Mucosal-associated invariant T cells are functionally impaired in pediatric and young adult patients following allogeneic hematopoietic stem cell transplantation and their recovery correlates with clinical outcomes

Abstract

Mucosal-associated invariant T (MAIT) cells are innate-like T cells implicated in the response to fungal and bacterial infections. Their contribution to restoring T-cell immunity and influencing hematopoietic stem cell transplant (HSCT) outcomes remains poorly understood. We retrospectively studied MAIT-cell recovery in 145 consecutive children and young adults with hematologic malignancies undergoing allogeneic (allo)-HSCT between April 2019 and May 2022, from unrelated matched donor (MUD, N=52), with standard graft-versus-host-disease (GvHD) prophylaxis, or HLA-haploidentical (Haplo, N=93) donor after in vitro αβT/CD19-cell depletion, without post-HSCT pharmacological prophylaxis. With a median follow-up of 33 months (range, 12-49 months), overall survival (OS), disease-free survival (DFS), and non-relapse mortality (NRM) were 79.5%, 72%, and 7%, respectively; GvHD-free relapse-free survival (GRFS) was 63%, while cumulative incidence of relapse was 23%. While αβT cells were reconstituted 1-2 years post HSCT, MAIT cells showed delayed recovery and prolonged functional impairment, characterized by expression of activation (CD25, CD38), exhaustion (PD1, TIM3) and senescence (CD57) markers, and suboptimal ex vivo response. OS, DFS, and NRM were not affected by MAIT cells. Interestingly, higher MAIT cells at day +30 correlated with higher incidence of grade II-IV acute GvHD (19% vs. 7%, P=0.06). Furthermore, a greater MAIT-cell count tended to be associated with a higher incidence of chronic GvHD (cGvHD) (17% vs. 6%, P=0.07) resulting in lower GRFS (55% vs. 73%, P=0.05). Higher MAIT cells also correlated with greater cytomegalovirus (CMV) reactivation and lower late blood stream infections (BSI) (44% vs. 24%, P=0.02 and 9% vs. 18%, P=0.08, respectively). Future studies are needed to confirm the impact of early MAIT-cell recovery on cGvHD, CMV reactivation, and late BSI.

Figure 1.

Mucosal-associated invariant T-cell recovery takes several years to be completed in pediatric and young adult patients after allogeneic-hematopoietic stem cell transplantation. (A) Representation of the experimental design. Absolute count (B) and frequency (D) of αβ T cells in peripheral blood mononuclear cells (PBMC) of patients after either matched unrelated donor (MUD) (red line) or HLA-haploidentical (Haplo) (blue line) hematopoietic stem cell transplantation (HSCT) at various time points. Absolute count (C) and frequency (E) of mucosal-associated invariant T (MAIT) cells in PBMC of patients after MUD or Haplo-HSCT at various time points. MAIT-cell gating strategies are detailed in Online Supplementary Figure S1. Results are shown as mean ± Standard Error of Mean. Full statistical analysis between groups and time points is reported in Online Supplementary Table S7. Light gray area shows the physiological interval of cells in 16 age-matched healthy donors (HD). Data were analyzed by Mann-Whitney test. *P<0.05; **P<0.01; ****P<0.001.

Figure 2.

Mucosal-associated invariant T-cell subset recovery in allogeneic-hematopoietic stem cell transplantation patients. (A) Uniform Manifold Approximation and Projection (UMAP) embedding of merged mucosal-associated invariant T (MAIT) cells derived from allogeneic-hematopoietic stem cell transplantation (allo-HSCT) samples of healthy donor (HD), and HLA-haploidentical (HAPLO) and matched unrelated donor (MUD) patients one year following hematopoietic stem cell transplantation (HSCT). UMAP were generated on MAIT cells by gating on CD45⁺CD3⁺TCRαβ⁺MR1-5OP-RU-tetramer⁺ cells, and using phenotypic and functional T-cell markers, defined in Online Supplementary Table S1. Distribution of CD161⁺, CD4⁺ or CD8⁺ MAIT-cell subsets is represented. (B) UMAP distribution of MAIT cells derived from peripheral blood mononuclear cells (PBMC) samples of HD (green), and Haplo- (Blu) and MUD- (red) HSCT patients one year following HSCT. Each plot is representative of 5-6 concatenated samples. (C) Frequency of CD161⁺ cells among MAIT cells at different time points in PBMC of Haplo- (blue line) or MUD- (red line) HSCT recipients. (D) Frequency of CD8⁺, CD4⁺, and CD8^-CD4^- double negative (DN) cells among MAIT cells in PBMC of Haplo- (blue line) or MUD- (red line) HSCT recipients. Results are shown as mean ± Standard Error of Mean. Light gray area shows the physiological interval of cells in 16 age-matched HD. (E) Percentage of MAIT cells expressing CD161, CD8, CD4, and CD8^- CD4^- DN, in bone marrow (BM) from HD (N=11), peripheral blood stem cells (PBSC) derived from the positive fraction of the TCRαβ/ CD19-depletion procedure (N=5), PBMC from aged-matched HD (N=16), and adult HD (N=11). Results show individual values and median (horizontal bar). Data were analyzed by Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001; ns: not significant.

Figure 3.

Peripheral mucosal-associated invariant T cells express activation and exhaustion markers one year post-hematopoietic stem cell transplantation patients. (A) Bubble diagram showing the expression of different activation markers among mucosal-associated invariant T (MAIT) cell subsets in peripheral blood mononuclear cells (PBMC) of patients one year post hematopoietic stem cell transplantation (HSCT). A gradient of violet to light yellow indicates an increase in expression. The size of the bubble indicates the number of cells. (B) Percentage of MAIT cells expressing CD28, CD25, CD38, CD57, PD-1, and TIM-3 among healthy donors (HD) (gray dots, N=16), and HLA-haploidentical (Haplo)-HSCT (blue dots, N=39) or matched unrelated donor (MUD)-HSCT (red dots, N=35) patients one year after transplantation. Results show individual values and median (horizontal bar). Gating strategies are shown in Online Supplementary Figure S3. Data were analyzed by Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001. d: day.

Figure 4.

Altered functional status of mucosal-associated invariant T cells in patients one year after hematopoietic stem cell transplantation patients. (A) Box plots summarizing the concentration (ng/mL) of cytokines (IFNγ, TNFα, Granzyme B, and Perforin) in the culture supernatants after E. coli stimulation for 24 hours of peripheral blood mononuclear cells (PBMC) derived from healthy donors (HD) (N=5) or HSCT patients (matched unrelated donor [MUD], N=5; HLA-haploidentical [Haplo], N=5) one year after transplantation. Total cytokine levels in the cell culture supernatants were assessed using ELISA assay. Results are shown as mean ± Standard Error of Mean. (B, C) Frequency of CD25⁺CD69⁺ cells among mucosal-associated invariant T (MAIT) cells following aCD3 / aCD28 (B) and prefixed E. coli (C) in vitro activation assay. Results show individual values; dots represent MAIT cells derived from PBMC of HD (N=10) or HSCT (MUD, N=5; Haplo, N=10) patients one year after transplantation. Data were analyzed by Wilcoxon matched pairs signed rank test per row, with individual ranks computed for each comparison. *P<0.05, **P<0.01, ***P<0.005, ns: not significant. d: day; MFI: median fluorescence intensity.

Figure 5.

Incidence of graft-versus-host disease in patients grouped according to post-transplant mucosal-associated invariant T-cell recovery. Curves show cumulative incidence of acute graft-versus-host disease (aGvHD) (A, B) and chronic GvHD (cGvHD) (C, D) in patients with higher- or lower-than median absolute numbers of mucosal-associated invariant T (MAIT) cells (red or black line, respectively) on day 30 post transplantation. (E) Curves show probability of GvHD relapse-free survival (GRFS) in patients with higher- or lower-than median absolute numbers of MAIT cells (red or black line, respectively) on day 30 post transplantation. MAIT cells were defined as CD3⁺T-CRαβ⁺MR1-5OP-RU-Tetramer⁺.

Figure 6.

Incidence of late blood-stream infection and cytomegalovirus reactivation in patients grouped according to post-transplant mucosal-associated invariant T-cell recovery. Curves show cumulative incidence of late blood-stream infection (BSI) (A) and cytomegalovirus (CMV) (B) in patients with higher- or lower-than median absolute numbers of mucosal-associated invariant T (MAIT) cells (red or black line, respectively) on day 30 post transplantation. MAIT cells were defined as CD3⁺TCRαβ⁺MR1-5OP-RU-Te-tramer⁺. HSCT: hematopoietic stem cell transplantation.