Brandon Moore

Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR

Significance The essence of the adaptive immune response depends on the specificity of antigen receptors. This report identifies shared alpha–beta T-cell receptor (TCR) pairs from the tissues of HLA-A2+ patients with breast cancer and control donors. Using an emulsion-based RT-PCR assay, we analyzed TCR sequences from tissues ex vivo. We identified multiple TCR pairs shared between tumors, but not control samples. Although recent reports have concluded that anticancer T-cell responses depend on patient-specific mutation-associated neoantigens, this study provides evidence that T cells also recognize shared antigens. This approach has broad application to a variety of research questions where the end goal is to examine T-cell repertoires and/or identify T-cell antigens. Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha–beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients’ tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.

TCR hypervariable regions expressed by T cells that respond to effective tumor vaccines

A major goal of immunotherapy for cancer is the activation of T cell responses against tumor-associated antigens (TAAs). One important strategy for improving antitumor immunity is vaccination with peptide variants of TAAs. Understanding the mechanisms underlying the expansion of T cells that respond to the native tumor antigen is an important step in developing effective peptide-variant vaccines. Using an immunogenic mouse colon cancer model, we compare the binding properties and the TCR genes expressed by T cells elicited by peptide variants that elicit variable antitumor immunity directly ex vivo. The steady-state affinity of the natural tumor antigen for the T cells responding to effective peptide vaccines was higher relative to ineffective peptides, consistent with their improved function. Ex vivo analysis showed that T cells responding to the effective peptides expressed a CDR3β motif, which was also shared by T cells responding to the natural antigen and not those responding to the less effective peptide vaccines. Importantly, these data demonstrate that peptide vaccines can expand T cells that naturally respond to tumor antigens, resulting in more effective antitumor immunity. Future immunotherapies may require similar stringent analysis of the responding T cells to select optimal peptides as vaccine candidates.

Improving Antigenic Peptide Vaccines for Cancer Immunotherapy Using a Dominant Tumor-specific T Cell Receptor

Background: Vaccination with mimotopes, peptide mimics of epitopes, stimulates a range of T cell protection. Results: Mimotopes identified from peptide libraries by T cells with common receptors increased immunity more than those with rare high affinity receptors. Conclusion: T cell prevalence must be considered when designing peptide vaccines. Significance: Optimizing mimotopes will improve antigen-specific vaccines for applications including cancer immunotherapies. Vaccines that incorporate peptide mimics of tumor antigens, or mimotope vaccines, are commonly used in cancer immunotherapy and function by eliciting increased numbers of T cells that cross-react with the native tumor antigen. Unfortunately, they often elicit T cells that do not cross-react with or that have low affinity for the tumor antigen. Using a high affinity tumor-specific T cell clone, we identified a panel of mimotope vaccines for the dominant peptide antigen from a mouse colon tumor that elicits a range of tumor protection following vaccination. The TCR from this high affinity T cell clone was rarely identified in ex vivo evaluation of tumor-specific T cells elicited by mimotope vaccination. Conversely, a low affinity clone found in the tumor and following immunization was frequently identified. Using peptide libraries, we determined if this frequently identified TCR improved the discovery of efficacious mimotopes. We demonstrated that the representative TCR identified more protective mimotopes than the high affinity TCR. These results suggest that targeting a dominant fraction of tumor-specific T cells generates potent immunity and that consideration of the available T cell repertoire is necessary for targeted T cell therapy. These results have important implications when optimizing mimotope vaccines for cancer immunotherapy.

Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model

Mechanisms of self-tolerance often result in CD8+ tumor-infiltrating lymphocytes (TIL) with a hypofunctional phenotype incapable of tumor clearance. Using a transplantable colon carcinoma model, we found that CD8+ T cells became tolerized in <24 h in an established tumor environment. To define the collective impact of pathways suppressing TIL function, we compared genome-wide mRNA expression of tumor-specific CD8+ T cells from the tumor and periphery. Notably, gene expression induced during TIL hypofunction more closely resembled self-tolerance than viral exhaustion. Differential gene expression was refined to identify a core set of genes that defined hypofunctional TIL; these data comprise the first molecular profile of tumor-specific TIL that are naturally responding and represent a polyclonal repertoire. The molecular profile of TIL was further dissected to determine the extent of overlap and distinction between pathways that collectively restrict T cell functions. As suggested by the molecular profile of TIL, protein expression of inhibitory receptor LAG-3 was differentially regulated throughout prolonged late-G1/early-S phase of the cell cycle. Our data may accelerate efficient identification of combination therapies to boost anti-tumor function of TIL specifically against tumor cells.

Antigen-presenting intratumoral B cells affect CD4+ TIL phenotypes in non-small cell lung cancer patients

B cells in non-small cell lung cancer tumors can present antigen. Activated or exhausted B cells were associated with effector or regulatory CD4+ T cells, respectively, which may make intratumoral B cells a viable target for immunotherapy. Effective immunotherapy options for patients with non–small cell lung cancer (NSCLC) are becoming increasingly available. The immunotherapy focus has been on tumor-infiltrating T cells (TILs); however, tumor-infiltrating B cells (TIL-Bs) have also been reported to correlate with NSCLC patient survival. The function of TIL-Bs in human cancer has been understudied, with little focus on their role as antigen-presenting cells and their influence on CD4+ TILs. Compared with other immune subsets detected in freshly isolated primary tumors from NSCLC patients, we observed increased numbers of intratumoral B cells relative to B cells from tumor-adjacent tissues. Furthermore, we demonstrated that TIL-Bs can efficiently present antigen to CD4+ TILs and alter the CD4+ TIL phenotype using an in vitro antigen-presentation assay. Specifically, we identified three CD4+ TIL responses to TIL-Bs, which we categorized as activated, antigen-associated, and nonresponsive. Within the activated and antigen-associated CD4+ TIL population, activated TIL-Bs (CD19+CD20+CD69+CD27+CD21+) were associated with an effector T-cell response (IFNγ+ CD4+ TILs). Alternatively, exhausted TIL-Bs (CD19+CD20+CD69+CD27−CD21−) were associated with a regulatory T-cell phenotype (FoxP3+ CD4+ TILs). Our results demonstrate a new role for TIL-Bs in NSCLC tumors in their interplay with CD4+ TILs in the tumor microenvironment, establishing them as a potential therapeutic target in NSCLC immunotherapy. Cancer Immunol Res; 5(10); 898–907. ©2017 AACR.

Antigen presentation by tumor infiltrating B cells influences CD4 T cell phenotype and function in primary lung cancer patient tumors

Meeting abstracts Despite improvements in surgical techniques and combined chemotherapies, the 5-year survival rate for all stages of non-small cell lung cancer (NSCLC) is only 18%. Understanding the function of tumor infiltrating lymphocytes (TILs) in NSCLC patient tumors will contribute to the

Abstract A11: The function of B cells in non-small cell lung cancer patients.

Lung cancer is the leading cause of cancer death in both men and women. Although novel therapies are emerging, we are interested in boosting tumor-specific immune responses in infiltrating lymphocytes. In a flow cytometric analysis of the immune infiltrate in non-small cell lung cancer (NSCLC) tumors (n=25), we found that the total number of infiltrating B cells (TIL-Bs) in the tumor versus the matched tumor-adjacent tissue was increased compared to other immune subsets, specifically T cells. Most NSCLC TIL-Bs were activated (CD69+ and CD86+) and had a memory phenotype (CD27+). Proliferation, antigen presentation, and antibody production studies were done to assess the range of function in TIL-Bs. Positively isolated TIL-Bs that were stimulated for three days through the antigen receptor had varied proliferation when compared to B cells from the tumor adjacent tissue, which suggests a spectrum of function in TIL-Bs at the site of the tumor. For antigen presentation, TIL-Bs stimulated CD4 tumor infiltrating lymphocytes (CD4 TILs) to proliferate without the addition of tumor lysate compared to the negative and positive controls (T cells alone and T cells activated through the T cell receptor, respectively). This suggests that TIL-Bs can present endogenous tumor antigens to CD4 TILs; however, antigen presentation is not as robust with TIL-Bs from other patient tumors, which is another example of a spectrum in TIL-B function. Finally, we assayed total antibody production (IgG) by TIL-Bs to determine if this is a function at the site of the tumor. Tumor and tumor adjacent B cells produce antibody in comparison to B cells from the periphery of a normal donor; this function is uniform unlike proliferation and antigen presentation. We predict that TIL-Bs have a range of function in their response against tumors. Our overall hypothesis is that tumor immunity in NSCLC is influenced by the molecular signature of TIL-Bs, which directs proliferation, presentation of tumor antigens to T cells, and production of anti-tumor antibodies. We are determining if proliferation, antigen presentation, and antibody production are tumor antigen-specific, particularly for the lung cancer-testis antigen XAGE-1b. This antigen was the most highly expressed in our patient tumor cells when analyzed by real time PCR and has been shown to play a role in NSCLC. We are performing experiments with TIL-Bs to test XAGE-1b specific proliferation and antigen presentation. Further, we generated a XAGE-1b ELISA to test the specificity of the antibodies generated by the TIL-Bs. Three out of twelve patient sera were positive for XAGE-1b-specific antibodies indicating that the XAGE-1b ELISA is working for the detection of XAGE-1b antibodies. Further, we are isolating cells from tumor, tumor adjacent, and normal (cancer-free) lung tissue to analyze the molecular signature of TIL-Bs to identify a pathway upstream of B cell signaling that may be altered and influencing TIL-B function. Results from this study will advance the understanding of the function of TIL-Bs in solid tumors, and which functions can be targeted. Ultimately, this project will provide a platform for a therapeutic vaccine for lung cancer patients, and more importantly, will help us understand why some patients do not respond well to immunotherapy. Such results may lead to the development of a prognostic tool to screen patients that will respond to vaccine. Citation Format: Tullia C. Bruno, Daniel Munson, Brandon Moore, Katherine Waugh, Jeffrey Kern, Jill E. Slansky. The function of B cells in non-small cell lung cancer patients. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A11.