High Frequencies of Phenotypically and Functionally Senescent and Exhausted CD56+CD57+PD-1+ Natural Killer Cells, SARS-CoV-2-Specific Memory CD4+ and CD8+ T cells Associated with Severe Disease in Unvaccinated COVID-19 Patients

Unvaccinated COVID-19 patients display a large spectrum of symptoms, ranging from asymptomatic to severe symptoms, the latter even causing death. Distinct Natural killer (NK) and CD4+ and CD8+ T cells immune responses are generated in COVID-19 patients. However, the phenotype and functional characteristics of NK cells and T-cells associated with COVID-19 pathogenesis versus protection remain to be elucidated. In this study, we compared the phenotype and function of NK cells SARS-CoV-2-specific CD4+ and CD8+ T cells in unvaccinated symptomatic (SYMP) and unvaccinated asymptomatic (ASYMP) COVID-19 patients. The expression of senescent CD57 marker, CD45RA/CCR7differentiation status, exhaustion PD-1 marker, activation of HLA-DR, and CD38 markers were assessed on NK and T cells from SARS-CoV-2 positive SYMP patients, ASYMP patients, and Healthy Donors (HD) using multicolor flow cytometry. We detected significant increases in the expression levels of both exhaustion and senescence markers on NK and T cells from SYMP patients compared to ASYMP patients and HD controls. In SYMP COVID-19 patients, the T cell compartment displays several alterations involving naive, central memory, effector memory, and terminally differentiated T cells. The senescence CD57 marker was highly expressed on CD8+ TEM cells and CD8+ TEMRA cells. Moreover, we detected significant increases in the levels of pro-inflammatory TNF-α, IFN-γ, IL-6, IL-8, and IL-17 cytokines from SYMP COVID-19 patients, compared to ASYMP COVID-19 patients and HD controls. The findings suggest exhaustion and senescence in both NK and T cell compartment is associated with severe disease in critically ill COVID-19 patients.

. CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 27, 2022.

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. CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 27, 2022. ; https://doi.org/10.1101/2022.07.26.501655 doi: bioRxiv preprint T cell Senescence in  6 Furthermore, the expression of CD57 aids in identifying the final stages of peripheral NK cell 96 maturation, and the expression of CD57 increases with age and chronic infections (16).

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Reports show that repeated T cell activation is associated with terminally differentiated cells 98 and the corresponding upregulation of CD57 (13, 17). It is observed that shortened telomeres are

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Our findings demonstrate that increased T cell exhaustion and senescence markers in 115 unvaccinated ASYMP COVID-19 patients compared to unvaccinated ASYMP COVID-19 patients and 116 Healthy Controls. Furthermore, T cell senescence markers were highly expressed on CD8 + T EM and 117 CD8 + T EMRA cells than on T Naive and T CM cells. These results suggest that the upregulation of 118 exhaustion and senescence pathways during symptomatic COVID-19 may affect both NK and T cell 119 compartments, leading to inefficient clearance of SARS-CoV-2 infection and severe disease. 120 121 . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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(Asymptomatic and Symptomatic) and ten unexposed Healthy individuals, who had never been 128 exposed to SARS-CoV-2 or COVID-19 patients, were enrolled in this study (

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. CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 27, 2022. ; https://doi.org/10.1101/2022.07.26.501655 doi: bioRxiv preprint T cell Senescence in  8 and subsequently acquired on a BD LSRII. Data were analyzed using FlowJo version 9.5.6 (Tree      was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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We analyzed the NK cell population following a gating strategy as shown in

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CD57 expression on NK cells defines a mature phenotype, and their expression of CD57 could also 208 be considered a marker of terminal differentiation, although not associated with senescence in this 209 population. It is highly expressed on CD56 dim cells, representing mature NK cells, whereas less than 210 1% of CD56 bright NK cells, considered immature, also express CD57 (Fig. 1C). There was a significant 211 . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 27, 2022. ; https://doi.org/10.1101/2022.07.26.501655 doi: bioRxiv preprint increase in the mature (CD56 dim /CD57 + ) and immature subset (CD56 bright /CD57 + ) (Fig. 1C

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The levels of CD4 + T-cell activation were also evaluated in SARS-CoV-2 infected ASYMP, SYMP 230 patients, and healthy controls. We found an increasing trend reflected by higher proportions of HLA-231 DR + CD38 + CD4 + T cells in SYMP patients compared to healthy controls, however with no statistical 232 significance (Fig. 2C).

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Furthermore, we evaluated the expression of the senescence/exhaustion molecules on CD4 + T cells 234 by analyzing markers CD57 and PD-1 (Fig. 2D). The proportion of CD57 + PD-1 + CD4 + T cells was 235 significantly higher in COVID-19 positive SYMP patients than in healthy controls and ASYMP patients.

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. CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 27, 2022. ; https://doi.org/10.1101/2022.07.26.501655 doi: bioRxiv preprint However, no statistical difference was detected in the proportion of CD57 + PD-1 + CD4 + T cells in 237 ASYMP and SYMP patients ( Fig. 2D; P = 0.03).

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Taken together, findings from COVID-19 positive SYMP individuals indicate the involvement of 239 activated CD4 + T cells and T cell exhaustion/senescence in the immunopathogenesis of SARS-CoV2 240 infection.

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Significantly higher percentages of CD57 + CD45RA high CCR7 low CD8 + T EMRA cells ( Controls. Within the CD8 + population, we analyzed markers commonly related to T cell activation 316 (HLA-DR and CD38). Our gating strategy was used to analyze markers related to activation status, 317 senescence, and exhaustion together within SARS-CoV-2 specific CD8 + T cells (Fig. 6A). The levels

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We evaluated the senescence/exhaustion molecules expression on circulating T cells by 322 analyzing markers CD57 and PD-1 (Fig. 6C). We found that the proportion of CD57 + PD-1 + CD8 + T     cytokines assessed in this study had different detection ranges, with IL-6 and IL-8 having the most 337 dynamic profile followed by TNF-α, IL-17, and IFN-γ. We found that TNF-α and IFN-γ (P = 0.001) were 338 . CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 27, 2022. ; https://doi.org/10.1101/2022.07.26.501655 doi: bioRxiv preprint significantly elevated in COVID-19 symptomatic patients compared to Healthy controls ( Fig. 7A and   339   7B). Similarly, we found that IL-6 and IL-8 (P = 0.01) were significantly elevated in COVID-19 340 symptomatic patients compared to Healthy controls ( Fig. 7C and 7D). Interleukin (IL)-17 is one of the 341 many cytokines released during SARS-CoV-2 infection. IL-17 plays a crucial role in neutrophil 342 recruitment and activation. Neutrophils subsequently can migrate to the lung and are heavily involved 343 in the pathogenesis of COVID-19. We found that SARS-CoV-2 positive ASYMP and SYMP individuals 344 had significantly higher levels of IL-17 than Healthy controls (P = 0.04) (Fig. 7E).

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The majority of prior studies into the biology of CD57 focused on the antigen's significance in 416 distinct T cell subsets. In the late phases of differentiation, CD57 has been found both on CD4 and 417 CD8 T cells (15). CD57 identifies terminally differentiated cells with decreased proliferative responses 418 in CD8 T lymphocytes. T cell senescent markers were more associated with CD8 + T cells than CD4 + T 419 cells, consistent with our results that accumulate at lower frequencies for CD4 + T cells in the human 420 periphery (44). Our findings herein indicate an increased expression of CD57 + T cell subsets in 421 symptomatic patients. It was previously shown that PD-1 + CD57 + CD8 + T cells had increased sensitivity

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. CC-BY-NC-ND 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made    was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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The phenotype of CD4 + T cells and the gating strategy shown in Fig. 2A

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The frequency of CD57 + on total CD8 + T cells and SARS-CoV-2 peptide/tetramer complex

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The phenotype of CD8 + T cells specific to S 1220-228 peptide/tetramer shown in  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 27, 2022. ; https://doi.org/10.1101/2022.07.26.501655 doi: bioRxiv preprint