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Persistent Cellular Immunity to SARS-CoV-2 Infection

SARS-CoV-2 is responsible for an ongoing pandemic that affected millions of individuals around the globe. To gain further understanding of the immune response in recovered individuals we measured T cell responses in paired samples obtained an average of 1.3 and 6.1 months after infection from 41 individuals. The data indicate that recovered individuals show persistent polyfunctional SARS-CoV-2 antigen specific memory that could contribute to rapid recall responses. In addition, recovered individuals show enduring immune alterations in relative numbers of CD4+ and CD8+ T cells, expression of activation/exhaustion markers, and cell division.

Careful studies of immune phenotypes in moderate, severe and recovered individuals revealed T cell responses, ranging from undetectable to robust CD8 + T cell and/or CD4 + T cell activation and proliferation , Peng et al., 2020, Braun et al., 2020, Neidleman et al., 2020.
During acute infection, T cells displayed a highly activated cytotoxic phenotype, whereas convalescent patients harbored polyfunctional SARS-CoV-2-specific T cells that display a stem like memory phenotype (Sekine et al., 2020, Neidleman et al., 2020. In addition, there is cross-reactivity with seasonal/common cold coronaviruses that suggests that these responses may be associated with a milder clinical course (Sekine et al., 2020, Le Bert et al., 2020.
Coronaviruses elicit variable levels of persistent Immunity to other coronaviruses. For example, individuals infected with MERS remain immune for only 1-3 years, while protection from seasonal coronaviruses is short-lived (Wu et al., 2007, Edridge et al., 2020, Tang et al., 2011. Although there is increasing evidence that cellular immunity plays a major role in resolution of COVID-19, little is known about the persistence of cellular immunity to SARS-CoV-2 (Rodda et al., 2020). This is a particularly important issue when considering an individual's ability to resist a second exposure to the virus. To determine whether cellular immunity to SARS-CoV-2 persists half a year after infection we examined paired samples obtained an average of 1.3 and 6.1 months after infection from a cohort of 41 COVID-19-convalescent volunteers . All of the individuals tested had RT-PCR confirmed SARS-CoV-2 infection or were close contacts that had seroconverted and were RT-PCR negative at the second time point . The 41 individuals were 63.4% male and 36.6% female, representing a range of different ages, 24-73 years old, and disease severity but skewed to mild forms of the disease (Table 1).

Changes in Circulating T cells After COVID-19
The phenotypic landscape of circulating T cells was determined by high-dimensional flow cytometry at both time points and compared to pre-COVID-19 samples from healthy individuals (n=20). Global high-dimensional mapping with t-distributed stochastic neighbor embedding (tSNE) revealed significant persistent alterations in SARS-CoV-2 recovered individuals (Fig. 1A). To further examine changes in the T cell compartment we queried the data by traditional gating (Suppl. Fig. 2). The relative proportions of circulating CD4 + T cells decreased significantly 1.3 months after infection whereas the circulating CD8 + T cells increased; but both returned to near physiologic levels by 6.1 months ( Fig. 2A and 2B). PD-1 expression is modulated on activated and exhausted T cells and is associated with acute and prolonged changes in T cell function after viral infection in mice (Schonrich andRaftery, 2019, Jubel et al., 2020). PD-1 expression was decreased on CD4 + and CD8 + T cells at both time points (Fig. 2C and 2D). Consistent with these alterations, TIGIT, Tim-3 and CD25 expression were also abnormal ( Fig. 2C and 2D). We conclude that there are persistent changes in the distribution of circulating CD4 + and CD8 + T cells and their expression of activation/exhaustion markers.
Consistent with the clustering analysis, central memory CD4 + and CD8 + T cells decreased, and this defect persisted throughout the observation period ( Fig. 2E and 2F). In addition, there was an increase in cycling CD4 + and CD8 + T cells (Ki67 + ) at both time points ( Fig. 2G and 2H). Both central memory and cycling cells also showed lower levels of PD-1 expression at both time points (Suppl. Fig. 3A-D). In contrast, we found no significant changes in circulating TFH or Treg cells (Suppl. Fig. 4). Thus, the more traditional gating strategy is generally consistent with the tSNE analysis.

SARS-CoV-2-antigen specific CD4 + T cells
To investigate SARS-CoV-2-antigen specific CD4 + T cells, we stimulated the cells with a collection of pooled SARS-CoV-2 peptides in vitro. COVID-19 convalescent individuals were compared to healthy donors by tSNE using high dimensional flow cytometry. Antigen-specific CD4 + T cells expressing memory markers as well as IL-2, IFN-g, TNF-a and CD154 were markedly increased in COVID-19 recovered individuals compared to healthy donors, but the relative frequency of these cells decreased at the 6.1 month time point (clusters 2, 3, 4 and 6, Fig. 3A-D and Suppl. Fig. 5A). The magnitude of the decrease in these responses between the time points varied between 22-32% depending on the cluster (Fig. 3D) and on the individual peptide pool (Suppl. Fig. 5B). In contrast, responses to CMV peptides remained unchanged between the 2 time points (Suppl. Fig. 6). We conclude that SARS-CoV-2 antigen specific CD4 + T cells are induced during acute infection and are only slightly reduced after 6.1 months.
To characterize SARS-CoV-2 antigen specific cytokine producing CD4 + T cells we analyzed the high dimensional flow cytometry data by traditional gating (Fig. 4 and Suppl. Fig. 2). IL-2, INFg, and TNFa responses to peptide pools corresponding to Spike (S1 and S2), NCAP, Memb, AP3a, and CMV control were measured independently at both time points (Fig. 4A-C). In all cases, responses to the individual peptide pools were elevated above control at both time points, and all but NCAP and Memb responses remained stable between the time points ( Fig. 4A-C). Among the individuals tested 97.5% and 95% responded to at least one of the antigens at 1.3 and 6.1 months respectively (Suppl. Fig. 7A-C). When all cytokines are considered together, we find a significant increase in antigen specific CD4 + T cell responses to all of the individual peptide pools at both time points compared to control (Fig. 4A). The increase in CD4 + T cell cytokine responses to the individual peptide pools and the overall combination of all SARS-CoV-2 antigens was not driven by any single cytokine but instead reflected increases in each of the three cytokines measured (Suppl. Fig. 7D). Moreover, when all antigen specific responses are considered in aggregate, the fraction of responding CD4 + T cells remains significantly elevated and is not different between the 2 time points (Fig. 4A, All). Antigen specific memory CD4 + memory T cells (CD45RA + CD27 + ) were similarly elevated at the two time points (Fig. 4B). Finally, there was no significant change in the CD4 + T cell response by the same individuals to the control CMV peptide pool ( Fig. 4A-C).
Polyfunctional cytokine responses are associated with effective cellular immune responses (Seder et al., 2008, Lin et al., 2015, Betts et al., 2006. Polyfunctional CD4 + T cells responses to each of the individual peptide pools were significantly elevated at the early time point and remained so after 6.1 months (Fig. 4C). The magnitude of these responses was directly correlated with antibodies to the SARS-CoV-2 receptor binding domain (Suppl. Fig. 8). However, there was a decrease in polyfunctional CD4 + T cell responses to NCAP and Memb antigens at 6.1 months which is also reflected in a 22% decrease in the overall tri-functional response to the combined SARS-CoV-2 peptide libraries ( Fig. 4C, All). In contrast, CD4 + T cells that produced only a single cytokine were only elevated in response to Memb and only at the early time point (Fig.   4C). We conclude that robust polyfunctional CD4 + T cell responses persist for 6.1 months after SARS-CoV-2 infection but decrease significantly when compared to an earlier time point.

SARS-CoV-2 antigen specific CD8 + T cells
To examine antigen specific CD8 + T cell responses we measured production of Mip-1b, CD107a, IL-2, INF-g and TNF-a in response to stimulation with SARS-CoV-2 peptide pools in vitro. In contrast to CD4 + T cells, CD8 + T cell responses were far more variable and generally less robust making tSNE analysis less reliable and therefore these responses were only analyzed by traditional gating (Fig. 5 and Suppl. Fig. 9). Although 95% of the donors tested responded to at least 1 of the peptide pools at both 1.3 and 6.1 months, the percentage of responding cells was low (Fig. 5A). When all peptide responses were pooled, we found significant Mip-1b, and INF-g responses at both time points, while CD107a was only increased above control at 1.3 months.
Polyfunctional responses that included at least 3 different cytokines were also elevated at both time points (Fig. 5B). We conclude that although we detect fewer antigen specific CD8 + than CD4 + T cells to SARS-CoV-2, they persist 6.1 months after infection.

Discussion
Most effective vaccines and anamnestic responses to pathogens are mediated by neutralizing antibodies (Plotkin, 2020, Inoue et al., 2018, Weisel and Shlomchik, 2017. Consistent with this notion, neutralizing antibodies are protective against SARS-CoV-2 infection in animal models  and appear to correlate with protection in vaccinated humans . However, antibody responses are dependent on specialized helper T cells that control the activation and selection of antibody producing plasma and memory B cells (Victora andNussenzweig, 2012, Crotty, 2015).
CD4 + and CD8 + T cells can also contribute directly to protection against SARS-CoV (Le Bert et al., 2020, Tang et al., 2011, Yang et al., 2006, Channappanavar et al., 2014 and other viral pathogens in animal models (Schmitz et al., 1999, Shoukry et al., 2003, Snyder, 2011. In addition, control of human viral pathogens such as HIV-1 is associated with CD8 + T cells in rare elite controllers (Collins et al., 2020). To gain further understanding into whether SARS-CoV-2 infection is associated with enduring responses we examined cellular immunity to SARS-CoV-2 in paired samples collected 1.3 and 6.1 months after infection. Similar to SARS-CoV and MERS infections, SARS-CoV-2 antigen specific memory CD4 + and CD8 + T cells persist and would be expected to play role in protection against re-exposure.
In addition to persistent antigen specific memory responses, chronic viral infections such as HIV are associated with lasting immune perturbations (Breton et al., 2013), but little is known about acute viral infections. SARS-CoV-2 is an acute infection that typically resolves after 2-3 weeks and in rare instances leads to severe lung disease and mortality (Richardson et al., 2020, O'Driscoll et al., 2020. The cohort we examined is biased toward milder forms of the disease, nevertheless, CD4 + and CD8 + T cell subset distribution, cell division and expression of activation/exhaustion markers remain altered 6 months after SARS-CoV-2 infection. These abnormalities were not directly associated with persistent symptoms and their impact on the overall immune health of the individual remains to be determined.
Previously enrolled study participants  were asked to return for a 6-month follow-up visit at the Rockefeller University Hospital in New York from August 31 through October 16, 2020. All of the individuals tested had RT-PCR confirmed SARS-CoV-2 infection or were close contacts that seroconverted. A summary of the participants' clinical characteristics is presented in Table 1 and has been extensively described elsewhere .
Cell preparation: Blood samples were collected an average of 1.3 and 6.1 months post infection (Table 1). Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation (Ficoll-Paque™) and cryopreserved in 90% heat-activated fetal bovine serum (FBS) plus 10% DMSO in liquid nitrogen. Thawed PBMCs were washed and resuspended at 2×10 6 cells/ml with RPMI 1640 supplemented with 10% heat inactivated human serum (GemCell) and 10 U/ml Benzonase. For cell stimulation experiments cells were rested at 37°C and 5% CO2 for 8 hours prior to stimulation with peptides for use in intracellular cytokine staining assays. The lymphocytes were gated for further analysis, as described in Figure S2, using Flowjo™ Software Version 9.9.6 (BD, USA). For cytokine expression, we subtracted the background in the negative control. To ensure equivalent fluorescence intensities (median fluorescence intensity or MFI) from one experiment to another, we used Rainbow beads (Spherotech, Inc, Lake Forest, IL, USA). For every run, rainbow beads were acquired first and the voltages were adjusted if necessary to accommodate both daily variations and larger changes in performance such as would be seen after cytometer maintenance and alignment.
Statistical analysis: Statistical analyses were performed using Prism 7.0 (GraphPad). Significances between matched groups were calculated using paired t-test whereas differences between unmatched groups were compared using unpaired t-test, or one-way ANOVA. Correlations were performed using the Spearman rank correlation test.

ACKNOWLEDGMENTS
We thank all study participants who devoted time to our research; Drs. Barry Coller and Sarah  Each dot represents an individual with COVID-19 at 1.3 months (dark blue for CD4 + T cells and dark red for CD8 + T cells) or 6.1 months (light blue for CD4 + T cells and orange for CD8 + T cells) as well as control individuals (green).

T cells and dark red for CD8 + T cells) or 6.1 months (light blue for CD4 + T cells and orange for CD8 + T cells) as well as control individuals (green). Significance determined by paired t test for comparisons between time points within individuals and unpaired t test for comparison
between controls and COVID-19 individuals. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.