Immunoprofiling reveals cell subsets associated with the trajectory of cytomegalovirus reactivation post stem cell transplantation

Abstract

Human cytomegalovirus reactivation is a major opportunistic infection after allogeneic haematopoietic stem cell transplantation and has a complex relationship with post-transplant immune reconstitution. Here, we use mass cytometry to define patterns of innate and adaptive immune cell reconstitution at key phases of human cytomegalovirus reactivation in the first 100 days post haematopoietic stem cell transplantation. Human cytomegalovirus reactivation is associated with the development of activated, memory T-cell profiles, with faster effector-memory CD4⁺ T-cell recovery in patients with low-level versus high-level human cytomegalovirus DNAemia. Mucosal-associated invariant T cell levels at the initial detection of human cytomegalovirus DNAemia are significantly lower in patients who subsequently develop high-level versus low-level human cytomegalovirus reactivation. Our data describe distinct immune signatures that emerged with human cytomegalovirus reactivation after haematopoietic stem cell transplantation, and highlight Mucosal-associated invariant T cell levels at the first detection of reactivation as a marker that may be useful to anticipate the magnitude of human cytomegalovirus DNAemia.

Fig. 1. Study design.

a HSCT recipients were divided into four groups: SN (HCMV-seronegative D-/R-; blue), SP-NR (HCMV-seropositive R + and/or D + , with no detected HCMV DNAemia in the first 100 days post-HSCT; red), LR (patients who developed low-level (<250 peak copies/mL) HCMV DNAemia; green), and HR (patients who developed high-level (> 830 peak copies/mL) HCMV DNAemia; purple). Graphs show HCMV DNA copies/mL plasma in the first 100 days post-transplant for LR (left) and HR (right). Each line represents one patient. The point of antiviral therapy initiation for each patient is indicated by the shift from solid line (pre-therapy) to dotted line (after therapy initiation). The horizontal dotted line at 150 copies/mL indicates the lower limit of quantitation (LLQ). b Representative HCMV reactivation profiles from one HR and one LR patient depicting the four time-points analysed with mass cytometry. Peripheral blood mononuclear cells (PBMC) collected prior to detection (T1), at the initial detection (T2), at the peak (T3) and near the resolution (T4) of HCMV DNAemia post-HSCT were analysed. The horizontal dotted line indicates the LLQ (150 HCMV DNA copies/mL). Samples from corresponding days post-transplant were analysed from HSCT recipients without HCMV reactivation. c HCMV DNA plasma copy load at each study time-point from patients with HCMV reactivation. Each line represents a patient. The horizontal dotted line at 150 copies/mL indicates the LLQ. Two-tailed Mann-Whitney test comparing HR and LR per time point (***p = 0.0003). d Days post-transplant for all PBMC samples analysed with mass cytometry. Lines indicate the mean. One-way ANOVA per time-point with Fisher’s Least Significant Difference test (ns, not significant). T1 (SN n = 11, SP-NR n = 5, LR n = 5, HR n = 9), T2 (SN n = 11, SP-NR n = 5, LR n = 6, HR n = 12), T3 (SN n = 10, SP-NR n = 5, LR n = 5, HR n = 12), T4 (SN n = 11, SP-NR n = 5, LR n = 5, HR n = 13). SN HCMV-seronegative, SP-NR Seropositive no reactivation, LR Low-level HCMV reactivation, HR High-level HCMV reactivation, D Donor, R Recipient, HCMV human cytomegalovirus, HSCT haematopoietic stem cell transplant. Source data are provided as a Source Data file.

Fig. 2. Reconstitution kinetics of peripheral blood mononuclear cell populations in the first 100 days post-HSCT.

a Absolute blood counts (x10⁹/L) of major immune cell subsets over the four study time-points in HSCT recipients with (LR, HR) or without (SN, SP-NR) HCMV reactivation. T1 is prior to detection of HCMV reactivation; T2, at initial detection of HCMV reactivation; T3, the peak; T4, near the resolution of HCMV reactivation. Blood samples from corresponding days post-HSCT were assessed in parallel from HSCT recipients without HCMV reactivation. Mean ± SEM is shown. Statistically significant comparisons are indicated (two-way mixed-effects model with Tukey’s multiple comparisons test applied to log-transformed data; *p < 0.05, **p < 0.01, ***p < 0.001). Horizontal lines indicate significant differences between pairs of time-points for the patient group(s) listed. Vertical brackets indicate significant differences between patient groups at T4. WBC: ^ap = 0.0020, ^bp = 0.0045, ^cp = 0.0041. Monocytes: ^ap = 0.0286. Lymphocytes: ^ap = 0.0440, ^bp = 0.0352, ^cp = 0.0431, ^dp = 0.0222. mDC: ^ap = 0.0477, ^bp = 0.0139. pDC: ^ap = 0.0113, ^bp = 0.0186, ^cp = 0.0459. NK cells: ^ap = 0.0347. B cells: ^ap = 0.0177, ^bp = 0.0366, ^cp = 0.0430, ^dp = 0.0137, ^ep = 0.0159, ^fp = 0.0296, ^gp = 0.0326. CD4⁺ T cells: ^ap = 0.0250, ^bp = 0.0099, ^cp = 0.0329, ^dp = 0.0393, ^ep = 0.0326. CD8⁺ T cells: ^ap = 0.0377, ^bp = 0.0166, ^cp = 0.0328, ^dp = 0.0042, ^ep = 0.0003, ^fp = 0.0205, ^gp = 0.0018. CD3⁺CD56⁺ cells: ^ap = 0.0213, ^bp = 0.0028. γδ T cells: ^ap = 0.0077, ^bp = 0.0336, ^cp = 0.0386, ^dp = 0.0019, ^ep = 0.0142. Tregs: ^ap = 0.0498, ^bp = 0.0347. b CD4:CD8 T cell ratio. Mean ± SEM is shown. Two-way mixed-effects model with Tukey’s multiple comparisons test after log-transformation. Horizontal line indicates significant difference between T2 and T3 in HR (p = 0.0250). T3: ^††p = 0.0100 for SN vs. LR; **p = 0.0045 for HR vs. SN. T4: *p = 0.0104 for HR vs. SN. For T1, T2, T3 and T4 respectively, SN (n = 11, n = 11, n = 10, n = 11), SP-NR (n = 5 for all), LR (n = 5, n = 6, n = 5, n = 5), HR (n = 9, n = 12, n = 12, n = 13). SN HCMV-seronegative, SP-NR Seropositive no reactivation, LR Low-level HCMV reactivation, HR high-level HCMV reactivation. mDC Myeloid dendritic cell, NK Natural killer, pDC plasmacytoid dendritic cell, Treg T regulatory cell, WBC White blood cells. Source data are provided as a Source Data file.

Fig. 3. Distinct immune reconstitution pattern in HSCT recipients with HCMV reactivation.

a Heat map rows display percentages of immune cell subsets that were significantly different between HSCT recipients who did (LR, HR) or did not (SN, SP-NR) experience HCMV reactivation in the first 100 days post-transplant. Percentages are expressed as percent of live cells and/or percent of parent subset. Significance was determined using two-class unpaired significance analysis of microarrays (SAM) involving 88 cell subsets per time-point (see Supplementary Table 4). Heat maps are coloured by the Z-score normalised per row (cell subset). Grey colour indicates missing data (too few cells in parent gate to accurately gate subpopulation). Each column represents a patient. b Heat map rows display cell subsets (absolute cell counts; x10⁹/L blood) that differ significantly between HCMV reactivators (LR, HR) and non-reactivators (SN, SP-NR) as determined by two-class unpaired SAM analysis involving 77 populations per time-point (see Supplementary Table 5). For T3 in (b), a FDR of 3.614% was used (0.831/23 subsets shown are false discoveries). Heat maps are coloured by the Z-score normalised per row (cell subset). Each column represents a patient. Patients with HCMV reactivation were subsequently stratified into ‘CD8^high’ (dark green box) or ‘CD8^low’ (black box) groups according to the presence or absence of the elevated CD8⁺ T cell dominated profile observed at T3. See Supplementary Fig. 3 for a schematic representation of cell subsets shown in Fig. 3a, b. T1 is prior to the detection of HCMV reactivation; T2, at the initial detection of HCMV reactivation; T3, the peak; T4, near the resolution of HCMV reactivation. Naive (CD45RA⁺ CD45RO^- CCR7⁺ CD27⁺), CM (central memory; CD45RA^- CD45RO⁺ CCR7⁺ CD27⁺), EM (effector memory; CD45RA^- CD45RO⁺ CCR7^- CD27^-), Mo (monocyte). T1 (SN n = 11, SP-NR n = 5, LR n = 5, HR n = 9), T2 (SN n = 11, SP-NR n = 5, LR n = 6, HR n = 12), T3 (SN n = 10, SP-NR n = 5, LR n = 5, HR n = 12), T4 (SN n = 11, SP-NR n = 5, LR n = 5, HR n = 13). SN HCMV-seronegative (blue), SP-NR Seropositive no reactivation (red), LR Low-level HCMV reactivation (green), HR High-level HCMV reactivation (purple), SD Standard deviation. Source data are provided as a Source Data file.

Fig. 4. Emergence of a CD8⁺ T cell dominated immune profile in a subset of HSCT recipients with HCMV reactivation.

a Absolute CD8⁺ T cell counts (x10⁹/L) in HSCT recipients with or without HCMV reactivation. Each line represents one patient. Dotted line indicates median CD8⁺ T cells across all patients at T3 (0.3116 × 10⁹/L). Reactivators were partitioned into ‘CD8^high’ (n = 10) and ‘CD8^low’ (n = 7) groups according to a CD8⁺ T cell-dominated signature at T3 in Fig. 3b. Graph on right: mean ± SEM, two-way mixed-effects model with Tukey’s multiple comparisons tests after log transform. On graph: *p = 0.0153 and ***p = 0.0002 for CD8^high (dark green) vs. CD8^low (black); ^†p = 0.0143 CD8^high vs. SN (HCMV-seronegative). Significant differences between pairs of time-points for patient groups are boxed: ^ap = 0.0118, ^bp = 0.0017, ^cp = 0.0043, ^dp = 0.0009, ^ep = 0.0384, ^fp = 0.0166. For T1, T2, T3 and T4 respectively: CD8^high (n = 7, n = 10, n = 10, n = 10), CD8^low (n = 5, n = 7, n = 7, n = 6), SN (n = 11, n = 11, n = 10, n = 11), SP-NR (n = 5 for all), in (a; right graph) and (b). b CD8⁺ T cells (percentage of live cells) in CD8^high reactivators (dark green), CD8^low reactivators (black), SN (blue) and SP-NR (seropositive no reactivation; red). Mean ± SEM. On graph: CD8^high vs. CD8^low (***p = 0.0005, *p = 0.0249), CD8^high vs. SN (^††p = 0.0025, ^†p = 0.0414), CD8^high vs. SP-NR (^##p = 0.0029). In box adjacent: ^ap = 0.0400, ^bp = 0.0071, ^cp = 0.0016, ^dp = 0.0022, ^ep = 0.0009, ^fp = 0.0177. In (b, c), two-way mixed-effects models with Tukey’s multiple comparisons tests. c Percentage of Granzyme B (GzmB⁺) CD8⁺ T cells in CD8^high reactivators, CD8^low reactivators, SN and SP-NR. Mean ± SEM shown. On graph: at T1 (**p = 0.0052, ***p = 0.0002), at T3 (**p = 0.0063), at T4 (CD8^low vs. SN, ***p = 0.0004; CD8^high vs. SN, ***p = 0.0005). In box: ^ap = 0.0446, ^bp = 0.0021, ^cp = 0.0027. For T1, T2, T3 and T4 respectively: CD8^high (n = 7, n = 10, n = 10, n = 10), CD8^low (n = 5, n = 7, n = 7, n = 6), SN (n = 10, n = 11, n = 10, n = 11), SP-NR (n = 4, n = 5, n = 5, n = 5). Reactivators without a T3 sample (n = 1 LR, n = 1 HR) are not shown in (a; right graph), (b) or (c). LR Low-level reactivation, HR High-level reactivation, T1 Before detection, T2 Initial detection, T3 Peak, and T4 Near resolution, of reactivation. Source data are provided as a Source Data file.

Fig. 5. CD8⁺ MAIT cell frequencies at the initial detection of reactivation distinguish low-level and high-level HCMV reactivators.

CD8⁺ MAIT cells were identified as Vα7.2⁺CD161^hiCD8⁺ T cells. a Absolute counts (x10⁹/L blood) of CD8⁺ MAIT cells. Mean ± SEM shown. On graph: *p = 0.0184, **p = 0.0026, ***p = 0.0002. b CD8⁺ MAIT cells as a percentage of live cells. Mean ± SEM shown. On graph: **p = 0.0040, *p = 0.0197, ***p = 0.0003. In (a, b), two-way mixed-effects models with Tukey’s multiple comparisons test after log transformation. For T1, T2, T3 and T4 respectively: SN (n = 11, n = 11, n = 10, n = 11), SP-NR (n = 5 for all), LR (n = 5, n = 6, n = 5, n = 5), HR (n = 9, n = 12, n = 12, n = 13). c Two-tailed Spearman correlations at T2 and T4 between CD8⁺ MAIT cells (x10⁹/L) in patients with HCMV reactivation (LR (green), HR (purple)) and the log₁₀ area under the curve of HCMV DNA copies/mL over 0-100 days post-HSCT (AUC_0-100). At T2, LR n = 6, HR n = 12. At T4, LR n = 5, HR n = 13. d Two-tailed Spearman correlations at T2 and T4 between CD8⁺ MAIT cell percentages in patients with HCMV reactivation (LR (green), HR (purple)) and the log₁₀ HCMV DNA AUC_0-100. e Median signal intensity (MSI) of CD38 on CD8⁺ MAIT cells. Mean ± SEM, two-way mixed-effects model with Tukey’s multiple comparisons test. For LR group, *p = 0.0105 between T2 and T4. At T4, **p = 0.006 for LR vs. HR; ^†p = 0.0188 SN vs. LR; ^#p = 0.0302 SP-NR vs. HR. For T1, T2, T3 and T4 respectively: SN (n = 9, n = 11, n = 10, n = 11), SP-NR (n = 4, n = 5, n = 5, n = 5), LR (n = 5, n = 6, n = 5, n = 5), HR (n = 7, n = 10, n = 12, n = 13). f, g Receiver-operating characteristic (ROC) curves for the performance of CD8⁺ MAIT cells (red), CD8⁺ T cells (blue) and CD4⁺ T cells (green) at T2 in discriminating LR and HR patients; (f) Shows absolute counts (x10⁹/L), (g) as percentages of live cells. Optimal CD8⁺ MAIT cell cut-offs were (f) 0.001337 × 10⁹ cells/L (91.67% sensitivity, 83.33% specificity) and (g) 0.0605% of live cells (100% sensitivity, 83.33% specificity). T1 is prior to detection; T2, at initial detection; T3, the peak; T4, near resolution of reactivation. SN HCMV-seronegative, SP-NR Seropositive no reactivation, LR Low-level reactivation, HR High-level reactivation. Source data are provided as a Source Data file.

Fig. 6. Expansion of effector-memory CD4⁺ T cells in HSCT recipients with low-level HCMV reactivation.

a Absolute counts (x10⁹/L blood) of effector-memory (EM; CD45RA^-CD45RO⁺CCR7^-CD27^-) CD4⁺ T cells. Mean ± SEM, two-way mixed-effects model with Tukey’s multiple comparisons test after log transformation. Horizontal lines display significant comparisons between indicated time-points for the patient groups shown: for LR, *T1-T2 p = 0.0213, **T1-T3 p = 0.0045, *T1-T4 p = 0.0447; for SP-NR, *T3-T4 p = 0.0428. Vertical brackets indicate significant differences between patient groups at T4 (*p = 0.0257 for LR vs. HR; **p = 0.0089 LR vs. SN). For T1, T2, T3 and T4 respectively: SN (n = 11, n = 11, n = 10, n = 11), SP-NR (n = 5 for all), LR (n = 5, n = 6, n = 5, n = 5), HR (n = 9, n = 12, n = 12, n = 13). b Percentage of CD4⁺ T cells with EM phenotype. Lines indicate mean ± SEM. Two-way mixed-effects model with Tukey’s multiple comparisons test. At T2, *p = 0.0399. At T3, *p = 0.0216 for SN vs. HR, *p = 0.0232 LR vs. HR, ***p = 0.0003 SN vs. LR. At T4, *p = 0.0483 LR vs. HR, **p = 0.0071 SN vs. HR, **p = 0.0013 SN vs. LR. For LR; ^aT1-T3 p = 0.0031, ^bT1-T4 p = 0.0178. For T1, T2, T3 and T4 respectively: SN (n = 9, n = 11, n = 10, n = 11), SP-NR (n = 4, n = 5, n = 5, n = 5), LR (n = 5, n = 6, n = 5, n = 5), HR (n = 8, n = 12, n = 12, n = 13). c Two-tailed Spearman correlation between log₁₀ area under the curve (AUC) of HCMV DNA copies/mL over 0–100 days post-transplant, and EM CD4⁺ T cell percentages (of total CD4⁺ T cells) at T3 (peak of HCMV reactivation) in HSCT recipients with HCMV reactivation (low-level (<250 copies/mL) reactivation (green; n = 5); high-level (>830 copies/mL) reactivation (purple; n = 12)). d Two-tailed Spearman correlation between peak HCMV copies/mL and EM CD4⁺ T cell percentages at T3. The vertical black dotted line at 150 HCMV copies/mL indicates the lower limit of quantitation of the HCMV DNA quantitative PCR assay. T1 is prior to detection of HCMV reactivation; T2, at initial detection; T3, the peak; T4, near resolution of HCMV reactivation. SN HCMV-seronegative (blue), SP-NR Seropositive no reactivation (red), LR Low-level HCMV reactivation (green), HR High-level HCMV reactivation (purple). Source data are provided as a Source Data file.