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

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.

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.

Improving antigenic peptide vaccines for cancer immunotherapy using a dominant tumor-specific T cell receptor.

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.

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 CDR3ß 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.

On the Significance of Systolic Flow Waveform on Aortic Valve Energy Loss.

This study aims to quantitatively and qualitatively assess energy dissipation in the aortic valve as a function of systolic aortic flow waveform representing pathologies where flow time-to-peak is delayed. A bioprosthetic valve was tested in the aortic position of a left-heart simulator under physiological pressure and flow conditions. The flow loop piston pump was programmed to generate three different flow waveforms each with a different peak time annotated as early peak (EP) with a rapid acceleration, mid peak (MP) and late peak (LP) with a rapid deceleration. Energy dissipation was calculated from flow and pressure measurements while sinus vorticity dynamics were evaluated using time-resolved planar particle image velocimetry. Average pressure gradients during systole are found 30.2 ± 0.19, 30.7 ± 0.25 and 32.9 ± 0.29 mmHg and average dissipation over systole is found 0.95 ± 0.026, 1.05 ± 0.034 and 1.25 ± 0.043 W for EP, MP and LP respectively. As systole's acceleration phase is slower, sinus vortices are more likely to form, necessitating more energy exchange from shear layers inducing more viscous dissipation. EP found in healthy individuals is superior in terms of reducing energy dissipation and increasing aortic valve efficiency. In the context of possible left ventricular dysfunction and aortic stenosis, this means that delayed time-to-peak in the aortic flow waveform seen is not compensatory.

Aortic sinus flow stasis likely in valve-in-valve transcatheter aortic valve implantation.

OBJECTIVE: Valve-in-valve procedures using transcatheter aortic valves are increasingly performed to treat degenerated bioprosthetic surgical aortic valves because they are less invasive than redo aortic valve replacement. The objective of this study is to quantify the changes in aortic sinus blood flow dynamics before and after a valve-in-valve procedure to gain insight into mechanisms for clinical and subclinical thrombosis of leaflets. METHODS: A detailed description of the sinus hemodynamics for valve-in-valve implantation was performed in vitro. A Medtronic Hancock II (Medtronic Inc, Minneapolis, Minn) porcine bioprosthesis was modeled as a surgical aortic valve, and Medtronic CoreValve and Edwards Sapien (Edwards Lifesciences, Irvine, Calif) valves were used as the transcatheter aortic valves. High-resolution particle image velocimetry was used to compare the flow patterns from these 2 valves within both the left coronary and noncoronary sinuses in vitro. RESULTS: Velocity and vorticity within the surgical valve sinuses reached peak values of 0.7 m/s and 1000 s-1, with a 70% decrease in peak fluid shear stress near the aortic side of the leaflet in the noncoronary sinus. With the introduction of transcatheter aortic valves, peak velocity and vorticity were reduced to approximately 0.4 m/s and 550 s-1 and 0.58 m/s and 653 s-1 without coronary flow and 0.60 m/s and 631 s-1 and 0.81 m/s and 669 s-1 with coronary flow for the CoreValve and Sapien valve-in-valve implantations, respectively. Peak shear stress was approximately 38% higher along the aortic side of the coronary versus noncoronary transcatheter aortic valve leaflet. CONCLUSIONS: Decreased flow and shear stress in valve-in-valve procedures indicate a higher risk of leaflet thrombosis secondary to flow stasis, perhaps more so in the noncoronary sinus.

Antigen-Presenting Intratumoral B Cells Affect CD4+ TIL Phenotypes in Non-Small Cell Lung Cancer Patients.

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.

Coronary Flow Impacts Aortic Leaflet Mechanics and Aortic Sinus Hemodynamics.

Mechanical stresses on aortic valve leaflets are well-known mediators for initiating processes leading to calcific aortic valve disease. Given that non-coronary leaflets calcify first, it may be hypothesized that coronary flow originating from the ostia significantly influences aortic leaflet mechanics and sinus hemodynamics. High resolution time-resolved particle image velocimetry (PIV) measurements were conducted to map the spatiotemporal characteristics of aortic sinus blood flow and leaflet motion with and without physiological coronary flow in a well-controlled in vitro setup. The in vitro setup consists of a porcine aortic valve mounted in a physiological aorta sinus chamber with dynamically controlled coronary resistance to emulate physiological coronary flow. Results were analyzed using qualitative streak plots illustrating the spatiotemporal complexity of blood flow patterns, and quantitative velocity vector and shear stress contour plots to show differences in the mechanical environments between the coronary and non-coronary sinuses. It is shown that the presence of coronary flow pulls the classical sinus vorticity deeper into the sinus and increases flow velocity near the leaflet base. This creates a beneficial increase in shear stress and washout near the leaflet that is not seen in the non-coronary sinus. Further, leaflet opens approximately 10% farther into the sinus with coronary flow case indicating superior valve opening area. The presence of coronary flow significantly improves leaflet mechanics and sinus hemodynamics in a manner that would reduce low wall shear stress conditions while improving washout at the base of the leaflet.