CD8 T cells compensate for impaired humoral immunity in COVID-19 patients with hematologic cancer

Abstract

Cancer patients have increased morbidity and mortality from Coronavirus Disease 2019 (COVID-19), but the underlying immune mechanisms are unknown. In a cohort of 100 cancer patients hospitalized for COVID-19 at the University of Pennsylvania Health System, we found that patients with hematologic cancers had a significantly higher mortality relative to patients with solid cancers after accounting for confounders including ECOG performance status and active cancer status. We performed flow cytometric and serologic analyses of 106 cancer patients and 113 non-cancer controls from two additional cohorts at Penn and Memorial Sloan Kettering Cancer Center. Patients with solid cancers exhibited an immune phenotype similar to non-cancer patients during acute COVID-19 whereas patients with hematologic cancers had significant impairment of B cells and SARS-CoV-2-specific antibody responses. High dimensional analysis of flow cytometric data revealed 5 distinct immune phenotypes. An immune phenotype characterized by CD8 T cell depletion was associated with a high viral load and the highest mortality of 71%, among all cancer patients. In contrast, despite impaired B cell responses, patients with hematologic cancers and preserved CD8 T cells had a lower viral load and mortality. These data highlight the importance of CD8 T cells in acute COVID-19, particularly in the setting of impaired humoral immunity. Further, depletion of B cells with anti-CD20 therapy resulted in almost complete abrogation of SARS-CoV-2-specific IgG and IgM antibodies, but was not associated with increased mortality compared to other hematologic cancers, when adequate CD8 T cells were present. Finally, higher CD8 T cell counts were associated with improved overall survival in patients with hematologic cancers. Thus, CD8 T cells likely compensate for deficient humoral immunity and influence clinical recovery of COVID-19. These observations have important implications for cancer and COVID-19-directed treatments, immunosuppressive therapies, and for understanding the role of B and T cells in acute COVID-19.

Extended Data Fig. 1 |. Inflammatory markers and blood cell counts in cancer patients with COVID-19.

Clinical laboratory values for (a) inflammatory markers and (b) cell counts in solid (n=62) and hematologic (n=21) cancer patients. (All) Significance determined by Mann Whitney test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Median and 95% CI shown.

Extended Data Fig. 2 |. SARS-CoV-2 antibody levels.

(a) Relative levels of SARS-CoV-2 IgG and IgM in non-cancer (n=108) and cancer (n=21) patients. (b) Relative IgG levels in cancer patients. Each dot represents a cancer patient (Heme: Red; Solid: Yellow). (All) Significance determined by Mann Whitney test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Median and 95% CI shown.

Extended Data Fig. 3 |. Dimensionality reduction and EMD clustering of MESSI cohort.

(a) UMAP projections of lymphocytes with indicated protein expression. (b) Frequencies of CD19+, CD3+, CD3+CD8+, and CD3+CD4+ cells of patients in each EMD cluster (Cluster 1 n=7; Cluster 2 n=16; Cluster 3 n=6; Cluster 4 n=10; Cluster 5 n=5). (All) Median and 95% CI shown.

Extended Data Fig. 4 |. Cellular phenotyping of COVID-19 patients with cancer.

(a) Frequencies of circulating T follicular helper cells (cTfh), plasmablasts, and CD138 expression on plasmablasts (HD n=33; non-cancer n=108; solid cancer n=7; heme cancer n=3). (b) UMAP projection of non-naïve CD8 T cells with indicated protein expression. (c) Heatmap showing expression patterns of various markers, stratified by FlowSOM clusters. Heat scale calculated as column z-score of MFI. (d) Frequencies of CD8 subsets: naive (CD45RA+CD27+CCR7+), central memory (CD45RA−CD27+CCR7+), transition memory (CD45RA−CD27+CCR7−), effector memory (CD45RA−CD27−CCR7−), and TEMRA (CD45RA+CD27−CCR7−) (HD n=33; non-cancer n=108; cancer n=9). (e) (Top) HLA-DR and CD38 coexpression in concatenated activated clusters (3, 4, and 5) and associated UMAP localization. (Bottom) Frequency of activated clusters (3, 4, and 5) in each patient (HD n=30; non-cancer n=110; solid-cancer n=8). (All) Significance determined by Mann Whitney test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Median and 95% CI shown.

Extended Data Fig. 5 |. Cellular, serologic, and clinical features in solid and hematologic cancer patients with COVID-19.

(a) Absolute counts of CD4, CD8, and CD19 expression in remission (n=11), solid cancer (n=23), and hematologic cancer (n=41) patients. (b) Relative levels of SARS-CoV-2 IgG and IgM in solid (n=11) and hematologic cancer (n=14) patients. (c) Severity (NIH ordinal scale for COVID-19 clinical severity) and RT-PCR cycle threshold (remission n=9; solid n=25; heme n=28) (Lower Ct: Higher viral load). (d) NIH ordinal scale for COVID-19 clinical severity. (All) Significance determined by Mann Whitney test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Median and 95% CI shown.

Extended Data Fig. 6 |. Dimensionality reduction and EMD clustering of MSKCC cohort.

(a) UMAP projections of lymphocytes with indicated protein expression. (b) Absolute counts of CD19+, CD3+, CD3+CD8+, and CD3+CD4+ cells of patients in each EMD cluster (Cluster 1 n=18; Cluster 2 n=6; Cluster 3 n=26; Cluster 4 n=7). (All) Median and 95% CI shown.

Extended Data Fig. 7 |. EMD Cluster 4 drives differences in mortality between hematologic and solid cancer patients.

(a) (Left) Number of patients with hematologic, solid, and remission cancer statuses within each EMD cluster. (Right) Mortality of patients within each EMD cluster for hematologic and solid cancers. (b) RT-PCR cycle threshold of solid and heme cancer patients in EMD cluster 4 (solid n=11; heme n=11). (c) Absolute CD8 and CD4 T cell counts for subjects in EMD cluster 4 stratified by solid (n=11) and heme (n=13) cancer. (d) Global UMAP projections of lymphocytes for subjects in EMD cluster 4: (Left) Hematologic cancer; (Middle) Solid cancer. (Right) Absolute B cell counts for subjects in EMD cluster 4 stratified by solid (n=11) and heme (n=13) cancer. (All) Significance determined by Mann Whitney test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Median and 95% CI shown.

Extended Data Fig. 8 |. Effect of cancer treatment on T cell differentiation in cancer patients with COVID-19.

(a) Absolute counts of CD4, CD8, and CD19 expressing cells. Frequencies of (b) CD4 and (c) CD8 T cell subsets in cancer patients treated with immune checkpoint blockade therapies, chemotherapies, and B cell depleting therapies. Naive (CD45RA+CCR7+), CM (CD45RA−CCR7+), EM (CD45RA−CCR7−), TEMRA (CD45RA+CCR7−). (All) Remission n=11, obs n=12, chemo only n=9, solid ICB n=7, and heme αCD20 n=10. Significance determined by Mann Whitney test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Median and 95% CI shown.

Extended Data Fig. 9 |. Association of mortality with cell counts and viral load.

(a) RT-PCR cycle threshold of patients treated with αCD20 therapy (alive n=7; dead n=3). (b) Absolute counts of CD8+, CD4+, and CD19+ cells in solid cancer patients (alive n=16; dead n=7). (All) Significance determined by Mann Whitney test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Median and 95% CI shown.

Fig. 1 |

Univariate analysis of potential risk factors in COVID-19 mortality.

Fig. 2 |. Hematologic cancer is an independent risk factor for COVID-19 related mortality.

(a) Kaplan Meier curve for COVID-19 survival of patients with solid (n=77) and hematologic (n=22) cancer. Cox regression-computed hazard ratio for mortality in hematologic vs solid cancer, adjusted for age, gender, smoking status, active cancer status, and ECOG performance status. (b) Ferritin, IL-6, and LDH in solid (n=62) and hematologic (n=15) cancer hospitalized for COVID-19. (All) Significance determined by Mann Whitney test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Median and 95% CI shown.

Fig. 3 |. High dimensional analyses reveal immune phenotypes associated with mortality and distinct phenotypes between solid and hematologic cancers.

(a) Demographic and mortality data for MESSI cohort at Penn. (b) Relative levels of SARS-CoV-2 IgG and IgM of solid (n=14) and hematologic (n=7) cancer patients and non-cancer patients (n=108). (c) (Left) Global UMAP projection of lymphocyte populations for all 45 patients pooled. (Right) Hierarchical clustering of Earth Mover’s Distance (EMD) using Pearson correlation, calculated pairwise for lymphocyte populations. (d) UMAP projection of concatenated lymphocyte populations for each EMD cluster. (Yellow: High Density; Black; Low Density) (e) Heatmap showing expression patterns of various markers, stratified by EMD cluster. Heat scale calculated as column z-score of MFI. (f) Mortality, disease severity, and SARS-CoV-2 antibody data, stratified by EMD cluster (Cluster 5 n=5; Cluster 1,2,3,4 n=40). Mortality significance determined by Pearson Chi Square test. Severity assessed with NIH ordinal scale for COVID-19 clinical severity (1: Death; 8: Normal Activity). (g) UMAP projections of concatenated lymphocyte populations for solid cancer, hematologic cancer, and non-cancer patients. (h) CD8 and CD4 T cell and B cell frequencies in healthy donors (HD) (n=33), non-cancer (n=108), solid cancer (n=7), and heme cancer (n=4). (i) UMAP projection of non-naive CD8 T cell clusters identified by FlowSOM. (j) (Top) UMAP projections of non-naïve CD8 T cells for non-cancer and cancer patients. (Bottom) UMAP projections indicating HLA-DR and CD38 protein expression on non-naive CD8 T cells for all patients pooled. (k) Frequency of activated FlowSOM clusters in HD (n=30), non-cancer (n=110), and cancer patients (n=8). (l) Representative flow plots and frequency of HLA-DR and CD38 co-expression in HD (n=30), non-cancer (n=110), solid cancer (n=7), and hematologic cancer (n=3) patients. (All) Significance determined by Mann Whitney test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Median and 95% CI shown.

Fig. 4 |. CD8 T cell counts associated with survival in hematologic cancer patients with COVID-19.

(a) Demographic and mortality data of MSKCC cohort. (b) (Left) Hierarchical clustering of Earth Mover’s Distance (EMD) using Pearson correlation, calculated pairwise for lymphocyte populations. (Right) Global UMAP projection of lymphocyte populations pooled. (c) UMAP projection of concatenated lymphocyte populations for each EMD cluster. (Yellow: High Density; Black: Low Density) (d) Mortality (Cluster 5 n=7; Cluster 1,2,4 n=50), severity, and RT-PCR cycle threshold (Cluster 1 n=14; Cluster 2 n=5; Cluster 4 n=24; Cluster 5 n=6) (Lower Ct: Higher viral load) stratified by EMD cluster. Mortality significance determined by Pearson Chi Square test. (e) Relative levels of SARS-CoV-2 IgG and IgM of patients with recent cancer treatments (solid tx n=9; heme αCD20 n=7; heme other tx n=5). (f) Mortality, severity, and RT-PCR cycle threshold stratified by cancer treatment (remission n=9; solid obs n=6; solid tx n=19; heme obs n=5; heme chemo n=4; heme αCD20 n=10). Severity assessed with NIH ordinal scale for COVID-19 clinical severity. (g) Recent cancer treatment of patients in each EMD cluster. (h) Mortality of patients treated with B cell depleting therapy in EMD cluster 1 (red) and EMD cluster 4 (blue). (i) Absolute CD8 and CD4 T cell counts in patients treated with B cell depleting therapy (alive n=7; dead n=4). (j) Absolute CD8 and CD4 T cell counts and B cell counts in hematologic cancer patients (alive n=17; dead n=18). (k) Kaplan-Meier curve for survival in hematologic cancer patients stratified by CD8 T cell counts (threshold = 55.9; log-rank hazard ratio) (>=55.9 n=28; <55.9 n=13). CD8 count threshold determined by Classification and Regression Tree (CART) analysis. (All) Significance determined by Mann Whitney test: *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Median and 95% CI shown.