Antigen-free adjuvant assists late effector CD4 T cells to transit to memory in lymphopenic hosts

Abstract

The events controlling the transition of T cells from effector to memory remain largely undefined. Many models have been put forth to account for the origin of memory precursors, but for CD4 T cells initial studies reported that memory T cells derive from IFN-γ-nonproducing effectors, whereas others suggested that memory emanates from highly activated IFN-γ-producing effectors. In this study, using cell proliferation, expression of activation markers, and production of IFN-γ as a measure of activation, we defined two types of effector CD4 T cells and investigated memory generation. The moderately activated early effectors readily transit to memory, whereas the highly activated late effectors, regardless of their IFN-γ production, develop minimal memory. Boosting with Ag-free adjuvant, however, rescues late effectors from cell death and sustains both survival and IFN-γ cytokine responses in lymphopenic hosts. The adjuvant-mediated memory transition of late effectors involves the function of TLRs, most notably TLR9. These findings uncover the mechanism by which late effector CD4 T cells are driven to transit to memory and suggest that timely boosts with adjuvant may enhance vaccine efficacy.

Figure 1. The effector phase includes moderately activated early and highly activated late cells that develop different recall IFNγ responses

CFSE-labeled naïve DO11.10 T cells were transferred into BALB/c mice and the hosts were immunized with OVAp in CFA. Three days later the LN cells were harvested and the number of cell divisions, the frequency of cytokine responders and expression of activation markers were analyzed. (A) shows the effector divisions as measured by CFSE dilution and presented in dot plot and histogram format. (B) shows intracellular and surface staining of cell divisions with isotype controls (left panel) and anti-IFNγ and anti-CD62L antibodies, respectively (right panel). (C) shows percent of CD4 T cells producing cytokines and MFI for the indicated markers compared to isotype control. (D) The LN cells in each effector division were sorted, transferred into naïve BALB/c mice (5 × 10⁴ cells/mouse) and the hosts were immunized with a suboptimal dose of OVAp in CFA. The mice were sacrificed at day 4 and 6 and the LN cells were stimulated with 10 μM OVAp and IFNγ responses were measured by ELISA. Immunized mice with no transfer of effector T cells were used as baseline control for IFNγ responses. The top plot shows the mean of IFNγ response of each division at day 4 and 6. Each point represents the mean ± SE. The bottom plot shows a summary of data comparing the mean ± SE memory IFNγ response of early (D2, D4 and D5) and late (D6, D7 and D8) divisions at the peak of their responses. ^***p<0.005. The results are representative of 4 independent experiments.

Figure 2. Transition to memory is more effective with early compared to late effectors

The BALB/c LN effectors described in Figure 1 were sorted from each division and transferred into Rag2^−/− mice for parking. Four months later, the hosts were divided into two groups, one of which was sacrificed for analysis of splenic memory precursor responses (A) and the other group was immunized with OVAp/CFA and three days after immunization the SP and LN cells were isolated and their IFNγ responses were analyzed (B and C). (A) The precursor cells were stimulated with graded amounts of OVAp (diamond) and IFNγ responses were measured by ELISA. Stimulation with HA peptide (10μM) was included in all experiments for control purposes (squares). (B) LN cells were stimulated with 10μM OVAp or HA peptide and IFNγ responses were measured by ELISA. (C) shows summary bar graph of data for SP and LN comparing the mean memory IFNγ response of early (D2, D4 and D5) and late (D6, D7 and D8) effector at 10 μM. (A-C) Each point represents the mean of at least three independent experiments. *p< 0.05.

Figure 3. Late effectors display survival disadvantage relative to early effectors

The BALB/c LN effectors were sorted from each division and the early (D4-5) and late (D6-8) effectors were transferred into Rag2^−/− mice. Four months later, the SP cells were harvested and in (A) the percentage and absolute number of surviving CD4⁺KJ1-26⁺ cells were measured by flow cytometry and their IFNγ responses were evaluated by ELISA. In (B) unseparated LN cells were cultured for 42hrs in the presence of OVAp and then stained with Annexin V. The cells were then gated on CFSE and Annexin V binding was analyzed on each division. The bars show the mean percent of Annexin V⁺ cells for early (D4-5) and late (D6-8) effectors. (C) Prior to transfer, the CFSE-positive D2-8 effectors were sorted and stimulated with PMA and Ionomycin (P+I), labeled and analyzed for IFN-γ production (left panel) and IFNγ^hi and IFNγ^lo cells of D6-8 and D4-5 cells were separated and re-analyzed for IFNγ to confirm the purity of separation. In (D) hosts of the sorted IFNγ^hi and IFNγ^lo late effectors were sacrificed 6 weeks post transfer and the percentage, absolute number and IFNγ production by the CD4⁺KJ1-26⁺ splenic cells were measured as in (A) and compared with D4-5 early effectors. The graphs represent the mean of at least three independent experiments. * p< 0.05, **p< 0.01, ***p< 0.005.

Figure 4. Continuous contact with CFA sustains superior memory

(A) Naïve CFSE-labeled DO11.10 T cells were transferred into Rag2^−/− and BALB/c mice and the hosts were immunized with 125μg OVA/CFA. Three days later the mice were divided into two groups, the Rag2^−/− mice remained as is (the T cells continue contact with CFA) and the BALB/c mice were used to harvest the LN effectors which were re-transferred into Rag2^−/− mice for parking (no CFA). In this group the T cells are no longer exposed to CFA. Four months later, the splenic cells from both groups were harvested, stimulated with graded concentrations of OVA peptide (triangle) or 10 μM HA peptide (rectangles) as control, and IFNγ responses were measured by ELISA. (B) The LN from the CFA (gray bars) and no CFA (black bars) groups were harvested after 4 or 16 weeks parking and the cells were analyzed for IL-7R expression and apoptosis by 7-AAD using flow cytometry. (C) Naïve CFSE-labeled DO11.10 T cells were transferred into BALB/c mice and the hosts were immunized with 125μg OVA/CFA. Three days later the LN effectors were isolated and re-transferred into Rag2^−/− hosts. One day later, one group of mice was given CFA and the other was left without CFA. After 120 days of parking the spleen cells were harvested and the percent KJ1-26⁺ CD4⁺ T cells were estimated among total splenic cells. The graph represents the mean of four independent experiments. *p< 0.05, **p< 0.01, ***p< 0.005.

Figure 5. Late effector T cells require stimulation with adjuvant for survival and generation of effective memory

(A) Early (D4-5) and late (D6-8) effectors were isolated from BALB/c mice (as in Figure 1) and transferred into Rag2^−/− hosts. D2 and D3 were also sorted and transferred to Rag2^−/− mice to serve for control purposes. One day later, the mice were given antigen-free CFA and parked for 3 months. The percentage, absolute number and IFNγ production by the CD4⁺KJ1-26⁺ splenic cells were then measured by flow cytometry and ELISA, respectively. Each bar represents the mean of four independent experiments. *p< 0.05, **p< 0.01. (B) shows the summary bar graph of data in (A) comparing the mean percent of CD4⁺KJ1-26⁺ splenic cells and the mean memory IFNγ response of early (D4 and D5) and late (D6, D7 and D8) effectors.

Figure 6. TLR-4 and -9 ligands improve the IFNγ memory response of effector T cells

CFSE-positive total (D2-8) lymph node effector T cells were sorted and re-transferred into Rag2^−/− hosts. On day 1, 5, and 8 after cell transfer, TLR ligands were administered i.p. The spleen cells were harvested 5 weeks after the last TLR injection and the percentage and the cell number as well as IFNγ production by the surviving CD4⁺KJ1-26⁺ cells were measured by flow cytometry and ELISA, respectively. The cell number was relative to PBS (set at 1 ± 0.1 × 10⁴). Each bar represents the mean of nine independent experiments. *p< 0.05, **p< 0.01.

Figure 7. TLR9 ligand supports transition of late effectors into memory

(A) Early (D4-5) and late (D6-8) effectors were isolated from BALB/c mice (as in Figure 3) and transferred into Rag2^−/− hosts. On day 1, 4, 7 and 10 after cell transfer, the hosts received TLR2, TLR4 or TLR9 ligand (optimal regimen) and the percentage and IFNγ production by the CD4⁺KJ1-26⁺ cells were analyzed 3 weeks after the last TLR ligand injection. Each bar represents mean of four independent experiments. (B and C) The hosts were transferred with separated IFNγ^lo or IFNγ^hi cells from D6-8 effectors or unseparated D4-5 cells (as in Figure 3) and the hosts were given TLR9-ligand as in (A) and splenic cells were harvested 3 weeks after the last TLR injection. The graphs in (B) show the percentage, absolute number, and IFNγ production by the CD4⁺KJ1-26⁺ cells while in (C) the plots show intracellular IFNγ. Each bar represents the mean of at least three independent experiments. (D) Purified naïve, effector and memory DO11.10 T cells were cultured in the presence of media alone (open bars), media supplemented with TLR9-ligand (black bars), or media from APC culture stimulated with TLR9-ligand (gray bars). 72 hours later, live cells were counted using trypan blue exclusion. For these assays, LN effector T cells (D2-8) were isolated on the basis of CFSE while naïve T cells and memory precursors (from Rag2^−/− hosts recipient of D2-8 LN effectors) were sorted based on KJ1-26, anti-CD3 and anti-CD4 staining. Purified splenic APCs were cultured simultaneously but separately from T cells in the presence of TLR9-ligand (CpG-ODN 1886; 50 μg/ml). 12 hours after the initiation of culture, APC supernatant was transferred to T cell culture. Each bar represents the mean of at least four independent experiments. *p< 0.05, ** p< 0.01. The cell numbers were relative to culture without TLR ligands (set at 1 × 10³).

Figure 8. TLR9 mRNA is expressed by naïve and memory DO11.10 CD4⁺ T cells

Naïve, effector and memory DO11.10 T cells were obtained as described in Figure 7D and RNA was isolated by Trizol. TLR expression was measured by real time-PCR as described in Methods. Fold changes were relative to the effector group (set at 1). Each point represents the mean of five independent experiments with ± SEM. *p < 0.05, **p< 0.01.