key: cord-1018170-8uvcpgev authors: Chen, Jennifer S.; Chow, Ryan D.; Song, Eric; Mao, Tianyang; Israelow, Benjamin; Kamath, Kathy; Bozekowski, Joel; Haynes, Winston A.; Filler, Renata B.; Menasche, Bridget L.; Wei, Jin; Alfajaro, Mia Madel; Song, Wenzhi; Peng, Lei; Carter, Lauren; Weinstein, Jason S.; Gowthaman, Uthaman; Chen, Sidi; Craft, Joe; Shon, John C.; Iwasaki, Akiko; Wilen, Craig B.; Eisenbarth, Stephanie C. title: High-affinity, neutralizing antibodies to SARS-CoV-2 can be made in the absence of T follicular helper cells date: 2021-06-11 journal: bioRxiv DOI: 10.1101/2021.06.10.447982 sha: 6533f258525dbcd28e894b405ff4f318dd5a7f9c doc_id: 1018170 cord_uid: 8uvcpgev T follicular helper (Tfh) cells are the conventional drivers of protective, germinal center (GC)-based antiviral antibody responses. However, loss of Tfh cells and GCs has been observed in patients with severe COVID-19. As T cell-B cell interactions and immunoglobulin class switching still occur in these patients, non-canonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both Tfh-dependent and -independent antibodies were induced against SARS-CoV-2 as well as influenza A virus. Tfh-independent responses were mediated by a population we call lymph node (LN)-Th1 cells, which remain in the LN and interact with B cells outside of GCs to promote high-affinity but broad-spectrum antibodies. Strikingly, antibodies generated in the presence and absence of Tfh cells displayed similar neutralization potency against homologous SARS-CoV-2 as well as the B.1.351 variant of concern. These data support a new paradigm for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GCs and even compensate for GCs damaged by viral inflammation. One-Sentence Summary Complementary pathways of antibody production mediate neutralizing responses to SARS-CoV-2. Antibodies are critical for protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID- 19) pandemic (1) . T follicular helper (Tfh) cells are the conventional drivers of protective antibody responses, as they support immunoglobulin class switching, germinal center (GC)-based affinity maturation, and long-lived humoral immunity (2) . Indeed, multiple studies have reported a correlation between circulating Tfh (cTfh) cells -in particular, type 1 CXCR3 + CCR6 − cTfh cells -and neutralizing antibody titers in COVID-19 patients (3) (4) (5) (6) (7) (8) (9) . While there have been mixed findings on the association of Tfh cells with disease severity, several groups have observed an absence of Tfh cells and GCs in severely ill patients (4, 5, 7, (9) (10) (11) (12) (13) (14) (15) . Despite a loss of Tfh cells and GC structures, T cell-B cell interactions and antibody class switching still occur in the secondary lymphoid organs of these patients (14) . Findings of enhanced extrafollicular B cell responses associated with severe disease further suggest that non-canonical pathways of antibody production may be operative in these individuals (14, 16) . However, a causal relationship between antibody provenance and disease severity cannot be established by existing human studies. Thus, it remains unclear whether antibodies produced through non-canonical pathways without Tfh cell help are also protective against SARS-CoV-2. No study has mechanistically evaluated the role of Tfh cells in antibody production to SARS-CoV-2. Previous work with SARS-CoV has shown that CD4 + T cells are required for neutralizing antibody titers (17) , but the nature of the T cell population has not been identified. For other viruses, as well as bacteria, Tfh cells are thought to be required for class-switched, pathogen-specific antibody production, especially at later timepoints (18) (19) (20) (21) (22) (23) (24) (25) (26) . In contrast, different studies have found conflicting results on the requirement of Tfh cells during vaccination in mice; certain vaccine strategies induce robust Tfh-independent antibody responses, though of lower affinity (27, 28) , while other strategies fail to induce durable, class-switched, or high-affinity antibodies in mice with Tfh cell deficiency or dysfunction (19, (29) (30) (31) . In humans though, cTfh cell populations in the blood correlate with the response to vaccination (32-34). Taken together, while non-Tfh CD4 + T cells can promote effective antibodies in certain contexts, protective antipathogen humoral immunity is largely thought to be Tfh cell-dependent. We therefore directly tested whether non-Tfh CD4 + T cells could compensate for Tfh cell loss in severe COVID-19, and perhaps during acute viral infection in general, by promoting classswitched antibodies. Based on prior work we hypothesized that antibodies induced through such non-canonical mechanisms would be lower in quantity and quality. To test this, we characterized the titer, isotype, longevity, affinity, and function of antibodies to SARS-CoV-2 as well as another clinically relevant respiratory virus, influenza A virus, from Tfh-deficient mice. We found that both infections induced substantial levels of Tfh-independent class-switched antibodies, likely driven by a population we call lymph node (LN)-Th1 cells. LN-Th1-driven antibodies to SARS-CoV-2 were durable and remarkably high-affinity. These antibodies were also broadly reactive against diverse SARS-CoV-2 epitopes and effective at neutralizing both homologous and heterologous viruses. In addition, LN-Th1 cells were present in the setting of intact GCs, suggesting that LN-Th1-driven responses are not just compensatory but also complementary with canonical Tfhdependent antibody production to multiple respiratory viruses. Thus, our study suggests a new paradigm for T cell-driven antibody responses to viruses in which a subset of Th1 cells complement Tfh cells in secondary lymphoid organs by interacting with and guiding B cell responses without emigrating to tissues to direct local cellular responses. Therefore, multiple types of CD4 + T cells in lymphoid organs coordinate the humoral response to viruses. This new understanding of T cell-B cell interaction could aid in approaches to develop effective protection against SARS-CoV-2 as well as other viruses. To study the cellular pathways that promote antibody production to SARS-CoV-2, we used mice that lack different CD4 + T cell subsets. Bcl6 fl/fl Cd4 Cre mice lack Tfh cells due to deletion of the Tfh lineage-defining transcription factor BCL6, and Ciita −/− mice lack all CD4 + T cells due to loss of MHC class II expression (30, 35). As SARS-CoV-2 is unable to efficiently interact with mouse ACE2, we overexpressed human ACE2 (hACE2) in the respiratory tract of these mice via intratracheal administration of AAV-hACE2 (36, 37). Two weeks after AAV-hACE2 transduction, we infected mice intranasally with SARS-CoV-2 (isolate USA-WA1/2020) and then assessed cellular and humoral responses at 14 days post infection (dpi) (Fig. 1A) . PD-1 hi CXCR5 hi Tfh cells were efficiently deleted in the mediastinal LN (medLN) of Bcl6 fl/fl Cd4 Cre mice (Fig. S1 , A to C). Consistent with the loss of Tfh cells, GC B cells were severely impaired, and plasmablast formation was reduced (Fig. S1, D to H ). Yet viral burden in the lungs of Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice was similar at 7 dpi (Fig. S1I ). Bcl6 fl/fl mice produced high levels of spike (S)-specific IgG antibodies at 14 dpi (Fig. 1B) . While Bcl6 fl/fl Cd4 Cre mice had reduced levels of S-specific IgG, they still produced substantially more compared to Ciita −/− mice. We next examined the requirement for Tfh cell help among different IgG subclasses. While S-specific IgG1 and IgG3 were completely Tfh-dependent, Sspecific IgG2b and IgG2c were promoted by both Tfh and non-Tfh CD4 + T cells (Fig. 1C ). Consistent with their divergent requirement for Tfh cell help, S-specific IgG2c was induced earlier than S-specific IgG1 (Fig. S1J ). S-specific IgM was unaffected by the absence of Tfh cells (Fig. S1K ). Together, these results suggest that both Tfh cells and non-Tfh CD4 + T cells promote the production of class-switched antibodies to SARS-CoV-2, though Tfh cells are uniquely able to induce certain subclasses. We next asked whether these findings were generalizable to other models of respiratory viral infection. We infected mice with mouse-adapted influenza virus A/PR/8/34 H1N1 (PR8) and assessed antibody production at 14 dpi (Fig. 1D ). Similar to SARS-CoV-2 infection, PR8 infection induced both Tfh-dependent and non-Tfh CD4 + T cell-dependent IgG antibodies (Fig. 1E) . Again, PR8-specific IgG1 demonstrated a complete dependence on Tfh cell help, while PR8-specific IgG2b and IgG2c were promoted by both Tfh-dependent and -independent pathways (Fig. 1F ). PR8-specific IgG3 and IgM were only partially dependent on Tfh and CD4 + T cell help ( Fig. 1F and S1L). Thus, both Tfh and non-Tfh CD4 + T cells contribute to antibody production in two distinct models of respiratory viral infection. To determine which non-Tfh CD4 + T cell populations promote antibody production, we analyzed the medLN at 7 dpi with SARS-CoV-2. While total CD4 + T cell counts were unaffected in Bcl6 fl/fl Cd4 Cre mice, activated CD44 + CD4 + T cell counts were reduced, consistent with the loss of Tfh cells (Fig. S2 , A and B) . We classified PD-1 lo CXCR5 lo CD44 + CD4 + T cells by their expression of PSGL-1 and Ly6C (Fig. S2C ). These markers have previously been used in acute LCMV and influenza virus infection to distinguish terminally differentiated PSGL-1 hi Ly6C hi Th1 cells from a heterogeneous PSGL-1 hi Ly6C lo Th1 compartment containing memory precursors along with other Th1-related functional subsets (38-40). We therefore defined PSGL-1 hi Ly6C hi cells as terminally differentiated Th1 cells and PSGL-1 hi Ly6C lo cells as mixed Th1 cells. We also observed a BCL6dependent PSGL-1 lo Ly6C lo population within the PD-1 lo CXCR5 lo gate, which has previously been described as pre-Tfh cells (Fig. S2 , D and E) (41) . Mixed Th1 and terminally differentiated Th1 cells increased in relative frequency among activated CD4 + T cells in Bcl6 fl/fl Cd4 Cre mice, consistent with the loss of Tfh and pre-Tfh cells; however, their absolute numbers were similar between Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice, indicating that BCL6 deficiency did not lead to an aberrant increase in Th1 populations (Fig. S2 , F to I). We next evaluated whether these Th1 populations produce CD40L and IL-21, effector molecules usually ascribed to Tfh cells (2) . CD40L and IL-21 act at multiple stages to support B cell activation, proliferation, differentiation, and antibody production (2) . While Tfh cells produced the highest levels of CD40L, mixed Th1 cells also produced substantial levels of this effector molecule (Fig. 2 , A and B) . Tfh cells and mixed Th1 cells comprised the majority of CD40Lexpressing CD4 + T cells in the medLN of Bcl6 fl/fl mice, while mixed Th1 cells became the main CD40L-expressing cells in Bcl6 fl/fl Cd4 Cre mice (Fig. 2C ). In Bcl6 fl/fl Cd4 Cre mice, the frequency of CD40L + cells among CD4 + T cells was decreased (Fig. S3A) , likely owing to the loss of CD40Lexpressing Tfh cells. However, mixed Th1 and terminally differentiated Th1 cells expressed higher levels of CD40L in Bcl6 fl/fl Cd4 Cre mice compared to Bcl6 fl/fl mice (Fig. S3 , B and C). In the absence of Tfh cells, Th1 subsets may have more opportunities to interact with antigen-presenting cells and experience T cell receptor signaling, which promotes CD40L expression (42) . In As mixed Th1 cells express IL-21 in addition to CD40L, they could support an alternative pathway of antibody production to that driven by Tfh cells. We therefore assessed whether mixed Th1 cells are sub-anatomically positioned to provide help to B cells. PSGL-1 mediates chemotaxis to CCL21 and CCL19, therefore helping naïve CD4 + T cells home to secondary lymphoid organs (45) . During their differentiation, Tfh cells downregulate PSGL-1 and upregulate CXCR5 to enable their migration into B cell follicles (41, 46) . However, as mixed Th1 cells continue to express PSGL-1 and do not upregulate CXCR5, it is unclear whether they can migrate to sites of B cell help. Immunofluorescence of medLN following PR8 infection showed that Th1 cells (PSGL-1 + CD4 + T-bet + ) co-localized with IgG2c + B cells at the T cell-B cell (T-B) border (Fig. 2J ). This was true in both Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice, suggesting that Th1 cells promote antibody production in parallel with Tfh cells as well as in their absence. Taken together, we found that a subset of mixed Th1 cells expressed CD40L and IL-21 and were positioned at the T-B border to help B cells during viral infection. To distinguish this population from the Th1 cells that function in peripheral tissues (e.g., the lung), we call these cells "LN-Th1" cells. We next measured the affinity of the antibodies generated during SARS-CoV-2 and PR8 infection. As GCs are the conventional site of somatic hypermutation and affinity maturation (2), we expected that Bcl6 fl/fl mice would generate high-affinity antibodies through Tfh/GC-dependent processes while Bcl6 fl/fl Cd4 Cre mice would generate low-affinity antibodies through LN-Th1-driven responses. Bcl6 fl/fl mice produced high-affinity IgG antibodies to S as well as the spike receptorbinding domain (RBD) (Fig. 3A) , a major target of neutralizing antibodies (47) . In contrast, S-and RBD-specific IgG antibodies from Ciita −/− mice displayed minimal affinity. However, we discovered that antibodies from Bcl6 fl/fl Cd4 Cre mice still demonstrated substantial affinity toward S and RBD, suggesting that LN-Th1 cells could promote high-affinity antibody production. To determine whether this was also true in a SARS-CoV-2 infection model that does not require AAV pre-transduction, we crossed Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice to K18-hACE2 transgenic mice, which express hACE2 in epithelial cells (48) . Both K18-hACE2 Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice produced high-affinity antibodies to S and RBD (Fig. 3B) , indicating that non-Tfh cells can support high-affinity antibody production in two separate models of SARS-CoV-2 infection. However, this was not the case with PR8 infection, as Bcl6 fl/fl Cd4 Cre mice produced IgG antibodies of minimal affinity to both PR8 and PR8 surface glycoprotein hemagglutinin (HA) (Fig. 3C ). Therefore, the ability of non-Tfh cells, likely LN-Th1 cells, to promote high-affinity antibodies may depend on the nature of the viral infection and the antigenic target. We also evaluated the durability of S-specific IgG antibodies produced by Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice following SARS-CoV-2 infection. Previous studies of viral infection have shown that antibody titers in mice with impaired Tfh cells are especially reduced at later timepoints (18, 19, 25) . In SARS-CoV-2 infection, S-specific IgG levels peaked at 14 dpi in Bcl6 fl/fl mice and were stable through 49 dpi (Fig. 3D ). In Bcl6 fl/fl Cd4 Cre mice, S-specific IgG antibodies were reduced tenfold at all timepoints measured yet remained stable between 14 dpi and 49 dpi (Fig. 3E ). Together, these results indicate that Tfh-independent antibodies to SARS-CoV-2 are both highaffinity and durable -two important qualities usually attributed to Tfh-dependent responses. We next characterized the antibody epitope repertoire of Tfh-versus LN-Th1-driven responses to SARS-CoV-2. Sera from AAV-hACE2 Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice at 14 dpi were profiled using a bacterial display library of 2410 linear peptides tiling the entire SARS-CoV-2 proteome (Fig. 4A ). We first compared the diversity of antibody epitope reactivity, calculating the Shannon entropy, Simpson's diversity index, and the repertoire focusing index within each sample (Methods). We observed that antibody diversity assessed by Shannon entropy and Simpson's diversity index was significantly decreased in Bcl6 fl/fl mice compared to Bcl6 fl/fl Cd4 Cre mice, while the degree of repertoire focusing was increased (Fig. 4B ). These findings were robust to variations in read counts (Fig. S4A ). These results suggest that Tfh cells help focus the antibody response to particular viral epitopes while LN-Th1 cells promote antibodies to a wider array of targets. Given these changes in antibody diversity, we next explored whether there were differences in antibody reactivity at the level of individual linear epitopes. After normalizing for read count variations using the median of ratios method (Fig. S4B) , we identified epitopes that were comparatively enriched or depleted in Bcl6 fl/fl versus Bcl6 fl/fl Cd4 Cre mice (Fig. 4C ). We found that seven epitopes were enriched in Bcl6 fl/fl mice, while one epitope was depleted (Fig. 4D) . Five of the seven enriched epitopes were derived from S: aa661-672 (proximal to S1/S2 cleavage site), To further investigate alterations in epitope reactivity, we converted the normalized counts to z-scores on a sample-by-sample basis, such that the z-scores would denote the relative rank of a specific epitope within a particular sample (Fig. S4C ). Consistent with our prior analyses, the average z-scores in Bcl6 fl/fl versus Bcl6 fl/fl Cd4 Cre mice were similar across most SARS-CoV-2 proteins, with the exception of regions within the S and ORF3a proteins (Fig. 4E ). In particular, the regression lines for non-S proteins all closely followed the line of identity, indicating that the relative ranks of epitopes from non-S proteins were largely similar in the presence or absence of Tfh cells. These analyses therefore indicate that, while LN-Th1 cells can promote antibody production against most SARS-CoV-2 epitopes, Tfh cells focus the antibody response against certain S-derived epitopes. Analyzing antibody epitope reactivity along the length of S, we observed that the majority of epitopes enriched in Bcl6 fl/fl mice (17/21 epitopes with differential average z-score > 1) were found in the S2 domain (aa686-1273; Fisher's exact test p = 0.0012) (Fig. 5A) , which mediates fusion of viral and target cell membranes (49) . These included most of the aforementioned S-derived epitopes that were significantly enriched (Fig. 4C ), as well as contiguous epitopes whose enrichment did not reach statistical significance in the epitope-level analysis. For example, several epitopes in the fusion peptide were enriched adjacent to the significantly enriched epitopes aa801-812 and aa817-828 (Fig. S4D ). Many of these epitopes are highly conserved across human coronaviruses (hCoVs) as well as the emerging variants of concern (Fig. 5A) . Interestingly, the enriched epitopes spanning FP1/FP2 and preceding HR2 have also been identified in numerous studies profiling the antibody epitope repertoire of COVID-19 patients (50) (51) (52) (53) (54) (55) (56) (57) (58) (59) (60) . Given their immunodominance and conservation across hCoVs, these epitopes have been proposed as targets for a pan-coronavirus vaccine, though it remains unclear whether antibodies directed against these regions are actually able to neutralize virus (51, 52, 55, 57, 58, 61) . In contrast, Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice demonstrated similar antibody reactivity to most epitopes within the RBD (Fig. 5B) , the target of most neutralizing antibodies (62) . However, as most antibodies to RBD likely recognize conformational epitopes (56, 57) , we also measured RBD-specific antibodies by ELISA using full-length RBD. RBD-specific IgG titers normalized by total S-specific IgG were similar between Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice (Fig. 5C) . Thus, while Tfh cells focus the antibody response against immunodominant S2 epitopes, LN-Th1 cells still promote antibodies against the primary target of neutralization, RBD. We next evaluated the function of antibodies from Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice following SARS-CoV-2 infection. While we had observed that Tfh-independent antibody responses lacked IgG1/IgG3 subclasses (Fig. 1C) and S2 epitope focusing (Fig. 5A) , these antibodies were still high-affinity (Fig. 3, A and B) and could target the RBD (Fig. 5C ). We therefore expected that Tfhindependent, LN-Th1-driven antibodies would demonstrate similar neutralizing function against homologous SARS-CoV-2 (USA-WA1/2020) as those generated with Tfh cell help. Using vesicular stomatitis virus (VSV) pseudotyped with USA-WA1/2020 S protein, we measured the neutralization titer (the reciprocal serum dilution achieving 50% neutralization of pseudovirus infection, NT50) of sera from Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre mice. Bcl6 fl/fl sera exhibited increased NT50 ( Fig. 6A) , which was expected given their higher levels of S-specific IgG antibodies (Fig. 1B) . However, by normalizing NT50 to S-specific IgG levels in each sample, we observed that the neutralization potency indices of Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre sera were similar and actually trended higher for Bcl6 fl/fl Cd4 Cre sera (Fig. 6A) . We next tested the same sera against VSV pseudotyped with S protein from the B.1.351 variant of concern. Multiple studies have shown that B.1.351 S mutations, particularly those in the RBD, disrupt binding by neutralizing antibodies and facilitate immune escape (63) (64) (65) (66) (67) (68) . We therefore hypothesized that increased focusing of Tfh-dependent antibodies against conserved S2 epitopes would enable Bcl6 fl/fl sera to better neutralize B.1.351 pseudovirus than Bcl6 fl/fl Cd4 Cre sera. While Bcl6 fl/fl sera exhibited greater NT50 than Bcl6 fl/fl Cd4 Cre sera, we found that the neutralization potency index of Bcl6 fl/fl Cd4 Cre sera was higher than that of Bcl6 fl/fl sera (Fig. 6B) . While protective antibodies are usually generated through Tfh/GC-dependent pathways, it is unclear what happens to the antibody response when these structures are disrupted by virusinduced inflammation. We found that certain class-switched antibodies were reduced but still present in Tfh-deficient mice during both SARS-CoV-2 and influenza virus infection. Tfhindependent antibodies to SARS-CoV-2, likely driven by LN-Th1 cells, were still high-affinity and durable while also demonstrating more diverse epitope reactivity compared to Tfh-dependent antibodies. Importantly, LN-Th1-driven antibody responses neutralized both homologous SARS-CoV-2 (USA-WA1/2020) and the B.1.351 variant of concern and were functional in vitro and in vivo (Fig. 7) . Though class-switched antibodies could be produced in the absence of Tfh cells, we observed that certain subclasses demonstrated a complete dependence on Tfh cell help. For example, IgG1 production was completely abrogated in Tfh-deficient mice infected with SARS-CoV-2 or influenza virus. This is consistent with previous studies analyzing antibody production by Tfh-deficient mice in the setting of vaccination or Zika virus infection (25, 27) . In contrast, IgG2c was still made without Tfh cells but generally required CD4 + T cell help. This divergent PSGL-1 expression on naïve CD4 + T cells is required for chemotaxis to CCL21 and CCL19, thus mediating homing to secondary lymphoid organs and ultimately the T cell zone (41, 45) . Once activated, T cells upregulate several glycosyltransferases that modify PSGL-1 such that it can interact with P-selectin and enable trafficking to sites of inflammation (74, 75) . These carbohydrate modifications of PSGL-1 also interfere with its ability to interact with CCL21 (45), providing a possible mechanism for how PSGL-1 hi LN-Th1 cells migrate from the T cell zone to the T-B border. Upregulation of chemokine receptors such as CXCR3 may also support LN-Th1 migration, as CXCR3 ligands CXCL9 and CXCL10 have been shown to regulate intranodal positioning and optimal differentiation of Th1 cells (76, 77) . However, in contrast to terminally differentiated Th1 cells that then emigrate from the LN to coordinate cellular responses in peripheral tissues, LN-Th1 cells stay in the LN to provide B cell help and promote antibody production. A surprising finding from our work was that SARS-CoV-2, but not influenza virus, induced high-affinity Tfh-independent antibodies. However, CD4 + T cell-independent antibodies demonstrated minimal affinity, indicating that either Tfh or LN-Th1 cells are required for highaffinity responses. While affinity maturation conventionally occurs in the GC through the iterative process of somatic hypermutation and competition for Tfh cell help (2) Epitope profiling revealed that Tfh cells focus the antibody repertoire against S2-derived epitopes that are highly conserved across human coronaviruses as well as the emerging variants of concern. These same epitopes spanning FP1/FP2 and preceding HR2 have been repeatedly identified in studies profiling the antibody repertoire of COVID-19 patients, suggesting that the immunodominance of these epitopes in humans is mediated by Tfh cells. However, it is less clear whether these S2-reactive antibodies are actually neutralizing. Analyses of S2-reactive antibodies from patient sera demonstrate that these antibodies have weak or absent neutralizing function (51, 52, 55, 89) . Additionally, vaccination studies in rabbits and mice with S2 or S2-derived epitopes have reached conflicting conclusions about the ability of the resultant sera to neutralize SARS-CoV-2 (57, 58, 90) . SARS-CoV-2 S2-cross-reactive antibodies have also been identified in pre-pandemic serum samples, likely induced by exposure to seasonal coronaviruses (53, 91) . While some studies have found that these cross-reactive antibodies can neutralize SARS-CoV-2 in vitro, others have not (61, 91, 92) . In patients, cross-reactive antibodies are also not associated with protection against SARS-CoV-2 infection or severe COVID-19 (61) . Our findings further suggest that S2-reactive antibodies are not protective, as Tfh-dependent antibodies enriched for S2 epitope reactivity displayed a similar or even lower neutralization potency index against SARS-CoV-2 than more diverse Tfh-independent antibodies. diversify Fc effector function, and focus the repertoire against immunodominant epitopes, LN-Th1 cells ensure the production of antibodies with activating Fc effector function and broader reactivity. LN-Th1-driven responses may serve as a parallel mechanism for producing protective antibodies in settings of Tfh/GC impairment, such as COVID-19-induced inflammation and old age (14, 93) . Understanding this additional axis of antiviral antibody production may therefore inform more effective vaccine design and help establish a new paradigm for how T cell-dependent humoral immunity is generated. We thank all members of the Eisenbarth and Wilen labs for helpful discussions. We would like to acknowledge Benhur Lee, BEI Resources, Joerg Nikolaus, and Yale West Campus Imaging Core for providing critical reagents, resources, and expertise. We thank Yale Environmental Health and Safety for providing necessary training and support for SARS-CoV-2 research. Illustrations were created with BioRender.com. Yale University (CBW) has a patent pending entitled "Compounds and Compositions for Treating, Ameliorating, and/or Preventing SARS-CoV-2 Infection and/or Complications Thereof." Yale University has committed to rapidly executable nonexclusive royalty-free licenses to intellectual property rights for the purpose of making and distributing products to prevent, diagnose, and treat COVID-19 infection during the pandemic and for a short period thereafter. All data are available in the main text or the supplementary materials. Figures S1 to S4 Data S1 to S10 (F) PR8-specific IgG1, IgG2b, IgG2c, and IgG3 antibody titers at 14 dpi with PR8. LOD, limit of detection of the assay. Statistical significance was assessed by either two-tailed unpaired t-test or Welch's t-test, based on the F test for unequal variance. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant. Data are expressed as mean ± standard error of mean (SEM) log10 arbitrary units (AU). Each symbol represents an individual mouse. Data are aggregated from at least two independent experiments. (E) Relative serum titers of S-specific IgG at 49 dpi, compared to S-specific IgG at 14 dpi. on the F test for unequal variance. *P < 0.05; **P < 0.01; ****P < 0.0001. ns, not significant. Data are expressed as mean ± SEM. Each symbol in (A to C and E) represents an individual mouse. Each symbol in (D) represents the mean of six mice. Data are aggregated from at least two independent experiments. Serum samples for epitope profiling were inactivated with UV light (250 mJ). The SERA platform for next-generation sequencing (NGS)-based analysis of antibody epitope repertoires has been previously described (101) . In brief, Escherichia coli were engineered with a surface display vector carrying linear peptides derived from the SARS-CoV-2 proteome (GenBank MN908947.3), designed using oligonucleotides (Twist Bioscience) encoding peptides 12 amino acids in length and tiled with 8 amino acids overlapping. Serum samples (0.5 µl each) were then diluted 1:200 in a suspension of PBS and bacteria carrying the surface display library (10 9 cells per sample with 3×10 5 fold library representation), and incubated so that antibodies contained in the serum would bind to the peptides on the surface of the bacteria. After incubating with protein A/G magnetic beads and magnetically isolating bacteria that were bound to antibodies contained in the serum, plasmid DNA was purified and PCR amplified for NGS. Unique molecular identifiers (UMIs) were applied during PCR to minimize amplification bias, designed as an 8 base pair semi-random sequence (NNNNNNHH). After preprocessing and read trimming the raw sequencing data, the resulting reads were filtered by utilizing the UMIs to remove PCR duplicates. The filtered UMI data (hereafter referred to as reads) were then aligned to the original reference of linear epitopes derived from SARS-CoV-2 and quantified. From the raw mapped read counts for each of the 2410 linear epitopes represented in the library, we first calculated the Shannon entropy and Simpson's diversity index of each sample using the diversity function in the diverse R package. To calculate the "repertoire focusing index", we used the formula: 1-(H'/log2(R)), where H' is Shannon entropy and R is richness (102) , defined here as the number of unique epitopes recognized by a given sample (read count > 0). Statistical differences in these various metrics were assessed by two-tailed unpaired Welch's t-test, comparing Bcl6 fl/fl and Bcl6 fl/fl Cd4 Cre conditions. For further analysis, we normalized the raw count data using the median ratio approach implemented in DESeq2 (103, 104) . Differential enrichment analysis was performed using the Wald test in DESeq2, comparing Bcl6 fl/fl vs Bcl6 fl/fl Cd4 Cre samples. Multiple hypothesis correction was performed by the Benjamini-Hochberg method, setting a statistical significance threshold of adjusted p < 0.05. After identifying differentially enriched epitopes, the normalized counts were log2 transformed (hereafter referred to as log2 normalized counts) for downstream visualization and analysis. For converting the log2 normalized counts into relative enrichment scores on a sample-bysample basis, we scaled the log2 normalized counts within each sample to z-scores. In this manner, a z-score of 0 would correspond to epitopes that exhibited an average level of enrichment in a given sample; a positive z-score would indicate that an epitope is relatively enriched in a Statistical significance was assessed by either two-tailed unpaired t-test or Welch's t-test, based on the F test for unequal variance. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, not significant. Data are expressed as mean ± SEM. Each symbol represents an individual mouse. Data are aggregated from three independent experiments. Correlates of protection against SARS-CoV-2 in rhesus macaques Helper Cell Biology: A Decade of Discovery and Diseases Antigen-dependent multistep differentiation of T follicular helper cells and its role in SARS-CoV-2 infection and vaccination Peripheral CD4+ T cell subsets and antibody response in COVID-19 convalescent individuals Spike-specific circulating T follicular helper cell and cross-neutralizing antibody responses in COVID-19-convalescent individuals Robust correlations across six SARS-CoV-2 serology assays detecting distinct antibody features Integrated immune dynamics define correlates of COVID-19 severity and antibody responses Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications Imbalance of Regulatory and Cytotoxic SARS-CoV-2-Reactive CD4+ T Cells in COVID-19 Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity Humoral and circulating follicular helper T cell responses in recovered patients with COVID-19 Single-cell multi-omics analysis of the immune response in COVID-19 The dichotomous and incomplete adaptive immunity in COVID-19 patients with different disease severity Deficiency of Tfh Cells and Germinal Center in Deceased COVID-19 Patients Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19 Cellular Immune Responses to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Infection in Senescent BALB/c Mice: CD4+ T Cells Are Important in Control of SARS-CoV Infection SAP is required for generating long-term humoral immunity SAP Is Required for Th Cell Function and for Immunity to Influenza Viral persistence redirects CD4 T cell differentiation toward T follicular helper cells Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development Transcription factor STAT3 and type I interferons are corepressive insulators for differentiation of follicular helper and T helper 1 cells Sustained T follicular helper cell response is essential for control of chronic viral infection Single-cell RNA sequencing unveils an IL-10-producing helper subset that sustains humoral immunity during persistent infection ZIKV infection induces robust Th1-like Tfh cell and long-term protective antibody responses in immunocompetent mice B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells and T-bet Distinguish Effector and Memory Th1 CD4+ Cell Properties during Viral Infection The transforming growth factor beta signaling pathway is critical for the formation of CD4 T follicular helper cells and isotype-switched antibody responses in the lung mucosa Opposing Signals from the Bcl6 Transcription Factor and the Interleukin-2 Receptor Generate T Helper 1 Central and Effector Memory Cells In Vivo Regulation of Bcl6 and T Follicular Helper Cell Development Regulation of CD40 ligand expression on naive CD4 T cells: a role for TCR but not co-stimulatory signals Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production Cytokine-secreting follicular T cells shape the antibody repertoire Interaction of the selectin ligand PSGL-1 with chemokines CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid organs Role of CXCR5 and CCR7 in Follicular Th Cell Positioning and Appearance of a Programmed Cell Death Gene-1High Germinal Center-Associated Subpopulation The antigenic anatomy of SARS-CoV-2 receptor binding domain Lethal Infection of K18-hACE2 Mice Infected with Severe Acute Respiratory Syndrome Coronavirus of Complementary Strategies to Understand Virus Structure and Function Identification of immunodominant linear epitopes from SARS-CoV-2 patient plasma Linear epitopes of SARS-CoV-2 spike protein elicit neutralizing antibodies in COVID-19 patients Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity SARS-CoV-2 Proteome Microarray for Mapping COVID-19 Antibody Interactions at Amino Acid Resolution Functional mapping of B-cell linear epitopes of SARS-CoV-2 in COVID-19 convalescent population Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses Linear epitope landscape of the SARS-CoV-2 Spike protein constructed from 1,051 COVID-19 patients The immunodominant and neutralization linear epitopes for SARS-CoV-2 Longitudinal antibody repertoire in "mild" versus "severe" COVID-19 patients reveals immune markers associated with disease severity and resolution Antibody affinity maturation and plasma IgA associate with clinical outcome in hospitalized COVID-19 patients Seasonal human coronavirus antibodies are boosted upon SARS-CoV-2 infection but not associated with protection Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies SARS-CoV-2 501Y.V2 variants lack higher infectivity but do have immune escape Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150) Mouse and human FcR effector functions Antibody potency, effector function, and combinations in protection and therapy for SARS-CoV-2 infection in vivo Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions for optimal therapeutic protection A Temporal Switch in the Germinal Center Determines Differential Output of Memory B and Plasma Cells Separable effector T cell populations specialized for B cell help or tissue inflammation Selectins in T-cell recruitment to non-lymphoid tissues and sites of inflammation CXCR3 Chemokine Receptor-Ligand Interactions in the Lymph Node Optimize CD4+ T Helper 1 Cell Differentiation Targeting Lymph Node Niches Enhances Type 1 Immune Responses to Immunization Liver Is a Generative Site for the B Cell Response to Ehrlichia muris Evolution of Autoantibody Responses via Somatic Hypermutation Outside of Germinal Centers Human neutralizing antibodies elicited by SARS-CoV-2 infection Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients Convergent antibody responses to SARS-CoV-2 in convalescent individuals Isolation of potent SARS-CoV-2 neutralizing antibodies and Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation Structural basis of a shared antibody response to SARS-CoV-2 Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals Antibody signature induced by SARS-CoV-2 spike protein immunogens in rabbits Absence of Severe Acute Respiratory Syndrome Coronavirus 2 Neutralizing Activity in Prepandemic Sera From Individuals With Recent Seasonal Coronavirus Infection How T follicular helper cells and the germinal centre response change with age A Critical Role for Dnmt1 and DNA Methylation in T Cell Development, Function, and Survival Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection Mx1 reveals innate pathways to antiviral resistance and lethal influenza disease Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein Preformed CD40 ligand exists in secretory lysosomes in effector and memory CD4+ T cells and is quickly expressed on the cell surface in an antigen-specific manner A structural basis for antibody-mediated neutralization of Nipah virus reveals a site of vulnerability at the fusion glycoprotein apex Antigenic conservation and immunogenicity of the HIV coreceptor binding site Antibody epitope repertoire analysis enables rapid antigen discovery and multiplex serology SARS-CoV-2-specific antibody rearrangements in prepandemic immune repertoires of risk cohorts and patients with COVID-19 Differential expression analysis for sequence count data Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 The EMBL-EBI search and sequence analysis tools APIs in 2019 Jalview Version 2-a multiple sequence alignment editor and analysis workbench PBS and stored in aliquots at -80°C. Virus titer was determined by plaque assay using Vero-E6 cells Influenza virus A/PR/8/34 H1N1 (PR8) expressing the ovalbumin OT-II peptide was grown for 2.5 days at 37°C in the allantoic cavities of 10-day-old specific-pathogen-free fertilized chicken For all infections, mice were anesthetized using 30% vol/vol isoflurane diluted in propylene glycol (30% isoflurane) and administered SARS-CoV-2 or PR8 intranasally in 50 μl PBS. AAV-hACE2 Bcl6 fl/fl , Bcl6 fl/fl Cd4 Cre , and Ciita −/− mice were infected with 1 K18-hACE2 mice in serum transfer experiments were infected with 100 PFU of SARS-CoV-2. Bcl6 fl/fl , Bcl6 fl/fl Cd4 Cre , and Ciita −/− mice were infected with 70 PFU of PR8. All work with SARS-CoV-2 was performed in a biosafety level 3 (BSL3) facility with approval from the office of Environmental Health and Safety and the Institutional Animal Care and Use Committee at Yale University. potential SARS-CoV-2. SARS-CoV-2 stabilized spike glycoprotein (BEI Resources, NR-53257) (97), SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) (BEI Resources, NR-52946), and influenza virus A/PR/8/34 H1N1 hemagglutinin (HA) protein (Sino Biological, 11684-V08Hdetected with anti-mouse IgG-HRP (1013-05) Single-cell preparations were resuspended in PBS with 2% FBS and 1 mM EDTA and preat 4°C: anti-CD4 (RM4-5), TCRβ (H57-597) Cells were preincubated with Fc block (10 μg/ml) for 10 min at room temperature. Cells were then stimulated with 50 ng/ml PMA and 1 μg/ml ionomycin in complete RPMI mercaptoethanol) in the presence of PE/Cy7 anti-CD40L (MR1, 4 μg/ml) for 30 min at 37°C. After washing, the remaining markers were stained as described above. For intracellular cytokine staining, cells were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin in complete IMDM (10% heat-inactivated FBS, 1% Penicillin/Streptomycin, 2 mM Lglutamine, 1mM sodium pyruvate, 25 mM HEPES, 1X MEM Non-Essential Amino Acids, 20 μM 2-mercaptoethanol) in the presence of BD GolgiPlug™ (1:1000) for 2 hr at 37°C. Cells were then washed, incubated with Fc block, and stained for surface markers PD-1, PSGL-1, and Ly6C as well as LIVE/DEAD™ Fixable Aqua. Cells were next fixed with 2% paraformaldehyde in PBS IFN-γ (XMG1.2), and IL-21 (1 μg of IL-21R-Fc Chimera Protein; R&D Systems, 596-MR-100) in permeabilization buffer for 40 min at 4°C, followed by secondary staining with PE anti-human IgG (Jackson ImmunoResearch, 109-116-098) for 30 min at 4°C. Samples from SARS-CoV-2-infected mice were fixed with 4% paraformaldehyde for 30 Beckman Coulter) and analyzed using FlowJo software (BD) Immunofluorescence MedLN were fixed with 4% paraformaldehyde in PBS for 4 hr at 4°C, followed by cryopreservation with 20% sucrose in PBS for 2 hr at 4°C. MedLN were snap-frozen in optimal cutting temperature compound and stored at -80°C. Tissues were cut into 7-μm sections and blocked with 10% rat serum in staining solution (PBS with 1% BSA and 0.1% Tween-20) for 1 hr at room temperature anti-B220 (RA3-6B2) and AF647 anti-GL7 (GL7) along with Fc block IgG2c (5.7), and rabbit anti-T-bet (E4I2K; Cell Signaling Technology, 97135S) overnight at 4°C Vector pCAGGS containing the SARS-CoV-2 Wuhan-Hu-1 spike glycoprotein gene was produced under HHSN272201400008C and obtained through BEI Resources The virus inoculum was then removed, and cells were washed with PBS before adding media with anti-VSV-G (8G5F11) to neutralize residual inoculum. Supernatant containing pseudovirus was collected 24 hours post inoculation, clarified by centrifugation, concentrated with Amicon Ultra Centrifugal Filter Units (100 kDa), and stored in aliquots at -80°Cof 1:50 for USA-WA1/2020 pseudovirus and 1:12.5 for B.1.351 pseudovirus, with up to eight two-fold serial dilutions Sera from SARS-CoV-2-infected AAV-hACE2 Bcl6 fl/fl or Bcl6 fl/fl Cd4 Cre mice at 14 days post infection (dpi) were pooled, and the resulting levels of spike-specific IgG were measured by ELISA Bcl6 fl/fl sera was diluted with naïve sera to match the spike-specific IgG titer of Bcl6 fl/fl Cd4 Cre sera. Serum samples were then diluted 1:1 with PBS, and 200 µl of diluted serum was transferred intravascularly by retro-orbital injection into K18-hACE2 mice under anesthesia with 30% In addition, 250 µl of lung homogenate was mixed with 750 µl of TRIzol™ LS Reagent (Invitrogen), and RNA was extracted with the RNeasy Mini Kit (Qiagen) following manufacturer's instructions. cDNA synthesis was performed using random hexamers and ImProm-II™ Reverse Transcriptase (Promega) The limit of detection was 100 SARS-CoV-2 genome copies/μl. Virus copy numbers were quantified using a control plasmid containing the complete nucleocapsid gene from SARS-CoV SARS-CoV-2 genome copies were normalized to Actb using Actb-specific oligonucleotides: Probe: 5' 6 Serum epitope repertoire analysis (SERA) with SARS-CoV-2 linear epitope library sample, while a negative z-score would denote a low-scoring epitope. Where applicable, statistical significance was assessed on the within-sample z-scores by two-tailed unpaired For visualization purposes, we also calculated the average z-scores in Bcl6 fl/fl or Bcl6 fl/fl Cd4 Cre groups. Pan-human coronavirus (hCoV) conservation scores were calculated through multiple alignment of several hCoV spike sequences using Clustal Omega (105). The following hCoV spike sequences were used Aminoacid level conservation scores for SARS-CoV-2 spike were extracted through JalView (106). Student's two-tailed Welch's two-tailed, unpaired t-test Mann-Whitney test; or two-tailed Wilcoxon signed-rank test, as indicated. P < 0.05 was considered statistically significant Adaptive immune response to SARS-CoV-2 in Tfh-sufficient and -deficient mice (A to H) Flow cytometric analysis of medLN from SARS-CoV-2-infected mice at 14 dpi. (A) Representative gating strategy to identify C) Frequency among CD44 + CD4 + T cells (B) and total number (C) of Tfh cells in Bcl6 fl/fl Representative gating strategy to identify germinal center (GC) B cells and plasmablasts (PB) E and F) Frequencies of GC B cells (E) and PB (F) in Bcl6 fl/fl (blue) or Bcl6 fl/fl Cd4 Cre (magenta) mice Total number of GC B cells (G) and PB (H) in Bcl6 fl/fl (blue) or Bcl6 fl/fl Cd4 Cre (magenta) mice Viral burden in lungs from Bcl6 fl/fl mice or Bcl6 fl/fl Cd4 Cre mice infected with SARS-CoV-2 at 7 Data are expressed as log10 N1 gene copy number by qPCR, normalized to Actb IgM titers in sera from Bcl6 fl/fl (blue), Bcl6 fl/fl Cd4 Cre (magenta), or Ciita −/− (green) mice at 14 dpi with SARS-CoV-2 Bcl6 fl/fl Cd4 Cre (magenta), or Ciita −/− (green) , not significant. Data are expressed as mean ± standard error of mean (SEM) CD40L and cytokine expression in CD4 + T cell subsets after viral infection (A to C) Flow cytometric analysis of medLN from Bcl6 fl/fl (blue) or Bcl6 fl/fl Cd4 Cre (magenta) mice at 7 dpi with SARS-CoV-2 C) Frequency (left) and MFI (right) of CD40L expression among mixed Th1 cells (B) and terminally differentiated Th1 cells (C) Flow cytometric analysis of medLN from Bcl6 fl/fl mice at 7 dpi with PR8 Frequency (left) and MFI (right) of CD40L expression among CD4 + T cell subsets Representative flow cytometric analysis of IL-21 and IFN-γ protein expression in distinct CD4 + T cell subsets from medLN of SARS-CoV-2-infected mice at 7 dpi G to I) Flow cytometric analysis of medLN from Bcl6 fl/fl (blue) or Bcl6 fl/fl Cd4 Cre (magenta) mice at 7 dpi with SARS-CoV-2. Frequencies of IL-21 + IFN-γ − , IL-21 + IFN-γ + , and IL-21 − IFN-γ + expression among CD44 + CD4 + T cells (G) Statistical significance was assessed by either two-tailed unpaired t-test or Welch's t-test, based on the F test for unequal variance 001. ns, not significant. Data are expressed as mean ± SEM. Each symbol represents an individual mouse. Data in (A to C and G to I) are