INTRODUCTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection–associated respiratory disease [coronavirus disease 2019 (COVID-19)] was declared a pandemic by the World Health Organization (WHO) on March 11, 2020.1 With the rise of the second wave, as of May 2, 2021, a total of 211 005 326 cases of COVID-19 were diagnosed worldwide resulting in 4 419 869 deaths, thus representing the greatest public health crisis of the last few decades.2 COVID-19 usually manifests with flu-like symptoms; however, a subset of patients, especially elderly patients with comorbidities, can deteriorate to severe disease (SD) with acute respiratory distress syndrome and multiorgan failure resulting in mortality.3-5 Several clinical trials are being conducted to treat the SD effectively, and identifying SD during the early phase of infection is essential to reduce mortality.6 Hence, clinically reliable biomarkers of SD during its early phase of the disease are urgently required.

S-protein and N-protein of SARS-CoV-2 have been described as the most immunogenic protein, and many studies have suggested a protective role of cell-mediated and humoral immune responses in COVID-19 infection.5, 7-10 Recent studies have suggested the central role of cellular immune response and inflammatory cytokines in the pathogenesis of COVID-19. An association of immunological parameters, such as absolute lymphocyte count, neutrophil-to-T-cell ratio, various T-cell subsets and increased proinflammatory cytokine levels, with the clinical course of COVID-19 has also been suggested.3, 5, 6, 11-14 More recently, studies have comprehensively reported the dysregulation in innate and adaptive immune responses in COVID-19.11, 15 However, reports elaborating the immune response parameters during the early phase of infection associated with SD in cancer patients are scarce. Hence, studies describing detailed cellular immune responses in the early phase of infection, providing early insight into the host immune response and possible clinical course of disease in patients with cancer, are needed. We prospectively studied about 200 immune response parameters, including a comprehensive immune cell profile, inflammatory cytokines and other laboratory parameters, in patients diagnosed with COVID-19 in the early phase of infection and their association with SD in cancer patients without intensive chemo/immunotherapy.

RESULTS Clinical characteristics

A total of 95 patients diagnosed with COVID-19 and 21 healthy donors (median age of 32 years, range 23–70 years) were included in this study. We included 58 (61.05%) patients without cancer and 37 (38.95%) cancer patients without intensive chemo/immunotherapy. Details of demographical characteristics and laboratory findings are given in Table 1. The median period between COVID-19 symptoms and admission/quarantine to the hospital was 2 days (range 0–14 days). Sixty-six (69.5%) patients were admitted within the first 4 days, and all except four patients (8–14 days) within the first 7 days of COVID-19 symptoms.

At the time of admission, 89/95 (93.68%) patients were classified into nonsevere disease (NsD) (SpO2 levels: median 98% and range 93–100%) and 6/95 (6.32%) into SD (SpO2 levels 88–90%). Patients were reclassified into NsD (n = 54) and SD (n = 41) based on their worst WHO score during the hospital stay.16, 17 The median time since first symptom for the first blood draw for immune monitoring was 2 days (range 0–14 days) in patients with NsD and 2.5 days (range 1–8 days) in those with SD. Notably, the median time since first day of symptom to the development of SD was 9 days (range 4–43 days).

The median age (range) for patients with NsD was 42 years (16–80 years) and for those with SD 63 years (27–84 years). Age over 59 years (receiver operating characteristic–based cut-off) was strongly associated with SD [odds ratio (OR) 16.23; P < 0.0001]. Frequency of SD in patients with comorbidities, including cancer, was high (50.7% versus 22.2%; OR 4.62, P = 0.01). There was no difference in SD frequency between patients with hematological and nonhematological cancer (P = 0.1). The median duration of follow-up was 31 days (range 4–92 days). The median time since admission to the hospital for developing SD was 5 days (range 1–17 days). Of the 41 patients with SD, 18 recovered from COVID-19, but 23/41 (56%) patients succumbed to acute respiratory distress syndrome.

Immune cell landscape

We studied absolute counts of immune cell subsets and their relative proportions in the respective compartments. Collectively, 175 immune cell parameters derived from 69 immune cell subsets were studied. Details of immune cell perturbations in COVID-19 patients (with or without cancer) compared with healthy donors are given in Supplementary table 1 (Figures 1-3). The details of sensitivity, specificity and OR of the absolute counts of the most significant immune cell parameters with area under the curve of 0.75 or higher are given in Table 2 (Supplementary tables 1 and 2).

The levels of various immune cell subsets in a healthy donor (HD; n = 21), in patients with nonsevere COVID-19 (NsD; n = 54) and in patients with severe COVID-19 (SD; n = 41). Patients with cancer are indicated with “C” (n = 37) and without cancer with “Nc” (n = 58). (a–c) The distribution of complete cell count parameters in healthy donors and COVID-19 patients. (d–o) The distribution of important immune cell subsets in healthy donors and COVID-19 patients. (p) Representative flow cytometry plots depicting the gating strategy for key CD4 T-cell subsets (Th1, Th2, Th9, Th17 and Tfh) after exclusion of T-regulatory cells. (q) Representative flow cytometry plots depicting the gating strategy for identifying basophils and dendritic cell subsets. ALC, absolute lymphocyte count; ANC, absolute neutrophil count; Baso, basophil; COVID-19, coronavirus disease 2019; EM, effector memory; fh, follicular helper; mDC, myeloid dendritic cell; Nc, nonclassical; NsD, nonsevere disease; NTC, neutrophil-to-T-cell ratio; pDC, plasmacytoid dendritic cell; reg, regulatory; SD, stable disease; Th, T helper cell; TLC, total leukocyte count.

Heatmap showing absolute counts of various statistically relevant immune cell subsets in healthy donors and COVID-19 patients. Each row in the heatmap represents an immune cell parameter, and each column represents a patient. The scale is normalized for each parameter. Patients with cancer are indicated with “C” (n = 37), without cancer with “Nc” (n = 58), healthy donors with “H” (H < 50, i.e. age < 50 years; n = 11 and H > 50, i.e. age ≥ 50 years; n = 10), nonsevere disease with “NsD” (n = 54) and severe disease with “SD” (n = 41). Act, activated; ALC, absolute lymphocyte count; ANC, absolute neutrophil count; B, B cell; Baso, basophils; C, classical; CM, central memory; COVID-19, coronavirus disease 2019; cPC, circulating plasma cell (plasmablasts); Eff, effector; EM, effector memory; Eo, eosinophil; Er, early; Exh, exhausted (PD1+ T cells); fh, follicular helper; GD, γδ T cells; I, intermediate; mDC, myeloid dendritic cell; MDSC, myeloid-derived suppressor cell; Mem, memory; Mono, monocytic; Nc, nonclassical; NsD, nonsevere disease; NTC, neutrophil-to-T-cell ratio; NtM, natural memory; NV, naïve; pDC, plasmacytoid dendritic cell; PMN, polymorphonuclear; reg, regulatory; SD, stable disease; T, T cells; t, terminal; TE, terminal effector; TLC, total leukocyte count; Tr, transitional.

The levels of absolute counts of statistically relevant immune cell subsets demonstrating the comparison between severe COVID-19 in patients with cancer (C, n = 18) and without cancer (C, n = 23) as well as age-wise categorized healthy donors. Healthy donors with age under 50 years are indicated with H < 50 (n = 11) and those aged 50 years or older are indicated with H > 50 years (n = 10). (a–e) The distribution of CD4 T-cell subsets in age-matched healthy donors and patients (cancer and non-cacner) with severe COVID-19. The distribution of γδT-cells (f), transitional B cells (g), intermediate-type monocytes (h), conventional (myeloid) dendritic cells (i) and plasmacytoid dendritic cells (j) in age-matched healthy donors and patients (cancer and non-cancer) with severe COVID-19. B, B cell; BTr, transitional B cells; C, classical; EM, effector memory; HD, healthy donor; I, intermediate; mDC, myeloid dendritic cell; mono, monocytic; Nc, nonclassical; NsD, nonsevere disease; pDC, plasmacytoid dendritic cell; SD, stable disease; T, T cell; TE, terminal effector; Th, T helper cell; Tr, transitional; Treg, regulatory T cell.

Table 1. Baseline demographic and clinical characteristics of patients with COVID-19 Characteristics Total (n = 95) Nonsevere disease (n = 54) Severe disease (n = 41) Age (years), median (range) 52 (16–84) 41.5 (16–80) 63 (27–84) Sex, n (%) Male 57 (60) 34 (63) 23 (56.1) Female 38 (40) 20 (37) 18 (43.9) Duration since first symptom and admission to hospital Median (range) in days 2 (0–14) 2 (0–14) 2.5 (1–7) Duration between diagnosis and admission to hospital Median (range) in days 0.5 (0–13) 1 (0–13) 0.5 (0–7) Duration since first symptom to severe disease Median (range) in days — — 8 (4–43) Comorbidities, n (%) Total 73 (69.4) 36 (68.5) 37 (35.2) Hematological cancer 17 (16.2) 11 (10.4) 6 (05.7) Nonhematological cancer 20 (19.0) 8 (7.6) 12 (11.4) Diabetes 23 (21.9) 9 (16.7) 14 (34.1) Hypertension 26 (24.7) 9 (18.5) 16 (39.0) Chronic pulmonary disease 4 (3.8) 1 (1.9) 3 (7.3) Other 1 (1.9) 1 (1.9) 1 (2.4) Two or more comorbidities 19 (18.1) 10 (18.1) 9 (22.0) Clinical symptoms and signs at diagnosis, n (%) Fever 87 (91.6) 47 (87.0) 40 (97.6) Cough 57 (60.0) 26 (48.1) 31 (75.6) Sore throat 72 (75.8) 33 (61.1) 39 (95.1) Headache 57 (51.6) 25 (46.3) 24 (58.5) Myalgia 56 (58.9) 23 (42.6) 33 (80.5) Rhinitis/Rhinorrhea 33 (34.7) 22 (40.7) 31 (75.6) Loss of taste or smell 29 (30.5) 14 (25.9) 15 (36.6) Nausea or vomiting 19 (18.1) 06 (11.1) 11 (26.8) Baseline RT-PCR Ct values, median (range) E gene 18.7 (10.4–36) 21.5 (10.4–36) 21.3 (13.3–32) RdRp gene 22.5 (9.7–36.6) 22.0 (9.7–33.4) 20.4 (14.8–36.5) SpO2 levels Median (range) % at admission 98 (70–100) 98 (96–100) 97 (70–100) Median (range) % at worst score 97 (92–100) 98 (95–100) 80 (40–94) WHO score Median (range) at admission 3 (2–6) 3 (2–3) 3 (2–5) Median (range) at worst score 3 (2–8) 3 (2–4) 7 (5–8) Blood counts at diagnosis, median (range) Hb (g mL−1) 120 (45–159) 129 (61–159) 107 (45–154) White blood cells (× 109 L−1) 5.31 (0.32–28.7) 4.59 (0.32–16.7) 9.9 (1–28.7) Platelet (×109 L−1) 208 (19–472) 198 (50–444) 215 (19–472) Coagulation profile at diagnosisa PT (s) 16 (13–149) 15 (14–19) 16 (13–149) aPTT (s) 32 (20–60) 33 (25–60) 31 (20–46) INR 1.0 (0.9–2.0) 0.9 (0.9–1.28) 0.99 (0.9–2.0) Fibrinogen (mg dL−1) 342 (143–810) 297(185–803) 458 (143–810) Other laboratory parameters at diagnosisa C-reactive protein (mg%) 2 (0.1–48) 0.7 (0.1–14.4) 9.7 (0.1–48) Procalcitonin (ng mL−1) 0.0 (0.0–59.8) 0.05 (0.05–0.48) 0.13 (0.05–59.8) aPTT, activated partial thromboplastin time; Hb, hemoglobin; INR, international normalized ratio; PT, prothrombin time; RT, reverse transcriptase; WHO, World Health Organization. a These tests were available in 65 patients (nonsevere = 41, severe = 24). Immune perturbations: nonsevere versus severe COVID-19 patients

We studied 200 parameters, including age, immune cell subsets, cytokine levels and other laboratory parameters (Supplementary table 1). A total of 79 parameters were found to be associated with severe COVID-19. The top 25 parameters demonstrating a strong association with the SD (area under the curve > 0.75) were analyzed further. Patients with severe COVID-19 predominantly belonged to the elderly age group. Hence, the levels of immune cell parameters significantly associated with SD were also compared with levels of age-adjusted healthy donors (median age 62 years, range 51–70 years) as shown in Table 3 and Figures 2 and 3.

Adaptive immune response compartment

In accordance with previous reports, our data also revealed a significant association of reduced CD3 T, CD4 T, CD8 T cells and transitional B cells, and increased neutrophil-to-T-cell ratio and plasmablasts count with SD (Figures 1d–h and 2).5, 7, 8, 15, 18, 19 Our data distinctly highlighted that within CD4 T-cell subsets, the decreased counts of effector memory CD4 T-cell (CD4 TEM), CD45 RA+ terminal effector CD4 T cell (CD4 TTE), Tregs, type 1 T helper (Th1) and Th9 were associated with severe COVID-19 (Figure 1i–l and Table 2). In cytotoxic T-cell subsets, in addition to previously reported increased proportion of activated CD8 T cells, the reduced counts of naïve-CD8 T (CD8 TNV), CD94+ γδ T and CD69+ γδ T cells were found to be associated with SD (Supplementary table 1).11, 12 Within the natural killer (NK) cells, an expanded proportion of effector NK cell (NKeff) subset was associated with SD (Supplementary table 2).

Innate immune response compartment

As reported previously, we also observed an association of higher levels of absolute neutrophil count and reduced levels of monocytes (classical, intermediate and nonclassical), eosinophils and basophils with SD (Supplementary tables 1 and 2).5, 8, 11, 13, 15, 18, 20, 21 By contrast, increased myeloid-derived suppressor cells monocyte type percentages in total leukocyte counts showed a tendency toward SD.

Notably, in the dendritic cell (DC) compartment, decreased levels of myeloid DCs (mDCs) showed a strong association with SD (absolute ≤ 3 μL−1; OR 15.2, P = 0.0001 and percentages ≤ 0.05%; OR 27.9, P = 0.0001). Likewise, reduced plasmacytoid DC levels were also associated with SD (absolute ≤ 1 μL−1; OR 6.28, P = 0.0001 and percentages ≤ 0.03%; OR 17.04, P = 0.0001) (Table 2 and Supplementary table 2).

Table 2. Levels of the most significant immune cell subsets of COVID-19 patients Immune cell subset Disease status Association with severe COVID-19 Nonsevere (n = 54) Severe (n = 41) Cut-off criteriaa Sensitivity (95% CI) Specificity (95% CI) Odds ratio (95% CI) BH-adjusted P-value Absolute cell counts (μL−1), median (range) CD4 TEM 156 (19–992) 44 (2–399) ≤ 68 68.29 (51.90–81.90) 81.48 (68.60–90.70) 9.47 (3.66–24.53) 0.0001 CD4 TTE 18 (0–249) 9 (0–152) ≤ 12 65.85 (49.40–79.90) 57.41 (43.20–70.80) 2.81 (1.21–6.52) 0.0165 Tregs 28 (2–94) 14 (2–76) ≤ 18 68.29 (51.90–81.90) 68.52 (54.40–80.50) 4.68 (1.96–11.23) 0.0006 Th1 61 (6–488) 21 (2–226) ≤ 33 70.73 (54.50–83.90) 70.37 (56.40–82.00) 5.74 (2.35–13.99) 0.0001 Th9 25 (3–127) 15 (2–169) ≤ 35 87.80 (73.80–95.90) 40.74 (27.60–55.00) 4.95 (1.68–14.60) 0.0038 BTr 4 (0–47) 1 (0–24) ≤ 2 63.41 (46.90–77.90) 66.67 (52.50–78.90) 3.77 (1.60–8.88) 0.0026 γδ-T 38 (1–277) 10 (0–81) ≤ 13 68.29 (51.90–81.90) 74.07 (60.30–85.00) 6.15 (2.51–15.08) 0.0001 Mono-I 37 (0–293) 13 (0–193) ≤ 15 56.10 (39.70–71.50) 83.33 (70.70–92.10) 6.39 (2.48–16.43) 0.0001 pDC 2 (0–12) 0 (0–10) ≤ 1 90.24 (76.90–97.30) 66.67 (52.50–78.90) 6.28 (2.56–15.43) 0.0001 mDC 6 (0–49) 1 (0–15) ≤ 3 75.17 (57.10–85.80) 78.52 (54.40–80.50) 15.16 (5.30–43.39) 0.0001 CD141+ mDCb 4 (0–32) 0 (0–5) ≤ 1 83.33 (62.60–95.30) 78.05 (62.40–89.40) 26.92 (6.71–107.90) 0.0001 CD1c−CD141− mDCb 1 (0–26) 0 (0–1) ≤ 0 91.67 (73.00–99.00) 56.10 (39.70–71.50) 5.81 (1.79–18.87) 0.0035 BH, Benjamini–Hochberg correction; BTr, transitional B cells; C, classical; CI, confidence interval; CM, central memory; Eff, effector; EM, effector memory; I, intermediate; mDC, myeloid dendritic cell; mono, monocytes; NC, nonclassical; pDC, plasmacytoid dendritic cell; reg, regulatory; Th, T helper cell; Tregs, regulatory T cells; TTE, terminal effector CD4 T cell. a Cut-off criteria were defined using ROC analysis. b Data were available in only 65 patients. Immune perturbations: cancer versus noncancer comorbidities

As comorbidities such as cancer can also result in immune dysregulation,21, 22 we compared the levels of immune cell parameters significantly associated with severe COVID-19 (area under the curve > 0.75) between the age-matched patients with and without cancer (Figures 1 and 3 and Table 3, Supplementary table 3). The levels of CD4 TTE, Tregs, Th9 cells, intermediate-type monocytes and mDC, and proportion of the NKeff subset within NK cells were significantly lower in cancer patients with NsD and SD compared with the corresponding groups of patients without cancer (Figure 3 and Table 3). By contrast, the levels of CD4 TEM were slightly higher in cancer patients with NsD. We also noted significantly lower B-cell levels in cancer patients than noncancer patients independent of the severity of COVID-19 (Figure 1g).

Table 3. The distribution of statistically significant immune cell subset levels in COVID-19 patients with and without cancer Immune cell subset Nonsevere disease Severe disease Cancer (n = 19) Noncancer (n = 35) BH-adjusted P-value Cancer (n = 18) Noncancer (n = 23) BH-adjusted P-value Absolute cell counts (μL−1), median (range) CD4 TEM 94 (19–992) 203 (25–621) 0.1640 55 (9–398) 33 (2–399) 0.1483 CD4 TTE 3 (0–234) 32 (2–249) 0.0078 4 (0–46) 23 (1–152) 0.0024 Tregs 16 (2–94) 32 (10–89) 0.0454 9 (2–38) 17 (2–76) 0.1510 Th1 47 (8–488) 67 (6–324) 0.5490 18 (5–226) 21 (2–179) 0.8604 Th9 12 (3–64) 32 (9–127) 0.0056 7 (2–31) 25 (3–169) 0.0077 BTr 0 (0–17) 6 (0–47) 0.0048 0 (0–2) 4 (0–24) 0.0016 γδ–T 33 (1–277) 38 (7–106) 0.5301 10 (0–81) 10 (2–68) 0.9686 Mono–I 27 (0–140)