The Effects of Tacrolimus on T-Cell Proliferation Are Short-Lived: A Pilot Analysis of Immune Function Testing

Abstract

Background: Optimal immunosuppression after organ transplant should balance the risks of rejection, infection, and malignancy while minimizing barriers to adherence including frequent or time-sensitive dosing. There is currently no reliable immune function assay to directly measure the degree of immunosuppression after transplantation.

Methods: We developed an immune function assay to mea//sure T-cell proliferation after exposure to immunosuppression in vivo. We tested the assay in mice, and then piloted the approach using single time point samples, 11 pediatric kidney transplant recipients prescribed tacrolimus, mycophenolate, and prednisone 6 months to 5 years posttransplant, with no history of rejection, opportunistic infection, or cancer. Twelve healthy adults were controls.

Results: We demonstrated that our assay can quantify suppression of murine T-cell proliferation after tacrolimus treatment in vivo. In humans, we found a mean 25% reduction in CD4 and CD8 T-cell proliferation in pediatric renal transplant recipients on triple immunosuppression compared with adult healthy controls, but the pilot results were not statistically significant nor correlated with serum tacrolimus levels. We observed that cell processing and washing reduced the effects of tacrolimus on T-cell proliferation, as did discontinuation of tacrolimus treatment shortly before sampling.

Conclusions: T-cell proliferation is currently not suitable to measure immunosuppression because sample processing diminishes observable effects. Future immune function testing should focus on fresh samples with minimal washing steps. Our results also emphasize the importance of adherence to immunosuppressive treatment, because T-cell proliferation recovered substantially after even brief discontinuation of tacrolimus.

FIGURE 1

Human PBMC respond differently to stimulation depending on the donor source. A and B, C57BL/6 mice were injected with 0.5 to 10 mg·kg⁻¹·d⁻¹ tacrolimus or DMSO vehicle control intraperitoneally for 4 days. Subsequently, splenocytes were obtained, CFSE labeled, and stimulated with anti-CD3ε mAb (1 μg to 125 ng·mL⁻¹) for 3 days. A, Exemplary (5 mg·kg⁻¹·d⁻¹ tacrolimus, 250 ng·mL⁻¹ CD3ε mAb). B, Cumulative data pooled from 2 independent experiments show markedly reduced CD4⁺ T-cell proliferation (one-way ANOVA). C, Human peripheral blood mononuclear cells (PBMC) were obtained from HDs and CD4⁺ T cells isolated, cryopreserved and thawed, and then CFSE labeled and costimulated with CD3ε/CD28 mAb-coated beads for 4 days. Human CD4⁺ T cells responded similar regardless of HD origin. The orange line indicates 80% proliferation. Data pooled from 15 independent experiments with samples from 12 individual donors. D-G, HD PBMC were frozen and thawed, CFSE-labeled, and stimulated with anti-CD3ε mAb, either coated on beads (D and E) or platebound (F and G). Either method produced different proliferation responses for CD4 (D and F) and CD8 (E and G) T cells depending on the donor source. Data pooled from 5 (D and E) and 6 (F and G) independent experiments. H, CD4⁺ T cells isolated from RTx patients on triple immunosuppression versus HD controls do not show differences in proliferation responses to anti-CD3ε/CD28 mAb-coated beads stimulation. Data representative of 5 independent experiments. RTx, renal transplant; ANOVA, analysis of variance.

FIGURE 2

Comparison of platebound mAb and mAb-coated beads for PBMC stimulation. A and B, HD PBMC were CFSE labeled and stimulated with platebound anti-CD3ε mAb for 4 days, the response to proliferation for CD4⁺ (A) and CD8⁺ (B) is not as constant as for isolated CD4⁺ T cells (Figure 1C). Data pooled from 7 independent experiments from 7 individual donors. C and D, Freshly isolated PBMC from HD were stimulated with anti-CD3ε mAb-coated beads to assess the response in CD4 (C) or CD8 (D) T-cell proliferation. Donor variability was somewhat more than with platebound anti-CD3ε mAb stimulation (A and B), and overstimulation artifacts were seen. Data pooled from 5 independent experiments.

FIGURE 3

In vitro T-cell proliferation is suppressed by low dose tacrolimus. Human HD PBMC were stimulated with anti-CD3ε mAb beads at a 3.5 beads per cell ratio after exposure to variable concentrations of tacrolimus, and proliferation was tracked using CFSE dilution. Even low concentration tacrolimus (3.125 ng·mL⁻¹) strongly reduced T-cell proliferation. Data representative of 2 independent experiments.

FIGURE 4

Serum tacrolimus levels do not correlate with ex vivo T-cell function. RTx recipient and HD PBMC were obtained on the same day as HD control PBMC, and stimulated with platebound anti-CD3ε mAb. Percent dividing T cells indicated at the top of each panel. Immunosuppressed transplant recipients showed reduced CD8⁺ T-cell proliferation with moderate (A) and high (B) tacrolimus serum levels obtained at the same time as the PBMC. C, Transplant recipient PBMC showed a trend to lower CD4⁺ and CD8⁺ T-cell proliferation compared with HD controls, 5 per group, paired Student t test. (D) Serum tacrolimus levels and relative T-cell suppression were not correlated, 5 per group, Pearson correlation. n.s., not significant.

FIGURE 5

The immunosuppressive effect of tacrolimus is lost shortly after drug removal. (A) Human PBMC from a HD were incubated with 200 ng·mL⁻¹ tacrolimus or DMSO vehicle control at 37°C for 1 hour, and then washed with phosphate-buffered saline, before being stimulated with CD3ε mAb-coated beads. T-cell proliferation was strongly suppressed by high dose tacrolimus, but the effects reduced by washing. Data representative of 2 independent experiments. B-D, C57BL/6 mice were randomly assigned to 4 groups for a 9-day course of intraperitoneal (i.p.) injections of tacrolimus (10 mg·kg⁻¹·d⁻¹) or vehicle control at the indicated protocol (B). Injection of tacrolimus just on the last day (C, row 2) was sufficient to suppress CD4+ T-cell proliferation to levels like the group that received the full 10-day course of tacrolimus (C, row 1), while missing just the last 2 doses of tacrolimus (C, row 3) produced no detectable suppression of T-cell proliferation, similar to what was seen in the control mice not receiving any tacrolimus (C, row 4). (C) Exemplary and (D) cumulative data (2-3/group, paired 1-way ANOVA), with *P < 0.05 and **P < 0.01, respectively.