MyD88 Shapes Vaccine Immunity by Extrinsically Regulating Survival of CD4+ T Cells during the Contraction Phase

Abstract

Soaring rates of systemic fungal infections worldwide underscore the need for vaccine prevention. An understanding of the elements that promote vaccine immunity is essential. We previously reported that Th17 cells are required for vaccine immunity to the systemic dimorphic fungi of North America, and that Card9 and MyD88 signaling are required for the development of protective Th17 cells. Herein, we investigated where, when and how MyD88 regulates T cell development. We uncovered a novel mechanism in which MyD88 extrinsically regulates the survival of activated T cells during the contraction phase and in the absence of inflammation, but is dispensable for the expansion and differentiation of the cells. The poor survival of activated T cells in Myd88-/- mice is linked to increased caspase3-mediated apoptosis, but not to Fas- or Bim-dependent apoptotic pathways, nor to reduced expression of the anti-apoptotic molecules Bcl-2 or Bcl-xL. Moreover, TLR3, 7, and/or 9, but not TLR2 or 4, also were required extrinsically for MyD88-dependent Th17 cell responses and vaccine immunity. Similar MyD88 requirements governed the survival of virus primed T cells. Our data identify unappreciated new requirements for eliciting adaptive immunity and have implications for designing vaccines.

Fig 1. Myd88 ^-/- mice fail to mount Th17 and Th1 cells and resistance after vaccination.

Wild type C57BL6 and Myd88 ^-/- mice were s.c. vaccinated with 10⁵ or 10⁶ live B.dermatitidis yeast and followed for survival (A). To circumvent dissemination, mice were vaccinated s.c. with 10⁶ heat-killed yeast and challenged i.t. with 2 x 10³ 26199 yeast. Two weeks post-infection, lung CFU were enumerated (B). Data are the mean ± SEM (n = 8–10 mice/group). Data are representative of three independent experiments. The number of Th17 and Th1 cells recalled to the lung were assessed at day 4 post-infection. Data are the mean ± SEM (n = 4–6 mice/group) (C). The numbers indicate the mean ± SEM of cytokine producing CD4⁺ T cells per mouse. * P <0.05 vs. vaccinated wild-type controls. Dot plots show concatenated samples of 4–6 mice/ group. Data are representative of three independent experiments. (D) Wild-type and Myd88 ^-/- mice were vaccinated s.c. with 10⁷ live H. capsulatum yeast and challenged with 5 x 10⁵ Hc G217B yeast. Two weeks post-infection, lung CFU were enumerated. * P <0.05 vs. vaccinated wild-type controls. (E) The frequencies of Th17 and Th1 cells were enumerated at day 4 post-infection. Data are the mean ± SEM (n = 4–6 mice/group).

Fig 2. Intrinsic MyD88 is dispensable for the development of Th17 and Th1 cells.

Naïve Myd88 ^-/- and Myd88 ^+/+ 1807 T cells (indicated by -/- and +/+) were adoptively transferred into wild type C57BL6 recipient mice prior to s.c. vaccination (Panels A-C). Expansion (A), contraction (B) and migration (C) of transferred 1807 cells and endogenous CD4⁺ T cells was determined by FACS in the skin draining lymph nodes (sdLN) on days 7 and 35 post-vaccination and in the lung at day 4 post-infection. Data are representative of five independent experiments. * P < 0.05 vs. wild type 1807 T cells. (D) Myd88∆T mice that are CD4-Cre⁺ Myd88fl/fl lack MyD88 in αβT cells and CD4-Cre^- Myd88fl/fl controls were vaccinated with 10⁶ heat-killed vaccine yeast and challenged with wild-type yeast. The number of activated (CD44⁺) and cytokine producing CD4⁺ T cells were enumerated at day 4 post-infection. Lung CFU were determined at day 4 and two weeks post-infection. Data from day 4 post-infection are an average of three independent experiments and day 14 post-infection data are representative of two independent experiments. The numbers in the graph indicate the n-fold change in lung CFU vs. unvaccinated control mice.

Fig 3. Extrinsic MyD88 regulates T cell contraction.

(A) 10⁶ CD4⁺ purified, CFSE-labeled, naïve 1807 Tg cells were transferred into wild type and Myd88 ^-/- mice that were then vaccinated s.c. with 10⁶ heat-killed vaccine yeast or not. At day 7 post-vaccination, the skin draining lymph nodes (sdLN) were harvested and the numbers of activated (CD44⁺) 1807 T cells and endogenous CD4⁺ T-cells enumerated by FACS. (B) T cell proliferation of 1807 T cells is indicated by CFSE loss. (C). BMDC from wild type and Myd88 ^-/- mice were co-cultured with vaccine yeast for 24 h and transcript analyzed by RT-PCR. * P < 0.05 vs. increase in transcript in wild type DC. (D) At day 11 post-vaccination, the frequency of cytokine producing 1807 T cells was determined at the site of vaccination. Dot plots show the sum of concatenated events from 6 mice/group and the numbers indicate the mean ± SEM of cytokine producing 1807 T cells. (E) Upon recall at day 4 post-infection, the frequency of IL-17 producing 1807 T cells was quantified in the lung. Data are representative of two independent experiments. (F) At day 35 post-vaccination, endogenous CD4⁺ T cells were purified from the skin draining lymph nodes (sdLN) and spleen of vaccinated wild type and Myd88 ^-/- mice and stimulated with CW/M antigen for 3 days. Cytokines were measured in the cell-culture supernatants by ELISA. *P < 0.05 vs. cytokine production by CD4⁺ T cells from vaccinated wild type controls. (G, H) 10⁶ purified naïve CD4⁺ 1807 T cells were transferred into wild type and Myd88 ^-/- mice that were vaccinated s.c. with 10⁶ heat-killed vaccine yeast or not. At serial time points post-vaccination, the sdLN and lung were harvested and the number of CD44⁺ 1807 T cells enumerated by FACS. * P < 0.05 vs. number of activated CD4⁺ T cells from vaccinated wild type controls. Data are representative of three independent experiments.

Fig 4. Extrinsic MyD88 regulates T cells during the contraction phase, but not the programming phase.

(A). Schematic: One million purified CD4⁺ T cells from naïve 1807 mice were transferred into Myd88 ^-/- and wild type mice prior to vaccination. Seven days post-vaccination, CD4⁺ T cells were magnetic bead-purified from the sdLN and spleen of the vaccinated mice and adoptively transferred into new sets of naïve Myd88 ^-/- and wild type mice as shown in the schematic and activated effector T cells were rested for 4wks. (B) Recipient mice were challenged and 1807 T cells enumerated in the lung at day 4 post-infection. Data are representative of two independent experiments. * P < 0.05 vs. groups with no asterisk. (C) Congenic Thy1.1⁺ wild type mice were vaccinated and at day 7 post-vaccination the CD4⁺ T were sorted for CD44⁺ T cells, transferred into naïve Myd88 ^-/- and wild type mice, and the mice rested for 4 wks as above. At day 4 post-challenge, the numbers of congenic Thy 1.1⁺ T cells were determined by FACS. Data are the mean ± SEM (n = 4–6 mice/group). Data are representative of two independent experiments. * P < 0.05 vs. corresponding wild type controls.

Fig 5. The role of extrinsic MyD88 for effector CD4⁺ T cell survival.

(A-C) Mice received 10⁶ purified CD4⁺ T cells from naïve 1807 mice and were vaccinated with heat-killed yeast. (A) 1807 T cell contraction. At serial time intervals post-vaccination, the numbers of activated 1807 T cells were enumerated from the sdLN and spleen by FACS. * P < 0.05 vs. wild type control recipient mice. (B) Caspase 3 expression. Numbers in the dot plot indicate the numbers (top line) and frequencies (2^nd line from top) of intracellular caspase 3 positive 1807 T cells within the CD4⁺ T cell gate at day 12 post-vaccination. * P < 0.05 vs. wild type control recipient mice. (C) The mean fluorescence intensity of Bcl2 and Bcl-xL expression in 1807 T cells transferred into wild type and Myd88 ^-/- recipients. Isotype controls are shown on the left of the graphs (unlabeled). (D) Adoptive transfer of naïve Bim^-/- 1807 T cells and Fas^-/- 1807 T cells into Myd88 ^-/- and wild type controls prior to vaccination. At days 7 and 35 post-vaccination, the numbers of adoptively transferred 1807 T cells were enumerated in the sdLN and spleen. Data are the mean ± SEM (n = 4–6 mice/group). Data are representative of two independent experiments. * P < 0.05 vs. corresponding wild type recipient mice. ** P < 0.05 vs. corresponding wild type 1807 cells.

Fig 6. TLR3, 7 and 9 upstream of MyD88 extrinsically regulate T cell contraction.

One million CD4⁺ T cells from naïve 1807 mice were transferred into wild type mice prior to vaccination. Seven days post-vaccination, effector CD4⁺ T cells were magnetic bead-purified from the sdLN and spleen of the vaccinated mice and adoptively transferred into naïve Myd88 ^-/-, IL-1R^-/-, TLR23479^-/-, TLR379^-/- and TLR24^-/- and wild type mice. After 4 weeks of rest, primed 1807 T cells harvested from the lymph nodes and spleen were enumerated by FACS. (A) Data are expressed as the mean ± SD of 8–12 mice/group. Data represent the average from two independent experiments. * P < 0.05 vs. wild type control mice. (B) shows the dot plots of concatenated events from 8–12 mice/group from panel A. * P < 0.05 vs. wild type control mice. (C) Myd88 ^-/-, IL-1R^-/-, TLR23479^-/-, TLR379^-/- and wild type mice were vaccinated with 10⁶ heat-killed vaccine yeast. At days 7 and 49 post-vaccination, the number of Cnx-specific CD4⁺ T cells in the sdLN and spleen was enumerated using tetramer enrichment and FACS detection. Tetramer positive cells are shown within the gate in each dot plot. The numbers represent the geometric mean ± SEM of tetramer-positive cells of 5 mice. Data are expressed from a single experiment representative of three independent experiments. * P < 0.05 vs. wild type control mice. (D) Myd88 ^-/-, IL-1R^-/-, TLR23479^-/-, TLR379^-/-, TLR24^-/- and wild type mice were vaccinated as above, challenged and lung CFU enumerated at day 14 post-infection when unvaccinated mice were moribund. The numbers indicate the n-fold change vs. unvaccinated mice. Data are the mean ± SD of 10–20 mice/group from a single experiment representative of three independent experiments. * P < 0.05 vs. vaccinated wild type control mice.

Fig 7. TLR379 upstream of MyD88 extrinsically regulate contraction of LCMV primed CD4⁺ T cells.

Congenic Thy 1.1⁺ wild type mice were infected with 2 x 10⁵ pfu of LCMV Armstrong. At day 8 post-infection, CD4⁺ T cells were purified from the spleen and adoptively transferred into naïve Myd88 ^-/-, IL-1R^-/-, TLR23479^-/-, TLR379^-/-, TLR24^-/- and wild type mice. After 4 weeks of rest, tetramer-positive, gp66-specific CD4⁺ T cells were enumerated in the spleen. (A, B) The numbers represent the geometric mean ± SEM of tetramer-positive cells from 5 mice. Data are expressed from a single experiment representative of two independent experiments. * P < 0.05 vs. vaccinated wild type control mice.