key: cord-0844109-58c825jx
authors: Tan, H.-X.; Lee, W. S.; Wragg, K. M.; Nelson, C.; Esterbauer, R.; Kelly, H. G.; Amarasena, T.; Jones, R. M.; Starkey, G.; Wang, B. Z.; Yoshino, O.; Tiang, T.; Grayson, M. L.; Opdam, H.; D'Costa, R.; Vago, A.; The Austin Liver Transplant Perfusionist Group,; Mackay, L. K.; Gordon, C. L.; Wheatley, A. K.; Kent, S. J.; Juno, J. A.
title: Adaptive immunity to human coronaviruses is widespread but low in magnitude
date: 2021-01-26
journal: nan
DOI: 10.1101/2021.01.24.21250074
sha: de984874b4472e748f4f42255592cbcb002061df
doc_id: 844109
cord_uid: 58c825jx

Endemic human coronaviruses (hCoV) circulate worldwide but cause minimal mortality. Although seroconversion to hCoV is near ubiquitous during childhood, little is known about hCoV-specific T cell memory in adults. We quantified CD4 T cell and antibody responses to hCoV spike antigens in 42 SARS-CoV-2 uninfected individuals. T cell responses were widespread within conventional memory and cTFH compartments but did not correlate with IgG titres. SARS-CoV-2 cross-reactive T cells were observed in 48% of participants and correlated with HKU1 memory. hCoV-specific T cells exhibited a CCR6+ central memory phenotype in the blood, but were enriched for frequency and CXCR3 expression in human lung draining lymph nodes. Overall, hCoV-specific humoral and cellular memory are independently maintained, with a shared phenotype existing among coronavirus-specific CD4 T cells. This understanding of endemic coronavirus immunity provides insight into the homeostatic maintenance of immune responses that are likely to be critical components of protection against SARS-CoV-2.

In contrast to the high pathogenicity of MERS-CoV, SARS-CoV and SARS-CoV-2 46 coronaviruses, endemic human coronaviruses (hCoV) circulate worldwide but typically cause common colds with only limited morbidity and mortality 1 . Endemic hCoV encompass two alpha-48 coronaviruses (aCoV), NL63 and 229E, and two beta-coronaviruses (bCoV), HKU1 and OC43 1 . 49

Sero-epidemiological studies suggest that infection and seroconversion to hCoV occurs during 50 early childhood (typically by 4 years of age) 2-4 , although there are discrepant reports on the 51 prevalence of each virus within distinct geographical cohorts 2,5 . Despite the early development of 52 immunity against multiple hCoV, most adults remain susceptible to periodic reinfection 6-8 , with 53 increased susceptibility among immunocompromised individuals 9-11 . This suggests the 54 magnitude and/or quality of hCoV-targeted immunity in adults is insufficient for sterilizing 55 protection but instead may limit the burden of disease to asymptomatic or mild infection 8 . 56

Defining the extent of serological and/or cellular immunity required to protect individuals from 57 reinfection or severe disease remains a key question in the SARS-CoV-2 pandemic. As 58 neutralizing responses wane after CoV infection, it is likely that a combination of serum 59 antibody and B cell / T cell memory provide longer-term protection from the recurrence of 60 disease 12,13 . The study of hCoV-specific T and B cell memory can therefore provide a key 61 preview into the development of durable, protective SARS-CoV-2 immunity. 62

Characterisation of population-level immunity to hCoV can also inform our understanding of 63 cross-reactive immune responses between high pathogenicity and endemic CoV. Studies of 64 SARS-CoV-2-specific immunity in uninfected individuals clearly demonstrate pre-existing 65 cross-reactive antibody 14-16 , B cell 16 and T cell responses [17] [18] [19] [20] [21] [22] . Nevertheless, it is currently 66 unclear what contribution, if any, cross-reactive immunity plays in modulating the response to 67 SARS-CoV-2 infection or vaccination 23 . Detailed analyses of cross-reactive T cells suggest the 68 majority of such responses are dominated by CD4 T cells and directed toward non-RBD epitopes 69 of the spike (S) protein 18,21,24 . To date, however, consensus regarding the origin of these cross-70 reactive responses is lacking, with evidence both for 18 and against 25 a major contribution from 71 hCoV-specific memory T cells. 72

Deconvolution of cross-reactive SARS-CoV-2 responses and de novo SARS-CoV-2 immunity 73 requires a more detailed understanding of hCoV-specific serological and cellular memory. 74

Relatively little is known about population-level T or B cell memory to hCoV in adults, despite 75 evidence suggesting an impact of recent hCoV infection on COVID-19 severity 26 . Several 76 groups find widespread but modest CD4 T cell responses to hCoV proteins, with estimates for 77 the prevalence of memory responses ranging from 70-100% of study participants 24, 25, 27 . 78

Detection of hCoV-specific CD8 T cell responses has been less reported 27 , and the prevalence of 79 cross-reactive SARS-CoV-2-specific responses in these cohorts varies substantially 24, 25 . 80

Furthermore, data comparing hCoV-specific T or B cell responses in the circulation with the 81 presence or absence of such responses in the respiratory tract or secondary lymphoid organs 82 (SLO) is lacking. Studies in animal models suggest that respiratory infections can generate long-83 lived T cell memory in lung draining lymph nodes (LDLN) 28 , raising the possibility of analogous 84 responses following hCoV infection. 85

To address these knowledge gaps, we assessed the prevalence and phenotypic characteristics of 86 hCoV spike-specific antibody, memory T cell and memory B cell responses in a cohort of 87 SARS-CoV-2 uninfected adults. We find that the magnitude of hCoV immunity is independent 88 of age and is characterized by robust antibody titres, widespread CD4 T cell memory within both 89

Tmem and cTFH populations, and an enrichment of T cell memory in LDLN. In contrast, 90

Results 95

We recruited a cohort of 42 SARS-CoV-2 uninfected adults (n=21 male, n=21 female), ranging 97 in age from 18-67 years with no recent cold or COVID-19 symptoms ( Figure 1A ). Consistent 98 with previous studies 15, 16 , we detected baseline plasma antibody responses to one or more hCoV 99 S antigens in all participants, with substantially lower reactivity toward SARS-CoV-2 S (herein 100

CoV-2; Figure 1B ). Plasma IgG endpoint titres for hCoV antigens ranged from 1:176 to 1:18268 101 To determine the distribution of CD4 T cell memory responses, we stimulated PBMC with 105 recombinant S antigens and quantified antigen-specific Tmem (CD3 + CD4 + CD45RA + CXCR5 -) 106 by measuring upregulation of the activation markers CD25 and OX-40 by flow cytometry (a 107 well-established activation-induced marker (AIM) assay 29-31 ) ( Figure 1C ; gating in Supplemental 108 Figure 1 ). Across the cohort, 88% of individuals exhibited a memory response greater than 109 0.01% above background 32 to any hCoV S antigen ( Figure 1D ). Interestingly, the prevalence of 110 responses was highest to HKU1 S (86% of participants), and lowest to NL63, with only 50% of 111 individuals exhibiting NL63 S-specific responses ( Figure 1D ). The magnitude of responses to 112 hCoV S antigens ranged from undetectable to a maximum of 0.84% of the Tmem compartment 113 ( Figure 1E ). Among individuals with above-background responses, median antigen-specific 114

Tmem frequencies were highest to HKU1 (median 0.133%, IQR 0.056-0.248, n=36), followed 115 by OC43 (median 0.106%, IQR 0.049-0.170, n=34), NL63 (median 0.093%, IQR 0.055-0.168, 116 n=21), and 229E (median 0.080%, IQR 0.050-0.124, n=27). Similar to other cohorts 17,19 , we find 117 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.24.21250074 doi: medRxiv preprint 48% of participants (n=20) demonstrated cross-reactive response to CoV-2 S with a median 118 frequency of 0.049% (IQR 0.027-0.160), despite no evidence of prior infection ( Figure 1D /E). T 119 cell responses were similar when measured using either CD25/OX-40 or CD137/OX-40 19 AIM 120 assays ( Supplementary Figure 2A-C) . Across the cohort, there was no relationship between the 121 total frequency of hCoV S-specific Tmem and age, or any association with gender 122 (Supplementary Figure 3A-B) . 123 124 hCoV-specific CD4 Tmem are predominately TCM cells with a CCR6 + phenotype 125

Given divergent host receptor specificity and possible differences in tissue tropism among 126 hCoV 1 , we assessed whether memory or chemokine receptor phenotypes differed among S-127 specific CD4 T cell populations (gating in Supplementary Figure 1B) . Similar to the parental 128 Tmem population, hCoV S-specific and CoV-2 cross-reactive CD4 T cells were predominately 129 CD27 + CCR7 + , classically defined as central memory T cells (TCM; Figure 2A -B). In contrast to 130 the bulk Tmem population, however, hCoV S-specific cells were substantially enriched for 131 CCR6 expression (with or without co-expression of CXCR3; Figure 2C -D). When comparing 132 intra-individual responses, hCoV S-specific Tmem phenotypes were generally similar across all 133 S antigens ( Figure 2E ). Prior studies have also described a dominant CCR6 phenotype of CoV-2 134 S-specific Tmem among convalescent COVID-19 subjects 33 , and here we find that CoV-2 cross-135 reactive responses are similarly highly CCR6 biased ( Figure 2D-E) . 136 137 hCoV reactivity is detected among circulating T follicular helper cell (TFH) memory 138

Circulating TFH cells (cTFH; CXCR5 + CD45RA -) comprise a clonally 34 and functionally 29,35 139 distinct memory CD4 T cell population identified by CXCR5 expression. Activated cTFH 140 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 26, 2021. ; correlate with antibody responses to infection or vaccination, and are thought to be surrogates of 141 germinal centre (GC) TFH activity 36,37 . Resting cTFH, in contrast, may represent a long-lived, 142 homeostatic memory population from which recall responses can be elicited even years after 143 antigen exposure 38-41 . Like conventional Tmem, hCoV-specific and cross-reactive cTFH 144 responses were widely detected across the cohort ( Figure 3A ). The frequency of donors 145 exhibiting cTFH responses above 0.01% to each antigen was similar to that observed for Tmem 146 responses (90% for HKU1, 88% for OC43, 69% for 229E, 59% for NL63, 43% for CoV-2). 147

Median frequencies among responding donors were highest to HKU1 (median 0.241%, IQR 148 0.147-0.531), followed by OC43 (median 0.213%, IQR 0.126-0.424), 229E (median 0.126%, 149 IQR 0.061-0.340), NL63 (median 0.096%, IQR 0.050-0.210) and CoV-2 (median 0.085%, IQR 150 0.050-0.195) ( Figure 3A) . 151

Interestingly, hCoV responses comprised a greater proportion of the cTFH population compared 152 to the Tmem compartment in a paired analysis (p<0.002 for all hCoV antigens), with some 153 donors exhibiting a greater than 9-fold enrichment of hCoV-specific cells in the cTFH gate (data 154 for HKU1 shown in Figure 3B ). Similar to Tmem, antigen-specific cTFH were highly enriched 155 for a CCR6 + CXCR3phenotype ( Figure 3C -D). The phenotypes of HKU1-and CoV-2-specific 156 cTFH in SARS-CoV-2-uninfected donors are consistent with phenotypes previously described in 157 COVID-19 convalescent subjects 29 . Comparison of antigen-specific cTFH and Tmem cells 158 revealed a significant enrichment of the CCR6 + CXCR3phenotype among cTFH, including the 159 CoV-2 cross-reactive population ( Figure 3E -F). These data suggest that while hCoV memory is 160 broadly observed among both Tmem and cTFH subsets, the frequency and phenotype of these 161 responses are, to a degree, subset-specific. 162 163 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.24.21250074 doi: medRxiv preprint

It is currently unclear whether CoV-2 cross-reactive T cell responses arise primarily from hCoV 165 memory or reflect cross-reactivity from a broad array of antigen specificities 18,25 . Among the 166 cohort, subjects with CoV-2 cross-reactive CD4 T cell responses frequently exhibited memory 167 responses to multiple hCoVs ( Figure 4A ). We assessed the relationship between the frequency of 168

CoV-2 and hCoV memory responses and found significant correlations only between βCoV and 169 CoV-2 cross-reactivity (p=0.006 for HKU1, p=0.018 for OC43; Figure 4B ). This association is 170 consistent with a greater sequence homology among βCoV strains (CoV-2, HKU1 and OC43) 171 compared to the aCoV 229E and NL63 42 . 42 Among the subset of donors with cross-reactive 172 responses, only HKU1 memory correlated with CoV-2 cross-reactivity (p=0.030, Figure 4B ). 173

Interestingly, while almost all individuals with CoV-2 cross-reactive responses exhibited HKU1 174 and OC43 memory, the converse was not observed. Indeed, individuals with relatively similar 175 patterns of hCoV reactivity could exhibit notably different CoV-2 reactivity ( Figure 4C ). There 176 was no significant association of demographic characteristics among individuals with or without 177 CoV-2 cross-reactive responses, although the cross-reactive group did exhibit a greater 178 representation of women compared to those without cross-reactivity (p=0.06, Supplemental 179 Figure 3C -D). Given the association between HKU1 and CoV-2 T cell frequencies, we assessed 180 whether a particular phenotype of HKU-specific Tmem was related to the presence or absence of 181 cross-reactive responses, but found no such distinctions (Supplemental Figure 3E CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted January 26, 2021. ; expected from a coordinated acute immune response. To assess whether such associations are 187 maintained in long-term hCoV immunity, we explored correlations between antibody and T cell 188 responses across the cohort. Surprisingly, there was no relationship for any antigen between 189 plasma IgG endpoint titre and the frequency of either S-specific CD4 Tmem or cTFH (p>0.05 for 190 all; data for HKU1 and NL63 shown in Figure 5A ). To gain greater insight into the coordination 191 of cellular and humoral hCoV memory, we undertook an in-depth interrogation of immunity to 192 NL63, which shares use of the cellular entry receptor ACE2 with SARS-CoV and CoV-2, and 193 therefore likely has similar tissue tropisms in vivo. 194

To assess S-specific MBC and quantify plasma neutralising activity, NL63 S-specific memory B 195 cell (MBC) probes were generated as described previously 29 , and a novel NL63 pseudovirus-196 based neutralization assay was performed with 293T cells stably expressing hACE2 as targets. 197 MBC specific for NL63 and CoV-2 S were detected infrequently among the cohort, particularly 198 in comparison to the frequency of CoV-2 S-specific MBC previously reported among COVID-19 199 convalescent donors 29 ( Figure 5B ). Accordingly, the frequency of NL63 S-specific MBC did not 200 correlate with plasma NL63 binding IgG titres ( Figure 5C ). Plasma neutralising activity against 201 NL63 pseudovirus was detected among all donors tested, with a median IC50 of 100.7 (n=12, 202 IQR 56.6-234.6). Neutralising activity strongly correlated with NL63 S-specific antibody titres 203 (p=0.006) but was not associated with NL63 S-specific MBC frequencies ( Figure 5D ). We did, 204 however, observe a trend toward a positive correlation of neutralization with NL63 S-specific 205 cTFH responses (p= 0.081; Figure 5E ). Given our prior observation that CCR6 + CoV-2 S-206 specific cTFH responses were inversely associated with neutralizing antibodies after COVID-207 19 29 , we assessed the correlation between NL63 neutralising activity and NL63 S-specific cTFH 208 phenotype (for donors with NL63 S-specific cTFH responses, n=7). Interestingly, the frequency 209 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Figure 6A ). Given the higher levels of background T cell activation in SLO compared to 220 peripheral blood, we validated the specificity of the hCoV responses by confirming that antigen 221 stimulation also drove expression of CD154 on OX-40 + cells ( Figure 6A ). Among the 5 donors 222 studied, the median frequency of HKU1 and NL63 S-specific Tmem was 1.2% (range 0.12-2.19) 223 and 1.12% (range 0.31-4.04), respectively ( Figure 6B ). Reactivity to CoV-2 S was substantially 224 lower, with a median of 0.07% (range 0.01-0.99). Similar antigen-specific responses were 225 observed within the CD4 + CD45RA -CXCR5 + population ( Figure 6B ). There was limited to no 226 evidence of ongoing hCoV S-specific GC TFH activity among the samples (data not shown). 227

In contrast to the high frequencies of hCoV-specific CD4 T cells in LDLN, we found only 228 modest hCoV reactivity among lung-derived CD4 T cells (Supplementary Figure 6A -B). These 229 data are consistent with reports that tissue resident T cells (TRM) in the lung are relatively short-230 lived compared to other tissues 44 . Furthermore, the majority of AIM + cells did not exhibit a 231 CD69 + CD103 + phenotype, suggesting they are unlikely to represent bona fide lung-resident T 232 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. It has been speculated that the dominant CCR6 + phenotype of CoV-2-specific CD4 T cells may 236 reflect preferential homing of these cells to the lung 32 . 32 We therefore compared the 237 CCR6/CXCR3 phenotypes of hCoV-specific T cells in LDLN to the peripheral blood obtained 238 from the unmatched healthy adult cohort presented in Figures 2-3. After adjustment for baseline 239 activation, we found that LDLN-derived hCoV S-specific Tmem exhibited a predominately 240 CXCR3 + phenotype, with a substantial population of CCR6 -CXCR3 + cells (median 37.7% for 241 HKU1, 37.5% for NL63; Figure 6C -D). In contrast, only 10.8% and 11.2% of circulating HKU1 242 and NL63 S-specific Tmem among the blood donor cohort were CCR6 -CXCR3 + ( Figure 2D ). As 243 we previously observed for cTFH in the periphery, CXCR5 + hCoV-specific T cells in the LDLN 244 remained more likely to express CCR6 than their Tmem counterparts ( Figure 6E -F). 245

Nevertheless, LDLN-derived hCoV-specific CXCR5 + cells were enriched for CXCR3 expression 246 (median 30.7% CCR6 -CXCR3 + for HKU1, 19.4% for NL63) compared to the phenotypes 247 observed among peripheral cTFH (median 6.6% CCR6 -CXCR3 + for HKU1, 9.8% for NL63; 248 Figure 6E -F, Figure 3D ). Collectively, these data suggest either differential retention or 249 formation of CXCR3 + hCoV S-specific CD4 T cells in LDLN compared to peripheral blood. 250 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.24.21250074 doi: medRxiv preprint

Despite periodic re-infection, most adults experience only mild or asymptomatic hCoV infection, 252

suggesting the presence of at least partially protective immune memory. We find that, in addition 253 to near-universal plasma antibody reactivity to hCoV, memory T cell responses to both aand 254 bCoV are widespread. In contrast, the relatively modest neutralization activity against NL63 and 255 low frequencies of S-specific MBC suggest that sterilizing humoral immunity is likely absent. Consistent with other cohorts 17,19 , we find evidence for CoV-2 cross-reactive CD4 T cells in 279 uninfected donors. In vitro expansion of CoV-2 cross-reactive T cell clones has demonstrated the 280 potential for shared specificity with all hCoV 18,24 . However at a cohort-wide level, we find the 281 frequency of CoV-2 cross-reactive cells correlates most strongly with HKU1 memory, although 282 no immediate immunological or demographic features distinguish HKU1-reactive individuals 283 with or without cross-reactive CoV-2 responses. Larger population-based studies will be required 284 to determine any associations between particular HLA class II alleles and cross-reactive CD4 285 responses. Although it has been speculated that pre-existing cross-reactive T cell immunity could 286 be beneficial in the context of SARS-CoV-2 vaccines 23 , it should be noted that only CXCR3 + , 287 but not CCR6 + , cTFH responses appear to correlate with neutralizing antibody titres during 288 COVID-19 convalescence 29,31,48 . While recall of the CCR6 + cTFH could induce expression of 289 CXCR3, currently available evidence suggests the highly CCR6-biased responses to hCoV may 290 not be beneficial in the context of vaccination or re-exposure. 291

Overall, these data clarify the characteristics of long-term immunity to endemic coronaviruses, 292 which have comparable magnitudes and share phenotypic features of S-specific antibody and T 293 cell memory across all four hCoV. Insight into the homeostatic maintenance of hCoV immunity 294 is likely to provide a preview of long-term CoV-2-specific immunity established in the 295 population after vaccination or wide-spread infection. 296

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.24.21250074 doi: medRxiv preprint 297

Subject recruitment and sample collection 299 SARS-CoV-2 uninfected controls were recruited as part of a previous COVID-19 study 29 , and 300 relevant demographic characteristics are indicated in Figure 1A . For all participants, whole blood 301 was collected with sodium heparin anticoagulant. Plasma was collected and stored at -80ºC, and 302

PBMCs were isolated via Ficoll Paque separation, cryopreserved in 10% DMSO/FCS and stored 303 in liquid nitrogen. The study protocols and sample use were approved by the University of Melbourne 304

Human Research Ethics Committee (#2056689) and all associated procedures were carried out in 305 accordance with the approved guidelines. All participants provided written informed consent in 306 accordance with the Declaration of Helsinki. 307

The use of tissue samples from human donors was approved by The University of Melbourne 308

Human Research Ethics Committee (#1954691) and all associated procedures were carried out in 309 accordance with approved guidelines. Tissues were collected from 6 donors: male, age 40-50, brain 310 death; female, 30-40, brain death; male, 30-40, circulatory death; male, 50-60, brain death; female, 311 60-70, brain death; female, 50-60, brain death. Tissues were passed through 70µM filters and 312 homogenised into single cell suspensions, which were subsequently cryopreserved in 10% DMSO/FCS. were tested in triplicate, with "virus+cells" and "virus only" controls included to represent 100% 385 and 0% infectivity respectively. After 48 hours, all cell culture media was carefully removed from 386 wells. Cells were lysed with 25µl of passive lysis buffer (Promega), incubated on an orbital shaker 387 for 15 mins and developed with 30µl britelite plus luciferase reagent (Perkin Elmer). 388

Luminescence was read using a FLUOstar Omega microplate reader (BMG Labtech). The relative 389 light units (RLU) measured were used to calculate %neutralisation with the following formula: 390 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 26, 2021. ; https://doi.org/10.1101/2021.01.24.21250074 doi: medRxiv preprint Neutralisation IC 50 (10 x ) S-specific cTFH CCR6 + S-specific cTFH S-specific IgG

Spearman correlation between HKU1 or NL63 S-specific IgG and the frequency of antigenspecific Tmem or cTFH (n=42). Representative staining of IgD-B cells with NL63 or CoV-2 probes and quanification of NL63 and CoV-2 S-specific MBC (n=18). (C) Spearman correlation of NL63 S-specific MBC and plasma binding IgG titres (n=18). MBC frequency was assigned a minimum value of 0.001%. (D) Spearman correlation between plasma NL63 neutralization activity and NL63 S-specific IgG titres or MBC (n=12). (E) Spearman correlation between NL63 neutralization activity and either total NL63 S-specific cTFH or the frequency of CCR6+ antigen-specific cTFH. 

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