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IL-2 starvation does not induce apoptosis in normal human IL-2 dependent T cell
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Abstracts
4.3 #51
IL-2 STARVATION DOES NOT INDUCE APOPTOSIS IN NORMAL HUMAN IL-2 DEPENDENT T CELLS. Lisa Geiselhart, Michael H. Gallichio and Brian M. Freed. Transplantation Immunology Laboratory. Albany Medical College, Albany, N. Y. IL-2 withdrawal has been shown to induce apoptosis in IL-2 dependent T-cell lines. We wished to study the effect ofIL-2 starvation in primary, non-transformed IL-2 dependent human T cells. To generate these cells we stimulated PBMC with PHA-M and then cultured the cells until they became IL-2 dependent (12-15 days). The proliferative response was 189-6,000 cpm without rIL-2 and 40,000-100,000 cpm with rIL-2. Flow cytometric analysis of the cells showed that they were >98% T cells «3% NK cells, <1 %CDI4, <1 % CD19). Surprisingly, the cells were predominantly (60-96%) CD8+CD25-. Further analysis of the cells over time revealed that 5 days post-stimulation the cells were >96% CD25+ with equal numbers ofCD4+ and CD8+ cells. After 10 days there was a significant reduction in CD25+ (IL2Ra) expression. The CD4+ (CD25+ and CD2S-) cells began to disappear after 10 days and were only 7-20% of the total after 15 days. Withdrawal of rIL-2 did not cause a significant loss in viability even after 5 days. This is in contrast to transformed IL-2 dependent cell lines, in which apoptosis has been shown to occur after 6 hours of IL-2 starvation. The proliferative response could recover from 4 days ofIL-2 starvation if the cells were re-incubated for at least 48 hours in rIL-2. Since these CD25- cells could proliferate after IL-2 starvation we wished to compare these cells to unstimulated PBMC. Unstimulated PBMC «7% CD25+) incubated with rIL-2 also became IL-2 dependent. CD25+ was induced (47% positive after 14 days) in these cells, but CD8+ cells were not preferentially selected. The PHA-generated, CD8+CD25", IL-2 dependent cells are nontransformed T-cells that have a strong proliferative response to IL-2 in the absence of the classical high affinity IL-2 receptor. These IL-2 dependent T -cells, in contrast to T-cell lines, do not undergo apoptosis after IL-2 starvation.
4.4 #52
ANALYSIS OF CORRELATION BETWEEN ALLOIMMUNE T HELPER RESPONSE GENERATED FROM UNRELATED DONORS AND SEVERITY OF ACUTE GRAFTVS-HOST DISEASE. J Pei, G Longton, C Anasetti, S Masewicz, JA Hansen, PJ Martin. Fred Hutchinson Cancer Research Center, Seattle, WA We have tested whether the risk of acute GVHD after allogeneic marrow transplantation correlates with T helper response specific for alloantigens of the host. A limiting dilution assay was used to measure the frequency ofHTLp (fHTLp). PBMC were used as primary stimulators and EBV-transformed LCL were used as restimulator cells on day 14. An HTLp "index" was calculated as the fold increase offHTLp with allogeneic-stimulator cells compared to autologous stimulator cells. Our previous data have demonstrated that HTLp index is very sensitive for detecting HLA class II disparities and less sensitive for detecting HLA class I, minor histocompatibility antigen and isolated HLA-DQ or DP disparities. HLA class I disparity stimulates only CD8+ cells. HLA class II disparity stimulates CD4+ cells, and also stimulates CD8+ cells to a limited degree. We analyzed the donor anti-host fHTLp and HTLp index in 48 patients who engrafted and survived for at least 30 days after transplantation of unmodified marrow from unrelated donors. Methotrexate and cyclosporine were administered for GVHD prophylaxis. For all tests, donor and patient cells cryopreserved before transplantation were used, and assays with HLA-disparate third party responder and stimulator cells were used as controls. GVHD was assessed by an independent observer as grade 0 in 5, grade I in 3, grade II in 17, grade III in 15 and grade IV in 8 patients. A non-parametric trend test yields suggestive evidence that the HTLp index is positively correlated with aGVHD grade (p=O.l), however, the relationship is not strong enough to effectively predict the risk of aGVHD when methotrexate and cyclosporine are given for prophylaxis in patients transplanted with unmodified marrow from unrelated donors.
Abstracts
4.3 #51
IL-2 STARVATION DOES NOT INDUCE APOPTOSIS IN NORMAL HUMAN IL-2 DEPENDENT T CELLS. Lisa Geiselhart, Michael H. Gallichio and Brian M. Freed. Transplantation Immunology Laboratory. Albany Medical College, Albany, N. Y. IL-2 withdrawal has been shown to induce apoptosis in IL-2 dependent T-cell lines. We wished to study the effect ofIL-2 starvation in primary, non-transformed IL-2 dependent human T cells. To generate these cells we stimulated PBMC with PHA-M and then cultured the cells until they became IL-2 dependent (12-15 days). The proliferative response was 189-6,000 cpm without rIL-2 and 40,000-100,000 cpm with rIL-2. Flow cytometric analysis of the cells showed that they were >98% T cells «3% NK cells, <1 %CDI4, <1 % CD19). Surprisingly, the cells were predominantly (60-96%) CD8+CD25-. Further analysis of the cells over time revealed that 5 days post-stimulation the cells were >96% CD25+ with equal numbers ofCD4+ and CD8+ cells. After 10 days there was a significant reduction in CD25+ (IL2Ra) expression. The CD4+ (CD25+ and CD2S-) cells began to disappear after 10 days and were only 7-20% of the total after 15 days. Withdrawal of rIL-2 did not cause a significant loss in viability even after 5 days. This is in contrast to transformed IL-2 dependent cell lines, in which apoptosis has been shown to occur after 6 hours of IL-2 starvation. The proliferative response could recover from 4 days ofIL-2 starvation if the cells were re-incubated for at least 48 hours in rIL-2. Since these CD25- cells could proliferate after IL-2 starvation we wished to compare these cells to unstimulated PBMC. Unstimulated PBMC «7% CD25+) incubated with rIL-2 also became IL-2 dependent. CD25+ was induced (47% positive after 14 days) in these cells, but CD8+ cells were not preferentially selected. The PHA-generated, CD8+CD25", IL-2 dependent cells are nontransformed T-cells that have a strong proliferative response to IL-2 in the absence of the classical high affinity IL-2 receptor. These IL-2 dependent T -cells, in contrast to T-cell lines, do not undergo apoptosis after IL-2 starvation.
4.4 #52
ANALYSIS OF CORRELATION BETWEEN ALLOIMMUNE T HELPER RESPONSE GENERATED FROM UNRELATED DONORS AND SEVERITY OF ACUTE GRAFTVS-HOST DISEASE. J Pei, G Longton, C Anasetti, S Masewicz, JA Hansen, PJ Martin. Fred Hutchinson Cancer Research Center, Seattle, WA We have tested whether the risk of acute GVHD after allogeneic marrow transplantation correlates with T helper response specific for alloantigens of the host. A limiting dilution assay was used to measure the frequency ofHTLp (fHTLp). PBMC were used as primary stimulators and EBV-transformed LCL were used as restimulator cells on day 14. An HTLp "index" was calculated as the fold increase offHTLp with allogeneic-stimulator cells compared to autologous stimulator cells. Our previous data have demonstrated that HTLp index is very sensitive for detecting HLA class II disparities and less sensitive for detecting HLA class I, minor histocompatibility antigen and isolated HLA-DQ or DP disparities. HLA class I disparity stimulates only CD8+ cells. HLA class II disparity stimulates CD4+ cells, and also stimulates CD8+ cells to a limited degree. We analyzed the donor anti-host fHTLp and HTLp index in 48 patients who engrafted and survived for at least 30 days after transplantation of unmodified marrow from unrelated donors. Methotrexate and cyclosporine were administered for GVHD prophylaxis. For all tests, donor and patient cells cryopreserved before transplantation were used, and assays with HLA-disparate third party responder and stimulator cells were used as controls. GVHD was assessed by an independent observer as grade 0 in 5, grade I in 3, grade II in 17, grade III in 15 and grade IV in 8 patients. A non-parametric trend test yields suggestive evidence that the HTLp index is positively correlated with aGVHD grade (p=O.l), however, the relationship is not strong enough to effectively predict the risk of aGVHD when methotrexate and cyclosporine are given for prophylaxis in patients transplanted with unmodified marrow from unrelated donors.