Keywords: T cells, T cell receptors, antigen presentation, aAPC
Effective immune responses are critical for control of a variety of infectious disease including bacterial, viral and protozoan infections as well as in protection from development of tumors. Central to the development of an effective immune response is the T lymphocyte which, as part of the adaptive immune system, is central in achieving sterilization and long lasting immunity. While the normal immune responses is tightly regulated there are also notable defects leading to pathologic diseases. Inactivity of tumor antigen-specific T cells, either by suppression or passive ignorance allows tumors to grow and eventually actively suppress the immune response. Conversely, hyperactivation of antigen-specific T cells to self antigens is the underlying basis for many autoimmune diseases including: multiple sclerosis; arthritis; and diabetes.
Secondary to their central role in a wide variety of physiologic and pathophysiologic responses my lab takes a broad-based approach to studying T cell responses. A critical interaction that helps initiate and direct T cells is the interaction between a specific T cell receptor and a cognate antigen-Human Leukocyte Antigen (HLA) complex. To facilitate these studies we have developed novel tools including soluble versions of HLA molecules, HLA-Ig complexes, and artificial Antigen Presenting Cells, aAPC. Several of them have been patented and are currently licensed and marked by BD under the product name of DimerX. Based on these and other tools we focus on a multiscale and multidimensional, analysis of the T cell responses which enhance our insights into both the basic biology of T cells responses and ultimately leads to development of novel therapeutic approaches.
Nanoscale Analyses- Each T cell uses its own unique T cell receptor, TCR, to recognize antigenic peptide in the context of a presenting complex, the HLA/MHC molecule. Interestingly, during the course of immune responses, activated T cells show enhanced sensitivity to antigen. This is not based on structural changes in the TCR but, in part, due to changes in the TCR organization which results in increased efficiency of TCR crosslinking on activated T cells that can be seen as increased avidity for dimeric peptide MHC complexes. Our studies show that TCR of naïve cells are monomeric or in small clusters while the TCR of activated cells are in larger clusters whose size allows divalent ligand binding. Currently we are actively involved in studying the biophysical, genetic and environmental control of TCR organization
Microscale analyses- To better understand and manipulate T cells immune responses, we developed an acellular aAPC. This lego-like system can be used to study T cells responses at a variety of different levels. Initially we used this approach to induce and expand clinically relevant amounts of highly enriched antigen-specific T cells. Using this approach it was possible to generate antigen-specific T cells directed at clinically relevant tumor antigens such as the melanoma self-antigen Mart-1 or infectious disease antigens such as CMVpp65. Using aAPC we can expand antigen-specific CTL, Mart-1 reactive cells, by at least a million fold in less than 2 month. Furthermore HLA-Ig based aAPC induced CTL directed at a subdominant peptides from the cancer testis antigen NY-ESO-1 successfully recognized and killed tumor cells in a antigen-specific fashion. Currently aAPC are being used to study manipulation of in vivo CTL responses and holds potential as a therapeutic approaches to augment both adoptive and active immunotherapy.
Macroscale analyses- The development of high-throughput technologies to address questions in cell biology has revolutionized our approach to medicine. In my lab we have focused on development of approaches to understand the breadth of the immune repertoire and a high-throughput approach to understanding genetic regulation of glycosylation. These projects are summarized below.
The development of high-throughput protein analyses for rapidly determining antigen-specific T-cell receptor repertoires of diverse T-cell populations can enable comprehensive, broad-based analyses of T-cell responses. Promising applications include medical diagnostics, vaccine development, treatment of autoimmune diseases, and detection of potential agents of bioterrorism. In the lab on-going studies examine the feasibility of using aAPC and/or peptide/HLA microarrays to selectively capture and enumerate antigen-specific T cells.
Glycosylation is among the most complex post-translational modifications with an extremely high level of diversity that has made it refractory to high-throughput analyses. Despite its resistance to high-throughput techniques, glycosylation is important in many critical cellular processes that necessitate a productive approach to their analysis. To facilitate studies in glycosylation, we developed a high-throughput lectin microarray for defining mammalian cell surface glycan signatures. Using the lectin microarray we analyzed cell surface glycan signatures and were able to predict mannose-dependent tropism using a model pathogen and used the glycan signatures to identify novel lectin biomarkers for cancer stem-like cells in a murine model. Thus, lectin microarrays are an effective tool for analyzing diverse cell processes including cell development and differentiation, cell-cell communication, pathogen-host recognition, and cell surface biomarker identification. On-going studies further explore use of this new technology in the identification of novel biomarkers.
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