Late Treatment With Autologous Expanded Regulatory T-cell Therapy After Alemtuzumab Induction Is Safe and Facilitates Immunosuppression Minimization in Living Donor Renal Transplantation

Abstract

Background.

The TWO Study (Transplantation Without Overimmunosuppression) aimed to investigate a novel approach to regulatory T-cell (Treg) therapy in renal transplant patients, using a delayed infusion protocol at 6 mo posttransplant to promote a Treg-skewed lymphocyte repopulation after alemtuzumab induction. We hypothesized that this would allow safe weaning of immunosuppression to tacrolimus alone. The COVID-19 pandemic led to the suspension of alemtuzumab use, and therefore, we report the unique cohort of 7 patients who underwent the original randomized controlled trial protocol. This study presents a unique insight into Treg therapy combined with alemtuzumab and is therefore an important proof of concept for studies in other diseases that are considering lymphodepletion.

Methods.

Living donor kidney transplant recipients were randomized to receive autologous polyclonal Treg at week 26 posttransplantation, coupled with weaning doses of tacrolimus, (Treg therapy arm) or standard immunosuppression alone (tacrolimus and mycophenolate mofetil). Primary outcomes were patient survival and rejection-free survival.

Results.

Successful cell manufacturing and cryopreservation until the 6-mo infusion were achieved. Patient and transplant survival was 100%. Acute rejection-free survival was 100% in the Treg-treated group at 18 mo after transplantation. Although alemtuzumab caused a profound depletion of all lymphocytes, including Treg, after cell therapy infusion, there was a transient increase in peripheral Treg numbers.

Conclusions.

The study establishes that delayed autologous Treg therapy is both feasible and safe, even 12 mo after cell production. The findings present a new treatment protocol for Treg therapy, potentially expanding its applications to other indications.

INTRODUCTION

Solid organ transplant recipients remain dependent on immunosuppressive therapy to prevent rejection of the transplanted organ. Unfortunately, long-term patient and transplant survival is limited by the significant side effects of immunosuppressive drugs, including increased rates of malignancy, infection, and cardiovascular disease.^1,2 Increasing evidence suggests that autologous cell-based therapies may effectively modulate the immune response to allow the safe reduction of conventional immunosuppression.³

Regulatory T cells (Treg) are a subset of T lymphocytes that are central to maintaining immune homeostasis. Therapeutically, Treg can modulate immune responses, for example preventing an inflammatory response to alloantigens. Experimental animal models have harnessed this ability to successfully prevent rejection of allografts.⁴ Progressive developments in cell culture and manufacturing techniques have allowed the ex vivo expansion of Treg from whole blood or leukapheresis products from prospective living donor kidney transplant recipients.^5,6 These cells may be cryopreserved with a negligible impact on their function.⁷ Several phase I clinical trials have reported the feasibility and safety of expanding recipient Treg and infusing the cell product safely into the transplant recipient posttransplantation.^3,8-11 Recently, we reported the successful expansion of autologous polyclonal Treg and safe infusion into 12 kidney transplant recipients as part of the ONE Study consortium.¹² The Treg-treated cohort had reduced immunosuppression requirements and no acute rejection episodes over 48 mo, as well as reduced episodes of opportunistic infection compared with a control cohort that received standard immunosuppression and had an acute rejection rate of 21.1%.

Evidence is now mounting to support the use of Treg therapy to minimize immunosuppression. However, it is not clear which immunosuppression protocol to use or when to infuse cells. Previous work has identified alemtuzumab to promote the enrichment of Treg populations during the late cell repopulation phase at around 6 mo posttreatment.^13-15 The 3C Study has also shown alemtuzumab induction to be associated with favorable early rejection rates.¹³ In this study, we report a novel approach to the use of autologous ex vivo–expanded naturally occurring polyclonal Treg therapy in a preliminary randomized controlled study where we have combined alemtuzumab induction with delayed Treg cellular therapy. The infusion of Treg was delayed to optimize the native excess proportion of natural Treg and transitional B cells that repopulate 6 mo posttransplantation, while providing additional Treg at the point where they may be needed during postlymphodepletion cell repopulation. Treg-treated patients further underwent mycophenolate mofetil (MMF) cessation and tacrolimus reduction, with control patients remaining on standard-of-care immunosuppression with MMF and tacrolimus. The patient cohort described here represents a unique cohort of patients who underwent the original TWO Study (Transplantation Without Overimmunosuppression) protocol (Supplemental File, SDC, http://links.lww.com/TP/D72; International Standard Randomised Controlled Trial Number registry 11038572). Because of the COVID-19 pandemic, the use of alemtuzumab was suspended nationally in the United Kingdom, and the TWO protocol was modified to use basiliximab in place of alemtuzumab. The outcomes of the 7 patients who completed the alemtuzumab-based protocol are reported here and provide insight into the combination of Treg therapy after alemtuzumab induction. Therefore, this study provides important proof-of-concept data for other trials that are considering lymphodepletion in conjunction with cellular therapy.

MATERIALS AND METHODS

The study was designed as a prospective randomized controlled trial in a single center in the United Kingdom to explore the role of delayed autologous Treg therapy combined with alemtuzumab induction, MMF cessation, and tacrolimus weaning in living donor kidney transplant recipients.¹⁴ The objective was to determine the safety and efficacy of delayed Treg therapy during lymphocyte repopulation after alemtuzumab induction, as well as the success of Treg treatment at facilitating minimization of immunosuppression. The Treg dose used was based on the ONE Study dose-escalation trial, where up to 10 × 10⁶ cells/kg was shown to be safe.¹² Control participants received a standard alemtuzumab-based immunosuppression regimen with long-term tacrolimus and MMF immunosuppression. Primary endpoints were graft survival and the incidence of biopsy-confirmed acute rejection events (Banff criteria) within 18 mo of transplantation. The TWO Study is registered on the International Standard Randomised Controlled Trial Number registry (11038572).

Ethical approval for the study was granted by the Health Research Authority, South Central, Oxford, a research ethics committee (Bristol Research Ethics Committee Centre, Whitefriars; oxforda.rec@hra.nhs.uk; ref no.: 18/SC/0054). The full trial protocol will be made available as a Supplemental Document (SDC, http://links.lww.com/TP/D72).

Patients

Potential living donor renal transplant recipients were recruited to include first primary transplant recipients and exclude high immunological risk participants (Table 1). Written informed consent was obtained at the previsit (V0) before eligibility and randomization, before any trial-specific protocols occurred. Information was provided and consent was obtained according to the trial protocol (Supplemental File, SDC, http://links.lww.com/TP/D72). Eligible participants were randomized to the Treg therapy arm, or standard immunosuppression with alemtuzumab induction and maintenance immunosuppression with tacrolimus and MMF. The cell therapy arm received the same alemtuzumab induction and immunosuppression regimen until week 12 when the MMF dose was progressively reduced until week 22 and a protocol transplant biopsy performed (Figure 1). At week 26, MMF was discontinued, provided the protocol biopsy showed no evidence suggesting acute rejection, and the recipients continued maintenance tacrolimus monotherapy (target trough level 5–10 ng/dL) until the result of a second protocol biopsy at week 38. If the second protocol biopsy revealed no evidence of acute rejection, the dose of tacrolimus was reduced (target trough level 4–6 ng/dL) until the trial was completed in week 78. The 78-wk duration of the trial was determined in order to capture 90% of primary endpoint events, which would have been expected within this time frame.¹⁵ Recruitment of living donor kidney transplant recipients and donors was performed in accordance with the inclusion and exclusion criteria outlined in Table 2. As this was designed as a phase II trial, we elected to exclude highly sensitized patients and included only first transplant recipients. Each enrolled patient had 19 trial study visits scheduled relative to the day of transplantation (day 0). Patient demographics are shown in Table 1. Clinical data collected included creatinine, estimated glomerular filtration rate, urine protein creatinine ratio, and tacrolimus level. Calculation of the estimated glomerular filtration rate was performed using the 2009 Chronic Kidney Disease Epidemiology Collaboration equation.

TABLE 1.

Demographics of patients included in the trial

Patient	101	102	103	104	105	106	107
Treatment group	Cell therapy	Cell therapy	Control	Control	Cell therapy	Control	Control
Age, y	60	32	57	30	32	35	38
Sex	Male	Female	Male	Male	Male	Male	Male
Race	White	White	White	White	White	White	White
Tx type	LRD	LRD	LRD	LRD	LRD	LRD	LURD
Donor CMV status	Neg	Pos	Neg	Pos	Neg	Neg	Neg
Recipient CMV status	Pos	Pos	Neg	Neg	Neg	Neg	Neg
No. of mismatches	0	2	2	2	3	1	2
cRF %	34	3	19	0	19	0	33

CMV, cytomegalovirus; cRF, calculated reaction frequency; LRD, living related donor; LURD, living unrelated donor; Neg, negative; Pos, positive; Tx, transplantation.

FIGURE 1.

Diagrammatic representation of the original TWO Study trial design before alemtuzumab was discontinued in transplant centers across the United Kingdom because of safety concerns. Both arms underwent alemtuzumab induction and the same initial immunosuppressive regimen. At 12 wk in the cell therapy group, MMF was gradually decreased until week 22, when a protocol biopsy was performed. At week 26, if there were no signs of acute rejection, MMF was stopped, and patients received an infusion of autologous Treg. Both groups underwent a protocol biopsy at week 38, and follow-up was concluded at 78 wk (18 mo) posttransplantation. MMF, mycophenolate mofetil; Treg, regulatory T cell; TWO, Transplantation Without Overimmunosuppression.

TABLE 2.

Inclusion and exclusion criteria

Donor criteria		Recipient
Inclusion	Exclusion	Inclusion	Exclusion
Eligible for live donation	Exposure to any investigational agents at or 28 d before trial induction	Chronic renal insufficiency necessitating transplantation	Any known contraindication to protocol-specific requirements
Age older than 18 y	Altruistic donor	Willing and able to give informed consent	ABO blood group incompatibility with donor
ABO blood group compatible with recipient	Paired exchange donor	Age older than 18 y	cRF >40% within 6 mo before transplant
Willing to provide personal, medical, and biological data	Any form of substance abuse, psychiatric disorder, or other cause of potential impaired judgment	Can comply with trial requirements	Any form of substance abuse, psychiatric disorder, or other cause of potential impaired judgment
Willing to provide blood samples for analysis		Able to commence immunosuppression at the specified time	Concomitant malignancy or history of malignancy within 5 y before study entry
Willing and able to give informed consent		Females of CBA/men with partners of CBA must be willing to use effective contraception for 18 mo after trial	Seropositive for HIV, HEPB, HCV, HTLV, or syphilis
		Willing to allow his/her GP and/or consultant to be informed of their participation in the trial	Significant liver disease (ALT >3× ULN)
			Participation in another trial within 28 d
			Female who is pregnant or lactating
			Any factors that may hamper compliance
			Any previous desensitization procedure

ALT, alanine transaminase; CBA, childbearing age; cRF, calculated reaction frequency; GP, general practitioner (primary care physician); HCV, hepatitis C virus; HEPB, hepatitis B virus; HTLV, human T-lymphotropic virus 1; ULN, upper limit of normal.

Manufacturing and Treg Therapy

Once randomized, whole blood from each potential living kidney transplant recipient in the Treg therapy arm was collected (370 mL) and transported to a good manufacturing practice unit at Guy’s and St. Thomas’ National Health Service Foundation Trust, London, United Kingdom. Treg were isolated and expanded in cell culture to a dose of 5–10 × 10⁶ cells per kg body weight using a previously published protocol.^6,16 Briefly, after blood volume reduction, Treg were isolated via CD8 depletion and CD25 enrichment using CliniMACS beads and a CliniMACS Plus Instrument (Miltenyi Biotech). Cells were then stimulated with anti-CD3/anti-CD28 beads (ExpAct Treg kit, Miltenyi Biotec) at a ratio of 4:1 (beads:cells). Rapamycin was added at the beginning of the culture and IL-2 (interleukin-2) was added at day 4. Rapamycin and IL-2 were replenished every 2–3 d. Three cycles of bead stimulation were performed. The final product was retrieved on day 36. Phenotypic and functional characterization was performed and then cells were cryopreserved in vapor phase liquid nitrogen.⁷ The cell product was transported in a temperature-monitored cold shipper (–180 °C), thawed at the bedside, and administered intravenously in 5% human albumin intravenously at week 26, which was 72 h after cessation of MMF. All clinical trial data up to 78 wk posttransplant were recorded in a bespoke electronic clinical trial database (Excelya, Germany).

Immune Profiling

Immune monitoring was performed using a protocol designed at trial initiation and, therefore, standardized for the duration of the study. For flow cytometry analysis, 100 μL (or 50 μL in the case of the Treg panel) of EDTA peripheral blood was stained using commercially available premixed, lyophilized antibody panels (Duraclone panels, Beckman Coulter) following the manufacturer’s protocol. For the B-cell panel, 300 μL of blood was washed twice with PBS before staining. Samples were acquired on a Navios flow cytometer (Beckman Coulter). As a quality control, FlowCheck Pro and FlowSet Pro beads (Beckman Coulter) were run before each immunomonitoring visit. Flow cytometry data were analyzed using Kaluza software (Beckman Coulter), followed by the calculation of absolute cell numbers based on clinical blood morphology results. Data visualization and statistical analysis were performed using GraphPad Prism 9.

Cytometry of the time of flight was performed using the MaxPar Direct Immune Profiling Assay (Fluidigm) and acquired at the Mass Cytometry Facility at the Kennedy Institute of Rheumatology on a third-generation Helios mass cytometer (Fluidigm). Briefly, whole blood was incubated with heparin to block Fc receptors, followed by incubation with a premixed cocktail of antibodies. Next, erythrocytes were lysed using ammonium-chloride-potassium Lysing buffer (Gibco), followed by 3 washes with staining buffer and fixation with 1.6% formaldehyde solution. After fixation, cells were cryopreserved in a freezing medium composed of 45% fetal calf serum, 45% RPMI, and 10% dimethyl sulfoxide and stored in liquid nitrogen for batch analysis. For analysis, samples were thawed, stained with MaxPar Intercalator-Ir (¹⁹¹Ir and ¹⁹³Ir), and resuspended in MaxPar water containing 10% EQ 4-element calibration beads, followed by acquisition on the cytometry of the time of flight system. Flow cytometry standard files were normalized with calibration beads by Helios software. Manual gating of flow cytometry standard files was performed using the Cytobank platform. Calibration beads and cell aggregates were excluded through manual gating, and the population of interest was gated and exported in a new flow cytometry standard file. Visualization stochastic neighbor embedding algorithm was used for analysis and visualization of peripheral B cells using the FlowSOM clustering algorithm, with Cytobank platform performed heatmaps to show the median expression of individual cellular markers.

RESULTS

The initial intention was to recruit 68 living kidney donor recipients and donors, but recruitment to the trial was halted in March 2020 because of the COVID-19 pandemic. At this stage, 9 recipients had been recruited, and we were able to continue to monitor the progress of these patients. Many transplant centers in the United Kingdom stopped performing organ transplants for several months during the first wave of the pandemic.^17,18 There was substantial concern about the potential adverse impact of immunosuppression on the severity and risk of COVID-19 infection, in particular profound lymphocyte depletion with alemtuzumab. In response to these concerns, we altered the protocol to avoid alemtuzumab induction thereafter.¹⁴

Here we report the results of the 9 living donor kidney transplant recipients in the preliminary phase of the TWO Study before its modification, although 2 participants recruited were withdrawn from the trial at an early stage as their transplant was delayed because of the COVID-19 pandemic. The remaining 7 participants have all now completed follow-up and form a unique cohort of patients receiving alemtuzumab induction, with 3 receiving delayed Treg therapy with immunosuppression minimization, and 4 contemporaneous control patients. One of the patients in the cell therapy arm had their protocol biopsy and cell infusion delayed because of the initial lockdown of the COVID pandemic from mid-May 2020 to mid-August 2020.

Clinical Outcomes

The demographics of the 7 participants are shown in Table 1. Clinical markers of renal function posttransplant are shown in Figure 2. There were no hemodynamic or inflammatory reactions to the infusion of the Treg product, and all 3 patients had the infusion as a day admission and were fit for discharge 4 h postinfusion. In both groups, there was 100% transplant survival at 18 mo. No acute rejection episodes occurred in the cell therapy patients, but one of the 4 control patients had a significant early acute rejection episode in week 1 posttransplant, successfully treated with 3 daily doses of methylprednisolone. One of the cell therapy patients and 3 of the control patients developed neutropenia, which responded to a temporary cessation of MMF and was thought to be secondary to the combination of alemtuzumab and the trial dose of MMF. Two of these individuals developed mild clinical cytomegalovirus disease requiring a 3-wk course of oral valganciclovir after completion of chemoprophylaxis, 1 from each treatment arm of the study. Both showed complete resolution of cytomegalovirus. One control patient developed a decline in transplant function 8 mo posttransplant with biopsy evidence of tacrolimus toxicity. Transplant function improved with cessation of tacrolimus and maintenance of prednisolone and MMF immunosuppression; however, it did not return to baseline. One cell therapy patient had a transient episode of proteinuria, which spontaneously resolved without explanation. Their kidney function was otherwise stable, and protocol biopsies did not show any pathology during the study period. There was no indication to perform a for-cause biopsy. This patient continues to have stable renal graft function. All 7 patients were fit and well at trial completion. In contrast to patients treated with Treg therapy in the ONE Study,¹² histopathological reporting did not identify infiltrates in the protocol biopsies 12 wk after Treg infusion on hematoxylin and eosin (H&E) staining (Table 3). All 3 cell therapy patients had minimization of immunosuppression to tacrolimus monotherapy and remained free from acute rejection throughout the planned follow-up period.

FIGURE 2.

Comparison of creatinine, eGFR, uPCR, and tacrolimus level across follow-up points in the Treg-treated arm and the control arm. A, Individual measurements of plasma creatinine in the 3 autologous Treg-treated patients and 4 control patients at 4, 12, 14, 24, 30, 38, 42, 44, 52, and 78 wk posttransplant. B, Individual measurements of eGFR in the 3 autologous Treg-treated patients and 4 control patients at 4, 12, 14, 24, 30, 38, 42, 44, 52, and 78 wk posttransplant. C, Individual measurements of uPCR in the 3 autologous Treg-treated patients and 4 control patients at 4, 12, 14, 24, 30, 38, 42, 44, 52, and 78 wk posttransplant. D, Individual measurements of tacrolimus levels in the 3 autologous Treg-treated patients and 4 control patients at 4, 12, 14, 24, 30, 38, 42, 44, 52, and 78 wk posttransplant. eGFR, estimated glomerular filtration rate; Treg, regulatory T cell; Tx, transplant; uPCR, urine protein creatinine ratio.

TABLE 3.

Results of biopsies obtained from cell therapy and control patients

	Cell therapy			Control arm
	Patient 1 (105)	Patient 2 (101)	Patient 3 (102)	Patient 1 (104)	Patient 2 (106)	Patient 3 (103)
Microscopic description	10 glomeruli	15 glomeruli	14 glomeruli	16 glomeruli	21 glomeruli	9 glomeruli
Glomerular sclerosis: Global	Neg	3/15	2/14	Neg	Neg	Neg
Glomerular sclerosis: segmental	Neg	NA	NA	Neg	Neg	Neg
Glomerulitis	Mild in 3 glomeruli	Neg	Neg	Mild in 1	Neg	Neg
Basement membrane	Normal	Normal	Normal	Normal	Normal	Normal
Tubulointerstitial fibrosis	5%	5%	10%	40%	10%	0%
Tubulitis	Mild focal lymphocytic	None	None	Mild focal lymphocytic	None	None
Vessels	Normal	Moderate fibroelastosis	Mild fibroelastosis	Normal	Moderate fibroelastosis	Normal
Rejection	No	No	No	No	No	No
C4d staining	Neg	Neg	Neg	Neg	Neg	Neg
SV40 staining	Neg	Neg	Neg	Neg	Neg	Neg
Comment	Minimal abnormality. No evidence of rejection	Minimal chronic damage. No evidence of rejection	Mild acute tubular injury. Mild chronic damage	Moderate chronic damage. Extensive mononuclear infiltrate in areas of fibrosis	Mononuclear infiltrate in areas of fibrosis	Minimal abnormality. No evidence of rejection

All 3 patients of the cell therapy group had protocol biopsies at 9 mo. Three of the control patients had biopsies as 1 declined the procedure.

C4d, complement split product, marker of antibody-mediated rejection in transplant; SV40, simian virus 40.

Immunological Outcomes

As expected, alemtuzumab treatment resulted in a prolonged depletion of T cells, especially CD4⁺ T cells (Figure 3; Figure S1, SDC, http://links.lww.com/TP/D72), with only 2 of 7 patients returning to a predepletion level of CD4⁺ T cells at the 18 mo visit (week 72). There were no statistically significant differences or obvious trends in levels of total T cells, or CD4⁺ and CD8⁺ T cells and their naive and memory subsets (Figure 3 and data not shown). Absolute cell numbers of peripheral blood Treg were reduced after alemtuzumab treatment (Figure 3G); however, a relative increase in Treg frequency and the Treg/Teff ratio in the first 12 wk after induction treatment was observed (Figure S1D, SDC, http://links.lww.com/TP/D72). A trend toward a transient increase in Treg numbers was observed 1 and 2 wk after cell infusion (Figure 3G; Figure S1D, SDC, http://links.lww.com/TP/D72; weeks 27 and 28 posttransplantation).

FIGURE 3.

Absolute numbers of T cells, CD4⁺ and CD8⁺ T cells, and FoxP3⁺CD4⁺ Treg in the peripheral blood over time. A, C, E, G, Absolute numbers of peripheral CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, and FoxP3⁺ CD4⁺ Treg in whole blood samples collected from patients before transplant, at 4, 12, 22, 24, 26, 27, 28, 30, 38, 44, 52, and 72 wk posttransplant (green arrow = week 26, Treg infusion, sample taken before cell infusion). Each patient presented as a separate point or point and line, cell therapy patients represented as closed triangles, control patients represented as open squares. B, D, F, H, Absolute numbers of peripheral CD3⁺ T cells, CD4⁺ T cells and CD8⁺ T cells and FoxP3⁺CD4⁺ Treg in the blood samples of the Treg therapy group (n = 3) pretransplant and at week 72 posttransplant (red) compared with the control group (n = 4) pretransplant and at week 72 (blue). Statistical significance was calculated by 1-way ANOVA with the Tukey test for multiple comparisons. ns = not significant, *P > 0.05. Data shown as absolute values (A, C, E, G) or mean ± SD. Treg, regulatory T cell; Tx, transplantation.

Naive and transitional B cells have been reported by others to be associated with operational tolerance,^19-21 and we have previously demonstrated an increase in naive and transitional B cells after alemtuzumab induction.¹ Similar trends can be observed in our limited patient cohort in the Treg-treated group (Figure 4; Figure S2, SDC, http://links.lww.com/TP/D72). Interestingly, in contrast to our previous data from induction-free Treg-treated patients from the ONE Study,¹² no increase in marginal zone B cells was observed. The original clinical trial protocol was designed to include donor-reactive T-cell quantification before and after infusion; however, we were unable to obtain these data for the cohort of 7 patients who underwent the original trial protocol because of challenges with the cell numbers required for these assays.

FIGURE 4.

Absolute numbers of B cells in the peripheral blood over time. A and C, Absolute numbers of total B cells and CD19⁺CD24^hiCD38^hi transitional B cells in whole blood samples collected from patients before transplant, at 4, 12, 22, 24, 26, 27, 28, 30, 38, 44, 52, and 72 wk posttransplant. Green arrow represents Treg infusion at 26 wk. Blood samples taken before infusion. B and D, Absolute numbers of total B cells and CD19⁺CD24^hiCD38^hi transitional B cells in the blood samples of the Treg therapy group (n = 3) pretransplant and at week 72 (red) compared with the control group (n = 4) pretransplant and at week 72 (blue). E, Absolute numbers of CD24^hiCD38^hi transitional B cells in the blood samples of the Treg therapy group (n = 3) before transplant and at 26, 27, and 72 wk posttransplant. F–I, Absolute numbers of naive B cells, marginal zone B cells, IgD^negCD27⁺ switched memory B cells, and IgD^negCD27^neg unconventional memory B cells in the blood samples of the Treg therapy group (n = 3) before transplant and at week 72 (red) compared with the control group (n = 4) pretransplant and at week 72 (blue). Each patient presented as a separate point or point and line, cell therapy patients represented as closed triangles, control patients represented as open squares. J, Representative viSNE analysis of peripheral B cells using the FlowSOM clustering algorithm from the peripheral blood of renal transplant recipients before transplant (n = 2), at 24 (n = 4), 30 (n = 4), and 38 (n = 4) wk posttransplant. K, Heatmap showing the median expression of cellular markers expressed in the clusters identified by FlowSOM. Cluster 1: naive 1, cluster 2: naive 2, cluster 3: marginal zone B cells 1, cluster 4: switched memory cells, cluster 5: marginal zone B cells 2, and cluster 6: transitional B cells. L, Percentages of each cluster within the B-cell compartment calculated by the FlowSOM algorithm pretransplant and at 24, 30, and 38 wk posttransplant. Dots represent individual samples. Statistical significance was calculated by 1-way ANOVA with the Tukey test for multiple comparisons; ns, not significant. Data shown as absolute values (A and C) or mean ± SD. Treg, regulatory T cell; Tx, transplantation; viSNE, visualization stochastic neighbor embedding algorithm.

DISCUSSION

The TWO Study was designed to test the hypothesis that polyclonal autologous Treg infusion would have maximum impact during the lymphocyte repopulation phase occurring typically 6 mo after infusion of alemtuzumab.²² There is evidence that the repopulating lymphocytes are rich in naive T cells and Treg with the theoretical potential that infused Treg could influence the Treg to effector T-cell balance favorably in terms of enhanced natural immune regulation. Here, we report the outcome of the patients recruited to the TWO Study original protocol, where we demonstrate the feasibility of expansion and cryopreservation of a viable Treg product for up to 12 mo and the safety of delayed Treg infusion posttransplantation. Unfortunately, the emergence of COVID-19 as a major public health concern resulted in the suspension of alemtuzumab use in transplant centers across the United Kingdom. The risk of COVID-19 superinfection after leukodepletion necessitated a switch to alternative induction immunosuppression. The TWO protocol was modified to use basiliximab instead of alemtuzumab, and as such, this cohort of 7 patients is a unique subset of patients undergoing this therapeutic combination. This study provides valuable data to support the potential use of Treg therapy in deceased donor kidney transplantation in the future. The only other study to report on this combination of immunotherapy induction and cell therapy is the report by Matthew et al, in 2018.²³ However, there were some key differences between this study and ours. First, Matthew et al infused autologous Treg at 60 d after alemtuzumab induction, whereas here, treatment was delayed for 26 wk, targeting the lymphocyte repopulation phase. Additionally, Matthew et al²⁴ did not stop MMF treatment and converted the tacrolimus to sirolimus. Previous data from our unit have shown conversion to sirolimus to be detrimental after alemtuzumab induction, and therefore, this trial was designed to achieve tacrolimus monotherapy. We achieved this goal in the 3 patients treated with cell therapy without incurring any acute rejection in the period up to 18 mo after transplantation. Tacrolimus levels were not significantly different between the control patients and the Treg-treated patients. However, in the Treg-treated patients, the withdrawal of MMF and maintenance on tacrolimus monotherapy represent a significant reduction in immunosuppressive burden. This adds further evidence to the potential value of cell therapy to reduce immunosuppression^8,9 Immune monitoring highlighted similar changes in peripheral immune phenotype as those observed in other Treg trials, with detectable live Treg postinfusion in a lymphodepleted environment and trends toward naive and transitional B-cell increases.

This study suggests that the delayed use of autologous Treg in living donor kidney transplant recipients, after lymphodepletion with alemtuzumab, is safe, feasible, and allows minimization of immunosuppressive therapy. Furthermore, our analysis shows that there was a transient increase in peripheral Treg numbers after cell therapy; however, it was not possible to determine whether this was because of the persistence of the infused Treg, or the expansion of endogenous Treg after cell therapy infusion. Although the trial protocol has since changed, this unique cohort outlines the promising potential for delayed Treg therapy with extended cell cryopreservation, with implications for future studies that may incorporate elements of the protocol.