Date of Award

January 2015

Document Type


Degree Name

Medical Doctor (MD)



First Advisor

Richard Edelson

Second Advisor

Robert Tigelaar

Subject Area(s)



Despite its clinical success, the mechanism underlying extracorporeal photopheresis (ECP) is not well understood, however, the plate-passage (PP) step appears integral to generating activated monocytes. In the first part of the project, we developed a functional assay to evaluate the efficacy of plate-passed myeloid cells (PPM) compared with freshly isolated, unstimulated monocytes (UM) and conventional dendritic cells derived from blood monocytes cultured with GM-CSF/IL4 (DC). Each of these three antigen presenting cell (APC) was co-cultured with purified, autologous CD8 cells, with or without CD4 cells. Cultures were carried out using melanoma antigen MART-1 "long peptide (LP)," a 25-amino acid peptide containing the binding sequences for the appropriate MHC class I and II, and for presentation to CD8+ and CD4+ cells. Results showed reliable expansions of freshly isolated naïve human T cells using the three types of APC, without any significant differences among the types, and the addition of CD4+ tended to enhance expansion of PPM and DC, but not UM. In the second part, we sought to develop a method for directly tracking early T cell responses during immunotherapy. Using calcium flux to indicate early T cell signaling, we focused mostly on the ovalbumin (OVA)-derived, SIINFEKL-specific transgenic mouse model (OT1). After a few protocol modifications, we were able to detect antigen-specific calcium flux (ASF) upon mixing naïve OT1 cells with SIINFEKL peptide-loaded DC compared with non-specific peptide counterparts. We could still detect ASF down to a peptide-loading concentration of ~10-3uM and at a frequency of ~0.1% OT1 cells among wild-type (WT), non-responding cells. We next identified the activation requirements of early effector and memory OT1 cells from the spleen, lymph nodes, and peripheral blood after adoptive transfer into WT recipients immunized with OVA. At 1 week, OT1 cells from all 3 tissues had become activated, effector cells (CD44hi and CD62 lo), and while detectable, ASF in all three tissues was reduced compared with naïve cells. At 6 weeks, only the peripheral blood OT1 cells had generated a memory response (CD127hi KLRG1lo), and ASF in all three tissues was further reduced. Herein, we have shown that ASF can be detected in naïve, and less so antigen-experienced and memory T cells in a single-antigen, transgenic system from which we hope to develop a multi-antigen tumor model.