Alonso Guallart, Paula
BOLEA BAILO, ROSA (dir.) ; DURAN-STRUUCK, RAIMON (dir.)
Universidad de Zaragoza,
2020
Resumen: Induction of transplantation tolerance to kidney allografts has been achieved through transient mixed hematopoietic chimerism in a non-myeloablative approach in both, non-human primates and humans. In order to make this approach applicable to other organs less tolerogenic such as lung or heart, we studied an approach to induce long-term mixed chimerism (instead of transient) through bone marrow transplantation (BMT) and infusion of recipient in vitro-expanded regulatory T cells (Tregs) in a non-human primate model (Cynomolgus macaque). Immunosuppression monotherapy was discontinued shortly after BMT. Donor-recipient pairs were major-histocompatibility-complex (MHC) mismatched in order to increase the applicability of this approach.
First, we studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed five in vitro Treg expansion protocols for translational studies that included the use of artificial antigen presenting cells (aAPCs), donor peripheral blood mononuclear cells (PBMCs) or a pool from different donors of CD40L-stimulated B cells (CD40L-sBc). Tregs from all protocols suppressed the proliferation of anti-CD2CD3CD28 bead-stimulated autologous PBMCs albeit with different potencies, varying from 1:2-1:4 Treg:PBMC ratios, up to >1:32. Treg expansion varied between protocols but at least 1,000 fold expansion was achieved with all of the them, up to >7,000 folds. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed suppression of PBMCs from autologous cells, cells from the same donor used for stimulation during the Treg cultures and from a third‐party PBMC responders, suggestive of the polyclonallity of these Tregs. Similar to humans, the Treg–specific demethylated region (TSDR) within the FoxP3 locus correlated with suppressive activity and expression of FoxP3. Contrary to humans, FoxP3 expression did not correlate with CD45RA or CD127 expression.
We then investigated the efficacy of ex vivo expanded Tregs to promote the induction of durable mixed chimerism along with BMT. A total of ten recipients received Tregs with different doses of bone marrow (BM) and outcomes were compared to five controls that did not receive Tregs. Prolonged chimerism was observed in Treg-treated recipients that received a high BM dose with infusion of Tregs compared to those that received low-dose BMT or did not received Tregs. Graft-versus-host disease (GVHD) was observed in four recipients, two controls and two animals that received Tregs expanded with CD40L-sBc. In those animals in which prolonged chimerism was observed, a higher number of peripheral Tregs was detected in blood compared to baseline levels and in vitro, the anti-donor response was decreased, suggestive of donor tolerance. BM rejection and chimerism loss was associated with an inversion of the CD4 and CD8 ratios and an increase in the CD8 absolute counts. Cytomegalovirus (CMV) was detected in all recipients post-BMT independently of the administration of Tregs. CMV reactivation was associated with an increase in the CD8 counts and with the loss of the BM graft. Therefore, promptly antiviral treatment was stablished for an early CMV control.
In conclusion, Tregs were able to expand the duration of chimerism (albeit transient) when administered with high-dose BMT across MHC barriers without immunosuppression.
Resumen (otro idioma):
First, we studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed five in vitro Treg expansion protocols for translational studies that included the use of artificial antigen presenting cells (aAPCs), donor peripheral blood mononuclear cells (PBMCs) or a pool from different donors of CD40L-stimulated B cells (CD40L-sBc). Tregs from all protocols suppressed the proliferation of anti-CD2CD3CD28 bead-stimulated autologous PBMCs albeit with different potencies, varying from 1:2-1:4 Treg:PBMC ratios, up to >1:32. Treg expansion varied between protocols but at least 1,000 fold expansion was achieved with all of the them, up to >7,000 folds. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed suppression of PBMCs from autologous cells, cells from the same donor used for stimulation during the Treg cultures and from a third‐party PBMC responders, suggestive of the polyclonallity of these Tregs. Similar to humans, the Treg–specific demethylated region (TSDR) within the FoxP3 locus correlated with suppressive activity and expression of FoxP3. Contrary to humans, FoxP3 expression did not correlate with CD45RA or CD127 expression.
We then investigated the efficacy of ex vivo expanded Tregs to promote the induction of durable mixed chimerism along with BMT. A total of ten recipients received Tregs with different doses of bone marrow (BM) and outcomes were compared to five controls that did not receive Tregs. Prolonged chimerism was observed in Treg-treated recipients that received a high BM dose with infusion of Tregs compared to those that received low-dose BMT or did not received Tregs. Graft-versus-host disease (GVHD) was observed in four recipients, two controls and two animals that received Tregs expanded with CD40L-sBc. In those animals in which prolonged chimerism was observed, a higher number of peripheral Tregs was detected in blood compared to baseline levels and in vitro, the anti-donor response was decreased, suggestive of donor tolerance. BM rejection and chimerism loss was associated with an inversion of the CD4 and CD8 ratios and an increase in the CD8 absolute counts. Cytomegalovirus (CMV) was detected in all recipients post-BMT independently of the administration of Tregs. CMV reactivation was associated with an increase in the CD8 counts and with the loss of the BM graft. Therefore, promptly antiviral treatment was stablished for an early CMV control.
In conclusion, Tregs were able to expand the duration of chimerism (albeit transient) when administered with high-dose BMT across MHC barriers without immunosuppression.
Resumen (otro idioma):
Pal. clave: inmunologia ; trasplante de organos ; cultivo celular
Titulación: Programa de Doctorado en Medicina y Sanidad Animal
Plan(es): Plan 498
Nota: Presentado: 07 01 2020
Nota: Tesis-Univ. Zaragoza, , 2020
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