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Modulation of MLR response using donor specific bone marrow cells
112
Abstracts
B-6.8 #211
B-6.8 #212
Ro 31-8220: A NOVEL IMMUNOSUPPRESSIVE AGENT THAT INHIBITS IL-2 PRODUCTION, SYNERGIZES WITH CYCLOSPORINE AND INHIBITS IL-2 DEPENDENT PROLIFERATION. LA Geiselhart and BM Freed, Transplantation Immunology, Albany Medical College, Albany, NY. Ro 31-8220 is a novel immunosuppressive agent that has been shown to inhibit protein kinase C (PKC) in vitro. We have found that Ro 31-8220 inhibits mitogen-induced IL-2 production in both normal and Jurkat T cells without inducing toxicity. Ro 31-8220 (800nM) had no effect on Jurkat T cell viability, growth, DNA or RNA synthesis. Ro 31-8220 (200nM) inhibited IL-2 production in both normal and Jurkat T cells by 75-85'%. The mixed lymphocyte response was also inhibited by Ro 31-8220 (IC.~,=80nM). In addition to inhibiting IL-2 production, Ro 31-8220 (like rapamycin) inhibits IL-2 dependent proliferation. Ro 31-8220 (600nM) inhibited IL-2 dependent proliferation in normal T cells by 80%. We also investigated its synergistic potential with immunosuppressive agents that inhibit the calcium-mediated activation pathway. Doses of Ro 31-8220 (25nM), FK506 (0.2nM) and cyclosporine (8nM) had little or no effect on Jurkat IL-2 production. When administered together, 25nM Ro 31-8220 and 8nM cyclosporine inhibited IL-2 production by 72%. Similarly, 25nM Ro 31-8220 and 0.2riM FK506 inhibited IL-2 production by 66%. In conclusion, Ro 31-8220 is a potent PKC inhibitor with a unique ability to inhibit two distinct events critical to lymphocyte activation, the synthesis of IL-2 and the cellular response to IL-2. Ro 31-8220 also synergizes with CsA and FK506, agents that inhibit calcium-mediated activation events.
M o d u l a t i o n of M L R r e s p o n s e u s i n g d o n o r specific bone m a r r o w cells. SA L a q o o - D e e n a d a y a l a n , J A Lemons, AS Lagoo, SA Shaneyfelt, HM Lorenz, DO McDaniel, A G Diethelm, WH Barber. D e p a r t m e n t s of Surgery and Medicine, U n i v e r s i t y of A l a b a m a at Birmingham, Birmingham, AL. P r e v i o u s studies have shown that p o s t - t r a n s p l a n t a t i o n donors p e c i f i c bone m a r r o w (BM) i n f u s i o n following a d m i n i s t r a t i o n of a nons p e c i f i c p o t e n t i m m u n o s u p p r e s s i v e agent such as ALG, can induce p a r t i a l t o l e r a n c e to allografts. W e have s t u d i e d the in v i t r o m o d u l a t i o n of m i x e d l y m p h o c y t e r e a c t i o n (MLR) by donor specific BM cells. F r e s h l y i s o l a t e d BM cells s i g n i f i c a n t l y d e c r e a s e d the M L R r e s p o n s e to i r r a d i a t e d PBMC s t i m u l a t o r s from the same donor. This s u p p r e s s i v e a c t i v i t y was m a r k e d l y d i m i n i s h e d when f r o z e n / t h a w e d BM cells were used. However, frozen cells c u l t u r e d for 7 days s u p p r e s s e d the M L R s i m i l a r to fresh BM cells. This r e s p o n s e is donor specific, w i t h the t h i r d p a r t y r e s p o n s e r e m a i n i n g normal. S u p e r n a t a n t s from the BM cell c u l t u r e s did not d e m o n s t r a t e any s u p p r e s s i v e effect. Culture of BM cells in the p r e s e n c e of 20% FCS, G M - C S F and acidic FGF not only r e s u l t e d in a h i g h e r y i e l d of v i a b l e BM cells, these cells d i s p l a y e d a s u p p r e s s i v e effect that was equal to or g r e a t e r than the effect m e d i a t e d by BM cells c u l t u r e d in m e d i a alone. Our studies have shown that if the p r o l i f e r a t i v e r e s p o n s e of r e c i p i e n t PBMs (responders) to d o n o r BM cells (stimulators) is less than the r e s p o n s e of recipient PBMs to d o n o r PBMs, then the d o n o r s p e c i f i c BM w o u l d be s u p p r e s s i v e in an M L R of the same d o n o r - r e c i p i e n t pair. D e p l e t i o n of CD3+ BM cells r e s u l t e d in an e v e n g r e a t e r s u p p r e s s i o n of the M L R response. C o m p a r i s o n of p r o l i f e r a t i v e r e s p o n s e s of r e c i p i e n t cells to donor PBMs and donor BM cells m a y be a p a r a m e t e r to g a u g e the e f f e c t i v e n e s s of bone m a r r o w as a b i o l o g i c a l inducer of t o l e r a n c e in a g i v e n donorr e c i p i e n t pair. I s o l a t i o n of cell subsets in BM cells that are c r i t i c a l to the p r o d u c t i o n of in v i t r o s u p p r e s s i o n may help o p t i m i z e the in vivo t r a n s f u s i o n protocol.
Abstracts
B-6.8 #211
B-6.8 #212
Ro 31-8220: A NOVEL IMMUNOSUPPRESSIVE AGENT THAT INHIBITS IL-2 PRODUCTION, SYNERGIZES WITH CYCLOSPORINE AND INHIBITS IL-2 DEPENDENT PROLIFERATION. LA Geiselhart and BM Freed, Transplantation Immunology, Albany Medical College, Albany, NY. Ro 31-8220 is a novel immunosuppressive agent that has been shown to inhibit protein kinase C (PKC) in vitro. We have found that Ro 31-8220 inhibits mitogen-induced IL-2 production in both normal and Jurkat T cells without inducing toxicity. Ro 31-8220 (800nM) had no effect on Jurkat T cell viability, growth, DNA or RNA synthesis. Ro 31-8220 (200nM) inhibited IL-2 production in both normal and Jurkat T cells by 75-85'%. The mixed lymphocyte response was also inhibited by Ro 31-8220 (IC.~,=80nM). In addition to inhibiting IL-2 production, Ro 31-8220 (like rapamycin) inhibits IL-2 dependent proliferation. Ro 31-8220 (600nM) inhibited IL-2 dependent proliferation in normal T cells by 80%. We also investigated its synergistic potential with immunosuppressive agents that inhibit the calcium-mediated activation pathway. Doses of Ro 31-8220 (25nM), FK506 (0.2nM) and cyclosporine (8nM) had little or no effect on Jurkat IL-2 production. When administered together, 25nM Ro 31-8220 and 8nM cyclosporine inhibited IL-2 production by 72%. Similarly, 25nM Ro 31-8220 and 0.2riM FK506 inhibited IL-2 production by 66%. In conclusion, Ro 31-8220 is a potent PKC inhibitor with a unique ability to inhibit two distinct events critical to lymphocyte activation, the synthesis of IL-2 and the cellular response to IL-2. Ro 31-8220 also synergizes with CsA and FK506, agents that inhibit calcium-mediated activation events.
M o d u l a t i o n of M L R r e s p o n s e u s i n g d o n o r specific bone m a r r o w cells. SA L a q o o - D e e n a d a y a l a n , J A Lemons, AS Lagoo, SA Shaneyfelt, HM Lorenz, DO McDaniel, A G Diethelm, WH Barber. D e p a r t m e n t s of Surgery and Medicine, U n i v e r s i t y of A l a b a m a at Birmingham, Birmingham, AL. P r e v i o u s studies have shown that p o s t - t r a n s p l a n t a t i o n donors p e c i f i c bone m a r r o w (BM) i n f u s i o n following a d m i n i s t r a t i o n of a nons p e c i f i c p o t e n t i m m u n o s u p p r e s s i v e agent such as ALG, can induce p a r t i a l t o l e r a n c e to allografts. W e have s t u d i e d the in v i t r o m o d u l a t i o n of m i x e d l y m p h o c y t e r e a c t i o n (MLR) by donor specific BM cells. F r e s h l y i s o l a t e d BM cells s i g n i f i c a n t l y d e c r e a s e d the M L R r e s p o n s e to i r r a d i a t e d PBMC s t i m u l a t o r s from the same donor. This s u p p r e s s i v e a c t i v i t y was m a r k e d l y d i m i n i s h e d when f r o z e n / t h a w e d BM cells were used. However, frozen cells c u l t u r e d for 7 days s u p p r e s s e d the M L R s i m i l a r to fresh BM cells. This r e s p o n s e is donor specific, w i t h the t h i r d p a r t y r e s p o n s e r e m a i n i n g normal. S u p e r n a t a n t s from the BM cell c u l t u r e s did not d e m o n s t r a t e any s u p p r e s s i v e effect. Culture of BM cells in the p r e s e n c e of 20% FCS, G M - C S F and acidic FGF not only r e s u l t e d in a h i g h e r y i e l d of v i a b l e BM cells, these cells d i s p l a y e d a s u p p r e s s i v e effect that was equal to or g r e a t e r than the effect m e d i a t e d by BM cells c u l t u r e d in m e d i a alone. Our studies have shown that if the p r o l i f e r a t i v e r e s p o n s e of r e c i p i e n t PBMs (responders) to d o n o r BM cells (stimulators) is less than the r e s p o n s e of recipient PBMs to d o n o r PBMs, then the d o n o r s p e c i f i c BM w o u l d be s u p p r e s s i v e in an M L R of the same d o n o r - r e c i p i e n t pair. D e p l e t i o n of CD3+ BM cells r e s u l t e d in an e v e n g r e a t e r s u p p r e s s i o n of the M L R response. C o m p a r i s o n of p r o l i f e r a t i v e r e s p o n s e s of r e c i p i e n t cells to donor PBMs and donor BM cells m a y be a p a r a m e t e r to g a u g e the e f f e c t i v e n e s s of bone m a r r o w as a b i o l o g i c a l inducer of t o l e r a n c e in a g i v e n donorr e c i p i e n t pair. I s o l a t i o n of cell subsets in BM cells that are c r i t i c a l to the p r o d u c t i o n of in v i t r o s u p p r e s s i o n may help o p t i m i z e the in vivo t r a n s f u s i o n protocol.