Xuemei Huang

The human "Treg MLR": immune monitoring for FOXP3+ T regulatory cell generation.

Background. Controversy exists about the conditions effecting the development of forkhead/winghead helix transcription factor P3 (FOXP3) expressing T cells and their relevance in transplant recipients. Methods. We generated carboxy-fluorescein diacetate succinimidyl ester-labeled CD4+CD25high FOXP3+ cells in mixed lymphocyte reactions (MLRs) (“the Treg MLR”), with varying human leukocyte antigen (HLA) disparities and cell components. Five color flow cytometry and 3H-thymidine uptakes were the readouts. Results. (1) Despite lower stimulation indices (SIs) than two DR-mismatched MLRs, 2 DR-matched MLRs generated more than twofold higher percentages when gating on proliferating CD4+CD25high FOXP3+ cells; (2) Even with low numbers of proliferating cells, autologous and HLA identical MLRs generated the highest FOXP3+:FOXP3− cell ratios; (3) Elimination of either non-CD3+ responding cells (resulting in “direct presentation” only) or responding CD25+ (Treg generating) cells increased the SI but inhibited proliferating CD4+CD25high FOXP3+ cell development; (4) MLR-generated CD4+CD25high FOXP3+ cells added as third components specifically inhibited the same freshly set MLR SI and caused recruitment of new CD4+CD25high FOXP3+ cells. As an example of the “Treg MLR” immune monitoring potential, addition of third component peripheral blood mononuclear cell containing high percentages of CD4+CD25high FOXP3+ cells from an HLA identical kidney transplant recipient (in a tolerance protocol) caused donor-specific Treg MLR inhibition or recruitment. This was similar to the third component MLR Tregs generated entirely in vitro. Conclusion. In the Treg MLR, the generation of CD4+CD25High FOXP3+ cells is more pronounced in the context of self-recognition (HLA matching, indirect presentation). These cells can be assayed for MLR inhibitory and Treg recruitment functions, so as to immunologically monitor the allospecific regulation after transplantation.

Allospecific regulatory effects of sirolimus and tacrolimus in the human mixed lymphocyte reaction.

Background. Tacrolimus (TAC) and sirolimus (SRL), two commonly used immunosuppressive agents, have demonstrated contrasting immunoregulatory effects. We recently described factors affecting the generation of allospecific CD4+CD25High forkhead/winged helix transcription factor P3 (FOXP3+) T-regulatory (Treg) cells in mixed lymphocyte reaction (Treg MLR) and now report additional findings on the effects of TAC and SRL. Methods. TAC, SRL, or media without agents were added separately to MLRs using human leukocyte antigen two DR-matched and -mismatched healthy volunteers and prekidney transplant donor/recipient pairs. Concentrations correlated with subtherapeutic and therapeutic blood levels. Stimulation indices of 3H-TDR uptake, cell proliferation, and the generation of carboxy-fluorescein diacetate succinimidyl ester (CFSE) labeled CD4+CD25HighFOXP3+ cells by flow cytometry were initially compared. Each group of (non-CFSE labeled) MLR-generated cells were then added as third components to CFSE-labeled responding cells in freshly prepared primary MLRs, to determine allospecific and nonspecific inhibitory and Treg recruitment effects. Results. TAC inhibited stimulation indices and CD4+CD25High FOXP3+ cell generation in both human leukocyte antigen DR-matched and -mismatched pairs, particularly at therapeutic levels (≥5 ng/mL). SRL had an equivalent effect in matched pairs but was associated with a significantly higher %generation of CD4+CD25HighFOXP3+ cells than TAC. SRL-MLR-generated Tregs added as third components allospecifically inhibited MLR proliferation and recruited additional CFSE-labeled autologous Tregs compared with addition of TAC- or media-MLR-generated Tregs. Conclusions. Calcineurin and mammalian target of rapamycin inhibitors have disparate effects on allospecific Treg generation using the Treg MLR. This assay can thereby be helpful in assessing allospecific regulatory effects of diverse immunosuppressive agents.

Inhibitory effects of belatacept on allospecific regulatory T-cell generation in humans.

Background It is unclear if new costimulatory blockade agents, such as the cytotoxic T lymphocyte–associated antigen 4-Ig molecule belatacept (BEL), promote or inhibit the potential for immunologic tolerance in transplantation. We therefore tested the in vitro effects of BEL on human regulatory T cells (Tregs) in mixed lymphocyte reactions (MLR) alone and in combination with maintenance agents used in transplant recipients. Methods BEL, mycophenolic acid (MPA), and sirolimus, either alone or in combination, were added to healthy volunteer Treg-MLR, testing (a) 3H-TdR incorporation for inhibition of lymphoproliferation and (b) flow cytometry to analyze for newly generated CD4+CD25highFOXP3+ Tregs in carboxyfluorescein succinimidyl ester–labeled MLR responders. In addition, the modulatory effects of putative Tregs generated in the presence of these drugs were also tested using the lymphoproliferation and flow cytometric assays. Results In comparison with medium controls, BEL dose-dependently inhibited both lymphoproliferation and Treg generation in human leukocyte antigen DR matched and mismatched MLRs either alone or in combination with MPA or sirolimus. However, MPA alone inhibited lymphoproliferation but significantly enhanced Treg generation at subtherapeutic concentrations (P<0.01). In addition, purified CD4+CD127- cells generated in MLR in the presence of MPA and added as third component modulators in fresh MLRs significantly enhanced newly developed Tregs in the proliferating responder cells compared with those generated with BEL or medium controls. Conclusions BEL alone and in combination with agents used in transplant recipients inhibits the in vitro generation of human Tregs. BEL might therefore be a less optimal agent for tolerance induction in human organ transplantation.

Favorable effects of alemtuzumab on allospecific regulatory T-cell generation

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(+)CD25(high)CD127(-)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(+)CD25(high) 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.

Generation and Characterization of Alloantigen-Specific Regulatory T Cells For Clinical Transplant Tolerance

Donor-specific CD4+CD127−CD25+FOXP3+ regulatory T cells (AgTregs) have the potential to induce clinical transplant tolerance; however, their expansion ex vivo remains challenging. We optimized a novel expansion protocol to stimulate donor-specific Tregs using soluble 4-trimer CD40 ligand (CD40L)-activated donor B cells that expressed mature antigen-presenting cell markers. This avoided the use of CD40L-expressing stimulator cells that might otherwise result in potential cellular contamination. Purified allogeneic “recipient” CD4+CD25+ Tregs were stimulated on days 0 and 7 with expanded “donor” B cells in the presence of IL-2, TGFβ and sirolimus (SRL). Tregs were further amplified by polyclonal stimulation with anti-CD3/CD28 beads on day 14 without SRL, and harvested on day 21, with extrapolated fold expansion into the thousands. The expanded AgTregs maintained expression of classical Treg markers including demethylation of the Treg-specific demethylated region (CNS2) and also displayed constricted TcR repertoire. We observed AgTregs more potently inhibited MLR than polyclonally expanded Tregs and generated new Tregs in autologous responder cells (a measure of infectious tolerance). Thus, an optimized and more clinically applicable protocol for the expansion of donor-specific Tregs has been developed.

Immunoregulatory Effects of Everolimus on In Vitro Alloimmune Responses

Everolimus (EVL) is a novel mTOR-inhibitor similar to sirolimus (SRL) that is used in organ transplant recipients, often in combination with tacrolimus (TAC) or mycophenolate (MPA). The current study aims to determine its effects on regulatory T cells. Increasing concentrations of EVL, MPA and TAC alone or in combination were added to MLRs of healthy volunteers. Lymphoproliferation by 3H-TdR incorporation and the percentage of newly generated CD4+CD127-CD25+FOXP3+ (total Treg) and CD4+CD127-CD25HighFOXP3+ (natural Treg) in CFSE labeled responder cells were assessed by flow cytometry. In comparison to medium controls, EVL and other agents dose-dependently inhibited 3H-TdR incorporation in HLA-2DR-matched and HLA-mismatched MLRs (n = 3–10). However, EVL significantly amplified newly generated total and natural Tregs in CFSE labeled responder cells (p<0.05) at all concentrations, while MPA and SRL did this only at sub-therapeutic concentrations and inhibited at therapeutic levels. In contrast, TAC inhibited newly generated Tregs at all concentrations. When tested in combination with TAC, EVL failed to reverse TAC inhibition of Treg generation. Combinations of EVL and low concentrations of MPA inhibited proliferation and amplified Treg generation in an additive manner when compared to medium controls or each drug tested alone (p<0.05). The relative tolerogenic effect from high to low was EVL > SRL> MPA > TAC. If the results from these in vitro studies are extrapolated to clinical transplantation, it would suggest EVL plus low concentrations of MPA may be the most tolerogenic combination.

DONOR SPECIFIC IMMUNOREGULATION IN HLA-IDENTICAL RENAL TRANSPLANT RECIPIENTS GIVEN ALEMTUZIMAB AND DONOR STEM CELLS: 1416

J. Miller1, J. Leventhal2, L. Gallon2, J. Friedewald3, J. Levitsky2, A. Tambur3, X. Huang1, J.M. Mathew1 1Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago/UNITED STATES OF AMERICA, 2Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago/IL/UNITED STATES OF AMERICA, 3Organ Transplantation, Northwetsern University, Chicago/UNITED STATES OF AMERICA

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

Historically, hepatitis B virus (HBV) liver transplantation (LT) recipients have less acute cellular rejection (ACR) than those without HBV. We questioned whether this has persisted in an era of decreased Hepatitis B immunoglobulin use (HBIG) given its in vitro immunoregulatory effects. We compared the incidence, risk factors and outcomes of ACR among 40,593 primary LT recipients with HBV, hepatitis C, steatohepatitis, and immune liver disease (OPTN 2000-2011). We also assessed the in vitro effect of HBIG on alloimmune lymphoproliferation and regulatory T cell generation using mixed lymphocyte reactions. In multivariate analysis, HBV status remained a strong independent predictor of freedom from ACR (OR 0.58, 95% CI: 1.5-2.1). Patient (67.7% vs 72.3%) and graft (60.8% vs 69.1%) survival were significantly lower in patients with ACR versus no ACR for all causes except HBV. HBIG use had no statistical association with ACR. In vitro, HBIG at concentrations equivalent to clinical dosing did not inhibit lymphoproliferation or promote regulatory T cell development. In summary, the incidence and impact of ACR is lower now for HBV LT and does not appear to be secondary to HBIG by our in vitro and in vivo analyses. Rather, it may be due to the innate immunosuppressive properties of chronic HBV infection.

A Phase I Clinical Trial with Ex Vivo Expanded Recipient Regulatory T cells in Living Donor Kidney Transplants

There is considerable interest in therapeutic transfer of regulatory T cells (Tregs) for controlling aberrant immune responses. Initial clinical trials have shown the safety of Tregs in hematopoietic stem cell transplant recipients and subjects with juvenile diabetes. Our hypothesis is that infusion(s) of Tregs may induce transplant tolerance thus avoiding long-term use of toxic immunosuppressive agents that cause increased morbidity/mortality. Towards testing our hypothesis, we conducted a phase I dose escalation safety trial infusing billions of ex vivo expanded recipient polyclonal Tregs into living donor kidney transplant recipients. Despite variability in recipient’s renal disease, our expansion protocol produced Tregs which met all release criteria, expressing >98% CD4+CD25+ with <1% CD8+ and CD19+ contamination. Our product displayed >80% FOXP3 expression with stable demethylation in the FOXP3 promoter. Functionally, expanded Tregs potently suppressed allogeneic responses and induced the generation of new Tregs in the recipient’s allo-responders in vitro. Within recipients, expanded Tregs amplified circulating Treg levels in a sustained manner. Clinically, all doses of Treg therapy tested were safe with no adverse infusion related side effects, infections or rejection events up to two years post-transplant. This study provides the necessary safety data to advance Treg cell therapy to phase II efficacy trials.

THE DIFFERENTIAL EFFECTS OF TACROLIMUS AND SIROLIMUS IN THE HUMAN “TREG-MLR”: 1023: IMMUNOLOGICAL MONITORING

J. Levitsky1, L. Gallon1, J. Miller2, J. Leventhal1, A. Tambur3, X. Huang2, C. Flaa2, E. Wang4, J.M. Mathew2 1Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago/IL/UNITED STATES OF AMERICA, 2Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago/UNITED STATES OF AMERICA, 3Organ Transplantation, Northwetsern University, Chicago/UNITED STATES OF AMERICA, 4Surgery, Northwestern University Feinberg School of Medicine, Chicago/UNITED STATES OF AMERICA