IL-2 and antigen dose differentially regulate perforin- and FasL-mediated cytolytic activity in antigen specific CD4+ T cells

Abstract

CD4 T cell effectors can promote survival against lethal influenza virus via perforin mediated cytolytic mechanisms; however, our understanding of how naïve CD4 cells differentiate into class II restricted killers remains obscure. To address this, TCR Tg CD4 cells were activated in vitro and examined for their ability to lyse target cells. We found that cytokine polarized CD4 T cell effectors displayed cytolytic activity with the hierarchy Th0>Th1>Th2. Further, IL-4 inhibited the generation of cytotoxic CD4 cells. LPS stimulated B cells and bone marrow derived dendritic cells (BMDC) both induced potent cytolytic activity; however, IL-6, TGF-beta, IL-10, IL-12 or TNF-alpha were not required for inducing cytolytic activity in CD4 effectors. Antigen dose had a marked effect on cytotoxicity: low concentrations of peptide induced more potent cytolytic activity than relatively high concentrations. At low peptide concentration, exogenous IL-2 was necessary to drive granzyme B (GrB) expression and perforin mediated lysis. Thus, low antigen dose and early activation signals via IL-2 direct the CD4 T cell response toward effectors with perforin mediated cytolytic potential. These data have implications for the design of vaccines that may induce cytolytic CD4 cells in vivo and improve cell-mediated immunity to viral and bacterial infections.

Figure 1. Cytolytic activity develops during Th1 differentiation, but does not require IFN-γ

TCR Tg CD4 cells from WT or IFN-γ ^−/− mice were cultured for 2, 3 or 4 days in the presence of polarizing conditions as described in the Materials and Methods. A) 4 hr in vitro cytolytic assay (JAM assay) comparing lytic activity of WT (left panel) or IFN-γ ^−/− (right panel) CD4 effectors to peptide pulsed (or unpulsed) A20 target cells. GrB levels were detected by FACS (D). Cytokine secretion was analyzed from WT (C) or IFN-γ ^−/− (D) supernatants using a Beadlyte multiplex cytokine assay after 2, 3, or 4d in culture and restimulation with anti-CD3 for an additional 36 hr. Shown is the amount of cytokine in ng/ml per 1 × 10⁶ input cells. Shown is a representative experiment of three performed.

Figure 2. Cytolytic activity correlates with increased GrB expression, Th1 cytokines and is inhibited by IL-4

TCR Tg CD4 cells were polarized to Th1 or Th2 or given IL-2 only for 4 days in culture and assayed for cytolytic activity by JAM assay (A). B) LU were calculated as described in the Materials and Methods for five separate experiments. * denotes significant differences by one way ANOVA analyses (p = .001) C) Th1, Th2 or IL-2 only effectors were restimulated with plate bound anti-CD3 for 48 hr and the supernatants analyzed for cytokines using a Beadlyte multiplex cytokine assay. Shown is the amount of cytokine in ng/ml per 1 × 10⁶ input cells. D) Th1, Th2 or IL-2 only 4 day effectors were stained for GrB and analyzed using FlowJo software. E) TCR Tg CD4 cells were incubated with peptide pulsed B cell blasts in the presence of IL-2 only or increasing concentrations of IL-4 for 4 days. Cells were then assayed for cytolytic activity as described. Th2 cells are shown as a negative control. This experiment was repeated twice with similar results.

Figure 3. APC induce peptide specific cytotoxicity that is not dependent on IL-10, IL-6, TNF-α, TGF-β or IL-12

TCR Tg CD4 cells were polarized to Th1 or Th2 effectors or cultured with IL-2 alone as described in the Materials and Methods. A) Th1, Th2 or IL-2 only effectors were stimulated with BMDC, A20 B cell lymphoma or anti-CD3 and anti-CD28 coated plates and assayed for cytolytic activity against peptide pulsed (closed symbols) or unpulsed (open symbols) target cells. A representative experiment of three performed is shown. B) B cell blasts or BMDC were stimulated with LPS for 48 h or 24 h respectively and the supernatants analyzed for cytokines using a Beadlyte multiplex cytokine assay. Shown is the amount of cytokine in pg/ml. C) Peptide pulsed WT B cells or B cells from IL-10^−/− mice were incubated with WT T cells or IL-10^−/− T cells for 4 days in the presence of IL-2 (left panel). TCR Tg CD4 cells were incubated with peptide pulsed B cell blasts in the presence of IL-2 only or isotype control antibody, anti-IL-6 or anti-TNF-α at 10 μg/ml for 4 days (middle panel) or anti-IL-12, anti-TGF-β or anti-TGF-β and anti-IL-6 at 10 μg/ml for 4 days (right panel). Cells were then assayed for cytolytic activity as described. Knock-out and blocking experiments were repeated with similar results.

Figure 4. Perforin and FasL mediated cytolytic activity inversely correlate with antigen dose

CD4 cells were isolated from HNT (A), OT-II (B) or HNT.Pfn^−/− (C) TCR Tg mice and stimulated with B cell blasts pulsed with various concentrations of peptide for 4 days. The same number of live cells in each group was plated in the JAM assay to account for differences in expansion. Cells were then assayed for cytolytic activity as described. D) LU (left panel) were calculated as described in the Materials and Methods from four to ten separate experiments. Fold expansion was also calculated at the end of the 4 day culture period and is shown in the right panel. *Means are statistically different by 1-way ANOVA. E) Supernatants from CD4 effectors (shown in A) incubated with plate-bound anti-CD3 for 48h were assayed for cytokines using a luminex bead array.

Figure 5. Exogenous IL-2 is necessary to induce perforin mediated cytotoxicity and GrB expression at low doses of peptide

CD4 cells were isolated from TCR Tg mice, labeled with CFSE and stimulated with B cell blasts pulsed with various concentrations of peptide in the presence or absence of IL-2 for 4 days. A) GrB expression and proliferation by loss of CFSE expression was analyzed by flow cytometry. Resulting histograms were gated on CD4 T cells in the culture. B) Cytolytic activity of CD4 effectors grown in the presence of IL-2 (closed symbols) or absence of IL-2 (open symbols) was determined by JAM assay. The same number of live cells in each group was plated in the JAM assay to account for differences in expansion. C) CD4 cells were isolated from WT or Pfn^−/− TCR Tg mice and stimulated with B cell blasts pulsed with 50 nM peptide in the presence (squares) or absence (circles) of IL-2. Cells were then assayed for cytolytic activity in the presence (open symbols) or absence (closed symbols) of anti-FasL antibody. D) GrB expression in gated CD4 cells cultured in the presence or absence of IL-2 was assayed by flow cytometry. Shown is a representative experiment of three performed.