T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. In the inactivated state, these cells circulate in the blood and accumulate in lymphoid tissues such as lymph nodes and spleen. Upon encounter with antigens on specialized presenting cells (dendritic cells), these resting T-cells become activated and differentiate into effector cells. The effector cells leave the lymphoid tissues and blood, entering sites of infection to combat pathogens. They can also cause autoimmune pathology. After elimination of an infecting organism, most activated T-cells die, but some remain as memory cells. Some memory T-cells recirculate in lymphoid compartments and others patrol peripheral tissues. Other lymphocytes such as regulatory T cells contribute to suppression of these T-cell responses. This project attempts to gain both a qualitative (especially tissue-specific 4 dimensional space and time) and a quantitative understanding of the activation, differentiation, migration, cell-cell interaction, memory status, and reactivation properties of both CD4 and CD8 T-cells. The movement of activated T-cells into non-lymphoid tissues is being analyzed using imaging techniques that allow high-resolution dynamic observation of how cells migrate, interact, and carry out their effector functions, while a finer grained analysis of tissue architecture, T cell localization, and functional state are being assessed using a newly developed method for quantitative multiplex static imaging called Histo-cytometry. Through this research, a better understanding of lymphocyte dynamics during an immune response to infection or after vaccination or during an autoimmune response will be established. These new insights can contribute to the more effective design of vaccines and to strategies for the amelioration of autoimmune processes. We have established a robust system for vaccination using the non-replicating pox vector MVA, to permit in situ analysis of immune cell behavior in response to a clinically used vaccine vector. Our studies indicate unexpected locations the cells initially infected by MVA within draining lymph nodes, distinct dendritic cells involvement in initial antigen presentation to CD4 vs. CD8 T-cells, a role for cross-presentation as well as direct presentation in the activation of CD8 T-cells, and different locations of naive vs. memory CD8 T-cells in the lymph node. After initial activation, both CD4 and CD8 T cells communicate with a single dendritic cell type, the XCR1+ (CD8alpha+) cell; this communication is essential for robust memory CD8 T cell responses. This project is also related to studies described in ZIA AI000545-27 LSB Multiscale Analysis of Immune Responses. A combination of intravital imaging and flow cytometry studies in a model of delayed-type hypersensitivity has revealed that effector CD4 T-cells in an inflamed site undergo a single round of activation to cytokine secretion status in concert with an arrest of migration, followed by a loss of cytokine production as the cells resume rapid movement in the tissue. This regain in motility occurs without the loss of antigen presentation. These data imply tuning of the antigen sensitivity of effector CD4 T-cells upon initial activation in the tissue that limits damaging cytokine production if the antigen load is not increasing, and also suggests that eventual elimination of a pathogen will depend on a constant influx of new antigen-sensitive effectors from secondary lymphoid tissue. Our data suggest this desensitization arises in part from a negative feedback loop involving PD-1, whose expression increases shortly after antigen stimulation of effector T-cells in the tissue. Engagement of PD-1 by PD-L1 that is also upregulated on antigen-presenting cells in the inflammatory tissue environment depresses signaling through the TCR and hence, desensitizes the T-cells with respect to the available antigen level. We propose that these events reflect the evolution of the adaptive immune system to a state that attempts to balance host defense against the possibility of inflammatory damage, by using antigen level as a representation of pathogen abundance and only activating effector cells to a level necessary to prevent that antigen level (pathogen load) from rising. Our data suggest that the in vitro paradigm of autocrine cytokine-induced polarization of CD4+ T-cells is an oversimplification of the in vivo reality. Under suitable conditions, Th1 and Th2 effector T-cells can be generated in the absence of the canonical cytokine (IFNgamma and IL-4, respectively), or the relevant STAT protein. A key determinant of T-cell fate is the strength of signaling through the T cell antigen receptor, and in vivo studies using 2 photon imaging show the fate choice (Th1 vs. Th2) is dominated by the duration of T-cell: dendritic cell interaction and the integrated strength of signal rather than by qualitative factors such as polarizing cytokines. Our most recent data show that the duration and strength of signaling establishes both the capacity of the T-cell to receive polarizing signals at a later time through upregulation of the IL-12Rb2 chain that is needed for IL-12p70 signaling for the induction of TH1 phenotype. The role of adjuvants in these models appears to be primarily that of determining the costimulatory state of the presenting cell; CD80/86 synergize with the TCR to determine the antigen-dependent strength of signal that determines whether or not IL-12Rb2 will be expressed, and possibly whether CD40L will b upregulated to further activate the DC for costimulatory molecule and IL-12 production. Adjuvants also secondarily provide the polarizing cytokine that TCR signaling regulates through cytokine receptor expression checkpoints, a very different model from the one that dominates current thinking and one with important implications for determining the dose, timing, and vehicles best suited to vaccine delivery. In a project related to studies described in ZIA AI000545-27 LSB Multiscale Analysis of Immune Responses, we have used Histo-cytometry to discover that Treg cluster around mostly migratory dendritic cells together with Tconv cells. The latter in some cases are activated to produce IL-2 locally that induces pSTAT5 formation and signaling in the co-localized Tregs. The Treg clustering is TCR-dependent and selective deletion of TCRalpha in mature Tregs results on loss of clustering and rapid autoimmune disease development. Imaging shows that the Tregs in these clusters are the highest in suppressive molecules such as CD73 and CTLA-4 and also that IL-2 sensing contributes to the suppressive action of these cells. Strikingly, the same pSTAT5+ Treg clusters are seen at comparable numbers in germ-free and conventional SPF mice, indicating that the activation of Tconv to the IL-2 producing state involves self-recognition, emphasizing that thymic negative election does not eliminate all T cells with TCR able to respond overtly to self-ligands. These studies, performed in collaboration with A. Rudensky, thus reveal the architectural basis for homeostatic control of T cell autoreactivity and the role of both Treg TCR signaling and a negative feedback loop involving Tconv production of IL-2 that together maintain the host in a non-diseased state. Finally, in collaboration with D. Mathis, we have imaged Treg in adipose tissue and documented the close association of these lymphocytes with MHC Class II+ cells in this tissue. Together with additional studies involving depletion of Tregs, ontogeny analysis, and examination of the role of IL-33 in adipose Treg function, these experiments have provided new insight into how tissue-specific subsets of regulatory T cells contribute to metabolic homeostasis.
NIH ExPorter
NIH ExPorter