Th1 and Th2 cells help CD8 T-cell responses

Abstract

Help from CD4 T cells is often important for the establishment of primary and memory CD8 T-cell responses. However, it has yet to be determined whether T helper polarization affects the delivery of help and/or whether responding CD8 T cells helped by Th1 or Th2 cells express distinct effector properties. To address these issues, we compared CD8 T-cell responses in the context of Th1 or Th2 help by injecting dendritic cells copulsed with the major histocompatibility complex class I-restricted OVA peptide plus, respectively, bacterial or helminth antigens. We found that Th2 cells, like Th1 cells, can help primary and long-lived memory CD8 T-cell responses. Experiments in interleukin-12 (IL-12)-/- and IL-4-/- mice, in which polarized Th1 or Th2 responses, respectively, fail to develop, indicate that the underlying basis of CD4 help is independent of attributes acquired as a response to polarization.

FIG. 1.

CD4 help is required for the development of IFNγ-producing CD8 T cells during immunization with peptide-pulsed DCs. DCs pulsed with P. acnes or SEA in combination with OVA_pep were injected into naive WT mice. One week after immunization, splenocytes were harvested, and IFN-γ production was measured by ICS and ELISA after ex vivo incubation with OVA_pep. (A and C) Splenocytes from mice primed with OVA_pep+P. acnes- or OVA_pep+SEA-pulsed DCs were restimulated for 5 h in medium alone (−) or with OVA_pep (+) and then stained for intracellular IFN-γ. The FACS plots are from a representative mouse, and the numbers in the quadrants are the percentages of CD8 T cells producing IFN-γ. The values in panel A are from mice treated with a control rat IgG; the values in panel C are from mice depleted of CD4 cells by injection with GK1.5. (B) Splenocytes from untreated mice primed with DCs alone (control) or with P. acnes+OVA_pep- or SEA+OVA_pep-pulsed DCs were restimulated for 5 h with OVA_pep. CD8 cells were stained for intracellular IFN-γ by ICS (upper panel), or splenocytes were stimulated for 72 h with OVA_pep, after which the culture supernatants were assayed for IFN-γ by ELISA (lower panel). The data represent mean values ± the standard error of the mean from seven separate experiments. (D) IFN-γ levels as measured by ELISA from groups of three individual DC-immunized (shown on the x axis) control (normal rat IgG treated) or CD4 T-cell depleted mice and restimulated with medium (hatched bars), OVA_pep (black bars), P. acnes (gray bars), or SEA (empty bars). Little or no detectable IFN-γ was made in medium- or SEA-restimulated cultures. Values are means ± the standard deviation of three analyses. These experiments were repeated twice with similar results.

FIG. 2.

Effect of Th1 and/or Th2 polarization on CD4 help for primary CD8 T-cell responses. Naive WT, IL-12p35-deficient, or IL-4-deficient mice were injected with Ag-pulsed DCs. One week after injection, splenocytes were harvested from individual mice and cultured in medium alone, Pa, SEA, or OVA_pep. Th1 responses are shown in panel A, and Th2 responses are shown in panel B. Panels C and D include IFN-γ production by CD8 T cells in response to restimulation with OVA peptide. Panels C and D utilize different scales compared to panel A, since the amounts of IFN-γ produced by CD8 T cells are so much greater than those produced by Th1 cells (A). Cytokine levels in 72 h culture supernatants were measured by ELISA. Two experiments were performed with similar results.

FIG. 3.

Th1 and Th2 cells provide help for memory CD8 T-cell development. Naive mice were primed with Ag-pulsed DCs. Sixty days later, mice were challenged with rLmOVA or injected with PBS. Six days after rLmOVA challenge, splenocytes were harvested and restimulated with OVA_pep for 5 h, and the IFN-γ and TNF-α levels were measured by ICS. Representative FACS plots show IFN-γ versus TNF-α (gated on live CD8 T cells) production by unstimulated (−) or OVA_pep-stimulated (+) splenocytes from unchallenged (A) and rLmOVA-challenged (B) mice. The numbers represent the percentages of CD8 T cells producing cytokines. (C) Bar graph showing mean ± the standard deviation of the numbers of splenic CD8 cells that made IFN-γ in response to in vitro stimulation with OVA_pep. Cells cultured in the absence of OVA_pep failed to make IFN-γ. These data were pooled from nine individual experiments.

FIG. 4.

Cytotoxicity of CD8 T cells helped by Th1 or Th2 cells. Naive mice were primed with Ag-pulsed DCs and >60 days later challenged with rLmOVA. At 5 days postchallenge, the mice were injected with CFSE^high-OVA_pep-pulsed syngeneic target cells and with unpulsed CFSE^low syngeneic control cells. One hour later individual spleens were removed, and in vivo cytotoxicity was measured directly by flow cytometry through a comparison of the ratio of CFSE^high to CFSE^low cells in individual animals. (A) Representative plots gated on CFSE-positive cells. The numbers indicate the percent lysis. (B) Mean values (± the standard error of the mean) from three mice per group for the percent lysis of target cells.