Follicular T cells optimize the germinal center response to SARS-CoV-2 protein vaccination in mice

Abstract

Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity.

Keywords: SARS-CoV-2; Tfh; Tfr; affinity maturation; aging; germinal center; somatic hypermutation; vaccines.

Graphical abstract

Figure 1

An adjuvanted SARS-CoV-2 subunit vaccine generates germinal center responses (A) Frequency of follicular T cells identified as CD4⁺ICOS⁺CXCR5⁺ (left) or CD4⁺PD-1^hiCXCR5⁺ (right) cells from the draining lymph node of naive mice or mice vaccinated with adjuvanted SARS-CoV-2 Spike protein vaccine 10 days prior. (B) Frequencies of Tfh and Tfr cells as a percentage of total CD4⁺ T cells (left and middle) or Tfr cells as a percentage of total follicular T (CD4⁺CXCR5⁺PD1⁺) cells (right). (C and D) Percentage of CD19⁺GL7⁺FAS⁺ GC B cells (C) or CD19⁺IgG1⁺CD38⁻ class-switched B cells (D) as a percentage of all CD19⁺ B cells. (E) Serological assessment of IgG for SARS-CoV-2 Spike, S1, S2, or RBD domains 10 days after vaccination. (F) Schematic of single GC B cell culture assays. Single GC B cells from mice vaccinated with SARS-CoV-2 vaccine 14 days prior were cultured with NB21 feeder cells for 6 days. Culture supernatants were screened for IgG positivity. IgG⁺ wells were investigated for SARS-CoV-2 Spike specificity. (G) Distribution of SARS-CoV-2 Spike specificity from IgG⁺ GC B cell cultures. Number in circle indicates total number of IgG⁺ cells analyzed. Data are from a single experiment and are representative of three independent repeats (A–D), are combined data from three independent repeats with n = 2–6 mice per group (E), or are combined data of individual single GC B cell cultures taken from at least six independent repeats and are representative of n = 15 mice total (G). Student's two-tailed unpaired t test (A–E). ^∗∗∗p < 0.001 and ^∗p < 0.05.

Figure 2

Tfh cells optimize early SARS-CoV-2 Spike-specific germinal center responses after adjuvanted protein vaccination (A) Assessment of total CD4⁺PD-1⁺CXCR5⁺ follicular T cells (left), CD4⁺PD-1⁺CXCR5⁻ T cells (middle), and CD19⁺ B cells (right) from draining lymph nodes of control (Cd4^CreCxcr5^wt) or Tfh-DTR (Cd4^CreCxcr5^{LoxSTOPLoxDTR}) mice that were vaccinated with an adjuvanted SARS-CoV-2 Spike protein vaccine, given DT, and harvested on day 14. (B) Frequencies of CD4⁺PD-1⁺CXCR5⁺FoxP3⁻ Tfh (left) and CD4⁺PD-1⁺CXCR5⁺FoxP3⁺ Tfr (right) cells. (C) Percentage of CD19⁺GL7⁺FAS⁺ GC B cells out of all CD19⁺ B cells. (D) Serological assessment of SARS-CoV-2 Spike and RBD-specific IgG from serum of vaccinated mice. (E) Schematic for single GC B cell cultures. (F) Assessment of SARS-CoV-2 Spike specificity for IgG⁺ GC B cells using single GC culture assays. Numbers in circles indicate total number of IgG⁺ GC B cells analyzed. ND, not determined to be specific for S1, RBD, or S2; S1, specific for S1 but not RBD; RBD, specific for RBD; S2, specific for S2. K_D indicates equilibrium dissociation constant for Spike-specific B cell clones measured by biolayer interferometry. (G) Assessment of SARS-CoV-2 Spike-specific IgG⁺ GC B cells using single GC culture assays separated by individual mice. (H) BCR somatic hypermutation analysis for V-heavy (VH) chain in ex vivo sorted GC B cells. (I) Mutation analysis for singleton and expanded (found at least twice) clones using smoothing spline fitting of data. (J) Analysis of clonal distribution from ex vivo GC B cells. Numbers indicate total number of clones and number of GC B cells analyzed. Orange indicates Spike specificity from published or single-cell GC B cell culture sequences. N75 index indicates the smallest number of clones to comprise 75% of sequenced cells and is represented on a per-mouse basis. Data are from a single experiment and are representative of three independent repeats with n = 3–4 mice per group (A–C, F), are concatenated data from two independent experiments with n = 6–12 mice per group (D), are concatenated data from two independent experiments (G), or are concatenated data from n = 3 mice per group from one experiment (H–J). Student's two-tailed unpaired t test (A–F) or Mann-Whitney test (I). ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05. See also Figure S1.

Figure 3

Tfr cells promote SARS-CoV-2 Spike-specific GC responses by limiting competition after vaccination (A) Assessment of total CD4⁺PD-1⁺CXCR5⁺ follicular T cells (right) from draining lymph nodes of control (Foxp3^Cre and Cxcr5^wt) or Tfr-DTR (Foxp3^CreCxcr5^{LoxSTOPLoxDTR}) mice that were vaccinated with an adjuvanted SARS-CoV-2 Spike-protein vaccine, given DT, and harvested on day 14. (B) Frequencies of CD4⁺PD-1⁺CXCR5⁺FoxP3⁺ Tfr, CD4⁺PD-1⁺CXCR5⁺FoxP3⁻ Tfh, CD4⁺PD-1⁺CXCR5⁻FoxP3⁻ T cells, CD4⁺PD-1⁺CXCR5⁺FoxP3⁺ Treg cells, and CD19⁺ B cells. (C) Percentage of CD19⁺GL7⁺FAS⁺ GC B cells out of all CD19⁺ B cells. (D) Percentage of CD19⁺IgG1⁺CD38⁻ class-switched B cells as a percentage of all CD19⁺ B cells. (E) Serological analysis of SARS-CoV-2 Spike or RBD-specific IgG. (F) Percentage of SARS-CoV-2 Spike-specific clones as a percentage of IgG⁺ GC B cells from single-cell GC cultures, along with individual epitope specificity. Number in the center of the circle plot indicates total number of IgG⁺ GC B cells analyzed. ND, not determined to be specific for S1, RBD, or S2; S1, specific for S1 but not RBD; RBD, specific for RBD; S2, specific for S2. (Right) Percentage of SARS-CoV-2 Spike-specific cells as a percentage of IgG⁺ GC B cells from individual mice. (G) Affinities of Spike-specific antibodies from (F). (H) BCR somatic hypermutation analysis for V-heavy (VH) chain in ex vivo sorted GC B cells, including analysis for singleton and expanded (found at least twice) clones using smoothing spline fitting of data. (I) Analysis of clonal distribution and diversity from ex vivo GC B cells. Numbers indicate total number of clones and number of GC B cells analyzed. N75 index is represented on a per-mouse basis. Data are from a single experiment and are representative of three independent repeats with n = 4–8 mice per group (A–D), are concatenated data from two independent experiments with at least six mice per group (E), are concatenated data from two independent experiments (F and G), or are concatenated data from n = 3 mice per group from one experiment (H and I). Student's two-tailed unpaired t test (A–G) or Mann-Whitney test (H). ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05. See also Figure S2.

Figure 4

Age-associated humoral immunoregulation alters SARS-CoV-2 GC responses after Spike protein vaccination (A) Assessment of total CD4⁺PD-1⁺CXCR5⁺ follicular T (left), CD4⁺PD-1⁺CXCR5⁺FoxP3⁻ Tfh (middle), and CD4⁺PD-1⁺CXCR5⁺FoxP3⁺ Tfr (right) cells from draining lymph nodes of young (8 weeks old) or aged (80 weeks old) mice that were vaccinated with an adjuvanted SARS-CoV-2 Spike protein vaccine and harvested on day 14. (B) Percentage of CD4⁺FoxP3⁺CXCR5⁻ Treg cells (left) and total CD19⁺ B lymphocytes (right). (C) Percentage of CD19⁺GL7⁺FAS⁺ GC B cells out of all CD19⁺ B cells (left) and CD19⁺IgG1⁺CD38⁻ class-switched B cells as a percentage of all CD19⁺ B cells (right). (D) Serological analysis of SARS-CoV-2 Spike or RBD-specific IgG. (E) Percentage of SARS-CoV-2 Spike-specific clones as a percentage of total IgG⁺ GC B cells from single-cell GC cultures (left). Number in the center of the circle plot indicates total number of IgG + GC B cells analyzed. ND, not determined to be specific for S1, RBD, or S2; S1, specific for S1 but not RBD; RBD, specific for RBD; S2, specific for S2. (F) Percentage of SARS-CoV-2 Spike-specific cells as a percentage of total IgG + GC B cells in individual mice. (G) BCR somatic hypermutation analysis for V-heavy (VH) chain in ex vivo sorted GC B cells, including analysis for singleton and expanded (found at least twice) clones using smoothing spline fitting of data. (H) Analysis of clonal distribution from ex vivo GC B cells. Numbers indicate total number of clones and number of GC B cells analyzed. N75 index is represented on a per-mouse basis. Data are from a single experiment and are representative of three independent repeats with n = 4–8 mice per group (A–C), are concatenated data from two independent experiments with n = 4–6 mice per group (D), are concatenated data from two independent experiments (E and F), or are concatenated data from n = 3 mice per group from one experiment (G and H). Student's two-tailed unpaired t test (A–D). ^∗∗∗p < 0.001. See also Figure S3.

Figure 5

Early Tfr humoral immunoregulation alters SARS-CoV-2 Spike-specific GC responses after vaccine boosting (A) Schematic of experiment. Control (Foxp3^Cre and Cxcr5^wt) or Tfr-DTR (Foxp3^CreCxcr5^{LoxSTOPLoxDTR}) mice were vaccinated with an adjuvanted SARS-CoV-2 Spike protein vaccine and given DT until day 11. The mice received a SARS-CoV-2 vaccine boost on day 30 and draining lymph nodes were harvested on day 38. (B) Assessment of total CD4⁺PD-1⁺CXCR5⁺ follicular T (left), CD4⁺PD-1⁺CXCR5⁺FoxP3⁺ Tfr (middle), and CD4⁺PD-1⁺CXCR5⁺FoxP3⁻ Tfh (right) cells on day 38. (C) Percentage of CD19⁺GL7⁺FAS⁺ GC B cells out of all CD19⁺ B cells. (D) Percentage of CD19⁺IgG1⁺CD38⁻ class-switched B cells out of all CD19⁺ B cells. (E) Serological analysis of SARS-CoV-2 Spike or RBD-specific IgG. (F) Percentage of SARS-CoV-2 Spike-specific clones out of the total IgG⁺ GC B cells from single-cell GC cultures. Number in the center of the circle plot indicates total number of IgG⁺ GC B cells analyzed. (G) Percentage of SARS-CoV-2 Spike-specific clones out of the total IgG⁺ GC B cells in individual mice. (H) Affinities of Spike-specific antibodies from single GC B cell cultures. (I) BCR somatic hypermutation analysis for V-heavy (VH) chain in ex vivo sorted GC B cells, including analysis for singleton and expanded (found at least twice) clones using smoothing spline fitting of data. (J) Analysis of clonal distribution and clonal diversity from ex vivo GC B cells. Numbers indicate total number of clones and number of GC B cells analyzed. N75 index is represented on a per-mouse basis. (K) Schematic of deletion during boosting experiment. Control (Foxp3^Cre and Cxcr5^wt) or Tfr-DTR (Foxp3^CreCxcr5^{LoxSTOPLoxDTR}) mice were vaccinated with a SARS-CoV-2 Spike protein vaccine and were boosted on day 30 followed by DT treatment. Draining lymph nodes were harvested on day 38. (L) Spike and RBD-specific IgG in serum from mice treated as in (K). (M) Single GC B cell cultures from GC B cells as in (K). Numbers indicate total numbers of IgG⁺ GC B cell clones analyzed. Data are from a single experiment and are representative of two independent repeats with n = 4–8 mice per group (A–D), are concatenated data from two independent experiments with n = 4–8 mice per group (E), are concatenated data from two independent experiments (E–H), are concatenated data from n = 3 mice per group from one experiment (I and J), or are concatenated data from two independent repeats with n = 4–6 mice per group (L and M). Student's two-tailed unpaired t test (A–E, I) or Mann-Whitney test (I). ^∗∗p < 0.01 and ^∗p < 0.05. See also Figure S4.

Figure 6

Early Tfh help optimizes somatic hypermutation during SARS-CoV-2 Spike protein vaccine boosting (A) Schematic of experiment. Control (Cd4^Cre and Cxcr5^wt) or Tfh-DTR (Cd4^CreCxcr5^{LoxSTOPLoxDTR}) mice were vaccinated with an adjuvanted SARS-CoV-2 Spike protein vaccine and given DT until day 11. The mice received a SARS-CoV-2 vaccine boost on day 30 and draining lymph nodes were harvested on day 38. (B) Assessment of total CD4⁺PD-1⁺CXCR5⁺ follicular T (left), CD4⁺PD-1⁺CXCR5⁺FoxP3⁻ Tfh (middle), and CD4⁺PD-1⁺CXCR5⁺FoxP3⁺ Tfr (right) cells on day 38. (C) Percentage of CD19⁺GL7⁺FAS⁺ GC B cells out of all CD19⁺ B cells. (D) Serological analysis of SARS-CoV-2 Spike or RBD-specific IgG. (E) Percentage of SARS-CoV-2 Spike-specific clones out of the total IgG⁺ GC B cells from single-cell GC cultures (left). Number in the center of the circle plot indicates total number of IgG⁺ GC B cells analyzed. (F) Affinities of Spike-specific antibodies from single GC B cell cultures. (G) BCR somatic hypermutation analysis for V-heavy (VH) chain in ex vivo sorted GC B cells, including analysis for singleton and expanded (found at least twice) clones using smoothing spline fitting of data. (H) Analysis of clonal distribution from ex vivo GC B cells. Numbers indicate total number of clones and number of GC B cells analyzed. N75 index is represented on a per-mouse basis. (I) Schematic of deletion during boosting experiment. Control (Cd4^Cre and Cxcr5^wt) or Tfh-DTR (Cd4^CreCxcr5^{LoxSTOPLoxDTR}) mice were vaccinated with a SARS-CoV-2 Spike protein vaccine and were boosted on day 30, followed by DT treatment. Draining lymph nodes were harvested on day 38. (J) Spike- and RBD-specific IgG in serum from mice treated as in (I). (K) Single GC B cell cultures from GC B cells as in (I). Numbers indicate total numbers of IgG⁺ GC B cells analyzed. Data are from a single experiment and are representative of two independent repeats with n = 4–8 mice per group (A–C), are concatenated data from two independent experiments with n = 4–8 mice per group (D), are concatenated data from two independent experiments (E and F), are concatenated data from n = 3 mice per group from one representative experiment (G and H), or are concatenated data from two independent repeats with n = 4–6 mice per group (J and K). Student's two-tailed unpaired t test (A–D, J) or Mann-Whitney Test (G). ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05. See also Figure S5.