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Preliminary in vitro evidence for regulatory cells in a miniature swine renal allograft model
ELSEVIER
Preliminary In Vitro Evidence for Regulatory Cells in a Miniature Swine Renal Allograft Model F.L. lerino, K. Yamada,
T. Hatch, and D.H. Sachs
P
REVIOUS studies in miniature swine have demonstrated that long term tolerance can be induced in recipients of Class II matched, class I mismatched renal allografts (SLAgg-+SLAdd) following a short course of Cyclosporine A (CyA).’ The thymus is required for stable tolerance induction, suggesting that central and peripheral mechanisms of tolerance exist (Yamada et al, current issue). In the present study, we analyzed peripheral mechanisms of tolerance using secondary cell-mediated lymphocytoxicity (CML) coculture assays and using peripheral blood lymphocytes (PBL) from thymectomized (thyx) tolerant swine, non-thymectomized (non-thyx) tolerant swine, and naive animals.
MATERIALS
AND
METHODS
Miniature swine (non-thyx and thyx), tissue culture media and PBL isolation techniques are described elsewhere’ (Yamada et al, current issue). Secondary coculture assays were performed in 2 phases. In phase 1, PBL from tolerant animals (4 X lO’/mL) were cultured with 4 X 106/mL of irradiated (25 Gy) stimulator PBL (SLAss or 3rd party PBL) for 6 days at 37°C in 7% CO,. Cultures were then harvested, washed, resuspended at 4 x 10h cells/ml and allowed to rest overnight at 37°C in 7% CO,. The following day (commencement of phase 2), the effecters from phase 1 (tolerant PBL anti-SLAsa or anti-3rd party) were washed before being set up in secondary cultures. Control mixed lymphocyte cultures were prepared containing 2 X 106/mL of naive responder PBL (SLAdd) or effecters from phase one and 4 X lO’/mL of irradiated stimulator PBL (SLApa or 3rd party PBL). Secondary cocultures contained naive responder PBL (SLA’“) (2 X lO?mL), cells from the effector groups from phase 1 (tolerant PBL anti-&A” or anti-3rd party) (2 x lO”/mL) and 4 x 106/mL of irradiated stimulator PBL (SLAa or 3rd party PBL). Cultures were incubated for 6 days at 37°C
in 7% CO, and tested for cytotoxicityas described previously.’
RESULTS
AND
DISCUSSION
Thyx Tolerant Animals
Effector PBL primed with donor antigen in the primary and/or secondary cultures suppressed the generation of CTL by naive responder SLAdd PBL in cocultures. PBL primed with 3rd party antigen in the primary and secondary cultures did not suppress the generation of CTL by naive responder SLA dd PBL to 3rd party antigens in cocultures. 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas,
New York, NY 10010
Non-Thyx Tolerant Animals
PBL from non-thyx tolerant animal stimulated by donormatched PBL in the primary and secondary cultures generated anti-donor CTL reactivity, and these effecters did not suppress the generation of CTL by naive PBL to donor-matched target cells. This pattern differed from thyx animals and may be due to a higher frequency ratio of anti-donor precursor CTL (pCTL) to regulatory cells in the non-thyx animals. Non-thyx animals would be capable of continuously producing anti-donor pCTL within the thymus (in contrast to thyx animals) which could make the inhibitory effect less pronounced in-vitro for non-thyx animals. PBL from non-thyx animals primed with donor-matched PBL once in either the primary or secondary culture inhibited the generation of CTL by naive SLAdd PBL in coculture assays. Effector PBL primed with 3rd party antigen in the primary and secondary cultures did not suppress the generation of CTL by naive responder SLAdd PBL to 3rd party antigens in cocultures.
CONCLUSIONS
The generation of mature CTL by naive SLAdd PBL was inhibited when cocultured with PBL from tolerant animals which were primed with the antigen to which they were tolerant. Regulatory cells can be demonstrated in vitro and may play a role in the maintenance of peripheral tolerance in thyx and non-thyx animals in vivo.
REFERENCE
1. Rosengard BR, Ojikutu CA, Guzzetta PC, et al: Transplantation 54:490, 1992
From the Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA. F.L. lerino is a recipient of a Don and Lorraine Jacquot Travelling Fellowship awarded by The Royal Australasian College of Physicians and The Australian and New Zealand Society of Nephrology. Address reprint requests to D.H. Sachs, MD, Transplantation Biology Research Center, Massachusetts General HospitaVHarvard Medical School, Building 149-9019, Boston, MA 02129.
0041-1345/97/!§17.00 PII SO041 -1345(96)00515-5
1165
Transplantation
Proceedings,
29, 1165 (1997)
Preliminary In Vitro Evidence for Regulatory Cells in a Miniature Swine Renal Allograft Model F.L. lerino, K. Yamada,
T. Hatch, and D.H. Sachs
P
REVIOUS studies in miniature swine have demonstrated that long term tolerance can be induced in recipients of Class II matched, class I mismatched renal allografts (SLAgg-+SLAdd) following a short course of Cyclosporine A (CyA).’ The thymus is required for stable tolerance induction, suggesting that central and peripheral mechanisms of tolerance exist (Yamada et al, current issue). In the present study, we analyzed peripheral mechanisms of tolerance using secondary cell-mediated lymphocytoxicity (CML) coculture assays and using peripheral blood lymphocytes (PBL) from thymectomized (thyx) tolerant swine, non-thymectomized (non-thyx) tolerant swine, and naive animals.
MATERIALS
AND
METHODS
Miniature swine (non-thyx and thyx), tissue culture media and PBL isolation techniques are described elsewhere’ (Yamada et al, current issue). Secondary coculture assays were performed in 2 phases. In phase 1, PBL from tolerant animals (4 X lO’/mL) were cultured with 4 X 106/mL of irradiated (25 Gy) stimulator PBL (SLAss or 3rd party PBL) for 6 days at 37°C in 7% CO,. Cultures were then harvested, washed, resuspended at 4 x 10h cells/ml and allowed to rest overnight at 37°C in 7% CO,. The following day (commencement of phase 2), the effecters from phase 1 (tolerant PBL anti-SLAsa or anti-3rd party) were washed before being set up in secondary cultures. Control mixed lymphocyte cultures were prepared containing 2 X 106/mL of naive responder PBL (SLAdd) or effecters from phase one and 4 X lO’/mL of irradiated stimulator PBL (SLApa or 3rd party PBL). Secondary cocultures contained naive responder PBL (SLA’“) (2 X lO?mL), cells from the effector groups from phase 1 (tolerant PBL anti-&A” or anti-3rd party) (2 x lO”/mL) and 4 x 106/mL of irradiated stimulator PBL (SLAa or 3rd party PBL). Cultures were incubated for 6 days at 37°C
in 7% CO, and tested for cytotoxicityas described previously.’
RESULTS
AND
DISCUSSION
Thyx Tolerant Animals
Effector PBL primed with donor antigen in the primary and/or secondary cultures suppressed the generation of CTL by naive responder SLAdd PBL in cocultures. PBL primed with 3rd party antigen in the primary and secondary cultures did not suppress the generation of CTL by naive responder SLA dd PBL to 3rd party antigens in cocultures. 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas,
New York, NY 10010
Non-Thyx Tolerant Animals
PBL from non-thyx tolerant animal stimulated by donormatched PBL in the primary and secondary cultures generated anti-donor CTL reactivity, and these effecters did not suppress the generation of CTL by naive PBL to donor-matched target cells. This pattern differed from thyx animals and may be due to a higher frequency ratio of anti-donor precursor CTL (pCTL) to regulatory cells in the non-thyx animals. Non-thyx animals would be capable of continuously producing anti-donor pCTL within the thymus (in contrast to thyx animals) which could make the inhibitory effect less pronounced in-vitro for non-thyx animals. PBL from non-thyx animals primed with donor-matched PBL once in either the primary or secondary culture inhibited the generation of CTL by naive SLAdd PBL in coculture assays. Effector PBL primed with 3rd party antigen in the primary and secondary cultures did not suppress the generation of CTL by naive responder SLAdd PBL to 3rd party antigens in cocultures.
CONCLUSIONS
The generation of mature CTL by naive SLAdd PBL was inhibited when cocultured with PBL from tolerant animals which were primed with the antigen to which they were tolerant. Regulatory cells can be demonstrated in vitro and may play a role in the maintenance of peripheral tolerance in thyx and non-thyx animals in vivo.
REFERENCE
1. Rosengard BR, Ojikutu CA, Guzzetta PC, et al: Transplantation 54:490, 1992
From the Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA. F.L. lerino is a recipient of a Don and Lorraine Jacquot Travelling Fellowship awarded by The Royal Australasian College of Physicians and The Australian and New Zealand Society of Nephrology. Address reprint requests to D.H. Sachs, MD, Transplantation Biology Research Center, Massachusetts General HospitaVHarvard Medical School, Building 149-9019, Boston, MA 02129.
0041-1345/97/!§17.00 PII SO041 -1345(96)00515-5
1165
Transplantation
Proceedings,
29, 1165 (1997)