Nonchimeric HLA-Identical Renal Transplant Tolerance: Regulatory Immunophenotypic/Genomic Biomarkers

Abstract

We previously described early results of a nonchimeric operational tolerance protocol in human leukocyte antigen (HLA)-identical living donor renal transplants and now update these results. Recipients given alemtuzumab, tacrolimus/MPA with early sirolimus conversion were multiply infused with donor hematopoietic CD34(+) stem cells. Immunosuppression was withdrawn by 24 months. Twelve months later, operational tolerance was confirmed by rejection-free transplant biopsies. Five of the first eight enrollees were initially tolerant 1 year off immunosuppression. Biopsies of three others after total withdrawal showed Banff 1A acute cellular rejection without renal dysfunction. With longer follow-up including 5-year posttransplant biopsies, four of the five tolerant recipients remain without rejection while one developed Banff 1A without renal dysfunction. We now add seven new subjects (two operationally tolerant), and demonstrate time-dependent increases of circulating CD4(+) CD25(+++) CD127(-) FOXP3(+) Tregs versus losses of Tregs in nontolerant subjects (p < 0.001). Gene expression signatures, developed using global RNA expression profiling of sequential whole blood and protocol biopsy samples, were highly associative with operational tolerance as early as 1 year posttransplant. The blood signature was validated by an external Immune Tolerance Network data set. Our approach to nonchimeric operational HLA-identical tolerance reveals association with Treg immunophenotypes and serial gene expression profiles.

Trial registration: ClinicalTrials.gov NCT00619528.

Keywords: Biomarker; bone marrow/hematopoietic stem cell transplantation; clinical research/practice; clinical trial; genomics; immunobiology; kidney transplantation/nephrology; monitoring: immune; tolerance: clinical; translational research/science.

Figure 1. Longitudinal therapy and biopsy plan in this non-chimeric HLA identical “operational” tolerance trial

The current milestone of the trial has been extended to a biopsy at 5 years (red circle). DHSC = Donor Hematopoietic Stem Cells; Al = Alemtuzumab; MPA = Mycophenolate; SRL = Sirolimus; TAC = Tacrolimus; Tx = Transplant.

Figure 2

STARD Flow Diagram for the Clinical Trial.

Figure 3

A. Patient #8 (Table 1) Non-Tol at 5 years. Banff 1A acute cellular rejection in H&E histopathology found at 5 years post-operatively. This patient (#8, Table 1) had been off all IS for three years, two years after no rejection was seen at the three-year biopsy milestone (one year after IS was withdrawn). There was no renal dysfunction accompanying this protocol biopsy (see Table 1). B. Patient #6 (Table 1) Tol at 5 years. No rejection was seen at five years post-operatively in patient #6 in contrast with patient #8 (Table 1) at a similar 5-year interval three years after IS was withdrawn (H&E stain). Note that the immunophenotypic Treg percentages remained high in this recipient compared with those of #8 (Figure 4B).

Figure 4. Marked depression in CD4⁺ T cell whole blood immunophenotyping brought about by the protocol

Note that this even persisted in several patients at 60 months post-transplant, but there were no significant differences between the Tol and non-Tol groups. Absolute numbers of CD4⁺ cells/cu mm of whole blood were expressed as a fold change from pre-operative baseline (log scale). Preoperative values were normalized to 1.0.

Figure 5. Increasing differences between the Tol and non-Tol groups in the fold change of the Treg phenotype percentages compared with preoperative values as time elapsed after transplantation (months)

Note that as early as one year post-transplant, while still on equivalent doses of IS, differences were established between the Tol and non-Tol groups, and that even at 5 years (60 months) postoperatively while the Tol group was IS-free for 3 years and the non-Tol group remained on IS, these differences persisted, with significantly higher percentages of CD4⁺CD25⁺⁺⁺CD127⁻FOXP3⁺ Treg phenotypes in those that remained tolerant (p≤0.001). Preoperative values were normalized to 1.0. However, Patients #1 and #2 did not yet have this assay standardized preoperatively early in the study. Therefore, preoperative values of these two were calculated from means of the other 18 (Table S1) All symbols represent individual patients (patient #8 is singled out).

Figure 6. Venn diagram showing shared genes in PBMC when combining Years 1 and 2 post-transplant, data

Combining the data for Tol vs non-Tol revealed 357 differentially expressed genes, 56 shared with Year 1 profiles and 82 shared with Year 2. These 357 represent genes unique to Tol subjects at both time points and potentially predictive of those patients that can be successfully weaned off IS to tolerance.

Figure 7

A. Tol vs non-Tol – Years 3 + 4 Combined. Venn diagram showing the 3526 genes (arrows) that are unique to Tol subjects at years 3 + 4 Years post-transplant. Tol vs non-Tol subjects were compared separately to PBMC collected from 20 normal healthy volunteers to control for the effect of IS in the non-Tol group and to identify genes uniquely expressed in Tol subjects not on IS. B. Tol vs non-Tol – Years 5 + 6 Combined. Venn diagram showing the 4529 genes (arrows) that are unique to Tol subjects at years 5 + 6 Years post-transplant. Tol vs non-Tol subjects were compared separately to PBMC collected from 20 normal healthy volunteers to control for the effect of IS in the non-Tol group and to identify genes uniquely expressed in Tol subjects not on IS.

Figure 8. Mapping of the 4529 genes in PBMC at years 5 + 6 post-transplant to show the differential regulation of multiple critical immune/inflammatory pathways

The shading of the bars clearly represent the specific suppression of these pathways, showing predominantly down-regulated genes (light grey) when compared to the up-regulated genes (dark grey). The lower X-axis represents the −log of the p-value, and the upper X-axis shows the percentage of genes in each pathway.

Figure 9

A. Molecular Scores in PBMC: Up-regulated Genes. Molecular scores calculated using the 357-gene signature based on raw signal intensities for the up-regulated genes for the Tol (grey bar) and the non-Tol group (black bar) while they were on full IS (Years 1+2). P-values were computed using a two-tailed Student’s t-test assuming equal variance. B. Molecular Scores: Down-regulated Genes. Molecular scores calculated using the 357-gene signature based on raw signal intensities for the down-regulated genes for the Tol (grey bar) and the non-Tol group (black bar) while they were on full IS (Years 1+2). ). P-values were computed using a two-tailed Student’s t-test assuming equal variance.

Figure 10

A. Up-regulated Scores in PBMC. Molecular scores calculated using the 357-gene signature based on raw signal intensiities for the up-regulated genes for the Tol group (grey bar) when off IS (Years 3–4 and Years 5–6) and the non-Tol group (black bar) while on full IS (Years 3–4 and Years 5–6). ). P-values were computed using a two-tailed Student’s t-test assuming equal variance. B. Down-regulated Scores in PBMC. Molecular scores calculated using the 357-gene signature based on raw signal intensiities for the down-regulated genes for the Tol group (grey bar) when off IS (Years 3–4 and Years 5–6) and the non-Tol group (black bar) while on full IS (Years 3–4 and Years 5–6). ). P-values were computed using a two-tailed Student’s t-test assuming equal variance.

Figure 11

A. Up-regulated Scores in Biopsies. Molecular scores calculated using the 416-gene signature based on raw signal intensiities for the up-regulated genes for the Tol group (grey bar) when off IS (Years 3–4 and Years 5–6) and the non-Tol group (black bar) while on full IS (Years 3–4 and Years 5–6). ). P-values were computed using a two-tailed Student’s t-test assuming equal variance. B. Down-regulated Scores in Biopsies. Molecular scores calculated using the 416-gene signature based on raw signal intensiities for the down-regulated genes for the Tol group (grey bar) when off IS (Years 3–4 and Years 5–6) and the non-Tol group (black bar) while on full IS (Years 3–4 and Years 5–6). ). P-values were computed using a two-tailed Student’s t-test assuming equal variance.

Figure 12. PBMC proliferation response to mitogens and a recall antigen

A. Note that pre-operatively there were no differences in reactivity between the Tol and non-Tol groups (with ESRD) versus normal laboratory volunteers (Patient #8 was still considered tolerant since the assays were performed between 3 and 5 years post-operatively (see Table 1 and Figure 3B). The pre-operative samples analyzed included patient #2, #5, #6, #9, #11, and #13 in the Tol group. The non-Tol group included #1, #3, #4, #8, #10, #12, #16, and #18. B. Note that there was no difference between the Tol and non-Tol groups post-operatively with each group having significantly lower responses than normal laboratory volunteers (*, p=0.03 and 0.02, at the base of the figure). This was despite all Tol recipients being IS-free for over one year. In the Tol group, the assays were performed on patient #2, #6, #9, #11, and #13. In the non-Tol group the assays were performed on patients #1, #3, #4, #8, and #12, all on IS for greater than one year. A log scale was used to emphasize the tetanus response differences vs normals.

Figure 13. Sequential pre- and post-operative ImmunKnow^®/Cylex^® (ATP consumption) assays measuring lymphoproliferative responses to the PHA mitogen up to 36 months post-operatively

The assays were recorded every three months using the same normal laboratory volunteer control for each patient (with few exceptions when unavailable). Note that the Tol and non-Tol groups could actually be distinguished at many time points at which lower responses were seen in the Tol group and statistical significance appeared to be reached between the two curves (p=0.049). Both were markedly lower than when simultaneous assays were performed on the normal controls (p=0.001). Only patients up to 36 months were studied with these assays. Analysis was performed on Tol patients #2, #5, #6, #9, #11, and #13. In the non-Tol group, the assays were performed on patients #1, #3, #4, #8, #12, and #16. Data represent n=6 per group up to 24 months and, n=4 per group at 24 – 36 months. (At the later time periods, reagents became unavailable.)