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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Cytokine. 2020 Sep 29;137:155313. doi: 10.1016/j.cyto.2020.155313

Lack of evidence supporting a role of IFN-β and TGF-β in differential polarization of Bordetella pertussis specific-T cell responses

Ricardo da Silva Antunes a,*, Lorenzo G Quiambao a, Ferran Soldevila a, Aaron Sutherland a, Bjoern Peters a,b, Alessandro Sette a,b
PMCID: PMC7726043  NIHMSID: NIHMS1634993  PMID: 33002739

Abstract

Bordetella Pertussis (BP) vaccine-induced immunity is waning worldwide despite excellent vaccine coverage. Replacement of the whole-cell inactivated vaccine (wP) by an acellular subunit vaccine (aP) is thought to play a major role and to be associated with the recurrence of whooping cough. Previously, we detected that the polarization towards a Th2 and Th1/Th17 response in aP and wP vaccinees, respectively, persists upon aP boosting in adolescents and adults. Additionally, IL-9 and TGF-β were found to be up-regulated in aP-primed donors and network analysis further identified IFN-β as a potential upstream regulator of IL-17 and IL-9. Based on these findings, we hypothesized that IFN-β produced following aP vaccination may lead to increased IL-9 and decreased IL-17 production. Also, due to the well characterized role of TGF-β in both Th17 and Th9 differentiation, we put forth that TGF-β addition to BP-stimulated CD4+ T cells might modulate IL-17 and IL-9 production. To test this hypothesis, we stimulated in vitro cultures of PBMC or isolated naive CD4+ T cells from aP vs wP donors with a pool of BP epitopes and assessed the effect of IFN-β or TGF-β in proliferative responses as well as in the cytokine secretion of IL-4, IL-9, IL-17, and IFN-γ. IFN-β reduced BP-specific proliferation in PBMC as well as cytokine production but increased IL-9, IL-4, and IFN-γ cytokines in naïve CD4+ T cells. These effects were independent of the childhood vaccination received by the donors. Similarly, TGF-β reduced BP-specific proliferation in PBMC but induced proliferation in naïve CD4+ T cells. However, stimulation was associated with a generalized inhibition of cytokine production regardless of the original aP or wP vaccination received by the donors. Our study suggests that key T cell functions such as cytokine secretion are under the control of antigen stimulation and environmental cues but molecular pathways different than the ones investigated here might underlie the long-lasting differential cytokine production associated with aP- vs wP-priming in childhood vaccination.

Keywords: T cell responses, IFN-β, TGF-β, Bordetella pertussis, vaccine

1. Introduction

Immune cell orchestration of responses is mediated by the concerted action of cytokines, which promotes recruitment, regulation, and activation or differentiation of key immune players into effector cells1. In particular, cytokines secreted by memory CD4+ T cells are crucial to mount an effective long-lived response against a pathogen and their generation regarded essential to achieve robust protection upon vaccination2–4. However, the complexity of studying T cell phenotypes or the patterns of cytokine secretion and understanding if naïve cells rather than fully differentiated memory cells play a more important role in vaccine efficacy is not yet fully characterized5,6. Indeed, in the case of responses to Bordetella pertussis (BP), the causative agent of whooping cough, it is now well established that the composition of the pertussis vaccine given to infants determines the long-term cell polarization or cytokine profile that persists into adulthood7–10. More importantly, the immunologic profile induced in infants in response to acellular (aP) vaccination, shaping a Th2 polarizing response during the first year of life, is being associated with the recurrence of whooping cough11,12. Those vaccinated with whole cellular vaccines (wP) in infancy developed a Th1 and Th17 polarizing response, mimicking immunity acquired by natural infection and are deemed more protected from disease8,13–15.

In our previous study, we have shown that IL-4, IL-9, and TGF-β drive polarization in aP recipients, whereas IFN-γ and IL-17 responses underlie immune polarization in wP recipients8. Based on these data and on reports in the literature, TGF-β can work as a master-switch regulator, promoting Th9, Th17, or regulatory T (Treg) cells differentiation based upon the presence of other cytokines such as IL-1 and IFN-β16–18. TGF-β is also capable of reprogramming fully differentiated Th2 cells to an IL-9 secreting phenotype19. We further identified IFN-β as a potential upstream negative regulator of IL-17, and positive regulator of IL-98. IFN-β can act as a counter-regulator of Th2 and Th17 responses and has been considered a vital ‘third signal’ for the memory and effector CD4+ T cell responses20. Other studies have also shown that high doses of IFN-β in T cell cultures induce a Th1 to Th2 shift and reduce T cell proliferation21–23. It has also been reported that IFN-β treatment causes professional APCs to produce less IL-12 and more IL-10 that would favor Th2 responses over Th124–26. Additionally, previous studies reported reciprocal Th17 and Treg cell differentiation mediated by retinoic acid27. This leads to the hypothesis that IL-9 production either induced by IFN-β or TGF-β may block Th17 differentiation in aP donors and eventually generate Tregs, which inhibit the development of later proliferation and long-lasting antibody responses to aP booster vaccines8. The experiments described herein were directed to test these hypotheses.

2. Materials and Methods

2.1. Study subjects

We recruited 24 healthy adults from San Diego, USA (Supplementary Table 1). In all groups, male and female subjects were included equally. Clinical data for each patient was collected by multiple approaches. Whenever possible, vaccination records were collected from study participants or parents/custodian as appropriate. For some donors, the original clinical vaccine record was not available or incomplete, including the brand and composition of the vaccines, in which case information was collected by the clinical coordinators through questionnaires, recording dates, and numbers of vaccination. All donors were from the San Diego area and followed the recommended vaccination regimen (which is also necessary for enrollment in the California school system), which entails five DTaP or DTwP doses for children under 7 years old (three doses at 2, 4 and 6 months and then two doses between 15–18 months and 4–6 years). For this primary series, each group received exclusively DTaP or DTwP vaccines and both groups received additional Tdap booster immunizations at 11–12 years and then every 10 years, but no boost was administered at least in the previous four years prior to this study. Individuals who had been diagnosed with BP infection or clinical disease at any given time in their life were excluded. Other exclusion criteria were pregnancy at the start of the study (no record of previous pregnancy or vaccination administered during pregnancy was collected); present severe disease or medical treatment that might interfere with study results; any vaccination in the last month and/or antibiotic use or fever [(>100.4F (38 C)]. The pertussis (P) compounds in these vaccines (“w” for whole-cell, also wP for short, and “a” for acellular, also aP for short) are co-administered with diphtheria toxoid (D) and tetanus toxoid (T). Also, the capital and lowercase letters denote higher or lower proportions of the overall components between vaccines.

2.2. Peptides and cytokines

Peptides were derived from Bordetella pertussis antigens included in the aP vaccines (FHA, Fim2/3, PRN, and PtTox) from the Tohama I and 18323 strains. Experimentally validated peptides were selected from a total of 785 peptides encompassing 16-mers overlapping by eight residues of the full length-coverage of all antigens. The top epitopes recognized by >5% donors corresponding to 132 peptides were chosen and the megapool (MG) of all combined peptides is described as PT (MG) hereafter7,8. Peptides were synthesized as crude material on a small (1 mg) scale by A and A (San Diego, CA), resuspended in dimethyl sulfoxide (DMSO), and pooled to equal amounts of each peptide followed by sequential lyophilization to reduce DMSO associated toxicity. This approach has been used to develop megapools specific for pertussis and tetanus among other pathogens28,29. Human recombinant IFN-β and TGF-β were obtained from R&D systems (Minneapolis, MN) and IL-6 and IL-23 from Peprotech (Rocky Hill, NJ).

2.3. PBMC isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood or leukapheresis by density gradient centrifugation according to the manufacturer’s instructions (Ficoll-Paque Plus, Amersham Biosciences, Uppsala, Sweden) as previously described30. Cells were cryopreserved in liquid nitrogen suspended in fetal bovine serum containing 10% (vol/vol) DMSO.

2.4. Naïve CD4+ T cell negative isolation

Freshly thawed PBMCs (1–1.5 × 108 cells) were subject to magnetic depletion of memory CD4+ T cells and non-CD4+ T cells through the utilization of the Naïve Human CD4+ T Cell Isolation Kit II (Miltenyi Biotec, San Diego, CA). PBMCs were centrifuged at 4°C, 1200 rpm for 7 minutes, then resuspended in MACS buffer (Miltenyi Biotec) at a volume of 40 μL MACS/1 × 107 cells. Naïve CD4+ T Cell Isolation Kit II biotinylated antibody cocktail (Miltenyi Biotec) was added to the resuspended cells at a volume of 10 μl cocktail/1 × 107 cells, then were incubated at 4°C for 5 minutes. Subsequent addition of MACS buffer was added post-incubation at a volume of 30 μL/1 × 107 cells, which was followed with the magnetic labeling through the addition of Anti-Biotin Microbeads (Miltenyi Biotec) for depletion. Anti-Biotin Microbeads were added at a volume of 20 μl/1 × 107 cells used. The solution was then incubated at 4°C for 10 minutes. Post-incubation, magnetically bound cells were washed with 40 ml MACS buffer and centrifuged at 4°C, 1200 rpm for 7 minutes. The cells were then resuspended at a concentration of 1 × 108 cells/ml. Preparation of the QuadroMACS Separator (Miltenyi Biotec) along with the component LS Column (Miltenyi Biotec) was conducted during incubation of the Anti-Biotin Microbeads and washing process. One LS Column was used per 1 × 108 cells in order to avoid clumping. LS Columns were equilibrated with 4 ml MACS buffer, then followed with addition of the magnetically labeled cells. The effluent through the LS Column and QuadroMACS Separator contained the negatively selected Naïve CD4+ T cells and were collected. Additional 4 ml MACS buffer was used to wash columns of remaining residual magnetically labeled cells and were also collected. The collected Naïve CD4+ T cells were then subject to centrifugation at 4°C, 1200 rpm for 7 minutes, then resuspended in 1 ml HR5 [RPMI media (Omega Scientific) supplemented with 5% human AB serum (Gemini Bioscience, Sacramento, CA), GlutaMAX (Gibco; Thermo Fisher Scientific), and penicillin/streptomycin (Omega Scientific), then taken to be counted. Naïve CD4+ T cells were then resuspended in a volume of HR5 to obtain a concentration of 5 × 106 cells/ml. Purity was assessed immediately from an aliquot of the preparation and the percentage of CD45RA+ CCR7+ double expression calculated from total cells. A purity higher than >95% was observed for all donors and considered satisfactory.

2.5. APC isolation through CD3+ depletion

Freshly thawed PBMCs (3 × 107 cells) were subject to CD3+ depletion in utilization of the EasySep Human CD3+ Positive Selection Kit II (STEMCELL Technologies). PBMCs were centrifuged at 4°C, 1200 rpm for 7 minutes, then resuspended in RoboSep Buffer (STEMCELL Technologies) at a volume to obtain a concentration of 1 ×108 cells/ml. Resuspended PBMCs were then added to a 5 ml round-bottom polystyrene tube (Falcon, STEMCELL Technologies). EasySep Human CD3+ Positive Selection Cocktail II (STEMCELL Technologies) was added to the PBMCs at a volume of 100 μl/ml of resuspended PBMC volume, then was incubated at 20°C for 6 minutes. Post-incubation, EasySep Dextran RapidSpheres (STEMCELL Technologies) were added in equal volume to the amount of positive selection cocktail used in the previous step with no incubation required. RoboSep Buffer was added to the solution to obtain a total volume of 2.5 ml. Magnetic separation was conducted in utilization of the EasyEights EasySep Magnet (STEMCELL Technologies). The 5 ml round-bottom polystyrene tubes were placed directly onto the magnets and were allowed to incubate at 20°C for 3 minutes. After 3 minutes, this intermeddle CD3+ depleted solution was carefully pipetted out of its original 5 ml round-bottom polystyrene tube and transferred into a new 5 ml round-bottom polystyrene tube. The new tube containing the intermeddle CD3+ depleted solution was placed back onto EasyEights EasySep Magnet and allowed to incubate for 10 minutes. Upon completion of the last incubation, the CD3+ depleted solution was then carefully transferred to a 15 ml conical centrifuge tube (Falcon, Thermo Fisher Scientific), which was then subject to centrifugation at 4°C, 1200 rpm for 7 minutes. The APCs isolated through CD3+ depleted cells were then resuspended in 1 ml HR5 and were counted. APCs were resuspended in a volume of HR5 to obtain a concentration of 5 × 106 cells/ml and combined with negatively isolated Naïve CD4+ T cells in a ratio of 1:1 of the same donor, to a total 10 × 106 cells. The purity of APC ranged from 97 to 99% (measured by subtracting the percent of CD3+ cell content contaminant in the preparations) and considered satisfactory. The APC yield from total PBMC preparation was 24.1% ± 5.3% (mean ± SD).

2.6. Cell culture and proliferation assay

PBMC or Naïve CD4+ T cells/APC were subject to centrifugation at 4°C, 1200 rpm for 7 minutes, then resuspended with 1 ml PBS. Cells were labeled with the CFSE Cell Proliferation Kit (Thermo Scientific) at a final concentration of 10 μM. CFSE labelling indicated a labelling efficiency higher than 99% (Supplementary Figure 1). Cells were supplemented with HR5 and stimulated with DMSO (2 μl/ml) or PT (MG) (1μg/ml) either alone or in the presence of IFN-β (1 or 10 ng/ml) or TGF-β (1 or 5 ng/ml). α-CD3/CD28 (1 μg/ml each) was used as a control. Cells were then incubated at 37°C, 5% CO2 for 7 days. For proliferation assessment, rounds of cell division were determined by sequential halving of CFSE-fluorescence intensity after additional surface phenotypic staining was performed. All the proliferation data presented throughout the manuscript is represented as NET proliferation and calculated by subtraction of stimuli-specific proliferation to unstimulated condition (media alone for PT stimulation or IFN-β/ TGF-β alone for PT+ IFN-β/TGF-β stimulation).

2.7. Peptide restimulation and intracellular cytokine staining (ICS) assay

Six hours prior to the conclusion of the 7-day culture, BFA (1 μg/mL; BD Biosciences) was added to the cultures, and stimulated with DMSO (2 μl/ml) or PT MG (1μg/ml). Cells were then washed and stained for extracellular markers for 30 minutes, and then washed, fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin (Sigma-Aldrich), and stained for intracellular IL-4, IL-9, IL-17, and IFNγ. ICS assay data together with phenotypic characterization was acquired on a BD LSRII Flow Cytometer and analyzed using FlowJo X software. All flow cytometry monoclonal antibody (mAb) reagents for surface or intracellular staining are listed in Supplemental Table 2.

2.8. Statistical analysis

Comparisons between groups were made using the nonparametric Two-tailed Unpaired Mann-Whitney or Paired Wilcoxon test. Prism 8.0.1 (GraphPad) was used for all these calculations. All data in all figures in which error bars are shown are presented as median ± interquartile range where each dot represents an individual donor. A p value <0.05 was considered statistically significant.

2.9. Study approval

This study was performed with approvals from the Institutional Review Board at La Jolla Institute for Immunology (protocols: VD-059 and VD-101). All participants provided written informed consent for participation and clinical medical history was collected and evaluated.

3. Results

3.1. Effects of IFN-β on BP-specific proliferation of whole CD4 versus naïve CD4+ T cells

Based on the network analysis described above, we formulated the hypothesis that IFN-β produced following aP vaccination may lead to increased IL-9 and decreased IL-17 production8. To test this hypothesis, we stimulated in vitro cultures of PBMC from aP vs wP donors with a pool of T cell epitopes derived from B. Pertussis [‘PT’ megapool (MP)7]. In previous studies8,31,32, we have successfully used this pool to stimulate PT antigen-specific memory T cells and characterize the phenotype of their response using an activation induced marker assay (AIM) and intracellular cytokine staining (ICS) or proliferation assays. As an initial read out, following IFN-β stimulation, we measured proliferation in response to PT (MP) stimulation. We reasoned that these IFN-β effects might not be detected using already differentiated (committed) T cells and that the addition of IFN-β to proliferating naïve T cells may have a different outcome as compared to whole PBMC stimulations, in terms of Th9 and/or a Th2 and Th17 polarization. For this reason, in parallel to the PBMC cultures, we also tested cultures utilizing naïve T cells and individually tested the responses of both aP- and wP-primed individuals since polarization is maintained for years after the original priming and imprinting is essentially life-long7,9. Naïve T cells were purified by negative selection of magnetic beads labelled PBMC as described in Materials and Methods.

PBMC or naïve CD4+ T cells cultured in combination with antigen-presenting cells (APCs) were left unstimulated or stimulated with PT (MP) antigen-specific stimuli in the absence or presence of IFN-β and proliferation determined by CFSE fluorescence loss using flow cytometry after 7 days of culture (Supplementary Figure 1). As a control, IFN-β was cultured alone and did not have any impact in proliferation (Supplementary Figure 2), and IFN-β and polyclonal stimulation with α-CD3/CD28 induced a higher proliferative response (data not shown). As shown in Figure 1A, IFN-β exerted a different effect in the cultures of PBMC versus purified naïve T cells. More specifically, it dramatically reduced BP-specific proliferation in PBMC but had no significant impact in the proliferation of naïve CD4+ T cells, although an increase was noted in 70% of the donors. The dividing naïve CD4+ T cells over the culture period further lost expression of CD45RA and differentiated into a central (Tcm; CD45RA-CCR7+) and effector (Tem; CD45RA-CCR7−) memory phenotype (Supplementary Figure 1A). Like observed for BP antigen-specific stimulation, IFN-β also significantly reduced α-CD3/CD28 induced proliferation in PBMC but not in naïve CD4+ T cells (data not shown). Interestingly, these effects were noted irrespective of the vaccine administered in infancy. Albeit lacking statistical power in PBMC samples (n=5 for each cohort), IFN-β exerted reduced BP-specific proliferation in all wP and in 4 out of 5 aP donors (Figure 1B).

Figure 1. IFN-β stimulation has different activities in BP-specific proliferation of whole CD4 versus naïve CD4+ T cells, independent of childhood vaccination.

Figure 1.

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The proliferative capacity of BP-specific cells was assessed by CFSE assay after 7 days of stimulation. (A) Graphs show % of dividing CD4+ T cells by CFSE quenching from whole PBMC or naïve CD4+ T cell cultures stimulated with PT (MP) in the presence or absence of IFN-β (10 ng/ml). (B) Graphs show data plotted for aP (orange) or wP (blue) primed cohorts separately. Each data point represents a single donor (n=10 and n=18 for PBMC and naïve CD4+ T cell cultures respectively). p value is shown according to Wilcoxon paired t-test.

3.2. IFN-β decreases cytokine responses to BP in memory CD4+ T cells

We further determined the effect of IFN-β stimulation in cytokine response to PT (MP) stimulation by performing an ICS assay after restimulation of dividing cells following the 7-day culture period. This strategy allows identification of the cytokine production of proliferating cells to antigen-specific stimulation while excluding non-dividing bystander responders (Supplementary Figure 3). In that regard, we compared the effect of IFN-β stimulation irrespective of the original vaccine priming (Figure 2), or by segregating the responses by childhood vaccination type (Supplementary Figure 4). Consistent with the inhibitory effect detected in the proliferation, we noted that IFN-β stimulation also decreased cytokine production of CD4+ T cells in PBMC cultures (Figure 2). These effects were noted at two different IFN-β concentrations (1 and 10 ng/ml) for IL-9 and IL-4, and at the highest IFN-β concentration for IFN-γ and IL-17 (Figure 2B). No significant effect was observed at both concentrations of IFN-β when aP- or wP-vaccinated donors were analyzed separately (Supplementary Figure 4).

Figure 2. IFN-β stimulation decreases cytokine responses in BP-specific memory CD4+ T cells.

Figure 2.

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IL-9, IL-4, IL-17, and IFN-γ cytokine production from proliferating CD4+ T cells was measured by ICS staining in PBMC cultures stimulated with PT (MP) in the presence or absence of IFN-β at (A) 1 ng/ml or (B) 10 ng/ml. Proliferating cells were identified by loss of CFSE after 7 days of stimulation. Graphs show individual cytokine secretion in proliferating cells as percentage of total CD4+ T cells. Each data point represents a single donor (n=12). p value is shown according to Wilcoxon paired t-test.

3.3. IFN-β increases cytokine responses to BP in proliferating naïve CD4+ T cells

An opposite effect to IFN-β stimulation was noted when cytokine production was assessed utilizing purified naïve CD4+ T cells (Figure 3). In this case, for at least one IFN-β concentration, cytokine production was significantly increased for all cytokines except for IL-17. Importantly, as shown in Supplementary Figure 5, the same effect was noted regardless of the original vaccination received by the donors in infancy. In summary, IFN-β increases IL-9, IL-4 and IFN-γ production from BP-stimulated naïve CD4+ T cells, but no difference was noted between aP- and wP-vaccinated cohorts.

Figure 3. IFN-β stimulation increases cytokine responses to BP naïve CD4+ T cells.

Figure 3.

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IL-9, IL-4, IL-17, and IFN-γ cytokine production from proliferating CD4+ T cells was measured by ICS staining in naïve CD4+ T cell cultures stimulated with PT (MP) in the presence or absence of IFN-β at (A) 1 ng/ml or (B) 10 ng/ml. Proliferating cells were identified by loss of CFSE after 7 days of stimulation. Graphs show individual cytokine secretion in proliferating cells as percentage of total naïve CD4+ T cells. Each data point represents a single donor (n=18). p value is shown according to Wilcoxon paired t-test.

3.4. No evidence for a role of TGF-β in differential response polarization

As mentioned above, TGF-β is a pleiotropic cytokine known to regulate the development, homeostasis, tolerance, and differentiation of T cells33,34. In particular, TGF-β promotes Th9, Th17, or Treg differentiation from naïve T cells upon the presence of other cytokines16,17. Also, Th9 cells can be derived from highly differentiated Th2 cells exposed to TGF-β17,19, and we have shown previously a clear IL-9 and TGF-β signature among BP-specific CD4+ T cells in donors originally primed with aP8. Accordingly, we were interested in testing how TGF-β addition to BP-stimulated CD4+ T cells might modulate IL-17 and IL-9 production. Initial experiments addressed the effect of addition of TGF-β on CD4+ T cell proliferation in responses to the BP epitopes (Figure 4 and Supplementary Figure 6). As shown in Figure 4A, TGF-β reduced BP-specific proliferation in PBMC but significantly increased BP-induced proliferation in naïve CD4+ T cells. These results are in accordance with literature35,36 and are remarkably similar to what was observed in the case of cultures stimulated with IFN-β. Likewise, these responses were not specific to the type of vaccine administered (Figure 4B), as the effect was observed in both cohorts, albeit significance was not reached in the wP cohort due to lack of statistical power (n=6).

Figure 4. TGF-β stimulation has opposite effect on BP-specific proliferation of whole CD4 versus naïve CD4+ T cells independent of childhood vaccination.

Figure 4.

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The proliferative capacity of BP-specific cells was assessed by CFSE assay after 7 days of stimulation. (A) Graphs show % of dividing CD4+ T cells by CFSE quenching from whole PBMC or naïve CD4+ T cell cultures stimulated with PT (MP) in the presence or absence of TGF-β (5 ng/ml). (B) Graphs show data plotted for aP (orange) or wP (blue) primed cohorts separately. Each data point represents a single donor (n=12 for both PBMC and naïve CD4+ T cell cultures). p value is shown as significant according to Wilcoxon paired t-test.

We further determined the effect of TGF-β stimulation in cytokine response to PT (MP) in isolated naïve CD4+ T cells. When we examined cytokine production in proliferating cells, we noted that PT(MP) stimulation of CD4+ T cells in the presence of TGF-β did not impact cytokine production, regardless of the dose, cytokine, or the original aP or wP vaccination received by the donors (Figure 5 and Supplementary Figure 7).

Figure 5. TGF-β stimulation does not affect cytokine responses to BP naïve CD4+ T cells.

Figure 5.

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IL-9, IL-4, IL-17, and IFN-γ cytokine production from proliferating CD4+ T cells was measured by ICS staining in naïve CD4+ T cell cultures stimulated with PT (MP) in the presence or absence of TGF-β at (A) 1 ng/ml or (B) 5 ng/ml. Proliferating cells were discriminated by loss of CFSE after 7 days of stimulation. Graphs show individual cytokine secretion in proliferating cells as percentage of total naïve CD4+ T cells. Each data point represents a single donor (n=12). p value is shown according to Wilcoxon paired t-test.

4. Discussion

Based on our previously published data, we detected a key intrinsic difference in vaccination responses associated with differential IL-9/IL-17 polarization as a function of the vaccine type administered in childhood8. Supported by transcriptomic and proteomic data, we hypothesized that T cell immune signatures would be under control of IFN-β and TGF-β, two powerful immune regulators of T cell responses18,20. The present series of experiments do not provide support for a role of the IFN-β/TGF-β pathways in determining the differential cytokine production observed as a result of the original aP versus wP vaccination8. They do however highlight a striking difference in outcome following addition of exogenous cytokines as a function of whether the CD4+ T cells responding to the BP epitope stimulation were derived from whole PBMC (thus being dominated from already mature differentiated T cells) versus purified naïve CD4+ T cells.

The hypothesis of the role of IFN-β/TGF-β pathways in determining the differential cytokine production was derived on the basis of the transcriptomic profiles of CD4+ T cells that responded to BP stimulation8. By definition, the transcriptomic events in T cells are downstream from those occurring in APCs. We are in the process of analyzing transcriptomic profiles in different cell types. It is possible that this strategy will reveal different and more precise pathways associated with differential cytokine production in aP vs wP donors.

It is also possible that the experimental strategy we employed failed to reveal an effect. Moreover, it cannot be ruled out that non-CD4+ T cells other than APCs (e.g., CD8+ T cells) can elicit suppression to CD4+ T cell responses after IFN-β/TGF-β stimulation. In this regard, we are also exploring whether developing cultures in different polarizing conditions might be informative. Many studies have increased our understanding of cytokine-induced polarization and share the view that once polarized, the phenotype acquired by CD4+ T cells remains stable 6,17. However, this dogma has been recently challenged and cytokine reprogramming of differentiated cells has been reported in many studies, highlighting the plasticity of CD4+ T cells5,37. By use of single-cell RNA-seq, we plan to be able to address additional issues relevant to the exact nature of the responding CD4+ T cell subpopulations to BP stimulation. For example, we will be able to address the relationship between the modulation of gene expression and the phenotypical response to vaccination.

The most striking effect we detected in the present study was the differential effects of both the IFN-β and TGF-β on PBMC versus purified naïve cells CD4+ T cells. This is in line with previous reports in the literature in both mice and humans that have demonstrated a broader role for IFN-β and TGF-β in CD4+ T cell functions by regulating the development and stability of long-lived memory cells through several different mechanisms such as cell cycle arrest and inhibition of proliferation or T cell activation20,35, but by also directly influencing the fate of naïve CD4+ T cells during the initial phases of antigen recognition37. Indeed, as suggested by our hypothesis of differential polarization to pertussis childhood vaccination8, a growing body of literature has highlighted the role of IFN-β in cross-regulating the differentiation and stability of both Th2 and Th17 cells20.

Our findings are also in accordance with a study that reports that IFN-β among other Type I IFNs enhance cytokine expression of IL-4 and IFN-γ by sub-optimally stimulating naïve CD4+ T cells38. The authors suggest that while IFN-β may enhance protective immunity by enhancing CD4+ T cell polarization and polyfunctionality, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation. This is would be of interest to explore in future studies, specifically in the context of BP vaccination to differential aP vs wP priming.

It has been recently published that Type I and Type III interferon play a very important role in lung inflammation in the murine model of BP39. Evaluating the effects of Type I/III interferon responses in different CD4+ T cells and PBMC populations from previously vaccinated adults can guide future research in this area. In future studies, it will be of interest to evaluate levels of other IFNs such as IFN-α and IFN-λ. Likewise, levels of IFN-β positively correlate with the levels of IL-5, IL-13, and CCL1140. As indicators of the T cell response, studying the levels of IL-5 and IL-13 on the different populations included in this study will be very beneficial to better understand the role of IFN-β in driving cytokines that shift towards a Th2 or Th1 phenotype. Finally, it would be very interesting to evaluate the changes in the response at different post-stimulation times in order to better understand the spatiotemporal response to IFN-β and TGF-β.

This study also confirmed that key T cell functions such as proliferation and cytokine secretion are under the control of antigen stimulation (i.e., BP effector memory responses) and environmental cues, and that these two factors can interact. Indeed, IFN-β was shown to inhibit T cell activation and proliferation directly from memory T cells induced by both mitogen- or antigen-specific stimulation21,41,42. However, studying the effector responses on memory T cells is challenging because memory cells comprise multiple subpopulations and high responsiveness that have been proposed recently to be influenced by an effectorness gradient37. Another challenging aspect of our study is the fact that different doses of IFN-β could have different outcomes in the statistical analysis of cytokine production. Future studies would benefit from a higher number of donors and a higher titration range of the dose-response to IFN-β and TGF-β stimulation. Also, one important limitation of this study is that cohorts of infant donors were not studied. It would certainly be interesting to include infants in future studies, even though all USA infants would be aP vaccinees, since wP is no longer approved by the FDA in the US, and hence a direct comparison would not be feasible.

In conclusion, our studies suggest that molecular pathways different than the ones investigated here underlie the long-lasting differential cytokine production associated with aP-vs wP-priming. They further illustrate how the differentiation status of CD4+ T cells dictate different outcomes following stimulation with IFN-β and TGF-β.

Supplementary Material

1

Highlights.

  1. Pertussis resurgence is associated with waning of vaccine-induced immunity

  2. Type of childhood pertussis vaccine defines long-term T cell polarization

  3. IFN-β increases cytokine responses to pertussis stimulated naïve CD4+T cells

  4. TGF-β increases proliferation of pertussis stimulated naïve CD4+T cells

  5. Lack of evidence on the role of long-lasting T cell polarization by IFN-β or TGF-β

5. Acknowledgments

Research reported in this publication was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Numbers U19AI142742 and U01AI141995. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors are also grateful to all the donors that participated in the study and the clinical studies group staff, particularly Shariza Bautista, Brittany Schwan and Gina Levi for all the support.

Footnotes

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Conflict of interest

The authors have declared that no conflict of interest exists

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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