Tolerance and withdrawal of immunosuppressive drugs in patients given kidney and hematopoietic cell transplants

Abstract

Sixteen patients conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA-matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T-cell subsets and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft-versus-host disease (GVHD), and eight with chimerism for at least 6 months were withdrawn from antirejection medications for 1-3 years (mean, 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and four patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naive CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, TLI and ATG promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.

Conflict of interest statement

Disclosure: The authors have no associations with commercial organizations to disclose. The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

Figure 1

Serum creatinine concentrations, immunosuppressive drug withdrawal, and chimerism among granulocytes in the blood of 12 patients followed for at least 18 months. A, shows serum creatinine concentrations, and percentage of donor type cells measured by STR analysis at serial time points in patients #1,7,8, and 12 with persistent chimerism throughout the observation period who were withdrawn from immunosuppressive drugs. Arrows show time points at which immunosuppressive drugs were discontinued (ISD). B, shows the data for patients #4, 5, 10, and 11 with loss of chimerism (<1% donor type cells) during the second year posttransplant who were withdrawn from immunosuppressive drugs before or after the loss of chimerism. C, shows the data for patient #2 with FSGS disease recurrence (DR) who never developed chimerism, and for patients #3, 6, and 9 who lost chimerism during the first or second year, and had an associated rejection episode (RE). Maintenance therapy for patient #3 is cyclosporine alone, for #9 is tacrolimus alone, for patient #2 is mycophenolate mofetil and cyclosporine, and for patient #6 is tacrolimus and mycophenolate mofetil. Testing for chimerism was stopped in B and C after failure to detect chimerism in 2 consecutive blood samples.

Figure 2

Chimerism among lymphocytes in the blood of 12 patients followed for 18 months. The percentage of donor type cells among purified T cells, B cells, and NK cells is shown before and at serial time points after kidney transplantation.

Figure 3

Serum creatinine concentrations, immunosuppressive drug withdrawal and chimerism among lymphocytes and granulocytes (G) in 4 patients followed for less than 18 months.

Figure 4

Early changes in T cell subsets in the blood of 12 transplant patients for at least 18 months. The posttransplant conditioning from days 1 through 10 induced severe lymphopenia, and there were insufficient cells to perform subset monitoring in all patients until day 28. A, shows the changes in mean absolute numbers of lymphocytes, mean percentages of CD3+, CD4+ and CD8+ T cells among lymphocytes, mean percentages of naive (CD62L+CD45RA+) cells among CD4+ and CD8+ T cells, and absolute numbers of naive CD4+ and CD8+ T cells at serial time points for patients before and at 28, 56, 90, 130, and 190 days after transplantation. Brackets show 90% confidence limits. B, shows the mean percentages of CD4+CD25+ T regs among CD4+ T cells, the mean ratios of Treg to naive CD4+ T cells, mean percentages of NKT cells among CD3+ T cells, and mean ratios of NKT cells to naive CD3+ T cells. All reductions in the day 0 versus day 28 and 190 means were significant for ALC (p=0.0004 and 0.006), percent T cells (p=0.003 and 0.012), percent naïve among CD4+ cells (p=0.004 and 0.002), percent naïve cells among CD8+ (p=0.001 and 0.02), absolute number of naïve CD4+ T cells (p=0.0004 and 0.0004), and absolute numbers of naïve CD8+ T cells (p=0.0004) with the exception of the latter at day 190 (p=0.12). All increases in the day 0 versus day 28 and 190 means were significant for percent Tregs (p=0.002 and 0.03), ratio of Tregs:naïve (p=0.0004 and 0.0004), ratio of NKT:naïve (p=0.0009 and 0.01), and percent NKT cells (p=0.01) with the exception of the latter at day 190 (p=0.72). C, shows representative examples of 2 color flow cytometry analyses at days 0 and 28 for naïve (CD62L versus CD45RA) T cells among gated CD4+ T cells, Treg (CD25 versus CD4) cells among gated CD4+ T cells, and NKT cells (Vβ11 versus Vα24) among gated CD3+ T cells. Boxes or ellipses enclose naïve, Treg, and NK T cells, and percentages of enclosed cells are shown.

Figure 5

Changes in the absolute numbers of T cell subsets and B cells for 1 to 4 years, and representative example of CD127 and intracellular FoxP3 staining of CD4+CD25+ Treg cells. A, The absolute numbers of total CD4+, CD8+, and CD3+ T cells and CD19+ B cells in the blood before and at serial time points after transplantation with follow-up from 400 to 1,400 days in the first 10 patients are shown. Y axis labels are variable to reflect variability in starting absolute counts. B, shows representative staining of Treg cells. CD4 versus CD3 among gated CD3+ T cells in a posttransplant blood sample with a high Treg/CD4 naive ratio is shown on the left panel, for CD4 versus CD25 among gated CD4+ cells in the adjacent panel, for CD25 versus FoxP3 among gated CD4+CD25+ cells in the adjacent panel, and for CD127 versus CD25 on gated CD4+ cells in the far right panel. Percentage of cells enclosed in each box or ellipse is shown.

Figure 6

In vitro immune responses of patients to donor alloantigens, third party alloantigens, and to microbial recall antigens. The Panels in A show representative responses from 2 (#4 and 8) of 4 patients who were withdrawn from immunosuppressive drugs, and had in vitro assays performed during the second or third year posttransplant. Assays in patients #4 and 8 were performed 17 and 13 months respectively posttransplant. The patient responses to HLA unmatched third party stimulator cells from normal individuals (allo#1 - allo#3) had recovered such that there were no statistically significant decreases (p>0.05) in the 3H-thymidine incorporation (mean cpm+/- SE) from 3 to 6 wells when the posttransplant samples were compared to the pretransplant samples as judged by the paired student t test. In contrast, the posttransplant responses to irradiated HLA-matched donor dendritic cells were significantly decreased (p=0.0007-0.01) as compared to pretransplant responses (right upper panels, asterisks show significant differences). The left and right lower panels show the mean cpm for responses of patient mononuclear cells obtained pre or posttransplant respectively to recall antigens including tetanus toxoid, influenza (Flu), and/or cytomegalovirus (CMV). There were no significant decreases (p>0.3) in posttransplant as compared to pretransplant recall responses. Control cultures with responder cells without stimulator cells or recall antigens (Medium) are shown for each assay. Note that the range of cpm values for stimulation with HLA matched dendritic cells is lower than that for stimulation with HLA unmatched 3rd party mononuclear cells. The pattern of responses from patients #4 and 8 were similar to those from patients #1 and 5 (not shown). Panels in B show responses from 2 (#2 and 3) patients who were not withdrawn from immunosuppressive drugs. The left upper panels for patient #2 show that during month 17 posttransplant, the patient responses to 3rd party stimulator cells from normal individuals (allo#1, - allo#2) were not statistically significantly decreased (p=0.5-0.9), but the response to allo#3 was significantly decreased (p=0.02) as compared to the pretransplant responses. The posttransplant response to irradiated donor dendritic cells was not significantly decreased (p=0.5) as compared to the pretransplant response (right upper panels). The left and right lower panels show the mean cpm for responses of patients’ mononuclear cells obtained pre or posttransplant respectively to recall antigens. There were no significant decreases in posttransplant as compared to pretransplant recall responses (p>0.3). The left upper panels for patient #3 show that during month 20 posttransplant, the response to third party allo#1 stimulator cells was significantly decreased (p=0.01), but the response to allo#2 was not (p=0.08). The posttransplant response to donor dendritic cells was not significantly decreased (p=0.8) nor were responses to recall antigens (p>0.3).