Phase I trial of donor-derived modified immune cell infusion in kidney transplantation

Abstract

BACKGROUNDPreclinical experiments have shown that donor blood cells, modified in vitro by an alkylating agent (modified immune cells [MICs]), induced long-term specific immunosuppression against the allogeneic donor.METHODSIn this phase I trial, patients received either 1.5 × 106 MICs per kg BW on day -2 (n = 3, group A), or 1.5 × 108 MICs per kg BW on day -2 (n = 3, group B) or day -7 (n = 4, group C) before living donor kidney transplantation in addition to post-transplantation immunosuppression. The primary outcome measure was the frequency of adverse events (AEs) until day 30 (study phase) with follow-up out to day 360.RESULTSMIC infusions were extremely well tolerated. During the study phase, 10 treated patients experienced a total of 69 AEs that were unlikely to be related or not related to MIC infusion. No donor-specific human leukocyte antigen Abs or rejection episodes were noted, even though the patients received up to 1.3 × 1010 donor mononuclear cells before transplantation. Group C patients with low immunosuppression during follow-up showed no in vitro reactivity against stimulatory donor blood cells on day 360, whereas reactivity against third-party cells was still preserved. Frequencies of CD19+CD24hiCD38hi transitional B lymphocytes (Bregs) increased from a median of 6% before MIC infusion to 20% on day 180, which was 19- and 68-fold higher, respectively, than in 2 independent cohorts of transplanted controls. The majority of Bregs produced the immunosuppressive cytokine IL-10. MIC-treated patients showed the Immune Tolerance Network operational tolerance signature.CONCLUSIONMIC administration was safe and could be a future tool for the targeted induction of tolerogenic Bregs.TRIAL REGISTRATIONEudraCT number: 2014-002086-30; ClinicalTrials.gov identifier: NCT02560220.FUNDINGFederal Ministry for Economic Affairs and Technology, Berlin, Germany, and TolerogenixX GmbH, Heidelberg, Germany.

Keywords: Clinical Trials; Tolerance; Transplantation.

Figure 1. TOL-1 study design.

On day –2 (groups A and B) or –7 (group C) before kidney transplantation, PBMCs were collected from donors by unstimulated standard leukapheresis. PBMCs were transferred to the good manufacturing practice (GMP) facility and incubated with the proliferation inhibitor mitomycin C. After washing out of mitomycin C and quality control, the final product (MICs) was administered to patients on the same day, approximately 12 hours after donor leukapheresis. In the intensive care unit, patients were intravenously administered either 1.5 × 10⁶ MICs per kg BW on day –2 (n = 3, group A) or 1.5 × 10⁸ MICs per kg BW on day –2 (n = 3, group B) or day –7 (n = 4, group C) before living donor kidney transplantation. After transplantation, patients received immunosuppressive therapy with CyA, EC-MPS, and corticosteroids according to the center’s protocol. The primary outcome measure was the frequency of AEs on day 30 (end of study).

Figure 2. Polyclonal, third-party, and donor-specific lymphocyte response of MIC-treated patients in vitro.

(A) Polyclonal stimulation (pokeweed mitogen [PWM], phytohemagglutinin [PHA], concanavalin A [ConA], CD3 mAb) of blood lymphocytes from patients in group C (R7, R11, R12, R14) on day 360 (black circles) versus before MIC treatment on day –7 (orange circles). Individual measurements and the median are shown. Values outside the normal range for healthy individuals are highlighted in gray. Patients showed preserved in vitro lymphocyte proliferation after transplantation. (B and C) Third-party and donor-specific stimulation of blood lymphocytes from patients in group C on day 360 (black circles) versus before MIC treatment on day –7 (orange circles) (B), or versus transplanted controls on triple immunosuppressive therapy (red circles) (C). Individual measurements and the median are shown. Values outside the normal range for healthy individuals are highlighted in gray. The findings indicate preserved immunological responsiveness of recipient T lymphocytes against irradiated third-party cells, with reduced responsiveness to donor cells after transplantation compared with before transplantation and MIC infusion (B). Transplanted control patients without MIC treatment showed higher responsiveness compared with MIC-treated patients on day 360 (C). (D–G) Third-party (black bars) and donor-specific (gray bars) stimulation of blood lymphocytes from individual patients in group C (R7, R11, R12, R14) was performed before (days –7, –6, and –1) and after transplantation (days 7, 30, 60, 90, 135, 180, 270, and 360), as well as before (day –7) and after (days –6 and –1) MIC infusion. In all experiments, stimulatory cells consisted of irradiated allogeneic PBMCs. T lymphocyte proliferation was assessed by CFSE staining. Plus sign indicates patient R14, who had no HLA-A, -B, or -DR mismatches with the donor; asterisk indicates infectious/inflammatory episodes; pound sign indicates at the time of steroid withdrawal.

Figure 3. Tregs in MIC-treated patients.

(A) Peripheral blood lymphocytes (PBLs) were gated in a forward scatter/side scatter (FSC/SSC) dot plot (gate R4). CD4⁺CD25⁺ PBLs were determined using a CD4/CD25 dot plot (R10) and further analyzed for coexpression of both Foxp3⁺ and CD127^– or only CD127^– in a Foxp3/CD127 dot plot (CD4⁺CD25⁺Foxp3⁺CD127^– and CD4⁺CD25⁺CD127^– Treg subsets). APC, allophycocyanin; PE, phycoerythrin; PerCP, peridinin-chlorophyll-protein. Individual measurements for the percentage of CD4⁺CD25⁺CD127^– Tregs (B) and CD4⁺CD25⁺FoxP3⁺CD127^– Tregs (C) in patients R7, R11, R12, and R14 from day –7 to day 360 are shown. Tregs were lowest on day 30 after kidney transplantation at the time of powerful immunosuppressive therapy.

Figure 4. Bregs in MIC-treated patients compared with Bregs in transplanted controls.

(A) PBLs were gated in a FSC/SSC dot plot (gate R1). B lymphocytes were gated using a CD19/SSC gate (gate R2). CD19⁺CD24⁺ cells were determined (gate R3) within the CD19⁺ B lymphocytes (gate R2). On the basis of gate R3, CD24^hiCD38^hi B lymphocytes were analyzed (gate R6) and assigned as CD19⁺CD24^hiCD38^hi transitional B lymphocytes (Bregs). IL-10 production of CD19⁺CD24^hiCD38^hi transitional B lymphocytes was further investigated using a CD38/IL-10 gate based on gate R6. (B) Individual measurements of the percentage of CD19⁺CD24^hiCD38^hi Bregs in patients R7, R11, R12, and R14 from day –7 to day 360. CD19⁺CD24^hiCD38^hi Bregs were low out to day 30 after kidney transplantation and increased out to day 180. (C) Individual measurements of the percentage of CD19⁺CD24^hiCD38^hi Bregs in patients in group C (black circles) were compared with measurements in transplanted controls (red circles). Individual measurements and the median are shown. Compared with transplanted controls, Breg percentages were 4, 3, 9, 19, 26, and 13 times higher in patients in group C on days 60, 90, 135, 180, 270, and 360 after transplantation, respectively. (D) Individual measurements of the percentage of CD19⁺CD24^hiCD38^hi Bregs in patients in group C were compared with measurements in a second independent cohort of transplanted controls stratified according to steroid dose (shown in parentheses). Individual measurements and the median are shown. Compared with transplanted controls without steroid treatment, Breg percentages were 68 and 44 times higher in patients in group C on days 180 and 360 after transplantation, respectively. (E) Percentage of CD19⁺CD24^hiCD38^hi Bregs in frozen cells from patients in groups A–C. The median and interquartile range are shown. In contrast to the percentage of CD19⁺CD24^hiCD38^hi Bregs from patients in groups B and C, the percentage of Bregs for patients in group A were 68 and 20 times higher, respectively, on day 180 after transplantation. (F) Percentage of IL-10–producing Bregs. Individual measurements and the median are shown for cumulative post-transplantation data for patients in group C. The majority of Bregs were producing the immunosuppressive cytokine IL-10.

Figure 5. Immune reaction patterns induced by MIC therapy based on proteome analysis.

IPA of proteome data on day –1 (after MIC infusion) versus days –2 and –7 (before MIC infusion) indicated significant downregulation of the biological function immunological disease/hypersensitive reaction after MIC administration but before kidney transplantation and application of immunosuppressive therapy. Upregulation (red shapes); downregulation (green shapes); predicted inhibition (blue shape); leads to inhibition (blue dashed lines); findings inconsistent (yellow dashed lines); findings not predicted (gray dashed lines) . CD74, HLA-DR; MMP3, stromelysin-1; PTPRC, receptor-type tyrosine-protein phosphatase C; SDC1*, syndecan-1; SPP1, osteopontin; TNFRSF8, TNF receptor superfamily member 8 (CD30); VDR, vitamin D3 receptor; CD2, T cell surface antigen CD2.