Vaccination Produces CD4 T Cells with a Novel CD154-CD40-Dependent Cytolytic Mechanism

Abstract

The discovery of new vaccines against infectious diseases and cancer requires the development of novel adjuvants with well-defined activities. The TLR4 agonist adjuvant GLA-SE elicits robust Th1 responses to a variety of vaccine Ags and is in clinical development for both infectious diseases and cancer. We demonstrate that immunization with a recombinant protein Ag and GLA-SE also induces granzyme A expression in CD4 T cells and produces cytolytic cells that can be detected in vivo. Surprisingly, these in vivo CTLs were CD4 T cells, not CD8 T cells, and this cytolytic activity was not dependent on granzyme A/B or perforin. Unlike previously reported CD4 CTLs, the transcription factors Tbet and Eomes were not necessary for their development. CTL activity was also independent of the Fas ligand-Fas, TRAIL-DR5, and canonical death pathways, indicating a novel mechanism of CTL activity. Rather, the in vivo CD4 CTL activity induced by vaccination required T cell expression of CD154 (CD40L) and target cell expression of CD40. Thus, vaccination with a TLR4 agonist adjuvant induces CD4 CTLs, which kill through a previously unknown CD154-dependent mechanism.

Figure 1. ID93+GLA-SE immunization produces cytolytic cells

(A) ID93+GLA-SE (black line) and unimmunized (gray fill) mice received a 1:1 mix of ID93 pulsed (CFSE^hi) and unpulsed (CFSE^lo) CD45.1 cells. 18 hours later CFSE^hi cells were specifically reduced in the ID93+GLA-SE immunized recipients. (B) The frequency of in vivo killing of CFSE^hi ID93-pulsed was determined for immunized animals relative to the unimmunized controls. (C) The frequency of granzyme A, granzyme B, and perforin was determined for naïve (CD44^lo) and memory (CD44^hi) CD8 T cells from unimmunized (black bars) and immunized (white bars) animals N=3–5 animals/group. Data are representative of eight experiments with similar results. Bars represent mean.

Figure 2. ID93+GLA-SE immunization elicits granzyme A expressing CD4 T cells

(A) ID93-specific CD4 T cells were identified by tetramer staining after immunization. The frequency of (B) granzyme A, (C) granzyme B, and (D) perforin or (E) MFI of Eomes was determined for naïve (CD44^lo), memory (CD44^hi) or Rv3619 tetramer specific CD4 T cells from unimmunized (black bars) and immunized (white bars) animals. N=5 animals. Data are representative of three experiments with similar results. Bars represent mean + s.d.

Figure 3. ID93+GLA-SE immunization produces cytolytic CD4 T cells

(A) ID93+GLA-SE (black line) and unimmunized (gray fill) mice received a 1:1 mix of ID93 pulsed (CFSE^hi) and unpulsed (CFSE^lo) cells. 18 hours later MHCII⁺ CFSE^hi cells were specifically eliminated in the ID93+GLA-SE immunized recipients. (B) The frequency of in vivo killing of MHCII⁺ and MHCII⁻ cells was determined for animals immunized with ID93 alone or ID93+GLA-SE. (C) Depletion of CD4 T cells, but not CD8 T cells and NK cells following vaccination with ID93+GLA-SE was sufficient to ablate the in vivo killing of ID93 pulsed MHCII⁺ cells. N=5 animals/group. Data are representative of three experiments with similar results. Bars represent mean + s.d.

Figure 4. In vivo cytotoxicity does not require granzyme A/B or perforin

(A and B) WT and GzmA/B−/− mice were immunized with ID93+GLA-SE or left unimmunized. (A) The frequency of granzyme A expression by memory (CD44^hi) and Rv3619 tetramer-specific CD4 T cells was increased in vaccinated WT but not GzmA/B−/− immunized animals. In vivo cytotoxicity of MHCII+ cells was not impaired by (B) granzyme A/B or (C) perforin deficiency relative to WT. N=5 animals/group. Data are representative of (A and B) two or (C) four experiments with similar results. Bars represent mean + s.d.

Figure 5. Development of cytolytic CD4 T cells by vaccination is independent of the Tbox transcription factors Eomes and Tbet

(A) The frequency of ID93 specific T_H1 cells and (B) in vivo cytotoxicity against MHCII⁺ ID93 pulsed target cells were determined in ID93-GLA-SE immunized Eomes^fl/fl and Eomes^0/0 mice. (C and D) WT and Tbet^−/− mice were immunized with ID93+GLA-SE. (C) Only the WT animals developed ID93-specific T_H1 cells, (D) but both populations killed MHCII⁺ ID93 pulsed target cells in vivo. N=5 animals/group . Data are representative of (A and B) four or (C and D) two experiments with similar results. Bars represent mean + s.d.

Figure 6. In vivo CD4 T cell cytotoxicity is independent of canonical cytolytic pathways

(A) WT and FasL^−/− immunized mice both kill MHCII⁺ CD45.1 cells. (B) WT and Pfr^−/− immunized mice both kill CD45.1 and Fas^−/− MHCII⁺ WT cells. (C) WT and Pfr^−/− immunized mice kill CD45.1 and DR5^−/− MHCII⁺ target cells. (D) WT immunized mice kill WT, Casp3^−/−, Casp7^−/−, Bax^−/− and TNFR1/2^−/− MHCII⁺ targets. N=5 animals/group. Data are representative of two to five experiments with similar results. Bars represent mean + s.d.

Figure 7. In vivo CD4 T cell cytotoxicity depends on CD40-CD154 interactions

Immunization with ID93+GLA-SE produced similar frequencies of ID93-specific CD4 T cells as determined by the frequency of (A) IFN-γ, TNF, or IL-2 producing cells upon ID93 stimulation or (B) staining with an I-A^b tetramer. (C) CD154^−/− immunized mice do not develop cytolytic CD4 T cells upon vaccination. Similarly immunized WT mice develop cytolytic CD4 T cells that kill WT but not CD40^−/− MHCII⁺ target cells. N=5 animals/group . Data are representative of four experiments with similar results. Bars represent mean + s.d