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Production of the glycosaminoglycan (GAG)-stimulatory factor by cloned in vitro activated human T lymphocytes
112
Annual AACHT Meeting, 198")
STUDIES ON THE GENERATION OF HUMAN ERYTHROID BURST PROMOTING ACTIVITY FROM ALLOREACTIVE T CELLS CLONES: ROLE OF MAJOR HISTOCOMPATIBILITY COMPLEX DETERMINANTS. A. Zeevi, K.F. Mangan, L. Divecchia, and R. D u q u e s n o y ;
Departments of Medicine and Immunopathy, University of Pittsburgh School of Medicine and Centrai Blood Bank of Pittsburgh, Pittsburgh, PA Studies were undertaken to determine the role of major histocompatibility complex (MHC) determinants in the generation o f human erythroid burst promoting activity (BPA) from pure (->99%) populations of alloreactive T cell clones. Specificity of the clones was determined by p r i m e d lymphocyte testing (PLT), cell mediated lympholysis (CML), and monoclonal antibody inhibition of PLT activity. 5 x 104 responder T cell clones ( + 5 0 0 R ) were cocultured in triplicate with 10 ~ irradiated (2000R) stimulator (S) cells, 105 null cells (-<5% T or B cells, ~-2% monocytes), and 2 IU/ml erythropoietin in a methyl-cellulose erythroid culture system. Benzidine positive BFU-E were scored on day 14. Two alloreactive T cell clones, H A 6 (anti-DR6) and HA-18 (anti-DR7), were tested for BPA activity. In the presence of original (S) cells, irradiated or unirradiated HA-6 T cells, but not HA-18 T cells, induced a 10- or 50-fold increase, respectively, in BFU-E compared to unstimulated controls (2 -+ 1 vs. 22 -+ 2 or 99-+ 22, mean + 1 SD, N = 3). Similar results were observed employing supernatants from HA-6 or HA-18 stimulated clones. In other experiments employing an anti-DR3 specific alloreactive T cell clone (DB-3), a twofold increase in BFU-E growth over controls (53-+ 7 vs. 25 + 4) was observed in the presence of original DR3 + (S) cells but not D R 3 - (S) cells. These data suggest that: (1) there are differences in the capacity o f class II specific alloreactive T cell clones to generate soluble erythroid BPA. (2) Release of erythroid BPA from alloreactive T cell clones is M H C restricted. PRODUCTION OF THE GLYCOSAMINOGLYCAN (GAG)-STIMULATORY FACTOR BY CLONED IN VITRO ACTIVATED HUMAN T LYMPHOCYTES. A. Zeevi, M. Tsao, L. DiVecchia, T. Whiteside, and R.J. Duquesnoy; Departments of Immunopathology, University of Pittsburgh and
Central Blood Bank of Pittsburgh, Pittsburgh, PA Supernatants of mitogen-activated mononuclear cells (MNC) contain a factor that stimulates up to 15-fold the synthesis o f glycosaminoglycan (GAG) by cultured normal dermal fibroblasts (DF). We have previously shown that the production o f the GAG-stimulatory factor by MNC, T lymphocytes, and T cell subsets depends on the presence o f monocytes. To demonstrate that the GAG-stimulatory factor is a product o f T lymphocytes, we cloned normal T lymphocytes that were activated in mixed lymphocyte culture. Selected alloreactive T celt clones were activated with irradiated stimulator cells (PBL or B cell line) and the supernatants collected after 72 hr. The G A G activity was determined by adding aliquots o f supernatants to confluent DF for 72 hr. ~H-glucosamine was added for the last 24 hr of culture and the newly synthesized G A G determined. Rep. resentative results are shown below. Clone
Stimulator
CPM GAG ~
HA6 HA6 HAl8 HA18 -Medium MNC+Con
-PBL -PBL PBL (neg. cont.) (pos. cont.)
13 50 21 104 17 24 235
A
~cpm G A G × 10/10 cells.
± -+ _+ +-+ + -+
1 4 l 13 2 2 16
Clone
Stimulator
310 310 -Medium MNC+Con
-BLCL BLCL m e g . cont.) (pos. cont.)
A
CPM GAG" 0 -~ 9 :' 42
:* *: * *: :
t () .2 2 1
Abstracts
113
Only supernatants from alloactivated T cell clones contained GAG-stimulatory factor, whereas supernatants from nonstimulated T clones or irradiated PBL and BLCL had no activity. Our results suggest that activated T cells release factors that modulate synthetic activities of dermal fibroblasts in vitro.
DISTINCT AUTOCYTOTOXIC AND AUTOSUPPRESSOR T CELL LINES GENERATED FROM IN VITRO AUTOLOGOUS MIXED LYPHOCYTE CULTURES. Karen Rosenkrantz, Bo Dupont, Donna Williams, and Neal Flomenberg; Memorial Sloan-Kettering Cancer Center. New York, N Y Previous studies using limiting dilution analysis have demonstrated both the generation and suppression of autocytotoxic cells following in vitro stimulation with autologous peripheral blood mononuclear leukocytes (PBL). At low responder cell number, cytolytic activity can be detected against both autologous phytohemaglutinin (PHA) activated lymphoblasts and autologous Epstein-Barr virus transformed B cells (BLCL). At higher responder cell numbers, the autocytotoxicity disappears. This biphasic response is consistent with the concept that cells capable of autoaggression exist and that these cells are suppressed by an autoregulatory population that is present in the peripheral blood at a lower frequency. Therefore, in order to isolate the autocytotoxic lymphocytes, cell lines were derived from an autologous mixed lymphocyte culture with 5000 responder PBL/well and 50,000 irradiated stimulator autologous PBL/well in autologous serum and an interleukin-2 containing human conditioned medium (HCM) The cultures were expanded and maintained using HCM and autologous irradiated PBL as feeder cells. The lines were screened for cytolytic activity against autologous PHA-activated lymphoblasts, autologous BLCL, and the natural killer target K562. O f 189 cell lines analyzed, 33 demonstrated cytotoxicity against an autologous target cell. One of the lines (N-43) appeared to be directed against self-HLA-DR/DQ molecules by panel studies and by blocking studies with monoclonal antibodies. This autocytotoxic line expressed the Leu 4+,Leu2+,Leu3-,Leu7-,Leullphenotype. Surprisingly, none of the 33 autocytotoxic cell lines appeared capable of lysing autocytotoxic cell lines or noncytotoxic cell lines. In order to study the autosuppressor population, cell lines were also screened for autoregulatory activity. Five cell lines were identified that inhibited autocytotoxicity generated in a limiting dilution assay. Three lines expressed the Leu 4 + ,Leu 5 +, Leu 11-phenotype. Two lines were Leu 4 +,Leu 5 +,Leu 11 +. None of the autoregulatory lines was capable of directly lysing autocytotoxic lines suggesting that they exert their inhibitory effect by a mechanism other than direct lysis of the autocytotoxic effector cell. These findings indicate that through the application of limiting dilution analysis and in vitro cell culture techniques, autocytotoxic and autosuppressor lymphocyte populations can be isolated and characterized in order to understand the cellular interactions involved in maintaining self-tolerance.
CLASS II POSITIVE HUMAN DERMAL FIBROBLASTS FUNCTION AS ANTIGEN PRESENTING CELLSFOR TNP-SPECIFIC CLONED T LYMPHOCYTES. David H. Maurer, Jeffrey H. Hanke, Robert R. Rich, and Marilyn S. Pollack; Baylor College of Medicine, Houston, T X Human dermal fibroblasts anomalously express HLA class II antigens in culture after induction with gamma interferon. We have previously shown that dermal fibroblasts expressing class II antigens will stimulate allospecific T cell clones and lines in the apparent absence of other antigen presenting cells. In order to further examine the antigen presentation capacity of class II positive human dermal
Annual AACHT Meeting, 198")
STUDIES ON THE GENERATION OF HUMAN ERYTHROID BURST PROMOTING ACTIVITY FROM ALLOREACTIVE T CELLS CLONES: ROLE OF MAJOR HISTOCOMPATIBILITY COMPLEX DETERMINANTS. A. Zeevi, K.F. Mangan, L. Divecchia, and R. D u q u e s n o y ;
Departments of Medicine and Immunopathy, University of Pittsburgh School of Medicine and Centrai Blood Bank of Pittsburgh, Pittsburgh, PA Studies were undertaken to determine the role of major histocompatibility complex (MHC) determinants in the generation o f human erythroid burst promoting activity (BPA) from pure (->99%) populations of alloreactive T cell clones. Specificity of the clones was determined by p r i m e d lymphocyte testing (PLT), cell mediated lympholysis (CML), and monoclonal antibody inhibition of PLT activity. 5 x 104 responder T cell clones ( + 5 0 0 R ) were cocultured in triplicate with 10 ~ irradiated (2000R) stimulator (S) cells, 105 null cells (-<5% T or B cells, ~-2% monocytes), and 2 IU/ml erythropoietin in a methyl-cellulose erythroid culture system. Benzidine positive BFU-E were scored on day 14. Two alloreactive T cell clones, H A 6 (anti-DR6) and HA-18 (anti-DR7), were tested for BPA activity. In the presence of original (S) cells, irradiated or unirradiated HA-6 T cells, but not HA-18 T cells, induced a 10- or 50-fold increase, respectively, in BFU-E compared to unstimulated controls (2 -+ 1 vs. 22 -+ 2 or 99-+ 22, mean + 1 SD, N = 3). Similar results were observed employing supernatants from HA-6 or HA-18 stimulated clones. In other experiments employing an anti-DR3 specific alloreactive T cell clone (DB-3), a twofold increase in BFU-E growth over controls (53-+ 7 vs. 25 + 4) was observed in the presence of original DR3 + (S) cells but not D R 3 - (S) cells. These data suggest that: (1) there are differences in the capacity o f class II specific alloreactive T cell clones to generate soluble erythroid BPA. (2) Release of erythroid BPA from alloreactive T cell clones is M H C restricted. PRODUCTION OF THE GLYCOSAMINOGLYCAN (GAG)-STIMULATORY FACTOR BY CLONED IN VITRO ACTIVATED HUMAN T LYMPHOCYTES. A. Zeevi, M. Tsao, L. DiVecchia, T. Whiteside, and R.J. Duquesnoy; Departments of Immunopathology, University of Pittsburgh and
Central Blood Bank of Pittsburgh, Pittsburgh, PA Supernatants of mitogen-activated mononuclear cells (MNC) contain a factor that stimulates up to 15-fold the synthesis o f glycosaminoglycan (GAG) by cultured normal dermal fibroblasts (DF). We have previously shown that the production o f the GAG-stimulatory factor by MNC, T lymphocytes, and T cell subsets depends on the presence o f monocytes. To demonstrate that the GAG-stimulatory factor is a product o f T lymphocytes, we cloned normal T lymphocytes that were activated in mixed lymphocyte culture. Selected alloreactive T celt clones were activated with irradiated stimulator cells (PBL or B cell line) and the supernatants collected after 72 hr. The G A G activity was determined by adding aliquots o f supernatants to confluent DF for 72 hr. ~H-glucosamine was added for the last 24 hr of culture and the newly synthesized G A G determined. Rep. resentative results are shown below. Clone
Stimulator
CPM GAG ~
HA6 HA6 HAl8 HA18 -Medium MNC+Con
-PBL -PBL PBL (neg. cont.) (pos. cont.)
13 50 21 104 17 24 235
A
~cpm G A G × 10/10 cells.
± -+ _+ +-+ + -+
1 4 l 13 2 2 16
Clone
Stimulator
310 310 -Medium MNC+Con
-BLCL BLCL m e g . cont.) (pos. cont.)
A
CPM GAG" 0 -~ 9 :' 42
:* *: * *: :
t () .2 2 1
Abstracts
113
Only supernatants from alloactivated T cell clones contained GAG-stimulatory factor, whereas supernatants from nonstimulated T clones or irradiated PBL and BLCL had no activity. Our results suggest that activated T cells release factors that modulate synthetic activities of dermal fibroblasts in vitro.
DISTINCT AUTOCYTOTOXIC AND AUTOSUPPRESSOR T CELL LINES GENERATED FROM IN VITRO AUTOLOGOUS MIXED LYPHOCYTE CULTURES. Karen Rosenkrantz, Bo Dupont, Donna Williams, and Neal Flomenberg; Memorial Sloan-Kettering Cancer Center. New York, N Y Previous studies using limiting dilution analysis have demonstrated both the generation and suppression of autocytotoxic cells following in vitro stimulation with autologous peripheral blood mononuclear leukocytes (PBL). At low responder cell number, cytolytic activity can be detected against both autologous phytohemaglutinin (PHA) activated lymphoblasts and autologous Epstein-Barr virus transformed B cells (BLCL). At higher responder cell numbers, the autocytotoxicity disappears. This biphasic response is consistent with the concept that cells capable of autoaggression exist and that these cells are suppressed by an autoregulatory population that is present in the peripheral blood at a lower frequency. Therefore, in order to isolate the autocytotoxic lymphocytes, cell lines were derived from an autologous mixed lymphocyte culture with 5000 responder PBL/well and 50,000 irradiated stimulator autologous PBL/well in autologous serum and an interleukin-2 containing human conditioned medium (HCM) The cultures were expanded and maintained using HCM and autologous irradiated PBL as feeder cells. The lines were screened for cytolytic activity against autologous PHA-activated lymphoblasts, autologous BLCL, and the natural killer target K562. O f 189 cell lines analyzed, 33 demonstrated cytotoxicity against an autologous target cell. One of the lines (N-43) appeared to be directed against self-HLA-DR/DQ molecules by panel studies and by blocking studies with monoclonal antibodies. This autocytotoxic line expressed the Leu 4+,Leu2+,Leu3-,Leu7-,Leullphenotype. Surprisingly, none of the 33 autocytotoxic cell lines appeared capable of lysing autocytotoxic cell lines or noncytotoxic cell lines. In order to study the autosuppressor population, cell lines were also screened for autoregulatory activity. Five cell lines were identified that inhibited autocytotoxicity generated in a limiting dilution assay. Three lines expressed the Leu 4 + ,Leu 5 +, Leu 11-phenotype. Two lines were Leu 4 +,Leu 5 +,Leu 11 +. None of the autoregulatory lines was capable of directly lysing autocytotoxic lines suggesting that they exert their inhibitory effect by a mechanism other than direct lysis of the autocytotoxic effector cell. These findings indicate that through the application of limiting dilution analysis and in vitro cell culture techniques, autocytotoxic and autosuppressor lymphocyte populations can be isolated and characterized in order to understand the cellular interactions involved in maintaining self-tolerance.
CLASS II POSITIVE HUMAN DERMAL FIBROBLASTS FUNCTION AS ANTIGEN PRESENTING CELLSFOR TNP-SPECIFIC CLONED T LYMPHOCYTES. David H. Maurer, Jeffrey H. Hanke, Robert R. Rich, and Marilyn S. Pollack; Baylor College of Medicine, Houston, T X Human dermal fibroblasts anomalously express HLA class II antigens in culture after induction with gamma interferon. We have previously shown that dermal fibroblasts expressing class II antigens will stimulate allospecific T cell clones and lines in the apparent absence of other antigen presenting cells. In order to further examine the antigen presentation capacity of class II positive human dermal