Faculty Bibliography

The current kidney allocation system (KAS) preferentially allocates kidneys from blood type A2 or A2B (A/A2B) donors to blood type B candidates. We used national data to evaluate center-level performance of A2/A2B to B transplants, and organ procurement organization (OPO) reporting of type A or AB donor subtyping, in 5-year time periods prior to (2009-2014) and following (2015-2019) KAS implementation. The number of centers performing A2/A2B to B transplants increased from 17 pre-KAS to 76 post-KAS, though this still represents only a minority of centers (7.3% pre-KAS and 32.6% post-KAS). For high-performing centers, the median net increase in A2/A2B to B transplants was 19 cases (range -2-72) per center in the 5 years post-KAS. The median net increase in total B recipient transplants was 21 cases (range -17-119) per center. Despite requirements for performance of subtyping, in 2019 subtyping was reported on only 56.4% of A/AB donors. This translates into potential missed opportunities for B recipients, and even post-KAS up to 2322 A2/A2B donor kidneys may have been allocated for transplantation as A/AB. Further progress must be made both at center and OPO levels to broaden implementation of A2/A2B to B transplants for the benefit of underserved recipients.

BACKGROUND:Cytomegalovirus (CMV) infection is a serious complication in immunosuppressed patients, specifically transplant recipients. Here, we describe the development and use of an assay to monitor the incidence and treatment of CMV viremia in a Cynomolgus macaque model of bone marrow transplantation (BMT) for tolerance induction. We address the correlation between the course of viremia and immune reconstitution. METHODS:Twenty-one animals received a nonmyeloablative conditioning regimen. Seven received cyclosporine A for 28 days and 14 received rapamycin. A CMV polymerase chain reaction assay was developed and run twice per week to monitor viremia. Nineteen recipients were CMV seropositive before BMT. Immune reconstitution was monitored through flow cytometry and CMV viremia was tracked via quantitative polymerase chain reaction. RESULTS:Recipients developed CMV viremia during the first month post-BMT. Two animals developed uncontrollable CMV disease. CMV reactivation occurred earlier in cyclosporine A-treated animals compared with those receiving rapamycin. Post-BMT, T-cell counts remained significantly lower compared with pretransplant levels until CMV reactivation, at which point they increased during the viremic phase and approached pretransplant levels 3 months post-BMT. Management of CMV required treatment before viremia reached 10 000 copies/mL; otherwise clinical symptoms were observed. High doses of ganciclovir resolved the viremia, which could subsequently be controlled with valganciclovir. CONCLUSIONS:We developed an assay to monitor CMV in Cynomolgus macaques. CMV reactivation occurred in 100% of seropositive animals in this model. Rapamycin delayed CMV reactivation and ganciclovir treatment was effective at high doses. As in humans, CD8 T cells proliferated during CMV viremia.

Reliable in vitro expansion protocols of regulatory T cells (Tregs) are needed for clinical use. We studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed four in vitro Treg expansion protocols for translational studies. Tregs expanded 3000-fold when artificial antigen presenting cells (aAPCs) expressing human CD80, CD58 and CD32 were used throughout the culture. When donor peripheral blood mononuclear cells (PBMCs) were used as the single source of APCs followed by aAPCs, Tregs expanded 2000-fold. 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. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed greater suppression of stimulation by anti-CD2CD3CD28 beads of PBMCs from the same donor used for stimulation during the Treg cultures and of autologous cells than of third-party PBMC responders. 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. In summary, we have characterized MCM Tregs and developed four Treg expansion protocols that can be used for preclinical applications.