key: cord-0926971-llyioqp8
authors: Ssemaganda, Aloysious; Nguyen, Huong Mai; Nuhu, Faisal; Jahan, Naima; Card, Catherine M.; Kiazyk, Sandra; Severini, Giulia; Keynan, Yoav; Su, Ruey-Chyi; Ji, Hezhao; Abrenica, Bernard; McLaren, Paul J.; Ball, T. Blake; Bullard, Jared; Van Caeseele, Paul; Stein, Derek; McKinnon, Lyle R.
title: Expansion of tissue-resident CD8+ T cells and CD4+ Th17 cells in the nasal mucosa following mRNA COVID-19 vaccination
date: 2021-05-09
journal: bioRxiv
DOI: 10.1101/2021.05.07.442971
sha: a57189571f7ae981b337d1911bc974be77f20894
doc_id: 926971
cord_uid: llyioqp8

Vaccines against SARS-CoV-2 have shown high efficacy in clinical trials, yet a full immunologic characterization of these vaccines, particularly within the upper respiratory tract, remains lacking. We enumerated and phenotyped T cells in nasal mucosa and blood before and after vaccination with the Pfizer-BioNTech COVID-19 vaccine (n =21). Tissue-resident memory (Trm) CD8+ T cells expressing CD69+CD103+ expanded ∼12 days following the first and second doses, by 0.31 and 0.43 log10 cells per swab respectively (p=0.058 and p=0.009 in adjusted linear mixed models). CD69+CD103+CD8+ T cells in the blood decreased post-vaccination. Similar increases in nasal CD8+CD69+CD103-T cells were observed, particularly following the second dose. CD4+ Th17 cells were also increased in abundance following both doses. Following stimulation with SARS-CoV-2 spike peptides, CD8+ T cells increased expression of CD107a and CD154. These data suggest that nasal T cells may be induced and contribute to the protective immunity afforded by this vaccine.

The COVID-19 pandemic has led to substantial mortality and caused major global disruption. At least six vaccines have demonstrated efficacy in preventing severe disease associated with SARS-CoV-2 infection 1 . While this is an impressive achievement guided by early immunogenicity data, a full understanding of how these novel vaccines interact with the host immune system is lacking. This is particularly the case in tissues such as the upper respiratory tract (URT), which are often more challenging to sample than peripheral blood 2 . Here we focus on characterizing nasal mucosal T cells in healthy individuals pre-and post-vaccination with the BNT162b2 mRNA-nanoparticle vaccine developed by Pfizer-BioNTech 3 .

Natural history studies of SARS-CoV-2 infection demonstrate significant cell-mediated and humoral immune responses [4] [5] [6] [7] , which in combination lead to a substantial decrease in re-infection risk 8 .

These responses can be readily detected for >8 months post-infection 9 , and appear to have a long halflife. In non-human primates, CD8+ T cell depletion led to increased susceptibility to re-infection 10 .

Studies that assess the quality and quantity of immune responses induced by vaccination in humans are beginning to emerge [11] [12] [13] [14] [15] [16] , adding depth to what was generated in early phase clinical trials. While these studies suggest a high magnitude of humoral and cell-mediated immunity to SARS-CoV-2 17, 18 , less is known regarding tissue and mucosal immunity induced by these vaccines, particularly in the URT, the primary site of viral entry.

We developed an ex vivo flow cytometry-based assay to enumerate and profile immune cells isolated from nasopharyngeal (NP) swabs used for SARS-CoV-2 diagnostic testing amongst healthy volunteers. We pilot-tested several types of NP swabs to gauge optimal immune cell recovery.

Strikingly, CD45+ immune cells were recovered from a certain type of swab (Flexible minitip flocked swab from BD), while CD326+ epithelial cells were the main cell type recovered from two other swabs that we tested, including the Copan FLOQ swab (Figure 1A &B) . In a pilot study (n=8), a median of 3,082 (IQR: 2,351-6,168) CD45+ cells were recovered per BD swab, ~80% of which were CD3+ T cells. Using CD69 and CD103 as markers of tissue-resident memory (Trm) T cells 19, 20 , virtually all CD8+ (>90%) and variable proportions of CD4+ T cells were Trm (10-80%; Figure 1C ).

We enrolled healthy volunteers scheduled to receive the BNT162b2 mRNA vaccine (n=21, Supplemental Table 1) , and collected baseline, pre-vaccine samples followed by peak response samples ~12 days after the first (visit 2) and second (visit 3) doses. Participants were a median age of 40 (IQR: 31-50) and predominantly female (14/21) . Most participants (19/21) received influenza vaccination in the previous few months, and two participants had prior COVID-19 infection. The median body mass index (BMI) was 24 (IQR: [22] [23] [24] [25] [26] [27] [28] [29] . Reporting of underlying medical conditions included allergies and/or asthma (n=6), type II diabetes (n=2), hypertension (n=2), and autoimmunity (n=1).

We analyzed both frequency of parent populations and total cell abundance, the latter by maximizing acquisition of cells from each swab; measuring abundance is critical as cell abundance can vary within mucosa across orders of magnitude 21, 22 . We observed significant increases in nasal CD8+ Trm at both peak vaccine response time points (Figure 2A) . In a linear mixed model adjusted for age, sex, BMI, prior COVID-19, influenza vaccination, and self-reported vaccine side effects, the number of CD8+ Trm increased by 0.31 and 0.44 log10 cells per nasal swab following the first and second vaccine dose, respectively (p=0.058 and p=0.009). No increases in CD4+ Trm defined by CD103 and/or CD69 expression were observed ( Figure 2B ). The absolute number of nasal CD8+CD69+CD103-T cells also increased at visit 2 and particularly at visit 3, by 0.06 and 0.48 log10 cells/swab, respectively (adjusted p=0.7 and p=0.004 for visits 2 and 3). The proportion of nasal CD8+ T cells that were Trm also increased, although not significantly (p=0.09 from visit 2 versus baseline, Supplemental Figure 1A -B). Interestingly, the low proportion of CD69+CD103+ T cells in the blood significantly declined at the peak vaccine response time points, suggesting that these cells may be migrating out of the blood into tissues ( Figure 2C-D) . We also observed that CD4+ T cells co-expressing the Th17 markers CD161 and CCR6 increased by 0.40 and 0.45 log10 cells/nasal swab at the peak vaccine response time points compared to baseline (adjusted p=0.017 and p=0.008, respectively) ( Figure 2E ). No increases in nasal Tfh cells were observed following either vaccine dose ( Figure 2F ). Similar increases were observed in analyses stratified by age and sex (Supplemental Table 2 ), and excluding participants with prior COVID-19 infection did not meaningfully change any outcomes.

Studies of other vaccines suggest that peak vaccine-elicited T cells can be captured by activation markers such as Ki67, HLA-DR, and CD38, which peak at 10-14 days following yellow fever and smallpox vaccination 23 . In our study, we did not observe any changes in the proportion of CD4+ or CD8+ T cells co-expressing HLA-DR and CD38, or any upregulation of Ki-67, in either nasal or blood samples (Supplemental Figure 2) .

We further analysed our flow data using the t-distributed stochastic neighbour embedding (tSNE), a dimensionality reduction method. Several substantial shifts in CD45+ nasal immune cell clusters were evident from baseline to visit 2, and from visit 2 to 3 (Supplemental Figure 3A) . These included a step-wise shift in CD8+ T cell populations across the three time points, but also shifts in CD3-and CD3+CD4-CD8-cells. In support of this, the abundance of CD45+, CD3+, and CD8+ T cells all increased at visit 3, compared to baseline (Supplementary Figure 3B-G) . While it was difficult to distinguish which phenotypic markers flow panel explained the observed divergent clustering patterns, these data suggest that multiple subsets of nasal immune cells may increase in number following COVID-19 vaccination.

To further assess the antigen-specificity of nasal T cells, we enrolled vaccinated individuals at 2 months following their second Pfizer-BNT dose, and either stimulated the NP swab cells with overlapping SARS-CoV-2 spike protein peptide pools (n=15), or left cells in media unstimulated (n=7).

Stimulated samples had increased expression of the cytotoxic marker, CD107a (p=0.016) and the activation marker CD154 (p=0.069, Figure 3 ). Similar trends were observed for TNF-α and perforin, though these were not statistically significant.

To understand the relationship between antibody titres and nasal T cells, we quantified levels of SARS-CoV-2 spike-specific IgG antibodies in plasma. Antibody titres increased at visit 2 (median titre 163, IQR: 67-376), and more dramatically at visit 3 (median 2,185, IQR: 826-3,652; Figure 4A ). Only one of two participants with prior COVID-19 was positive for SARS-CoV-2 nucleocapsid IgG ( Figure   4B ). We next correlated antibody titres to the number of nasal CD8+ Trm and CD4+ Th17 at visit 3.

Nasal CD8+ Trm and CD4+ Th17 increases were similar in participants with varying increases in spike titres, with no correlations observed (Figure 4C-D) . These data suggest that tissue T cell responses to vaccination may be independent of the antibody response and may provide an additional layer of immunity against SARS-CoV-2 infection.

Trm are a vital form of surveillance in mucosal tissues, where they can mount rapid immune responses to pathogens upon re-exposure. In animal models, T cells at peak activation up-regulate homing markers that distribute these cells to relevant tissues 24 , where a high proportion is retained for extended periods as a form of sentinel surveillance against re-infection 25 . Trm have been more difficult to study in humans, given challenges in sampling the hard-to-reach tissues and in proving residency 26, 27 . Our data suggest that certain types of NP swabs commonly used for COVID-19 diagnostics are effective in recovering immune cells from nasal tissue. Multiple populations of nasal CD4+ and CD8+ T cells increased in frequency by ~0.5 orders of magnitude following vaccination with the BNT162b2 mRNA vaccine. This is a critical observation, as most human mucosal COVID-19 studies have focused either on antibodies 28 or lung T cells 29, 30 ; while a tissue population, the latter may not represent the front-line defense against infection. It has been hypothesized that Trm may be the most effective form of T cell response against SARS-CoV-2 infection 31 , capable of preventing viral dissemination beyond the URT, where more virus-induced damage can occur 32 . Data from mice have suggested that parenteral vaccination induces nasal Th17 Trm that protect against bacterial colonization 33, 34 . However, our study is the first, to our knowledge, to suggest nasal T cells may be induced by parenteral vaccination against COVID-19.

Our study has some limitations. We enrolled a relatively small sample size, restricting our ability to extensively stratify our data. The age range does not include the elderly, a group most in need of protection by COVID-19 vaccination 35 . The immune cell recovery from NP swabs, while sufficient for phenotyping and enumeration, is challenging for extensive functional characterization, in particular in assays that required splitting samples for paired analyses.

In summary, nasal T cell studies are feasible in humans, but this is highly dependent on the type of NP swab that is used. A high proportion of nasal T cells express markers of tissue residency, and these cells increase significantly in number and function following SARS-CoV-2 mRNA immunization. Further work should be performed to understand the role that local T cells may play in the protection against SARS-CoV-2 infection. Informed consent was obtained and a comprehensive socio-demographic and health assessment questionnaire administered to all study participants.

Sample collection and processing. In pilot work, we compared the BD Flex-mini and Copan Cells were subsequently washed, permeabilized with fixation/permeabilization solution (eBiosciences) and stained in a cocktail of pre-titrated monoclonal antibodies (Supplementary Table 1 ). Cells were then washed, acquired using a BDLSR Fortessa (BD Biosciences) and analyzed using FlowJo TM version 10.7.1 (Becton Dickinson Life Sciences).

Concentrations of SARS-CoV-2-specific IgG were determined using commercial assays. At visit 3, plasma was diluted 1:10 because of the high magnitude of the responses. IgG recognizing the SARS-CoV-2 spike protein was quantified using the Diasorin Trimeric Spike ELISA, while nucleocapsid antibodies were measured using the ELISA from Abbott.

Clinical and demographic data are presented as proportions or medians and interquartile ranges. Linear mixed models were used to compare changes in log10 transformed immune cell abundance (in nasal mucosa, typically cells/swab; in blood, % of CD3+CD4+ or CD8+ T cells). In these models, visit as a categorical variable was the main predictor, with visit 2 and 3 compared to baseline. Multivariate models were adjusted for a range of covariates. Raw p-values, beta coefficient and 95% confidence intervals were reported. Statistical analyses were performed using SPSS v. 27 .0 while all the graphing and was performed using Prism version 9 (GraphPad Software, LLC).

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Nasal immune cell clustering and cell counts per swab following SARS-COV-2 vaccination. (A) t-distributed stochastic neighbour embedding (tSNE) plots of integrated nasal CD45+ immune cells showing shifts in CD3+CD4+, CD3+CD8+, CD3-CD4-CD8-and CD3+CD4-CD8-immune cell clusters from vaccinated individuals at baseline, visit 2 and visit 3. (B) CD45+ (C) CD45+CD3+ and (F) CD3+CD8+ cells were significantly increased following the 2 nd vaccination dose while no changes were observed for (D) CD3+CD4-C8

Research. Special thanks to the study volunteers whose time and willingness to provide repeated specimens made this project possible. All authors reviewed and approved the submitted manuscript.