Favorable effects of alemtuzumab on allospecific regulatory T-cell generation

Abstract

We studied the effects of alemtuzumab on T-regulatory cells (Tregs) during alloactivation, first by differences in depletion of various naive versus alloactivated cell subsets in peripheral blood of healthy volunteers, then by adding serial concentrations to human leukocyte antigen (HLA)–DR–matched and –mismatched responding and stimulating cells in mixed lymphocyte reaction (MLR). Lymphoproliferation inhibition and the development of proliferating carboxyfluorescein succinimidyl ester (CFSE)–diluted CD4+CD25highCD127−FOXP3+ (phenotypic) Tregs by flow cytometry were measured. Also, the ability of alemtuzumab-treated versus nontreated MLR generated CD4+CD127− cells to allospecifically inhibit MLRs and recruit additional responding Tregs was tested. We found a more pronounced refractoriness of alloactivated versus naive CD4+CD25high cells to alemtuzumab induced lymphodepletion. Alemtuzumab dose dependently inhibited lymphoproliferation while amplifying percentages of MLR-generated Tregs. This was somewhat augmented by human complement addition. CD127−CD4+ cells immunoselected after 7 days from alemtuzumab-treated MLRs allospecifically inhibited lymphoproliferation and recruited additional Tregs in fresh MLR-responding cells, similar to modulators derived from MLRs without drug addition (media). Addition of tacrolimus and sirolimus to alemtuzumab further inhibited MLR proliferation. However, Treg percentages were markedly higher with sirolimus. These results support the notion that alemtuzumab induces immunoregulation in naïve T cells undergoing alloactivation absent presensitization, especially used in conjunction with maintenance SRL.

Low incidence of acute rejection in hepatitis B virus positive liver transplant recipients and the impact of hepatitis B immunoglobulin

“…4B and C). The percentage of CD127 − CD25 + FOXP3 + total Tregs in the medium controls without HBIG comprised 10–40% of the CD4 + cells in the proliferating fraction depending on individual MLR combinations in each experiment, with the percentage of CD127 − CD25 High FOXP3 + nTregs being slightly lower, also consistent with previous observations [11–13]. There was no significant effect of HBIG in any concentration on the generation of these Tregs in culture over that of media controls, in contrast to other immunoregulatory agents tested previously [12,13].…”

“…4A. As previously reported with other agents [11–13], we tested the influence of HBIG on the development of newly generated Tregs in the proliferating fraction (Fig. 4B and C).…”

“…In this approach, we studied the effects of HBIG on the generation of regulatory T cells (Tregs) using our validated assays (Treg-MLR) with laboratory volunteer peripheral blood mononuclear cells (PBMC) [11–13]. We tested whether HBIG (supplied by Cangene©, Winnipeg, Canada) at indicated concentrations would inhibit MLRs but simultaneously generate CD4 + CD127 − CD25 + FOXP3 + total Tregs and CD4 + CD127 − CD25 High FOXP3 + natural Tregs (nTregs) in contrast to cultures without HBIG.…”

“…They were stimulated with x-irradiated (3,000 RADS) PBMC labeled with PKH26 from 2 HLA-DR matched (Bx) vs. mismatched (Ix) volunteer donors, in 15% normal human AB serum containing complete media. The cultures were incubated at 37°C in a 5% CO 2 humidified atmosphere for 5, 7 ± 9 days before processing [11–13]. …”

Ex Vivo-Expanded Cynomolgus Macaque Regulatory T Cells Are Resistant to Alemtuzumab-Mediated Cytotoxicity

“…The mechanism responsible for the initial increase in Treg is not understood, but de novo generation was suggested and could be explained by the induction of Treg in vivo (22). In vitro studies of Treg proliferation have shown that Treg expand preferentially in allogeneic MLR in the presence of alemtuzumab (23). However, in a similar study, incubation of human PBMC with anti-thymocyte globulin was associated with Treg expansion whereas, by contrast, alemtuzumab depleted CD25 + Treg (17).…”