Obtaining regulatory T cells from uraemic patients awaiting kidney transplantation for use in clinical trials

Abstract

Adoptive transfer of regulatory T cells (T(regs)) has been proposed for use as a cellular therapy to induce transplantation tolerance. Preclinical data are encouraging, and clinical trials with T(reg) therapy are anticipated. In this study, we investigate different strategies for the isolation and expansion of CD4(+) CD25(high) CD127(low) T(regs) from uraemic patients. We use allogeneic dendritic cells (DCs) as feeder cells for the expansion and compare T(reg) preparations isolated by either fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) that have been expanded subsequently with either mature or tolerogenic DCs. Expanded T(reg) preparations have been characterized by their purity, cytokine production and in-vitro suppressive ability. The results show that T(reg) preparations can be isolated from uraemic patients by both FACS and MACS. Also, the type of feeder cells used in the expansion affects both the purity and the functional properties of the T(reg) preparations. In particular, FACS-sorted T(reg) preparations expanded with mature DCs secrete more interleukin (IL)-10 and granzyme B than FACS-sorted T(reg) preparations expanded with tolerogenic DCs. This is a direct comparison between different isolation techniques and expansion protocols with T(regs) from uraemic patients that may guide future efforts to produce clinical-grade T(regs) for use in kidney transplantation.

Keywords: cell therapy; clinical trials; kidney transplantation; regulatory T cells; tolerance.

Fig. 1

Overview of the regulatory T cell (T_reg) production process. Step 1: the starting material is comprised of peripheral blood mononuclear cells (PBMCs) collected from both the intended organ donor and recipient. We prefer the use of leukapheresis, because this greatly increases the yield. Step 2: the PBMCs are purified further over a Ficoll density gradient. Step 3: the PBMCs are separated with regard to their adherent properties by which monocytes (attaching to plastic) and lymphocytes (non-plastic adherent) are separated. Step 4: T_regs with a phenotype of CD4⁺CD25^highCD127^low are sorted from the non-plastic adherent cells of the potential organ recipient using fluorescence activated cell sorting (FACS), with the possibility of performing a pre-enrichment with magnetic activated cell sorting (MACS) for CD4⁺ T cells prior to FACS. Monocytes from the potential organ donor are stimulated with granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4 and lipopolysaccharide (LPS) by which the cells differentiate into mature dendritic cells (mDC). Some of these cells are cryopreserved for use in possible restimulations and functional analyses. Step 5: T_regs from the potential organ recipient are mixed with irradiated mDC from the potential organ donor. The culture is supplemented with anti-CD3 antibody and IL-2. Steps 6–8: the T_reg product is assessed for sterility, function, etc. If the cells meet the set release criteria for the particular study they can be administered to the patient.

Fig. 2

Sorting CD4⁺CD25^highCD127^low cells from a representative uraemic patient. (a) First, the lymphocyte population was selected using their characteristics in forward- (FSC) and side-scatter (SSC). Next, the CD25^high expressing cells were selected. As evident in the dot-plot, these cells expressed on average slightly lower levels of CD4 and the inferior boundary of the gate was set to include this putative population. Finally, cells expressing low levels of CD127 were selected. Only cells present in all three gates were sorted. (b) Post-sort analysis was performed after the sorting. In general, post-sort purity was satisfying, but we occasionally noted decreased cell viability, as evident by an increased amount of cell debris.

Fig. 3

Expansion of CD4⁺CD25^highCD127^low T cells using dendritic cells (DCs). The number of fluorescence activated cell sorting (FACS)-sorted CD4⁺CD25^highCD127^low cells and the corresponding number of cells after expansion with mature DCs (mDC). There is no apparent correlation between the number of sorted regulatory T cells (T_regs) and the total yield after expansion.

Fig. 4

Phenotype of expanded regulatory T cells (T_regs). (a) The expanded cells constituted a coherent population. Here, a representative example of T_regs expanded with mature dendritic cells (mDC) is shown. (b) T_reg preparations sorted by fluorescence activated cell sorting (FACS) and expanded with mDC were purer than T_reg preparations expanded with DC-10 (P < 0·05). The T_reg preparations sorted by magnetic activated cell sorting (MACS) dropped occasionally to purities <20%, whereas T_reg preparations sorted by FACS were, on average, 72% pure.

Fig. 5

Short-term functional assessment of regulatory T cells (T_regs) from uraemic patients. (a) Gating for CD69-expressing responder cells. Unstained responder cells (top row) and stained, non-stimulated responder cells (second row) were included as controls. In the third column (CD69⁺ gate) only the CD25^– cells are shown. Suppression was calculated as the ratio of CD69 expression on stimulated responder cells without (third row) and with (fourth row) the addition of autologous T_regs. (b) T_reg preparations sorted by fluorescence activated cell sorting (FACS) and expanded with mDC were superior to T_reg preparations sorted by magnetic activated cell sorting (MACS) and expanded with either mature dendritic cells (mDC) or DC-10 (P < 0·05 and P < 0·001, respectively). Furthermore, suppression of CD69 expression in FACS-sorted and mDC-expanded T_reg preparations was consistently more than 20%.

Fig. 6

Long-term functional assessment of regulatory T cells (T_regs) from uraemic patients. (a) In the long-term suppression assay, assessed by inhibition of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled responder cells, T_reg preparations sorted by fluorescence activated cell sorting (FACS) and expanded with mature dendritic cells (mDC) were superior to T_reg preparations sorted by magnetic activated cell sorting (MACS) and expanded with DC-10. (b) Titration of the T_reg : T_resp ratio. A representative titration of the ratio between T_regs, sorted by FACS and expanded with mDC, and autologous CD4⁺ cells. About 50% suppression was observed even at T_reg : T_resp ratios of 1:32. Titrations beyond 1:32 were not performed.

Fig. 7

Cytokine secretion of expanded regulatory T cells (T_regs). (a) T_reg preparations sorted by fluorescence activated cell sorting (FACS) and expanded with mature dendritic cells (mDC) secreted low levels of interferon (IFN)-γ, less than cells sorted by magnetic activated cell sorting (MACS) (P < 0·05). (b) T_reg preparations sorted by FACS and expanded with mDC secreted more interleukin (IL)-10 than T_reg preparations sorted by FACS and expanded with DC-10 (P < 0·05). (c–e) There was a trend (P < 0·1) towards higher secretion of tumour necrosis factor (TNF)-α, IL-5 and IL-13 in T_reg preparations sorted by MACS compared to T_regs sorted by FACS. (f) The secretion of IL-17 was low in general and did not differ between T_reg preparations. (g) T_reg preparations sorted by FACS and expanded with mDC secreted more granzyme B than T_reg preparations expanded with DC-10 (P < 0·05).