The antibody repertoire of each individual is continuously updated by the evolutionary process of B cell receptor mutation and selection. It has recently become possible to gain detailed information concerning this process through high-throughput sequencing. Here, we develop modern statistical molecular evolution methods for the analysis of B cell sequence data, and then apply them to a very deep short-read data set of B cell receptors. We find that the substitution process is conserved across individuals but varies significantly across gene segments. We investigate selection on B cell receptors using a novel method that side-steps the difficulties encountered by previous work in differentiating between selection and motif-driven mutation; this is done through stochastic mapping and empirical Bayes estimators that compare the evolution of in-frame and out-of-frame rearrangements. We use this new method to derive a per-residue map of selection, which provides a more nuanced view of the constraints on framework and variable regions.

The antibody repertoire of each individual is continuously updated by the evolutionary process of B-cell receptor (BCR) mutation and selection. It has recently become possible to gain detailed information concerning this process through high-throughput sequencing. Here, we develop modern statistical molecular evolution methods for the analysis of B-cell sequence data, and then apply them to a very deep short-read dataset of BCRs. We find that the substitution process is conserved across individuals but varies significantly across gene segments. We investigate selection on BCRs using a novel method that side-steps the difficulties encountered by previous work in differentiating between selection and motif-driven mutation; this is done through stochastic mapping and empirical Bayes estimators that compare the evolution of in-frame and out-of-frame rearrangements. We use this new method to derive a per-residue map of selection, which provides a more nuanced view of the constraints on framework and variable regions.

The Bruton's tyrosine kinase inhibitor ibrutinib is a highly effective, new targeted therapy for chronic lymphocytic leukemia (CLL) that thwarts leukemia cell survival, growth, and tissue homing. The effects of ibrutinib treatment on the T cell compartment, which is clonally expanded and thought to support the growth of malignant B cells in CLL, are not fully characterized. Using next-generation sequencing technology, we characterized the diversity of TCRβ-chains in peripheral blood T cells from 15 CLL patients before and after 1 y of ibrutinib therapy. We noted elevated CD4⁺ and CD8⁺ T cell numbers and a restricted TCRβ repertoire in all pretreatment samples. After 1 y of ibrutinib therapy, elevated peripheral blood T cell numbers and T cell-related cytokine levels had normalized, and T cell repertoire diversity increased significantly. Dominant TCRβ clones in pretreatment samples declined or became undetectable, and the number of productive unique clones increased significantly during ibrutinib therapy, with the emergence of large numbers of low-frequency TCRβ clones. Importantly, broader TCR repertoire diversity was associated with clinical efficacy and lower rates of infections during ibrutinib therapy. These data demonstrate that ibrutinib therapy increases diversification of the T cell compartment in CLL patients, which contributes to cellular immune reconstitution.

In this cross-sectional and longitudinal analysis of mapping the T-cell repertoire in kidney transplant recipients, we have investigated and validated T-cell clonality, immune repertoire chronology at rejection, and contemporaneous allograft biopsy quantitative tissue injury, to better understand the pathobiology of acute T-cell fraction, T-cell repertoire and antibody-mediated kidney transplant rejection. To follow the dynamic evolution of T-cell repertoire changes before and after engraftment and during biopsy-confirmed acute rejection, we sequenced 323 peripheral blood samples from 200 unique kidney transplant recipients, with (n=100) and without (n=100) biopsy-confirmed acute rejection. We report that patients who develop acute allograft rejection, have lower (p=0.01) T-cell fraction even before transplantation, followed by its rise after transplantation and at the time of acute rejection accompanied by high TCR repertoire turnover (p=0.004). Acute rejection episodes occurring after the first 6 months post-transplantation, and those with a component of antibody-mediated rejection, had the highest turnover; p=0.0016) of their T-cell repertoire. In conclusion, we validated that detecting repertoire changes in kidney transplantation correlates with post-transplant rejection episodes suggesting that T-cell receptor sequencing may provide recipient pre-transplant and post-transplant predictors of rejection risk.

Targeting immune inhibitory receptors has brought excitement, innovation and hope to cancer patients. Our recent work revealed the immunological effects of blocking the CTLA4 and PD-1 immune checkpoints on T cell receptor usage among peripheral blood cells, and further uncovers how the expansion of the T cell repertoire matches the immunotoxicity profile of the therapy.

Although immunotherapeutic strategies are emerging as adjunctive treatments for cancer, sensitive methods of monitoring the immune response after treatment remain to be established. We used a novel next-generation sequencing approach to determine whether quantitative assessments of tumor-infiltrating lymphocyte (TIL) content and the degree of overlap of T-cell receptor (TCR) sequences in brain tumors and peripheral blood were predictors of immune response and overall survival in glioblastoma patients treated with autologous tumor lysate-pulsed dendritic cell immunotherapy. A statistically significant correlation was found between a higher estimated TIL content and increased time to progression and overall survival. In addition, we were able to assess the proportion of shared TCR sequences between tumor and peripheral blood at time points before and after therapy, and found the level of TCR overlap to correlate with survival outcomes. Higher degrees of overlap, or the development of an increased overlap following immunotherapy, was correlated with improved clinical outcome, and may provide insights into the successful, antigen-specific immune response. Cancer Immunol Res; 4(5); 412-8. ©2016 AACR.

Purpose

To evaluate the immunomodulatory effects of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) blockade with tremelimumab in peripheral blood mononuclear cells (PBMC).

Experimental design

We used next-generation sequencing to study the complementarity-determining region 3 (CDR3) from the rearranged T-cell receptor (TCR) variable beta (V-beta) in PBMCs of 21 patients, at baseline and 30 to 60 days after receiving tremelimumab.

Results

After receiving tremelimumab, there was a median of 30% increase in unique productive sequences of TCR V-beta CDR3 in 19 out of 21 patients, and a median decrease of 30% in only 2 out of 21 patients. These changes were significant for richness (P = 0.01) and for Shannon index diversity (P = 0.04). In comparison, serially collected PBMCs from four healthy donors did not show a significant change in TCR V-beta CDR3 diversity over 1 year. There was a significant difference in the total unique productive TCR V-beta CDR3 sequences between patients experiencing toxicity with tremelimumab compared with patients without toxicity (P = 0.05). No relevant differences were noted between clinical responders and nonresponders.

Conclusions

CTLA4 blockade with tremelimumab diversifies the peripheral T-cell pool, representing a pharmacodynamic effect of how this class of antibodies modulates the human immune system.

In early-stage breast cancer, the degree of tumor-infiltrating lymphocytes (TIL) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 × 106 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared with monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T-cell responses to therapy and for the study of cryoimmunotherapy in early-stage breast cancer. Cancer Immunol Res; 4(10); 835-44. ©2016 AACR.

Therapies that target the programmed death-1 (PD-1) receptor have shown unprecedented rates of durable clinical responses in patients with various cancer types. One mechanism by which cancer tissues limit the host immune response is via upregulation of PD-1 ligand (PD-L1) and its ligation to PD-1 on antigen-specific CD8(+) T cells (termed adaptive immune resistance). Here we show that pre-existing CD8(+) T cells distinctly located at the invasive tumour margin are associated with expression of the PD-1/PD-L1 immune inhibitory axis and may predict response to therapy. We analysed samples from 46 patients with metastatic melanoma obtained before and during anti-PD-1 therapy (pembrolizumab) using quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and next-generation sequencing for T-cell antigen receptors (TCRs). In serially sampled tumours, patients responding to treatment showed proliferation of intratumoral CD8(+) T cells that directly correlated with radiographic reduction in tumour size. Pre-treatment samples obtained from responding patients showed higher numbers of CD8-, PD-1- and PD-L1-expressing cells at the invasive tumour margin and inside tumours, with close proximity between PD-1 and PD-L1, and a more clonal TCR repertoire. Using multivariate analysis, we established a predictive model based on CD8 expression at the invasive margin and validated the model in an independent cohort of 15 patients. Our findings indicate that tumour regression after therapeutic PD-1 blockade requires pre-existing CD8(+) T cells that are negatively regulated by PD-1/PD-L1-mediated adaptive immune resistance.

The efficacy of PD-1/PD-L1 targeted therapies in addition to anti-CTLA-4 solidifies immunotherapy as a modality to add to the anticancer arsenal. Despite raising the bar of clinical efficacy, immunologically targeted agents raise new challenges to conventional drug development paradigms by highlighting the limited relevance of assessing standard pharmacokinetics (PK) and pharmacodynamics (PD). Specifically, systemic and intratumoral immune effects have not consistently correlated with standard relationships between systemic dose, toxicity, and efficacy for cytotoxic therapies. Hence, PK and PD paradigms remain inadequate to guide the selection of doses and schedules, both starting and recommended Phase 2 for immunotherapies. The promise of harnessing the immune response against cancer must also be considered in light of unique and potentially serious toxicities. Refining immune endpoints to better inform clinical trial design represents a high priority challenge. The Cancer Immunotherapy Trials Network investigators review the immunodynamic effects of specific classes of immunotherapeutic agents to focus immune assessment modalities and sites, both systemic and importantly intratumoral, which are critical to the success of the rapidly growing field of immuno-oncology.