Distinctive features of SARS-CoV-2-specific T cells predict recovery from severe COVID-19

Abstract

Although T cells are likely players in SARS-CoV-2 immunity, little is known about the phenotypic features of SARS-CoV-2-specific T cells associated with recovery from severe COVID-19. We analyzed T cells from longitudinal specimens of 34 COVID-19 patients with severities ranging from mild (outpatient) to critical culminating in death. Relative to patients that succumbed, individuals that recovered from severe COVID-19 harbored elevated and increasing numbers of SARS-CoV-2-specific T cells capable of homeostatic proliferation. In contrast, fatal COVID-19 displayed elevated numbers of SARS-CoV-2-specific regulatory T cells and a time-dependent escalation in activated bystander CXCR4+ T cells. Together with the demonstration of increased proportions of inflammatory CXCR4+ T cells in the lungs of severe COVID-19 patients, these results support a model whereby lung-homing T cells activated through bystander effects contribute to immunopathology, while a robust, non-suppressive SARS-CoV-2-specific T cell response limits pathogenesis and promotes recovery from severe COVID-19.

Figure 1.. De novo IFNγ-producing SARS-CoV-2 spike-specific CD4+ and CD8+ T cells are elicited during mild, moderate, and severe COVID-19.

(A) Pseudocolor plots of CyTOF datasets reflecting percentages of CD4+ or CD8+ T cells producing IFNγ at baseline or in response to a 6-hour stimulation with overlapping peptides from SARS-CoV-2 spike in representative donors from mild, moderate, and severe cases. Results are gated on live, singlet CD4+ or CD8+ T cells. (B) Percentages of CD4+ and CD8+ T cells producing IFNγ in response to spike peptide stimulation in all COVID-19 cases. ns: non-significant as determined by one-way analysis of variance with a Bonferroni post-test. (C) Evidence for CD4+ and CD8+ T cell lymphopenia in specimens from moderate and severe COVID-19 relative to mild cases. ** p < 0.01 as determined by one-way analysis of variance with a Bonferroni post-test. (D) Proportion of IFNγ-producing cells among T cells from ICU patients with or without SARS-CoV-2 infection. Even in the uninfected specimen with the highest response to spike stimulation (red dot), the proportion of IFNγ-producing cells was only 0.01% (inset), suggesting that the responses we detect in COVID-19 specimens correspond to de novo SARS-CoV-2-specific T cells. *** p < 0.001 as determined by a Student’s unpaired t-test. See also Fig. S1, S2 and S3.

Figure 2.. Subset distribution of total and SARS-CoV-2-specific T cells during mild, moderate, and severe COVID-19 by manual gating.

(A, B) Distribution of baseline and SARS-CoV-2-specific CD4+ (A) and CD8+ (B) T cells from COVID-19 patients among memory (CD45RO+CD45RA−) and a mixture of Tn, Temra, and Tscm (CD45RO−CD45RA+) cells. Breakdown into further subsets was achieved by manually gating on subset marker combinations. (C) Proportions of Tregs (CD45RO+CD45RA−CD25+CD125^low) and cTfh (PD1+CXCR5+) cells among baseline and SARS-CoV-2-specific CD4+ T cells from COVID-19 patients. (D) The proportion of apoptosis-prone (PD1+CD95+) cells among CD4+ and CD8+ T cells is increased in severe relative to mild COVID-19 cases for both total and SARS-CoV-2-specific T cells. (E) The proportion of exhausted cells (PD1+TIGIT+) among total and SARS-CoV-2-specific CD8+ T is increased in severe relative to mild COVID-19. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as determined by one-way analysis of variance with a Bonferroni post-test.

Figure 3.. Clustering reveals enrichment in activated and exhausted SARS-CoV-2-specific CD4+ T cells during severe COVID-19.

(A, C) FlowSOM clustering of total (Baseline, A) and SARS-CoV-2-specific (C) CD4+ T cells from all infected patient groups, visualized as a t-SNE plot. Ten clusters were identified. Each cluster name begins with a “B” (for baseline) or an “S” (for SARS-CoV-2-specific), followed by a “4” (for CD4). (B, D) Total (B) and SARS-CoV-2-specific (D) CD4+ T cells from mild (grey), moderate (blue), and severe (red) COVID-19 map to different areas of the panel A t-SNE plot, indicating that they have different phenotypes. (E) Proportion of total CD4+ T cells in the B4.9 cluster in the three patient groups. B4.9 enrichment in the severe relative to the mild group was significant (p < 0.05) prior to adjustment for multiple testing by Bonferroni. (F) High expression levels of memory marker CD45RO, activation marker HLADR, and chemokine receptor CCR6 in cells from the B4.9 cluster (yellow trace) relative to total CD4+ T cells (grey trace). (G) Proportion of SARS-CoV-2-specific CD4+ T cells in the S4.5 and S4.10 clusters across the three patient groups. Cluster S4.5 was significantly enriched in the severe relative to the mild group, and cluster S4.10 in the mild relative to the severe group. *** p < 0.001 and **** p < 0.0001 as determined by Student’s unpaired t-testand adjusted for multiple testing using Bonferroni for FDR. (H) Cluster S4.5 cells express elevated levels of the activation markers HLADR and CD38 and the exhaustion markers PD1 and CTLA4, but diminished levels of the IL7 receptor alpha chain CD127 (orange traces) relative to all SARS-CoV-2-specific CD4+ T cells (grey traces). Cluster S4.10 cells (maroon traces) do not. (I) Increased frequencies of exhausted (PD1+CTLA4+) cells among SARS-CoV-2-specific CD4+ T cells in severe relative to mild COVID-19 samples, as validated by manual gating. * p < 0.05 and ** p < 0.01 as determined by one-way analysis of variance with a Bonferroni post-test.

Figure 4.. Clustering reveals enrichment of activated, exhausted SARS-CoV-2-specific CD8+ T cells during severe COVID-19.

(A, C) FlowSOM clustering of total (Baseline, A) and SARS-CoV-2-specific (C) CD8+ T cells from all infected patient groups, visualized as t-SNE plots. Ten clusters were identified. Each cluster name begins with a “B” (for baseline) or an “S” (for SARS-CoV-2-specific), followed by a “4” (for CD4). (B, D) Total (B) and SARS-CoV-2-specific (D) CD8+ T cells from mild (grey), moderate (blue), and severe (red) COVID-19 map to different areas of the t-SNE plots, indicating that they have different phenotypes. (E) Clusters B8.5, B8.6, B8.7 and B8.8 of total CD8+ T cells are significantly enriched in severe relative to mild group, while there is a trend for cluster B8.1 enrichment in mild relative to severe group. * p < 0.05, ** p < 0.01, and **** p < 0.0001 as determined by Student’s unpaired t-testand adjusted for multiple testing using Bonferroni for FDR. Of note, when not adjusting for multiple testing, cluster B8.1 was significantly enriched (p < 0.05) in mild vs. severe groups. (F) Cells from the B8.5, B8.6, B8.7 and B8.8 (severe-enriched) clusters all express high levels of the activation marker HLADR (colored traces) relative to total CD8+ T cells (grey trace), while activation marker CD38 is elevated in only a subset of these clusters (colored traces) relative to total CD8+ T cells (grey trace). Cells from the B8.1 (mild-enriched) cluster express low levels of both activation markers. (G) Cluster S8.4 is enriched in the severe and moderate groups relative to the mild group. ** p < 0.01 as determined as assessed by Student’s unpaired t-testand adjusted for multiple testing using Bonferroni for FDR. (H) Cluster S8.4 cells express elevated levels of the activation markers HLADR and CD38 and the exhaustion markers PD1 and CTLA4, but diminished levels of the IL7 receptor alpha chain CD127 (green traces) relative to all SARS-CoV-2-specific CD8+ T cells (grey traces). (I) Increased frequencies of exhausted (PD1+CTLA4+) cells among SARS-CoV-2-specific CD8+ T cells in severe relative to mild COVID-19 validated by manual gating. * p < 0.05 as determined by one-way analysis of variance with a Bonferroni post-test.

Figure 5.. Distinct features of SARS-CoV-2-specific T cells associate with recovery from severe COVID-19.

(A) Higher frequencies of SARS-CoV-2-specific T cells in individuals with severe COVID-19 that survived (“survivor”) than in those that did not (“non-survivor”). Left: Representative pseudocolor plots reflecting the percentage of SARS-CoV-2-specific CD4+ or CD8+ T cells from representative infected individuals in the ICU that did or did not survive COVID-19. Right: Quantification of the data shown in (A) across all infected ICU individuals. * p < 0.05, ns: non-significant as determined by a Student’s unpaired t-test. (B) SARS-CoV-2-specific CD8+ T cells from non-survivors produce elevated levels of IL6. Left: Pseudocolor plots of baseline and spike-stimulated samples from the two patients harboring the highest levels of SARS-CoV-2-specific CD8+ T cells in panel A, only one of whom survived. Top right: Histogram plots showing expression levels of IL6 in non-survivor and survivor CD8+ T cells. Bottom right: Proportion of IL6-producing cells among total and SARS-CoV-2-specific T cells in all non-survivor and survivor patients. (C) SARS-CoV-2-specific Tregs but not cTfh cells are significantly more abundant in non-survivors of severe COVID-19. See also Fig. S4 – S8.

Figure 6.. Clustering reveals enrichment of long-lived, activated SARS-CoV-2-specific CD4+ T cells in patients that successfully recover from severe COVID-19.

(A, B) The phenotypes of total (A) and SARS-CoV-2-specific (B) CD4+ T cells differ between non-survivors and survivors of severe COVID-19. Left: tSNE plot and FlowSOM clusters identified in Fig. 3A (A) and Fig. 3C (B). Right: Distribution of CD4+ T cells from non-survivors (pink), survivors (purple), and mild and moderate patient groups (grey). (C) Activated memory CD4+ T cells are elevated in survivors of severe COVID-19. Left: Clusters B4.1 and B4.9 are enriched in the survivor relative to the non-survivor group. * p < 0.05 as determined by a Student’s unpaired t-test. Right: Cells from clusters B4.1 and B4.9 are memory (CD45RO+) cells and express elevated levels of activation marker HLADR. Other activation markers (CD38, CD69, CCR5) are preferentially elevated only in cluster B4.1. Expression levels are displayed in grey for total CD4+ T cells and in color for clusters. (D) Survivors tend to harbor more activated (HLADR+CCR5+) CD4+ T cells. (E) Left: Cluster S4.5 is enriched in non-survivors, and cluster S4.9 in survivors. Right: Cells from cluster S4.5 co-express exhaustion markers PD1 and CTLA4, while cells from cluster S4.9 express high levels of cTfh marker CXCR5, low levels of exhaustion markers PD1 and CTLA4, high levels of IL7 receptor alpha chain CD127, and high levels of activation marker CD69. Expression levels from each cluster (colored traces) are shown against expression levels from all SARS-CoV-2-specific CD4+ T cell clusters combined (grey traces). * p < 0.05 as determined by a Student’s unpaired t-test. (F, G) SARS-CoV-2-specific CD4+ T cells expressing the homeostatic proliferation marker CD127 (F) or co-expressing CD127 with the activation marker CD69 (G) are more abundant in survivors than in non-survivors. * p < 0.05, ** p < 0.01, as determined by a Student’s unpaired t-test. See also Fig. S9 and S10.

Figure 7.. Escalating numbers of SARS-CoV-2-specific and diminishing numbers of lung-homing bystander CXCR4+ T cells correlate with survival of severe COVID-19.

(A, B) Longitudinal analysis of a patient that survived (A) and did not survive (B) severe COVID-19, demonstrating an increasing number of SARS-CoV-2 T cells producing IFNγ over time in the survivor but not non-survivor. (C) Activated CXCR4-expressing T cells decrease over time in survivors but not non-survivors. Left: Pseudocolor plots showing gating for CD69+CXCR4+ and CD69−CXCR4− cells among total CD4+ and CD8+ T cells in longitudinal specimens from a representative survivor of severe COVID-19. Right: Longitudinal analyses of 7 cases of severe COVID-19, 4 of which survived infection. While the frequencies of CXCR4−CD69− T cells increase over time in survivors (blue lines), they decrease over time in non-survivors (red lines). Not shown are the opposite trend for CXCR4+CD69+ cells which over time decrease in survivors and increase in non-survivors. (D-I) Higher levels of infiltrating CXCR4+CD69+ T cells are present in lungs of severe/critical vs. moderate COVID-19 patients as determined by mining of public BAL scRNAseq datasets. Higher levels of SARS-CoV-2 viral reads in T and epithelial cells were detected during severe COVID-19 (D), which were also associated with higher levels of CXCR4 in T cells as depicted by violin plots (E). Note viral reads detected in T cells likely reflect cell-surface virion sticking as these cells do not express ACE2. UMAP visualization of T and epithelial cells from the scRNAseq datasets (F) revealed elevated expression of CXCR4 and CD69 on subsets of T cells (G) separate from T cells expressing high levels of the Trm marker CD103 (H). The CXCR4 ligand HMGB1 was expressed in T and epithelial cells, but especially concentrated among a subset of CXCR4−CD69− Trm CD8+ T cells outlined in purple (I). See also Fig. S11.