IL-4 directs both CD4 and CD8 T cells to produce Th2 cytokines in vitro, but only CD4 T cells produce these cytokines in response to alum-precipitated protein in vivo

Abstract

While IL-4 directs CD4 T cells to produce Th2 cytokines (including IL-4, IL-13, IL-5) in vitro it has been shown that production of these cytokines can be induced in vivo in the absence of IL-4/IL-13/STAT-6 signaling. The present report shows that CD8 as well as CD4 T cells activated through their TCR, in vitro upregulate the Th2-features - IL-4, IL-13, IL-5, and GATA-3. However, in vivo while alum-precipitated antigen strongly and selectively induces these Th2-features in CD4 T cells, CD8 T cells mount a markedly different response to this antigen. This CD8 response is associated with strong proliferation and production of IFN-gamma, but no Th2-features are induced. Alum-protein formulations are widely used in human vaccines and typically induce strong antibody responses characterized by the differentiation of IL-4-producing CD4 T cells and immunoglobulin class switching to IgG1. Nevertheless, the mechanism responsible for CD4 Th2 and follicular helper T cell commitment triggered by these alum-protein vaccines is still poorly understood. Analysis of the in vivo response to alum-precipitated protein shows that while subsets of CD4 T cells strongly upregulate Th2 and follicular helper T cell features including the surface markers OX40, CXCR5, PD-1, IL-17RB and the transcription factor c-Maf, CD8 T cells do not. These discrete differences between responding CD4 and CD8 T cells provide further insight into the differences between Th2 polarization of CD4 T cells directed by IL-4 in vitro and the induction of IL-4 production by CD4 T cells in vivo in response to alum-precipitated protein.

Fig. 1

Th2 cytokines are induced in OTI and OTII cells by OVA-peptide with IL-4 in vitro, but only in OTII cells responding to alumOVA in vivo. (A) LN cell suspensions from OTI or OTII mice were cultured for 6 days with OVA-peptide (SIINFEKL or 323-339, respectively), IL-4, anti-IL-12 and anti-IFN-γ. The cultured cells were then restimulated in anti-CD3-coated culture wells with soluble anti-CD28 for 5 h before testing for cytokine production by intracellular flow cytometry. Dot plots (left) show gated CD8⁺ OTI cells (top row) or CD4⁺ OTII cells (bottom row). The graph (right) shows the proportion of cytokine-producing OTI cells (open squares) or OTII cells (closed circles) in five independent experiments. (B) CD45.2⁺ C57BL6 mice received congenic CD45.1⁺ OTI cells or CD45.1⁺ OTII cells. One day later the chimeras were immunized with alumOVA in both footpads. After 7 days draining popliteal LN cell suspensions were restimulated with OVA-peptide (SIINFEKL or 323-339) and assessed as in (A). Dot plots (left) are gated on OTI cells (CD45.1⁺/CD8⁺) (top row) or OTII cells (CD45.1⁺/CD4⁺) (bottom row). The graph (right) shows the percentage of cytokine-producing OTI or OTII cells from the popliteal LN of eight mice in two independent experiments. Parallel studies (not shown) indicate that <0.1% OTI and OTII cells from non-immunized chimeric mice produce any of these cytokines. Allophycocyanin (APC) or Phycoerythrin (PE) anti-cytokine or isotype control (Neg) antibodies were used. n8 indicates the number of observations where <0.1% cells contained the cytokine. Horizontal lines and italicized numbers indicate the medians of groups. Two-tailed Mann-Whitney non-parametric statistical differences between OTI cell and OTII cell values are shown in the graphs.

Fig. 2

Th2-cytokine mRNAs are induced in OTI and OTII cells by OVA-peptide with IL-4 in vitro, but only in OTII cells responding to alumOVA in vivo. (A) LN cell suspensions from OTI (open squares) or OTII (closed circles) mice were cultured for 6 days with OVA-peptide (SIINFEKL or 323-339, respectively), IL-4, anti-IL-12 and anti-IFN-γ. The cultured cells were then restimulated in anti-CD3-coated culture wells with soluble anti-CD28 for 5 h. They were then analyzed for Th2 and TFh cytokine mRNA expression by real time RT-PCR. Data are derived from four independent experiments. (B) CD45.2⁺ C57BL6 mice received congenic CD45.1⁺ OTI cells or CD45.1⁺ OTII cells or both OTI and OTII cells. One day later the chimeras were immunized with alumOVA in both footpads. After 7 days OTI cells (CD45.1⁺/CD8⁺) or OTII cells (CD45.1⁺/CD4⁺) were FACS-sorted from draining popliteal LN cell suspensions. Cytokine mRNA expression was assessed in the sorted cells by real time RT-PCR. Data are derived from two independent experiments each with four mice in each group. Statistical differences between groups indicated in the graph were assessed by the two-tailed Mann–Whitney non-parametric test.

Fig. 3

Differences in transcription factor and cell surface molecule mRNA levels between OTI and OTII cells responding in the in vitro or in vivo Th2 cytokine-inducing responses. (A) LN cell suspensions from OTI mice (open squares) or OTII mice (closed circles) were cultured for 6 days with OVA-peptide (SIINFEKL or 323-339, respectively), IL-4, anti-IL-12 and anti-IFN-γ. The cultured cells were then restimulated in anti-CD3-coated culture wells with soluble anti-CD28 for 5 h. The mRNA expression was then assessed by real time RT-PCR for the transcription factors: GATA-3 and c-Maf and the cell surface molecules OX40, IL-17RB and CXCR5. These data are derived from two independent experiments each with four mice in each group. (B) mRNA expression was also assessed by real time RT-PCR in LN suspensions from the chimeric mice described in Fig. 2B. The mRNA levels are shown for sorted OTI cells’ or OTII cells’ from the chimeras constructed with OTI cells only, or OTII cells only, or both OTI and OTII cells 7 days’ responding to alumOVA. These mRNA levels are compared to levels in OTI or OTII cells from non-immunized chimeras.

Fig. 4

Phenotypic changes related to proliferation among OTI and OTII cells responding to alumOVA in the same popliteal draining LN. CD45.1⁺ OTI cells plus CD45.1⁺ OTII cells, were CFSE-labeled and co-injected into congenic CD45.2⁺ C57BL6 mice. One day later they were immunized with alumOVA in both footpads. (A) Representative flow cytometric dot plots at various times post-immunization. The plots in the left hand column show analysis of the total LN cells with a gate round the CFSE⁺ CD45.1⁺ donor cells. This donor cell gate is resolved in next column of plots into CD8α⁺ OTI cells and CD8α⁻ OTII cells. In the next three paired columns of dot plots CD69, CXCR5, and OX40 expression in relation to cell division assessed by CFSE dilution is shown for OTI cells (left) and OTII cells (right). (B) Graphs show the percentage OTI and OTII cells expressing CXCR5 or OX40 at different times after immunization. Each symbol represents OTI or OTII cells from the two popliteal LN of 1 mouse. Data are derived from two independent experiments for days 0-4 and three independent experiments for day 7. (C) PD-1 expression in OTI and OTII cells 7 days after immunization assessed by flow cytometry in six mice in two independent experiments.