Cellular and molecular profiling of T-cell subsets at the onset of human acute GVHD

Abstract

The cellular and molecular processes involved in acute graft-versus-host disease (aGVHD) development early after allogeneic hematopoietic cell transplantation (HCT) in humans remain largely unknown. We have performed multiparameter immunophenotyping and molecular profiling of CD4+ and CD8+ T cells in 2 independent cohorts of patients undergoing HCT, as well as in their HLA-identical sibling donors. Cellular profiling using spectral flow cytometry showed an incomplete reconstitution of the T-cell compartment in recipients without aGVHD early after transplantation, as well as a shift toward an effector memory phenotype, paralleled by depletion of the naive T-cell pool. Molecular profiling of T-cell populations in donors vs recipients without aGVHD revealed increased pathway activity of >40 gene modules in recipients. These pathways were associated in particular with T-cell activation, adhesion, migration, and effector functions. Cellular profiles from recipients developing aGVHD displayed an enrichment of cells with a T memory stem cell-like phenotype compared with recipients without aGVHD. Comparison of gene profiles from these recipients revealed that transforming growth factor-β (TGF-β) signaling was most significantly downregulated, whereas the pathway activity of NF-κB-associated transcription factors and signaling pathways were increased, at aGVHD onset. This study suggests that the integration of cellular and molecular profiles provides new insights into the development of aGVHD in humans.

Graphical abstract

Figure 1.

T-cell compartment in recipients without aGVHD after HCT. Shown are results for donor/recipient couples without aGVHD from cohort 1. (A) Frequency of CD3⁺ T cells within the lymphocyte population (left panel) and frequencies of CD4⁺ (middle panel) and CD8⁺ (right panel) T cells within CD3⁺ T cells. (B) Ratio of CD4^+/CD8⁺ T cells in recipients (R) at day 90 post-HCT compared with their respective sibling donors (D). (C) Gating strategy used to identify naive and memory subsets within the CD4⁺ and CD8⁺ T-cell compartments in donors and recipients after transplantation by polychromatic flow cytometry. (D) Frequencies of T_Naive, T_SCM-like, T_CM, T_EM, and T_EMRA cells within the CD4⁺ (upper panels) and CD8⁺ (lower panels) T-cell compartments in recipients (R) at day 90 post-HCT compared with their respective sibling donors (D). (E) Frequency of proliferating Ki-67⁺ cells in total CD4⁺ and CD8⁺ T cells and in the different naive and memory subsets in CD4⁺ (left panel) and CD8⁺ (right panel) compartments in donors and recipients. The frequency of Ki-67⁺ cells is represented as the percentage of Ki-67–expressing cells within total CD4⁺ or CD8⁺ T cells and as the percentage Ki-67–expressing cells within the parent gate for T_Naive, T_CM, T_EM, and T_EMRA cell subsets. Horizontal lines indicate the median. P values were calculated using the Wilcoxon matched-pairs Student t test (donor vs respective recipient). Differences are considered significant for P < .05.

Figure 2.

Treg homeostasis in recipients without aGVHD after HCT. Shown are results for donor/recipients couples without aGVHD from cohort 1. (A) Frequency of FOXP3⁺ cells within the CD4⁺ population in donors (D) and recipients (R) at day 90 post-HCT. (B) Frequencies of Ki-67⁺, within the CD4⁺ FOXP3⁺ Treg population, in donors (D) and recipients (R) at day 90 post-HCT. (C) Frequency of proliferating (Ki-67⁺) cells within the CD4⁺FOXP3⁺ Treg and CD4⁺FOXP3⁻ Tconv populations in donors (D) and recipients (R) (left panel) and ratios of proliferating (Ki-67⁺) Treg/Tconv in donors (D) and recipients (R) at day 90 post-HCT (right panel). (D) Frequencies of CTLA4⁺, ICOS⁺, and PD1⁺ cells within the CD4⁺FOXP3⁺ Treg population in donors (D) and recipients (R) at day 90 post-HCT. Horizontal lines indicate the median. P values were calculated using a Wilcoxon matched-pairs Student t test or a Mann-Whitney U test. Differences are considered significant for P < .05.

Figure 3.

Molecular signature of T cells in recipients without aGVHD after HCT compared with their donors. QuSAGE of CD4⁺ (A) and CD8⁺ (B) T-cell gene-expression profiles in recipients at day 90 post-HCT compared with their respective donors before transplant in cohort 1. For each pathway, the mean fold change and the 95% confidence intervals are plotted and color-coded according to their FDR-corrected P values compared with 0. Red and green bars indicate a statistically significant increased or decreased pathway activity, respectively, in recipients compared with donors. Correlation between cohorts 1 and 2 for QuSAGE in CD4⁺ (C) and CD8⁺ (D) T cells. Red dots correspond to modules significantly enriched (FDR ≤ 0.05) in both cohorts, blue dots correspond to modules enriched in only 1 cohort, and black dots correspond to modules not enriched. The concordance correlation coefficient (CCC) is displayed to quantify the reproducibility of the results between the 2 cohorts. Relative gene-expression levels of genes in selected enriched modules in recipients at day 90 post-HCT compared with their respective donors before transplant in cohort 1 in CD4⁺ (E) and CD8⁺ (F) T cells. In the heat maps, columns represent samples and are ordered by hierarchical clustering, whereas rows represent genes and are ranked by fold change. Yellow indicates high levels of expression, and blue indicates low levels of expression. Ag, antigen; CTL, cytotoxic T lymphocyte; MHC, major histocompatibility complex; TCR, T-cell receptor; TF, transcription factor; TGF, transforming growth factor; TLR, Toll-like receptor; TNF, tumor necrosis factor.

Figure 3.

Molecular signature of T cells in recipients without aGVHD after HCT compared with their donors. QuSAGE of CD4⁺ (A) and CD8⁺ (B) T-cell gene-expression profiles in recipients at day 90 post-HCT compared with their respective donors before transplant in cohort 1. For each pathway, the mean fold change and the 95% confidence intervals are plotted and color-coded according to their FDR-corrected P values compared with 0. Red and green bars indicate a statistically significant increased or decreased pathway activity, respectively, in recipients compared with donors. Correlation between cohorts 1 and 2 for QuSAGE in CD4⁺ (C) and CD8⁺ (D) T cells. Red dots correspond to modules significantly enriched (FDR ≤ 0.05) in both cohorts, blue dots correspond to modules enriched in only 1 cohort, and black dots correspond to modules not enriched. The concordance correlation coefficient (CCC) is displayed to quantify the reproducibility of the results between the 2 cohorts. Relative gene-expression levels of genes in selected enriched modules in recipients at day 90 post-HCT compared with their respective donors before transplant in cohort 1 in CD4⁺ (E) and CD8⁺ (F) T cells. In the heat maps, columns represent samples and are ordered by hierarchical clustering, whereas rows represent genes and are ranked by fold change. Yellow indicates high levels of expression, and blue indicates low levels of expression. Ag, antigen; CTL, cytotoxic T lymphocyte; MHC, major histocompatibility complex; TCR, T-cell receptor; TF, transcription factor; TGF, transforming growth factor; TLR, Toll-like receptor; TNF, tumor necrosis factor.

Figure 4.

Cellular correlates of aGVHD onset. (A) Ratios of the frequency of CD3⁺ T cells within lymphocytes (left panel), CD4⁺ T cells within CD3⁺ T cells (middle panel), and CD8⁺ T cells within CD3⁺ T cells (right panel) between recipients and donors (R/D) in aGVHD vs No aGVHD couples. (B) Ratios of the frequency of T_Naive, T_SCM-like, T_CM, T_EM, and T_EMRA cells within CD4⁺ cells (upper panels) and CD8⁺ cells (lower panels) between R/D in aGVHD vs No aGVHD couples. Horizontal lines indicate the median. For this analysis, samples from the 2 cohorts were pooled. All recipients without aGVHD or with grade ≥2 aGVHD were included in the analysis (No GVHD, n = 36; GVHD, n = 22). P values were calculated using the Mann-Whitney U test; differences are considered significant for P < .05.

Figure 5.

CD4⁺T-cell gene-expression signature at aGVHD onset. For this analysis, samples from the 2 cohorts were pooled. All recipients without aGVHD or with grade ≥2 aGVHD were included in the analysis (No GVHD, n = 35; GVHD, n = 24). (A) Gene-expression profiles of CD4⁺ T cells from 24 patients at aGVHD onset compared with 35 patients without aGVHD in cohorts 1 and 2. Orange and light blue dots represent transcripts that were significantly up- and downregulated in recipients at aGVHD onset compared with No GVHD recipients, respectively, with FDR < 0.05. P values were calculated using an unpaired Student t test. Adjusted P values (FDR) were calculated using the Benjamini-Hochberg method to correct for multiple comparisons. (B) Results of QuSAGE conducted on CD4⁺ T-cell gene-expression profiles from aGVHD and No GVHD recipients. Plotted is the negative logarithm to the base 10 of the P values for the 6 modules with significantly different pathway activity (FDR < 0.05) at GVHD onset compared with No GVHD recipients. (C) Transcript levels of TGFBR1, SMAD3, and IGF2R in CD4⁺ T cells from recipients without aGVHD (No GVHD) and at aGVHD onset (GVHD). These genes showed decreased expression at aGVHD onset compared with recipients without GVHD (P < .05; FDR < 0.1). (D) Transcript levels of BCL3, BCL10, ICOS, and PSMD7 in CD4⁺ T cells from recipients without aGVHD and at aGVHD onset. These genes showed increased expression at aGVHD onset compared with recipients without aGVHD (P < .05; FDR < 0.1). P values were calculated using an unpaired Student t test. Adjusted P values (FDR) were calculated using the Benjamini-Hochberg method to correct for multiple comparisons. Nominal P values are shown in panels B and D.

Figure 6.

CD8⁺T-cell gene-expression signature at aGVHD onset. For this analysis, samples from the 2 cohorts were pooled. All recipients without aGVHD or with aGVHD grade ≥2 were included in the analysis (No GVHD, n = 36; GVHD, n = 22). (A) Gene-expression profiles of CD8⁺ T cells from 22 patients at aGVHD onset compared with 36 patients without aGVHD in cohorts 1 and 2. Orange and light blue dots represent transcripts that were significantly up- or downregulated in recipients at aGVHD onset compared with No GVHD recipients, respectively, with FDR < 0.05. P values were calculated using an unpaired Student t test. Adjusted P values (FDR) were calculated using the Benjamini-Hochberg method to correct for multiple comparisons. (B) Transcript levels of NFKB1, BCL3, NFKBIA, ICOS, CD28, MIF, and GAPDH in CD8⁺ T cells from recipients without aGVHD and at aGVHD onset. These genes showed increased expression at aGVHD onset compared with recipients without GVHD (P < .05; FDR < 0.05). (C) QuSAGE of CD8⁺ T-cell gene-expression profiles in recipients at aGVHD onset compared with recipients without aGVHD from cohorts 1 and 2. For each pathway, the mean fold change and the 95% confidence intervals are plotted and color-coded according to their FDR-corrected P values compared with 0. Red and green bars indicate a statistically significant increased or decreased pathway activity, respectively, in recipients at aGVHD onset compared with recipients without GVHD. (D) Transcript levels of TGFBR2, SMAD3, IGFR2, LAIR1, BTLA, KLRG1, and IL10RA in CD8⁺ T cells from recipients without aGVHD and at aGVHD onset. These genes showed decreased expression at aGVHD onset compared with recipients without aGVHD (P < .05; FDR < 0.05). P values were calculated using an unpaired Student t test. Adjusted P values (FDR) were calculated using the Benjamini-Hochberg method to correct for multiple comparisons. Nominal P values are shown in panels C and D. Tfh, T follicular helper cell.