Enriched CXCR3⁺ CXCR5⁺ CD8⁺ T cells in SARS-CoV-2 infected and vaccinated individuals effectively respond to the antigen in recall

T cell-mediated effector and memory response are essential to tackle the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Activated CD4⁺ T cells, in particular follicular helper T (T_FH) cells, are critical to coordinate the optimal humoral antibody response following the SARS-CoV-2 infection or vaccination. It is, however, inconclusive regarding the phenotypic changes, memory potential, and functionality of CD8⁺ T cells following the SARS-CoV-2 infection and vaccination. Some early studies reported that activated CD8⁺ T cells were hyperactivated or exhausted during the acute SARS-CoV-2 infection. These exhausted CD8⁺ T cells showed impaired abilities to efficiently clear the pathogens and might cause bystander activation-induced tissue injury.^,  While longitudinal analysis revealed that a proportion of CD8⁺ T cells expressing distinct signature of genes can persist 1 year post infection in COVID-19 convalescents. Studies in Journal of Infection recently reported the serologic analysis of the persistent T cell response induced by multiple doses of mRNA vaccine.^,  While the cellular information regarding the long-lived human CD8⁺ T cells that can respond effectively to the SARS-CoV-2 antigen re-exposure is poorly understood.

To interrogate the existence of a functional subpopulation of human CD8⁺ T cells that may exhibit superior memory potential to the SARS-CoV-2, we reexamined our cohort of COVID-19 convalescents. CD8⁺ T cells from peripheral blood mononuclear cell (PBMC) were assessed using a multicolor flow cytometry panel with an antibody combination targeting key lymphocyte lineage and activation molecules. To unbiasedly evaluate the CD8⁺ T cells in convalescents, we performed Unsupervised Uniform Manifold Approximation and Projection (UMAP) and FlowSOM analysis to group CD8⁺ T cells into six clusters (Fig. 1A). A distinct embedding pattern with enriched cluster 3 and cluster 4 was found in COVID-19 convalescents, where CD8⁺ T cells showed higher expression of activation markers CD25 and PD-1 (Fig. 1B and C). Interestingly, in these convalescents-enriched clusters, activated CD8⁺ T cells also expressed high levels of CXCR5 and CCR6 in cluster 3; and CXCR3, CCR7, and CCR6 in cluster 4 (Fig. 1C and D).

CXCR5 expressing CD8⁺ T cells exhibit “stem-like” memory T cell phenotypes that undergo self-renewal, migrate to the tissue niches, and control the pathogens during viral infection. CXCR3 is critical in positioning CD8⁺ T cells in the tissue sites such as airway and skin for the control of the local infection. We next set out to confirm the expression of CXCR3 and CXCR5 on CD8⁺ T cells in convalescents with manual biaxial gating of the blood samples (Fig. 1E). Interestingly, we found a co-expression pattern of CXCR5 and CXCR3 on CD8⁺ T cells and these cells were around 2.5-fold higher in the convalescents (Fig. 1E). MILLIPLEX assay was used to analyze the cytokine productions in the patients’ peripheral. The results were further incorporated into the principal component analysis (PCA) of 14 CD8⁺ T cell-related variables to phenotype the CXCR3⁺ CXCR5⁺ CD8⁺ T cells. Intriguingly, these cells were closely associated with the expression of CD25, CCR6 and CD127 expression, while to a less extent, Perforin, Granzyme A and Granzyme B production. Notably, CXCR3⁺ CXCR5⁺ CD8⁺ T cells were not correlated with the expression of Tim3 and CTLA4 as well as the soluble level of CD137 and Fas in peripheral (Fig. 1F). Collectively, these data have depictured an enriched subpopulation of CD8⁺ T cells in SARS-CoV-2 infected individuals, where they express less cell-exhaustion markers but are in close correlation with the molecules defining memory T cell identity.

To investigate how CXCR3⁺ CXCR5⁺ CD8⁺ T cells persist and respond to the recall exposure of SARS-CoV-2 antigen, we took the advantage of our reasonably sized cohort of 53 healthy subjects that have received 3 doses of CoronaVac vaccine. We longitudinally analyzed these T cells from PBMCs that were collected at 5 different time points (pre-vaccination, 1 week after the 1st vaccination-1st Vax, 2 weeks after the 2nd vaccination-2nd Vax, 6–8 months after the 2nd vaccination-6 m 2nd Vax, and 2 weeks after the 3rd vaccination-3rd Vax) (Fig. 1G). Around 5% of polyclonal peripheral CD8⁺ T cells were found as CXCR3⁺ CXCR5⁺ CD8⁺ T cells in individuals before and after their 1st vaccination (Fig. 1H). While this frequency was significantly increased to an average of 10% after the 2nd vaccination (Fig. 1H and I). Although the peripheral level of CXCR3⁺ CXCR5⁺ CD8⁺ T cells waned over 6 months post the 2nd Vax, a 3rd vaccine booster significantly increased the peripheral frequency of CXCR3⁺ CXCR5⁺ CD8⁺ T cells to around 10% of total polyclonal CD8⁺ T cells (Fig. 1I). Activation-induced marker (AIM) assay was used to further interrogate the antigen specificity of CXCR3⁺ CXCR5⁺ CD8⁺ T cells. To do so, PBMCs from 53 vaccinated donors at five different time points were ex vivo stimulated with SARS-CoV-2 spike protein pool (2 µg/mL, SinoBiological PP003) in 5% CO₂ at 37 °C for 24 h. Comparing to the dimethyl sulfoxide (DMSO, negative control) and staphylococcal enterotoxin B (SEB, positive control) treated cells, SARS-CoV-2 spike-induced robust antigen-specific CD8⁺ T cell response, defined as HLA-DR⁺ CD38⁺ CD8⁺ T cells (Fig. 1J). Consistent with the polyclonal CD8⁺ T cell results shown in Fig. 1I, the 2nd vaccination markedly increased the frequency of antigen-specific AIM⁺ CXCR3⁺ CXCR5⁺ CD8⁺ T cells (Fig. 1K). Unlike the kinetics of polyclonal CD8⁺ T cells, 6 months after the 2nd Vax, the magnitude of antigen-specific CXCR3⁺ CXCR5⁺ CD8⁺ T cells remained close to that of 1 week after the 2nd vaccine administration. These results indicate that the antigen-specific CXCR3⁺ CXCR5⁺ CD8⁺ T cells indeed persist very long and respond efficiently to the antigen re-exposure. Moreover, a 3rd Vax dose further increased the antigen-specific CXCR3⁺ CXCR5⁺ CD8⁺ T cells comparing to the 2nd Vax dose (Fig. 1K).

In summary, here we provided evidence showing that a proportion of CD8⁺ T cells expressing CXCR3 and CXCR5 were increased in both SARS-CoV-2 infected and CoronaVac-vaccinated individuals. This result is in line with other studies showing that CXCR5 or CXCR3 expressing CD8⁺ T cells are enriched in surviving COVID-19 patients compared to the deceased patients and may confer protection against infection. We further found that both polyclonal and antigen-specific CXCR3⁺ CXCR5⁺ CD8⁺ T cells responded rapidly to the inactivated SARS-CoV-2 vaccine. And the antigen-specific CXCR3⁺ CXCR5⁺ CD8⁺ T cells persist at least 6 months post the 2nd dose of vaccination. These results help us understand the complex nature of memory CD8⁺ T cells in response to the emerging pathogens. The mechanistic understanding of the generation and function of CXCR3⁺ CXCR5⁺ CD8⁺ T cells in further studies may aid to the design of future vaccines and novel immuno-therapeutics to the pandemic-threatening pathogens.