylated IL-10 (pegilodecakin) induces systemic immune activation, CD8+ T-cell invigoration and polyclonal T-cell expansion in cancer patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A016.

Comprehensive Analysis of TCR-\u03b2 Repertoire in Patients with Neurological Immune-mediated Disorders

In this study we characterized the TCR repertoire profiles in patients with chronic progressive inflammatory neurological disorders including HAM/TSP, associated with human T-cell lymphotropic virus type I (HTLV-I) infection, and multiple sclerosis (MS), an inflammatory, demyelinating disease of the CNS of unknown etiology. We hypothesized that a T-cell receptor (TCR) clonal repertoire ‘signature’ could distinguish HAM/TSP patients from healthy controls, as well as from patients with a more heterogeneous CNS-reactive inflammatory disease such as MS. In this study, we applied an unbiased molecular technique – unique molecular identifier (UMI) library-based strategy to investigate with high accuracy the TCR clonal repertoire by high throughput sequencing (HTS) technology. cDNA-TCR β-chain libraries were sequenced from 2 million peripheral mononuclear cells (PBMCs) in 14 HAM/TSP patients, 34 MS patients and 20 healthy controls (HC). While HAM/TSP patients showed a higher clonal T-cell expansion compared to MS and HC, increase of the TCR clonal expansion was inversely correlated with the diversity of TCR repertoire in all subjects. In addition, longitudinal analysis of TCR repertoires from HAM/TSP patients demonstrated a correlation of the TCR clonal expansion with HTLV-I proviral load. Surprisingly, MS patients showed a higher diversity of TCR repertoires than other groups. Despite higher TCR clonal expansions in HAM/TSP patients, no disease-specific TCRs were shared among patients. Only non-shared or “private” TCR repertoires was observed. While no clones that shared the same CDR3 amino acid sequences were seen in either HC or MS patients, there was a cluster of related CDR3 amino acid sequences observed for 18 out of 34 MS patients when evaluated by phylogenetic tree analysis. This suggests that a TCR-repertoire signature may be identified in a subset of patients with MS.

Enhanced interaction between SEC2 mutant and TCR Vβ induces MHC II–independent activation of T cells via PKCθ/NF-κB and IL-2R/STAT5 signaling pathways

SEC2, a major histocompatibility complex class II (MHC II)-dependent T-cell mitogen, binds MHC II and T-cell receptor (TCR) Vβs to induce effective co-stimulating signals for clonal T-cell expansion. We previously characterized a SEC2 mutant with increased recognition of TCR Vβs, ST-4, which could intensify NF-κB signaling transduction, leading to IL-2 production and T-cell activation. In this study, we found that in contrast to SEC2, ST-4 could induce murine CD4+ T-cell proliferation in a Vβ8.2- and Vβ8.3-specific manner in the absence of MHC II+ antigen-presenting cells (APCs). Furthermore, although IL-2 secretion in response to either SEC2 or ST-4 stimulation was accompanied by up-regulation of protein kinase Cθ (PKCθ), inhibitor of κB (IκB), α and β IκB kinase (IKKα/β), IκBα, and NF-κB in mouse splenocytes, only ST-4 could activate CD4+ T cells in the absence of MHC II+ APCs through the PKCθ/NF-κB signaling pathway. The PKCθ inhibitor AEB071 significantly suppressed SEC2/ST-4-induced T-cell proliferation, CD69 and CD25 expression, and IL-2 secretion with or without MHC II+ APCs. Further, SEC2/ST-4-induced changes in PKCθ/NF-κB signaling were significantly relieved by AEB071 in a dose-dependent manner. Using Lck siRNA, we found that Lck controlled SEC2/ST-4-induced phosphorylation of PKCθ. We also demonstrated that the IL-2R/STAT5 pathway is essential for SEC2/ST-4-induced T-cell activation. Collectively, our data demonstrate that an enhanced ST-4-TCR interaction can compensate for lack of MHC II and stimulate MHC II-free CD4+ T-cell proliferation via PKCθ/NF-κB and IL-2R/STAT5 signaling pathways. Compared with SEC2, intensified PKCθ/NF-κB and IL-2R/STAT5 signals induced by ST-4 lead to enhanced T-cell activation. The results of this study will facilitate better understanding of TCR-based immunotherapies for cancer.

Local mutational diversity drives intratumoral immune heterogeneity in non-small cell lung cancer

Combining whole exome sequencing, transcriptome profiling, and T cell repertoire analysis, we investigate the spatial features of surgically-removed biopsies from multiple loci in tumor masses of 15 patients with non-small cell lung cancer (NSCLC). This revealed that the immune microenvironment has high spatial heterogeneity such that intratumoral regional variation is as large as inter-personal variation. While the local total mutational burden (TMB) is associated with local T-cell clonal expansion, local anti-tumor cytotoxicity does not directly correlate with neoantigen abundance. Together, these findings caution against that immunological signatures can be predicted solely from TMB or microenvironmental analysis from a single locus biopsy.

Chimeric Antigen Receptor T-Cell Therapy Shapes T Cell Repertoire in Chinese Patients with B-Cell Acute Lymphocyte Leukemia

Introduction: Chimeric antigen receptor (CAR) T-cell therapy has displayed potent anti-leukemia activity in refractory/relapsed acute lymphocytic leukemia (ALL). However, the influence of CAR-T therapy on host systemic and local immunity has not been well examined. We therefore applied high-throughput T cell receptor β chain sequencing to track dynamic change of T-cell repertoire in vivo induced by CAR-T therapy in B-cell acute lymphocytic leukemia patients.Patients and Methods: Six patients with 45 samples were under observation. The samples obtained to be tested were the peripheral blood mononuclear cell (PBMC) samples and bone marrow mononuclear cell (BMMC) samples before and after CAR-T administration, as well as the CAR-transduced autologous T cell samples on the day when they were to be infused to patients. The information of samples and patients was summarized in Table 1. The TCR full length mRNAs of these samples were deeply sequenced using the ImmunHubTM TCR profiling system (ImmunQuad Biotech). Briefly, a 5'RACE unbiased amplification protocol was used. An algorithm was applied to raw sequencing data for PCR and sequencing errors correction and V, D, J, C gene segments mapping with IMGT®. The inverse Simpson index and the clonality index was calculated to estimate TCRβ clonotype diversity and the state of clonal proliferation of T cells. The donut chart and clone tracking heat map were generated by R.Result: Compared to preinfusion TCR diversity, we observed inverse Simpson's index of 4 of 6 patients' PBMC samples (Fig. 1A) and of 3 of 4 patients' BMMC samples (Fig. 1B) had been increasing to day 7 after CAR-T treatment. As time went by, a decline of TCR diversity from day 7 to day 28 was detected in both PBMC and BMMC samples (Fig. 1). Of note, a relative rising tide of TCR diversity was observed after the decline in PBMC samples (Fig. 1A). The decreased TCR diversity leaded us to test the change of TCR clonality. As shown by Fig. 2A and 2B, in comparison with the pre-treatment and the CAR-T samples, we detected highly clonal proliferation of T cells in both blood and bone marrow. Next, we applied the clonality index to quantitatively define T cell clonal expansion (Fig. 2C and 2D). The clonality index was raised from 0.16±0.07 and 0.14±0.05 before treatment to 0.27±0.09 and 0.3±0.15 at last time point post infusion in PBMCs and BMMCs, respectively (mean ± SD, student's t test, P=0.03 and P=0.17, respectively).CAR-T-induced T-cell clonal expansion triggered us to trace back the original clonal source. For tracking clonal evolution, we selected the 100 most prevalent T cell clonotypes found at the last time point in both PBMCs and BMMCs and tracked their frequencies at earlier time points. As was displayed by the heat map, the top 100 T cell clones dominating in PBMCs and BMMCs at the last time point were barely found in CAR-T cell pool and were the low-frequency clonotypes in preinfusion samples (Fig. 3A and 3B). In order to quantitatively describe this phenomenon, we compared the total frequency of top 10 T cell clones in PBMC and BMMC samples at the last time point to their corresponding total frequency in preinfusion samples and the infused CAR-T pool (Fig. 3C, 3D, 3E and 3F). The total frequency of the top 10 T cell clones was 23%±11% and 27%±14% at last time point, 4%±6% and 8%±7% before treatment and 0.05%±0.09% and 0.01%±0.01% in the infused CAR-T samples in PBMCs and BMMCs, respectively (mean ± SD, Fig. 3C, 3D, 3E and 3F; student's t test, P=0.01, P=0.01, P=0.08 and P=0.03, respectively). Only one patient's (patient 6's) top 10 T cell clones in PBMCs at last time point were not found in both CAR-T pool and preinfusion sample, which may be caused by the limitation of sequencing detection (Fig. 3C).Conclusion: For the first time, we demonstrated CAR-T therapy could stimulate the clonal proliferation of endogenous non-CAR T cells in patients. Along with other groups' animal results (Barber A et al. J Immunol. 2009) indicating that CAR-T therapy could facilitate the infiltrating of tumor antigen-specific T cells, these expanded T cell clones of patients in our trial are most likely tumor antigen specific, which could provide synergistic anti-tumor effect following CAR-T adoptive transfer. The trial was registered in Chinese Clinical Trial Registry and the registration number is ChiCTR-OCC-15007008. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

PEGylated IL-10 (Pegilodecakin) Induces Systemic Immune Activation, CD8+ T Cell Invigoration and Polyclonal T Cell Expansion in Cancer Patients

Tumor-reactive Tcell exhaustion prevents the success of immune therapies. Pegilodecakin activates intratumoral CD8+ Tcells in mice and induces objective tumor responses in patients. Here we report that pegilodecakin induces hallmarks of CD8+ Tcell immunity in cancer patients, including elevation of interferon-γ and GranzymeB, expansion and activation of intratumoral CD8+ Tcells, and proliferation and expansion of LAG-3+ PD-1+ CD8+ Tcells. On pegilodecakin, newly expanded Tcell clones, undetectable at baseline, become 1%-10% of the total Tcell repertoire in the blood. Elevation of interleukin-18, expansion of LAG-3+ PD-1+ Tcells and novel Tcell clones each correlated with objective tumor responses. Combined pegilodecakin with anti-PD-1 increased the expansion of LAG-3+ PD-1+ CD8+ Tcells.

Clonal Expansion of Antigen Specific T-Cells during Radiation Therapy for HPV Associated Cervical Cancers Is Regulated By the Vaginal Microbiome

Cervical cancers are known to be highly radiation sensitive; however, the rate of response to treatment is variable and poor responders are more likely to recur. The microbiota of colon cancers has been reported to play a role in response to chemotherapy and immune activation. We hypothesized that the cervical microbiome may impact radiation response and T-cell expansion. To address this, we completed prospective serial T-cell receptor (TCR) and microbiome sequencing of 30 patients with CCa throughout chemoradiation (CRT). Patients with newly diagnosed CCa were enrolled on a prospective study to characterize changes in the TCR profile and vaginal microbiome during CRT. Cervical samples were obtained from 30 patients before and during (weeks 1, 3, and 5) radiation. TCR profiling was performed through ImmunoSEQ to amplify and sequence the TCR CDR3b regions. Total unique templates, clonality, maximum productive frequency (MPF) and MPF of the top 10 clones (MPF10) were quantified. Disease response was categorized as exceptional response (ER), standard response (SR) or poor response (PR) based on week 5 MRI and 3 month PET/CT. Microbiome was characterized via 16S rDNA sequencing. Shannon diversity index (SDI) was used to compare microbiome diversity. Impact of specific microbial species were assessed using Linear Effect Size (LEFSe) analysis. Among all patients, the total templates (p=0.22), sample clonality (p=0.3) and MPF (p=0.07) were similar among SR, ER and PR at baseline. At week 5, total templates was similar among all groups (p=0.13); however, ER samples demonstrated higher clonality (ER median 0.05 [0.04-0.06] vs SR median (0.01 [0.009-0.02]; p=0.02). MPF10 decreased over time for PR/SR and increased over time for ER, with a higher fold change in MPF10 from week 3 to week 5 for ER (1.51 [1.10-2.61]) vs PR (0.60 [0.30-0.94]; p=0.02) or SR (0.80 [0.54-1.10]; p=0.03). Alpha diversity of the cervical microbiome was similar among responders (SR/ER) and non-responders (PR); however, distinct genus-level differences existed between SR/ER and non-responders PR, with enrichment of Porphyromonas in SR/ER and enrichment of Lactobacilli in PR. A Lactobacillus:Porphyromonas ratio (LPR) >= 1 (N=7) vs. LPR <1 (N=23). A Lactobacillus:Porphyromonas ratio (LPR) >= 1 was associated with decreased RFS (12.3 months vs NR; p=0.004) with failure rate of 43% (3/7) versus 4% (1/23) noted in the low LPR (<1; N=23). High LPR at baseline was also associated with decreased fold change in MPF10 by week 5 (p=0.015). Exceptional responders to radiation treatment are more likely to have clonal expansion of antigen specific T-cells within CCa. Predominance of vaginal Lactobacillus is associated with poor radiation response and lack of clonal T-cell expansion. Loss of E6/7 expression driven by lactobacillus may provide a mechanism to account for this observation. Further validation will be needed prior to development of interventions targeting the local tumor microbiome.