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. 2017 Feb;101(2):567-576.
doi: 10.1189/jlb.4A0616-271R. Epub 2016 Aug 26.

Coordination between T helper cells, iNKT cells, and their follicular helper subsets in the humoral immune response against Clostridium difficile toxin B

Pragya Rampuria  1 , Gillian A Lang  1 , T Scott Devera  1 , Casey Gilmore  1 , Jimmy D Ballard  1 , Mark L Lang  2
Affiliations

Coordination between T helper cells, iNKT cells, and their follicular helper subsets in the humoral immune response against Clostridium difficile toxin B

Pragya Rampuria et al. J Leukoc Biol. 2017 Feb.

Abstract

Activation of iNKT cells with the CD1d-binding glycolipid adjuvant α-galactosylceramide (α-GC) enhances humoral immunity specific for coadministered T-dependent Ag. However, the relationship between the iNKT cell and the classic T helper (Th) or T follicular helper (Tfh) function following this immunization modality remains unclear. We show that immunization with the C-terminal domain (CTD) of Clostridium difficile toxin B (TcdB), accompanied by activation of iNKT cells with α-GC, led to enhanced production of CTD-specific IgG, which was CD1d- and iNKT cell-dependent and associated with increased neutralization of active TcdB. Immunization with CTD plus α-GC followed by NP hapten-linked CTD increased NP-specific IgG1 titers in an NKT-dependent manner, suggesting that iNKT activation could enhance Th or Tfh function or that iNKT and iNKTfh cells could provide supplemental, yet independent, B cell help. Th, Tfh, iNKT, and iNKTfh cells were, therefore, examined quantitatively, phenotypically, and functionally following immunization with CTD or with CTD plus α-GC. Our results demonstrated that α-GC-activated iNKT cells had no direct effect on the numbers, phenotype, or function of Th or Tfh cells. However, CD4+ T cell-specific ablation of the Bcl6 transcription factor demonstrated that Tfh and iNKTfh cells both contributed to B cell help. This work extends our understanding of the immune response to vaccination and demonstrates an important contribution by NKTfh cells to humoral immunity.

Keywords: CD1d; T follicular helper; humoral immunity; iNKT follicular helper.

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Figures

Figure 1.
Figure 1.. Contribution of CD1d-restricted iNKT cells to the protective humoral immune response against TcdB B6 and CD1d−/−.
Mice were immunized s.c. with 50 μg CTD in sterile PBS ± 4 µg α-GC. The mice were then boosted with 25 μg CTD on d 28. Sera were collected on d 28 and 35. For all parts of the experiment, 7–9 mice were used in each group. (A) The endpoint serum anti-CTD IgG1 titers for B6 and CD1d−/− mice. Each data point represents an individual mouse, and geometric titer means are indicated. (B) Means ± sd percentage of in vitro neutralization of TcdB for each of the sera tested in (A). (C) Mice depicted in (A) and (B) were challenged on d 60 with 500 ng whole TcdB administered via the i.v. route. Naïve B6 and CD1d−/− controls (n = 3 per group) were used to demonstrate TcdB efficacy. Kaplan-Meier survival plots show the percentage of survival of the naïve or immunized B6 mice (left panel) and the CD1d−/− mice (right panel). Data are representative of 2 independent experiments. Statistical significance is indicated by asterisks, ***P < 0.001.
Figure 2.
Figure 2.. Priming of an anti-hapten IgG1 response following immunization with CTD plus α-GC.
As represented in the schematic, B6 and Jα18−/− mice were s.c. immunized with 50 μg CTD or 50 μg CTD plus 4 μg α-GC; 14 d after immunization, the mice received a booster vaccine consisting of 25 µg NP-conjugated CTD in sterile PBS. Sera were collected on d 28 (14 d after booster). Graph shows the means ± sd of endpoint anti-NP IgG1 titers measured by ELISA. Data are representative of 2 independent experiments using 5–6 mice per group. Statistical significance is indicated by the asterisk, *P < 0.05.
Figure 3.
Figure 3.. Expansion of iNKTfh cells but not Tfh cells following activation of iNKT cells with α-GC.
Cells were harvested from lymph nodes of B6 mice 7 d after s.c. immunization with 50 μg CTD in the presence or absence of 4 μg α-GC. Cells were then stained and analyzed by flow cytometry. (A) Contour plots show CD1d tetramer−/TCRβ+ T cells (left panels), CD4+/CD44hi activated Th cells (middle panels), and CXCR5+/PD-1+ Tfh cells (right panels). (B) Contour plots show CD1d tetramer+/TCRβ+ T cells (left panels) and CXCR5+/PD-1+ NKTfh cells (right panels). Inset image depicts background staining using isotype controls for CXCR5 and PD-1, which were used to set double-positive population gates in (A) and (B). (C) Graphs show the means ± sd frequencies of Tfh cells (left panel) and NKTfh cells (right panel). Data are representative of 3 independent experiments using 5 mice per group. Statistical significance is indicated by the asterisks, ***P < 0.001.
Figure 4.
Figure 4.. Effect of α-GC on IL-4 and IL-21 secretion.
(A) B6 and CD1d−/− mice were immunized s.c. with 50 μg of CTD or 50 μg of CTD plus 4 μg of α-GC; 7 d after immunization, splenocytes were isolated. Cells were then cultured with or without CTD or OVA (25 μg/ml final concentration), and the number of cytokine-secreting cells was examined by ELISPOT. (B and C) Graphs show the means ± sd number of IL-4– and IL-21–secreting cells per million splenocytes. Data are representative of 2 independent experiments using 4 mice per group. Statistical significance is indicated by asterisks, *P < 0.05; **P < 0.01.
Figure 5.
Figure 5.. iNKT cell-derived and -facilitated secretion of IL-4 and IL-21 following activation with α-GC.
(A) B6 mice were immunized s.c. with 50 μg of CTD plus 4 μg of α-GC; 3 or 7 d after immunization splenocytes, were isolated and subjected to mock-depletion (control) or to NKT cell depletion (depleted). Cells were then cultured with or without CTD, and cytokine secretion was examined by ELISPOT (B) Contour plots show CD1d tetramer+/TCRβ+ NKT cells for control (left panel) and for depleted cells (right panel). (C) Graphs show the means ± sd number of IL-4–secreting cells at d 3 (left panel) and d 7 (right panel). (D) Graphs show the means ± sd number IL-21–secreting cells at d 3 (left panel) and d 7 (right panel). A paired t test was used to determine significance. Data are representative of 3 similar experiments.
Figure 6.
Figure 6.. No effect of α-GC immunization on CD4+ T cell–mediated B cell help.
(A) CD45.1+ B6 donor mice were immunized s.c. with 50 μg of CTD or 50 μg of CTD plus 4 μg of α-GC; 7 days after immunization, splenocytes were harvested, and Th cells were isolated using magnetic separation. Six million Th cells were then adoptively transferred into TCRα−/− recipient mice (expressing CD45.2 congenic marker). Recipient mice were immunized i.p. with 50 μg CTD and 50 μg OVA 24 h after cell transfer. Sera were collected on d 14, and CTD-specific IgG1 titers were measured by ELISA. (B) Means ± sd of endpoint anti-CTD IgG1 titers (left panel) and endpoint anti-OVA IgG1 titers (right panel) observed in the recipient mice. Data are representative of 2 independent experiments using 6 mice per group. Statistical significance is indicated by asterisks, *P < 0.05; **P < 0.01.
Figure 7.
Figure 7.. Contribution of iNKTfh cells to production of CTD-specific IgG1.
(A) Bcl6+/+;Cd4-Cre+ control mice and Bcl6fl/fl;Cd4-Cre+ mice were administered 4 μg α-GC. After 7 d, lymph node cells were harvested and examined by flow cytometry. Plots on the left depict TCRβ+/CD1d-tetramer+ iNKT cells, whereas plots on the right depict CXCR5+/PD-1+ iNKTfh cells. The inset image depicts background staining using isotype controls for CXCR5 and PD-1, which were used to set double-positive population gates. (B) Bcl6+/+;Cd4-Cre+ control mice and Bcl6fl/fl;Cd4-Cre+ mice were immunized s.c. with 50 μg CTD (open symbols) or 50 μg CTD plus 4 μg α-GC (closed symbols); 28 d after immunization, sera were collected, and anti-CTD IgG1 titers were measured by ELISA. (C) Bcl6fl/fl;Cd4-Cre+ were mock transferred (with PBS) or transferred with 5 × 106 whole or NKT-depleted, naïve CD4+ cells from B6 donors (upper panel) or transferred with 106-enriched NKT cells. Graphs in (B) and (C) show the endpoint of anti-CTD IgG1 titer means. Each symbol represents an individual mouse, and statistical significance is indicated by asterisks, *P < 0.05. As described in Materials and Methods, a hypothetical control titer of 1 was used for upper graph (C) due to having 2 recipients in control group.
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