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. 2016 Jun 22:6:28287.
doi: 10.1038/srep28287.

CD4 T cell epitope specificity determines follicular versus non-follicular helper differentiation in the polyclonal response to influenza infection or vaccination

Zackery A G Knowlden  1 , Andrea J Sant  1
Affiliations

CD4 T cell epitope specificity determines follicular versus non-follicular helper differentiation in the polyclonal response to influenza infection or vaccination

Zackery A G Knowlden et al. Sci Rep. .

Abstract

Follicular helper T cells (Tfh) are essential for B cell production of high-affinity, class-switched antibodies. Much interest in Tfh development focuses on the priming environment of CD4 T cells. Here we explored the role that peptide specificity plays in the partitioning of the polyclonal CD4 T cell repertoire between Tfh and NonTfh lineages during the response to influenza. Surprisingly, we found that CD4 T cells specific for different epitopes exhibited distinct tendencies to segregate into Tfh or NonTfh. To alter the microenvironment and abundance, viral antigens were introduced as purified recombinant proteins in adjuvant as native proteins. Also, the most prototypical epitopes were expressed in a completely foreign protein. In many cases, the epitope-specific response patterns of Tfh vs. NonTfh persisted. The functional TcR avidity of only a subset of epitope-specific cells correlated with the tendency to drive a Tfh response. Thus, we conclude that in a polyclonal CD4 T cell repertoire, features of TcR-peptide:MHC class II complex have a strong deterministic influence on the ability of CD4 T cells to become a Tfh or a NonTfh. Our data is most consistent with at least 2 checkpoints of Tfh selection that include both TcR affinity and B cell presentation.

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Figures

Figure 1
Figure 1. Tfh and NonTfh cells exhibit prototypical phenotype and kinetics following influenza infection.
(a) Example of sorting strategy for isolating antigen-experienced Tfh and NonTfh cells after infection. SJL mice were infected intranasally with 50,000EID50 of A/New Caledonia/20/1999. Lymphocytes were isolated from the draining mediastinal lymph node on day 9 post infection and FACS sorted based upon expression of the following markers: CD4 + CD44hiCXCR5 + PD-1 + (Tfh) or CD4 + CD44hiCXCR5negPD-1neg (NonTfh). (b,c) Kinetics of effector CD4 T cell and B cell expansion following infection depicting Tfh and NonTfh populations, as well as GC (CD19 + B220 + GL-7 + CD95+) and plasmablasts (CD19 + B220intCD138+) (n = 2–5 mice per timepoint, representative of 2 experiments). (d) Expression of Bcl6 and (e) CCR7 in Tfh (Filled Grey) and NonTfh (Black Open) populations. (f) RT-PCR analysis of total RNA from Tfh (Filled) and NonTfh (Open) showing relative expression of various transcripts isolated from the indicated sorted T cell populations, normalized to B2m expression. Data shown is from a single experiment, representative of four.
Figure 2
Figure 2. Influenza-specific effector cells distribute between Tfh and NonTfh depending on epitope specificity.
Single epitope reactivity of sorted CD4 T cell populations at day 9 post infection, Tfh (Filled) and NonTfh (Open) from mediastinal lymph nodes pooled from 20–25 mice. CD4 T cell populations were assayed for epitope reactivity in IL-2 ELISPOT assays by restimulation with influenza-derived epitopes from HA (a) and NP (c). The epitope-specific IL-2 spots per million Tfh or NonTfh cells are shown. Relative distribution of antigen-reactive CD4 T cells between Tfh and NonTfh cells is depicted by the ratio of antigen-specific cells in the two populations for HA-specific T cells (b) and NP-specific cells (d). Mean and S.D. of 4 experiments. Statistical significance determined by two-tailed paired t test (a,c) and 1-way ANOVA with Tukey post test (b,d). *p < 0.05; **p < 0.01.
Figure 3
Figure 3. Vaccination with influenza proteins maintains epitope specific patterns in effector distribution between Tfh and NonTfh.
Single epitope reactivity of sorted CD4 T cell populations at day 9 post vaccination, Tfh (Filled) and NonTfh (Open) from popliteal lymph nodes pooled from 10 mice. Mice were vaccinated subcutaneously with 5 μg HA and NP proteins together in IFA/LPS emulsion. CD4 T cell populations were assayed for epitope reactivity in IL-2 ELISPOT assays by restimulation with influenza-derived epitopes from HA (a) and NP (c). The epitope-specific IL-2 spots per million Tfh or NonTfh cells are shown. Relative distribution of antigen-reactive CD4 T cells between Tfh and NonTfh cells is depicted by the ratio of antigen-specific cells in the two populations for HA-specific T cells (b) and NP-specific cells (d). Mean and S.D. of 3 experiments. Statistical significance determined by two-tailed paired t test (a,c) and 1-way ANOVA with Tukey post test (b,d). *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4
Figure 4. Epitope locations within the native protein context of hemagglutinin and nucleoprotein and the heterologous protein MalE133.
Protein structures of MalE133 (a), hemagglutinin (b) and nucleoprotein (c). Influenza epitopes and their locations are indicated by coloration and are denoted by first amino acid associated with the 17-mer peptide used for testing of CD4 T cell reactivity. For MalE133 protein, a heterologous peptide is shown for illustrative purposes at the insertion site within the protein structure. The structures used to model peptide epitope location were sufficiently homologous to H1N1 A/New Caledonia/20/1999 proteins to overlay New Caledonia epitopes: Hemagglutinin (HA0) from A/South Carolina/1/18 (H1N1); Nucleoprotein from A/England/256/2009 (H1N1).
Figure 5
Figure 5. Epitope-specific reactivity for Tfh and NonTfh following vaccination with a heterologous protein containing influenza-derived epitopes.
Shown are the responses of sorted Tfh and NonTfh cells to HA-derived epitopes (a) and NP-derived epitopes (b). As before, mice were vaccinated with the indicated individual MalE133 constructs (5 μg protein) in an IFA/LPS emulsion. Cells were isolated from popliteal lymph nodes pooled from 5 mice per protein at day 9 post vaccination. Eight different MalE133 proteins containing influenza epitopes were constructed. Mean and S.D. of 3–5 experiments. Statistical significance determined by one-tailed paired t test. *p < 0.05; **p < 0.01.
Figure 6
Figure 6. Context-independent relationship between epitope-specificity and effector function.
Epitope-specific reactivity for individual specificities were compared as ratios of reactive Tfh:NonTfh as shown previously for (a) infection (Fig. 2) and (b) HA/NP vaccination (Fig. 3). Data from previous figures on infection and vaccination were duplicated below for comparison of the response to the inserted epitope in MalE. (c) The ratio of reactive cells (Tfh:NonTfh) for each epitope is depicted for MalE133 vaccination, as calculated from enumeration of IL-2 producing cells in each population (Fig. 5). Statistical significance determined by 1-way ANOVA with Tukey post test. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7
Figure 7. The impact of functional avidity in partitioning of polyclonal CD4 T cells between Tfh and NonTfh.
Cells isolated from either MLN (open symbol) or spleen (closed symbol) at day 9 or 10 post-infection were evaluated by ELISPOT assay for reactivity to the indicated influenza peptide epitopes. The affinity of the polyclonal CD4 T cell response to various influenza epitopes was determined as the peptide dose at which 50% maximum reactivity was reached, and is indicated here as the negative log10 of this value (a). Mean of MLN and spleen derived values in independent assays (n = 3–8) is indicated. For reference, the dotted line at 0.5 is associated with a 0.32 μM peptide dose, 1.2 with 0.063 μM and 1.9 with 0.013 μM, roughly approximating low, medium and high affinity, respectively. Hybridomas derived from CD4 T cells isolated from infected mice were also evaluated to approximate TCR affinity, measured as the peptide concentration at which reactivity was first detectable in an ELISPOT assay. Mean values (red symbol) were determined from 3 (HA 162, 386, NP 342), 7 (NP 97), or greater than 10 (HA 144, NP 270) individual clones. Data from panels Fig. 2b,c have been combined and reproduced here (b) for comparison between Tfh/NonTfh ratio and CD4 T cell affinity.
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