key: cord-0992708-e99hu26i
authors: Newell, Krista L.; Waldran, Mitchell J.; Thomas, Stephen J.; Endy, Timothy P.; Waickman, Adam T.
title: Simultaneous analysis of antigen-specific B and T cells after SARS-CoV-2 infection and vaccination
date: 2021-12-09
journal: bioRxiv
DOI: 10.1101/2021.12.08.471684
sha: fb1c4d4759cf2cccd81ffda8a660e263f5cb3f21
doc_id: 992708
cord_uid: e99hu26i

Conventional methods for quantifying and phenotyping antigen-specific lymphocytes can rapidly deplete irreplaceable specimens. This is due to the fact that antigen-specific T and B cells have historically been analyzed in independent assays each requiring millions of cells. A technique that facilitates the simultaneous detection of antigen-specific T and B cells would allow for more thorough immune profiling with significantly reduced sample requirements. To this end, we developed the B And T cell Tandem Lymphocyte Evaluation (BATTLE) assay, which allows for the simultaneous identification of SARS-CoV-2 Spike reactive T and B cells using an optimized Activation Induced Marker (AIM) T cell assay and dual-color B cell antigen probes. Using this assay, we demonstrate that antigen-specific B and T cell subsets can be identified simultaneously using conventional flow cytometry platforms and provide insight into the differential effects of mRNA vaccination on B and T cell populations following natural SARS-CoV-2 infection.

The identification, quantification, and characterization of antigen-specific 38 lymphocytes is a critical prerequisite for understanding the breadth, magnitude, and 39 functional potential of vaccine and/or natural pathogen-elicited immunity. Multiple The frequency of spike-specific B cells was expressed as a percentage of total B 139 cells (CD19 + , CD3 / CD14/CD56 / LIVE/DEAD -, lymphocytes), or as number per 140 Software, La Jolla, CA). End-point titers of SARS-CoV-2 antigen-reactive serum 161 samples were determined as the reciprocal of the final dilution at which the optical 162 density (OD) was greater than 2× of control SARS-CoV-2 naïve serum. Study design and assay workflow schematics were created with BioRender.com. 182

We developed an assay workflow (Figure 1a) for the simultaneous detection of 186 SARS-Cov-2 spike-specific T and B cells from cryopreserved PBMC. Cryopreserved 187 PBMC were thawed and stimulated for 24 hours with overlapping peptide pools 188 spanning SARS-CoV-2 spike to stimulate SARS-CoV-2 spike protein-reactive T cells, 

To demonstrate the utility of our new approach, we used the BATTLE assay to 203 examine the impact of SARS-CoV-2 mRNA vaccination on spike protein-specific 204 adaptive immunity following natural infection. We selected a cohort of subjects from a 205 large study of convalescent plasma donors who experienced symptomatic but mild 206 infection and had a peripheral blood draw between 30 and 75 days of symptom 207 resolution (Figure 2a) . This window was selected to capture the profile of antigen-208 specific memory lymphocytes proximally to natural infection, but following resolution of 209 acute inflammatory responses to infection. We included subjects who were vaccinated 210 with two doses of mRNA vaccine at least 9 months following a negative SARS-CoV-2 211 PCR test and had a subsequent blood draw more than 30 days, but no more than 22 212 weeks following vaccination. These time constraints were chosen to ensure that 213 immune memory was stable prior to vaccination, and to capture the profile of antigen-214 specific memory lymphocytes proximally to vaccination, but following resolution of acute 215 vaccine-driven inflammation. 216

To validate and extend the clinical utility of our findings, we evaluated the 217 serological profiles of each donor from the same visits used in the BATTLE assay. As CD4 + and CD8 + T cell compartments following natural infection. 243

We next sought to determine whether spike-reactive CD4 + and CD8 + T cells 244 within the same PBMC vial were similarly enhanced by mRNA vaccination after natural 245 SARS-CoV-2 infection. We quantified the expression of activation-induced markers on 246 these T cell subsets and found that, unlike for B cells targeting spike protein, the 247 frequency of CD4 + spike peptide-activated T cells was not significantly enhanced 248 following mRNA vaccination compared to infection alone (Figure 3b, e) . Although 249 significant changes were not observed for the full cohort, the frequency and number of 250 AIM + CD4 + T cells was increased following vaccination for some individuals, particularly 251 for those with a lower initial spike-specific adaptive response. The same trend was observed for subjects with low AIM + CD8 + T cell frequencies following natural infection, 253 but unlike the spike-specific CD4 + T cell compartment, CD8 + T cells reactive to spike 254 were marginally enhanced in frequency following mRNA vaccination (Figure 3c, f) . The 255 same patterns were present when adjusting for the number of cells used as input for the 256 assay, although for AIM + CD8 + T cells differences between the groups no longer 257 reached statistical significance (Supplementary Figure 3b, c) . As observed for spike-258 specific B cells, few AIM + T cells were present in PBMC samples collected prior to the 259 onset of the SARS-CoV-2 pandemic, and unlike T cells from exposed subjects, those 260 from pre-SARS-CoV-2 samples were largely unresponsive to spike peptide pool 261 stimulation (Supplementary Figure 3d, e) . Unlike other studies, we did not detect substantial frequencies or numbers of 296 SARS-CoV-2 spike-binding T cells in non-exposed individuals (Grifoni et al., 2020;  potentially to demographic variability between our sampling region and populations and 299 the those sampled in other studies. It is also possible that the sensitivity of our assay 300 was lower, although we consider this unlikely to be the case, as the quantities of The observation of increased quantities of spike-specific B cells following mRNA 305 vaccination after natural infection supports the idea that mRNA vaccination is beneficial 306 even after natural adaptive immunity has developed. Additionally, our results suggest 307 that spike-reactive B cells may possess a capacity for vaccine-elicited expansion 308 beyond that of spike-reactive CD4 + T cells. The mechanism by which this process may 309 occur is still an active area of investigation, but an attractive hypothesis is that B and T 310 cell populations each achieve an independent immunological setpoint following 311 resolution of acute infection or vaccine-induced activation. As the epitopes recognized 312 by B cells, CD4 + T cells, and CD8 + T cells, the timing and settings in which these events 313 occur, and the environmental signals these population receive can differ considerably, it 314 is tempting to speculate that uncoordinated dynamic expansion and contraction of B 315 and T cells can lead to the generation of immunological memory in a compartmentalized 316 manner. This interpretation would be supported with recent work describing coordinated 317 and timely B and T cell responses that lead to the generation of superior adaptive 318 immunity, and importantly that the lack thereof can have pathological consequences 319 (Lucas et al., 2021; Painter et al., 2021; Zohar et al., 2020) . BATTLE is an ideal 320 approach that can be harnessed to explore these concepts. 321

Our study did not exclude the possibility that co-culture during T cell stimulation 322 and/or BCR baiting has an impact on each cell population at the transcriptional level. 

Phenotypic analysis of antigen-specific T 470 lymphocytes

Techniques to Study Antigen-Specific 472 B Cell Responses

Immune correlates of protection by 475 mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates

A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific 479 antibody-secreting cells

A 482 Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal

Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood

Immunological memory to SARS-CoV-2 487 assessed for up to 8 months after infection

Persistent COVID-19 Symptoms Minimally Impact the 490 Development of SARS-CoV-2-Specific T Cell Immunity

Direct access to CD4+ T cells specific for defined antigens 493 according to CD154 expression

Targets of T

Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and 497 Unexposed Individuals

Demonstration that antigen-499 binding cells are precursors of antibody-producing cells after purification with a 500 fluorescence-activated cell sorter

Loss of Bcl

Expressing T Follicular Helper Cells and Germinal Centers in COVID-19

CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping 508 pentadecapeptides

Delayed production of neutralizing antibodies 511 correlates with fatal COVID-19

Use of overlapping 514 peptide mixtures as antigens for cytokine flow cytometry

Selective and cross-reactive 518 SARS-CoV-2 T cell epitopes in unexposed humans

Antigen-specific B cell detection reagents: use 520 and quality control

Switched and unswitched 523 memory B cells detected during SARS-CoV-2 convalescence correlate with limited 524 symptom duration

Rapid induction of 527 antigen-specific CD4+ T cells is associated with coordinated humoral and cellular 528 immunity to SARS-CoV-2 mRNA vaccination

Functional SARS-CoV-2-specific immune memory persists after mild COVID-19 532 (Infectious Diseases

Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with 536 Age and Disease Severity

Adaptive immunity to SARS-CoV-2 and COVID-19

SARS-CoV-2 infection induces long-541 lived bone marrow plasma cells in humans

Naturally 544 enhanced neutralizing breadth against SARS-CoV-2 one year after infection

Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients 549 with acute respiratory distress syndrome

Compromised Humoral Functional Evolution 552 Tracks with SARS-CoV-2 Mortality

S.J.T. reports compensation from Pfizer, during the conduct of the study; personal fees 357 from Merck, Sanofi, Takeda, Themisbio, and Janssen, outside the submitted work. All 358 other authors declare that the research was conducted in the absence of any 359 commercial or financial relationships that could be construed as a potential conflict of 360 interest. 361

Flow cytometry data has been submitted under ID FR-FCM-Z4PB and annotated at 363 http://flowrepository.org. All other data is available upon request. 364