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Determination of surface immunoglobulin density on frozen-thawed human mononuclear cells analyzed by the fluorescence-activated cell sorter
606
ABSTRACTS,
16th ANNUAL
6”C/min. On the other hand, when cooled at the same ranges of rates, followed by slow warming, maximum survival was obtained at a cooling rate of -l”C/min. These results suggest that rapid warming can “rescue” some cells frozen intracellularly. Using the “twostep” procedure of Farrant et a/., we also found that high survival of precursor B cells could be obtained by cooling and holding the cells at -25°C to equilibrate osmotically before plunging them into liquid nitrogen for storage. Preliminary experiments using spleen cell suspensions from normal C57BL mice suggested that precursors of B and T cells respond differently to the same freezing procedures. However, using fluorescent labeling to identify the cells, we were unable to demonstrate a change in the ratio of B to T cells caused by freeze -thawing. 75.
Serological Lymphocytes Functions.
Identification that Exert
of Subsets Different in
of
TVitro
F. PANDOLFI, D. M. STRONG, AND G. D. BONNARD (Laboratory of Immunodiagnosis, NCI, and CEID, Naval Medical Research Institute, Bethesda, Maryland 20014).
Two different rabbit specific anti-human T-cell sera were studied. Anti-HTY was raised by injecting fetal thymocytes and absorbed (abs) on several B hematopoietic cell lines (HCL). Anti-CTC was obtained against human cultured T-cells (CTC) and abs on the autologous Epstein-Barr virus transformed B-HCL. Both sera reacted in the cytotoxicity test with T-cells in the different tissues studied: 60-80% of normal peripheral blood lymphocytes (PBL), 20-45% of tonsil lymphocytes, and high percentages of thymocytes, CTC, and PHA and Con A blasts. The sera were also positive on T-HCL but nonreactive against normal B-cells, B-HCL, and B or null leukemic blasts. The sera were abs on the cells of a case of chronic T lymphocytic leukemia (T-CLL) with surface markers and functional activities of suppressor cells or on one of four T-HCL (CCRF-CEM, HSB-2, MOLT-4, RPMI-8402). Two of these absorptions provided reagents that could discriminate subsets of T-PBL. (1) The anti-HTY abs with T-CLL cells. This serum reacted with 40-50% of PBL. It was also positive against T-PBL which are not inhibited by theophylline in their ability to rosette with sheep erythrocytes, i.e., cells with helper function. This serum exerted a suppressive activity on lymphoproliferative response of normal PBL to lectins and in MLC, but did not affect the response to tetanus toxoid. (2) Anti-CTC abs with MOLT-4. This serum reacted with about 30% of normal PBL and with the majority of the cells of our suppressor T-CLL. It slightly but consistently enhanced the responses of PBL to lectins, tetanus toxoid, and MLC. The sera could be used as discriminative reagents for the study of patients with immunological disorders (such as patients undergoing allografts), conditions in which an unbalance of T-cell subsets has been postulated.
MEETING
76. Determination of Surface Immunoglobulin Density on Frozen -Thawed Human Mononuclear Cells Analyzed by the Fluorescence-Activated Cell Sorter. R. B. SLEASE, D. M. STRONG, R.
WISTAR, AND I. SCHER (Department of Clinical and Experimental Immunology, Naval Medical Research Institute, Bethesda, Maryland 20014). Recent data have demonstrated that differences in surface immunoglobulin (slg) density on Blymphocytes distinguish functionally distinct subpopulations of these cells. Other reports suggest that cryopreservation may change the frequency of sIgbearing lymphocytes. In order to determine if cryopreservation alters either the frequency of sIg cells or the distribution of sIg density, peripheral blood mononuclear cells (PBM) from normals and patients with chronic lymphocytic leukemia (CLL) were analyzed using the fluorescence-activated cell sorter (FACS). Aliquots of Ficoll-Hypaque-separated PBM were controlled-rate frozen (1”Cimin) in 7.5% Me,SO in RPM1 1640 and thawed in a 37°C water bath on the same day. Fresh and frozen-thawed PBM aliquots were labeled with fluorescein conjugates of F(ab’) fragments of affinity chromatography-purified anti-Fab or class-specific anti-p, anti-i& anti-y, or anti-o. Histograms of relative cell fluorescence, reflecting sIg density, were prepared for each aliquot with the FACS, which utilizes an argon-ion laser to rapidly assess relative cellular fluorescence. The frequency of sIg-bearing PBM labeled with each reagent was not significantly altered by freezing. Likewise, FACS profiles demonstrated that the distribution of sIg density on normal and CLL PBM was unchanged after freezing. However, the fluorescence peak produced by frozen-thawed unlabeled cells was occasionally somewhat broader than that of fresh cells, suggesting slightly increased autofluorescence induced by freezing. These data indicate that frozen cell preparations may be utilized for the study of B-lymphocyte subsets as determined by sIg density. 77. Renal Ailograft Rejection due to HLA-BB Antibody Follo,~~ing a Negative T-Cell, Positive B-Cell Crossmatch. S. METZ, D. STRONG, M.
GOLDMAN, AND J. LIGHT (Walter Reed Army Medical Center, Washington, D.C. 20012, and Naval Medical Research Institute, Bethesda, Maryland 20014). Controversy exists as to whether a positive B-cell crossmatch is in fact due to B-cell antibody and, moreover, whether it is a contraindication to transplantation. An Is-year-old male (Aw24, All; B13, Bw15.1; Cw3, Cw6) received a third cadaver renal allograft (Al, A3: B8, B 13) after his second graft (A2, All; B5, Bw15) chronically rejected over a 5-year period. His first allograft (Al, AlO; Bw35, B8) had functioned for less than 1 year. Prior to the third trans-
ABSTRACTS,
16th ANNUAL
6”C/min. On the other hand, when cooled at the same ranges of rates, followed by slow warming, maximum survival was obtained at a cooling rate of -l”C/min. These results suggest that rapid warming can “rescue” some cells frozen intracellularly. Using the “twostep” procedure of Farrant et a/., we also found that high survival of precursor B cells could be obtained by cooling and holding the cells at -25°C to equilibrate osmotically before plunging them into liquid nitrogen for storage. Preliminary experiments using spleen cell suspensions from normal C57BL mice suggested that precursors of B and T cells respond differently to the same freezing procedures. However, using fluorescent labeling to identify the cells, we were unable to demonstrate a change in the ratio of B to T cells caused by freeze -thawing. 75.
Serological Lymphocytes Functions.
Identification that Exert
of Subsets Different in
of
TVitro
F. PANDOLFI, D. M. STRONG, AND G. D. BONNARD (Laboratory of Immunodiagnosis, NCI, and CEID, Naval Medical Research Institute, Bethesda, Maryland 20014).
Two different rabbit specific anti-human T-cell sera were studied. Anti-HTY was raised by injecting fetal thymocytes and absorbed (abs) on several B hematopoietic cell lines (HCL). Anti-CTC was obtained against human cultured T-cells (CTC) and abs on the autologous Epstein-Barr virus transformed B-HCL. Both sera reacted in the cytotoxicity test with T-cells in the different tissues studied: 60-80% of normal peripheral blood lymphocytes (PBL), 20-45% of tonsil lymphocytes, and high percentages of thymocytes, CTC, and PHA and Con A blasts. The sera were also positive on T-HCL but nonreactive against normal B-cells, B-HCL, and B or null leukemic blasts. The sera were abs on the cells of a case of chronic T lymphocytic leukemia (T-CLL) with surface markers and functional activities of suppressor cells or on one of four T-HCL (CCRF-CEM, HSB-2, MOLT-4, RPMI-8402). Two of these absorptions provided reagents that could discriminate subsets of T-PBL. (1) The anti-HTY abs with T-CLL cells. This serum reacted with 40-50% of PBL. It was also positive against T-PBL which are not inhibited by theophylline in their ability to rosette with sheep erythrocytes, i.e., cells with helper function. This serum exerted a suppressive activity on lymphoproliferative response of normal PBL to lectins and in MLC, but did not affect the response to tetanus toxoid. (2) Anti-CTC abs with MOLT-4. This serum reacted with about 30% of normal PBL and with the majority of the cells of our suppressor T-CLL. It slightly but consistently enhanced the responses of PBL to lectins, tetanus toxoid, and MLC. The sera could be used as discriminative reagents for the study of patients with immunological disorders (such as patients undergoing allografts), conditions in which an unbalance of T-cell subsets has been postulated.
MEETING
76. Determination of Surface Immunoglobulin Density on Frozen -Thawed Human Mononuclear Cells Analyzed by the Fluorescence-Activated Cell Sorter. R. B. SLEASE, D. M. STRONG, R.
WISTAR, AND I. SCHER (Department of Clinical and Experimental Immunology, Naval Medical Research Institute, Bethesda, Maryland 20014). Recent data have demonstrated that differences in surface immunoglobulin (slg) density on Blymphocytes distinguish functionally distinct subpopulations of these cells. Other reports suggest that cryopreservation may change the frequency of sIgbearing lymphocytes. In order to determine if cryopreservation alters either the frequency of sIg cells or the distribution of sIg density, peripheral blood mononuclear cells (PBM) from normals and patients with chronic lymphocytic leukemia (CLL) were analyzed using the fluorescence-activated cell sorter (FACS). Aliquots of Ficoll-Hypaque-separated PBM were controlled-rate frozen (1”Cimin) in 7.5% Me,SO in RPM1 1640 and thawed in a 37°C water bath on the same day. Fresh and frozen-thawed PBM aliquots were labeled with fluorescein conjugates of F(ab’) fragments of affinity chromatography-purified anti-Fab or class-specific anti-p, anti-i& anti-y, or anti-o. Histograms of relative cell fluorescence, reflecting sIg density, were prepared for each aliquot with the FACS, which utilizes an argon-ion laser to rapidly assess relative cellular fluorescence. The frequency of sIg-bearing PBM labeled with each reagent was not significantly altered by freezing. Likewise, FACS profiles demonstrated that the distribution of sIg density on normal and CLL PBM was unchanged after freezing. However, the fluorescence peak produced by frozen-thawed unlabeled cells was occasionally somewhat broader than that of fresh cells, suggesting slightly increased autofluorescence induced by freezing. These data indicate that frozen cell preparations may be utilized for the study of B-lymphocyte subsets as determined by sIg density. 77. Renal Ailograft Rejection due to HLA-BB Antibody Follo,~~ing a Negative T-Cell, Positive B-Cell Crossmatch. S. METZ, D. STRONG, M.
GOLDMAN, AND J. LIGHT (Walter Reed Army Medical Center, Washington, D.C. 20012, and Naval Medical Research Institute, Bethesda, Maryland 20014). Controversy exists as to whether a positive B-cell crossmatch is in fact due to B-cell antibody and, moreover, whether it is a contraindication to transplantation. An Is-year-old male (Aw24, All; B13, Bw15.1; Cw3, Cw6) received a third cadaver renal allograft (Al, A3: B8, B 13) after his second graft (A2, All; B5, Bw15) chronically rejected over a 5-year period. His first allograft (Al, AlO; Bw35, B8) had functioned for less than 1 year. Prior to the third trans-