Abstract
T cells are either naïve, having never encountered cognate antigen, or memory, with a history of activation, proliferation, and acquistion of effector specialization including tissue specific homing properties. We hypothesized that memory T cells would contain a subpopulation of alloantigen specific cells that might have acquired tissue specific homing characteristics such as upregulation of α4β7 integrin that directs T cells to gut lymphoid and non-lymphoid tissue. In mice, α4β7 integrin dependent migration to the peyer’s patch is essential to instigate lethal GVHD in MHC mismatched BMT models. To test this hypothesis, immunomagnetic sorting for CD45RO and CD45RA was used to obtain populations either depleted or enriched for naïve T cells respectively. Using CFDA dye dilution and CD69 upregulation, alloantigen specific T cells were tracked with flow cytometry at days 3, 5, and 7 of a mixed lymphocyte reaction, with an autologous control. ModFit LT software was used to estimate precursor frequencies, which showed that approximately 1 in 200 CD4 and CD8 positive T cells are alloreactive (n=9) regardless of whether they were CD45RO or CD45RA selected, demonstrating that alloreactivity could be potentially recruited from either the naïve or memory donor pool in a GVHD reaction. Analysis of α4β7 integrin coexpression before alloantigen stimulation demonstrated that for both CD4 and CD8 positive T cells, the memory pool had two-fold higher levels of coexpression that were statistically significant compared to the naive pool, with CD8 memory T cells demonstrating the highest coexpression (24 +/− 12%). When examined at day 5 of the MLR, both CD4 and CD8 positive alloantigen specific T cells from the CD45RO selected group maintained levels of α4β7 integrin coexpression similar to baseline (14% and 25% respectively). In contrast, CD4 and CD8 positive alloantigen specific T cells from the CD45RA selected group had levels of α4β7 integrin coexpression that were four-fold higher compared to baseline, with 20% and 48% coexpression respectively (n=6). The relative contribution of α4 and β7 integrins was examined by comparing the mean flourescent intensity (MFI) of the alloantigen specific T cells to the resting T cells within the same day 5 MLR. The greatest increase in expression was seen for β7 integrin on the CD45RA selected CD4 and CD8 positive T cells with 3.1 and 3.2 times higher expression respectively (both p<.001). In contrast, the CD45RO selected CD4 and CD8 positive T cells had 1.78 (p<.01) and 1.35 (p<.05) times higher β7 expression levels respectively. With regard to α4 integrin expression, CD45RA selected CD4 and CD8 positive T cells had 2.2 and 2.0 times higher expression respectively (both p<.01). The α4 integrin expression on CD45RO selected CD4 and CD8 positive T cells was 2.0 (p<.05) and 1.43 (p= NS) times higher respectively. These data suggest that both naïve and memory CD4 and CD8 positive T cells may contribute to alloreactivity, although there are differences in the regulation of α4β7 integrin expression which could significantly affect in vivo T cell homing and therefore instigation of GVHD. Based upon murine studies, it has been demonstrated that memory T cells do not contribute to GVHD; our data suggest in contrast that human memory T cells could contribute in a significant way to GVHD and further study is necessary before development of successful T cell manipulation strategies aimed at attenuating GVHD.
The Roles of CD4+ T Cells in Tumor Immunity