Abstract A80: Functionally significant heterogeneity in apparently homogenous naive CD8 T cell populations
Authors: Balyan Renu , Gund Rupali , Ebenezer Chitra , Krishnamurthy Thyagarajan , Chawla Amanpreet Singh , Durdik Jeannine , George Anna , Bal Vineeta , Rath Satyajit 
Published: March 2017

Functional heterogeneity in apparently homogenous T cell populations has been well reported. However, what factors lead to such variations within a T cell population is still unclear. Depending upon the time of egress from the thymus and duration of residence in the periphery, individual cells would be of relatively different ages, however, consequences of such cellular aging have not been addressed. We observed that naive CD8 T cells from aged mice have lower coreceptor levels than naive CD8 T cells of young mice. Further, when naive T cells from young mice are separated into coreceptor-hi and coreceptor-lo subsets, we find that coreceptor-lo cells are smaller, respond poorly and are more susceptible to death. Similar coreceptor-lo associated defects were observed even in TCR-transgenic monoclonal CD8 T cell population. However, this phenotype is an outcome of peripheral residence, as coreceptor-lo and coreceptor-hi subsets of single-positive (SP) thymocytes do not show these differences. We show that coreceptor levels reduce over time, as a result of MHC-I mediated tonic signals and lower CD8 levels can be used as a surrogate marker to identify relatively aged cells in a naive CD8 T cell population. Thus, our data show that apparently unimodally distributed coreceptor heterogeneity among naive T cells, simplistically expected to be due to intrinsic noise, is not only correlated with major functional variation but is modulated by extrinsic microenvironmental cues in vivo. These findings indicate that ensemble averages may not represent individual cell function, even for a unimodal normal distribution of single cell measurements, highlighting the importance of distinguishing between population versus individual T cell responses to antigen encounter. At the population level, CD8 T cell response magnitude increases with ligand dose as well as with ligand affinity. However, whether ligand density and affinity only modulate the frequency of responding T cells and/or alters responses at the single cell level is unclear. We observed that the early activation response of individual naive CD8 T cell is binary such that the frequency of CD69 expressing cells increases with increment in ligand density or affinity, but the levels of CD69 expression in individual responding cells remain constant and variations in baseline cellular metabolic status between individual T cells may contribute to the differences in their activation thresholds. We show that ligand density and affinity determine the extent of TCR internalization in responding CD8 T cells, which in turn regulates the time to cell cycle entry. TCR internalization, which is commonly thought of as a mechanism to attenuate TCR-mediated signaling, was found to be associated with sustained TCR signaling via PI3K, leading to rapid metabolic reprogramming and when the degradation of internalized TCR complex is prevented, the duration of signaling is extended possibly from endosomal compartments, leading to better T cell response. Collectively, our findings reveal that heterogeneity among T cells for signal receptivity as well as microenvironmental influences experienced during their life history shape individual T cell responses and understanding the basis of heterogeneity within putatively homogenous T cell populations is significant for understanding of immune T cell responses.

Citation Format: Renu Balyan, Rupali Gund, Chitra Ebenezer, Thyagarajan Krishnamurthy, Amanpreet Singh Chawla, Jeannine Durdik, Anna George, Vineeta Bal, Satyajit Rath. Functionally significant heterogeneity in apparently homogenous naive CD8 T cell populations. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A80.