Physiological numbers of CD4+ T cells generate weak recall responses following influenza virus challenge

Abstract

Naive and recall CD4(+) T cell responses were probed with recombinant influenza A viruses incorporating the OVA OT-II peptide. The extent of OT-II-specific CD4(+) T cell expansion was greater following primary exposure, with secondary challenge achieving no significant increase in numbers, despite higher precursor frequencies. Adoptive transfer experiments with OT-II TCR-transgenic T cells established that the predominant memory set is CD62L(hi), whereas the CD62L(lo) precursors make little contribution to the recall response. Unlike the situation described by other investigators, in which the transfer of very large numbers of in vitro-activated CD4 effectors can modify the disease process, providing CD62L(hi) or CD62L(lo) OT-II-specific T cells at physiological levels neither enhanced virus clearance nor altered clinical progression. Some confounding effects of the transgenic model were observed, with decreasing primary expansion efficiency correlating with greater numbers of transferred cells. This was associated with increased levels of mRNA for the proapoptotic molecule Bim in cells recovered following high-dose transfer. However, even with very low numbers of transferred cells, memory T cells did not expand significantly following secondary challenge. A similar result was recorded in mice primed and boosted to respond to an endogenous IA(b)-restricted epitope derived from the influenza virus hemagglutinin glycoprotein. Depletion of CD8(+) T cells during secondary challenge generated an increased accumulation of OT-II-specific T cells but only at the site of infection. Taken together, significant expansion was not a feature of these secondary influenza-specific CD4 T cell responses and the recall of memory did not enhance recovery.

Figure 1

Primary and secondary expansion of transgenic OT-IIT cells. (A) 6×10⁵ OT-IIT cells were transferred i.v. into wt B6 mice, which were challenged i.n. soon after with H3wt (white) or H3ova (black) and the spleens harvested after a further 10d. The values show the relative expansion of the OT-IIT cells (comparing H3ova vs. H3wt infected). Cell number was determined by FACS analysis of Thy1.1⁺CD4⁺ cells. (B) Shows the % total CD4+ (left) and CD4⁺OT-IIT (right) cells in the BAL after primary or secondary infection. (C) 6×10⁵ OT-IIT cells were given i.v. to wt B6 mice, which were infected i.p. with H1wt or H1ova and rested for 50d, when these “memory” mice were harvested for analysis. Identically primed mice were challenged i.n. with either H3wt or H3ova and the spleen 8d later. (D) Representative FACS plots of OT-IIT cells (right panels) in the BAL following primary (upper) or secondary (lower) challenge. Control BAL samples (left panels) from B6 mice infected with wt viruses are shown for comparison. Each panel is representative of two independent experiment with 5 mice per group. The * indicates p<0.05 by ANOVA (Kruskal-Wallis, Tukey's post-test).

Figure 2

CD62L phenotype correlates with proliferative capacity but not protection. The experimental protocol is illustrated in (A). 1×10⁵ CD44^hiCD62L^hi and CD44^hiCD62L^lo OT-IIT cells were sorted from the spleens of H1ova i.n. primed mice 30d after infection and transferred into naïve recipients. Other mice were given in equivalent numbers of naïve OT-IIT cells. Representative plots of sorted cells are displayed in (B), with the CD4⁺Thy1.1⁺CD8⁻MHC Class II⁻ cells in gated in from the left panel displayed in the right panel stained for CD44 and CD62L (sorting on hi and lo cells). All groups were then challenged i.n. with either a potentially lethal (CD) or sublethal (EF) dose of H1ova (10⁴ EID₅₀) or H3ova (10⁶ EID₅₀) , respectively . The OT-IIT cell counts in the spleen (C,E) for each cell population show greater expansion for the naïve and antigen-experience CD62L^hi vs CD62L^lo phenotype. (D,F) Relative weight loss (initial weight normalized to 1) after infection of the different groups of mice. Each panel is representative of at least two independent experiments with 4–5 mice per group. The * indicates p<0.05 by ANOVA (Kruskal-Wallis, Tukey's post-test), compared to naïve transfer.

Figure 3

Increasing transfer numbers decreases per cell proliferative capacity. (A) 10⁵, 10⁴, or 10³ naïve OT-IIT cells were transferred into naïve mice. Mice were primed with H1ova, rested for thirty days and either analyzed for splenic OT-IIT memory responses (memory) or challenged with H3ova (secondary) and analyzed eight days later for splenic OT-IIT expansion. (B) Time course of OT-IIT expansion following secondary infection. 10³ naive OT-IIT cells were transferred into naive mice, which were subsequently primed, rested and challenged as in (A). OT-IIT cell were enumerated in the spleen, BAL and MLN on the indicated days after infection. (C) Bim expression is increased in memory OT-IIT cells from high dose transfer. Relative quantitation (ddCT) of Bim levels in d30 memory splenic OT-IIT cells compared to splenic cells isolated on d5 following primary infection, with data normalized to internal controls. Data represent averages of at least three mice per group and are representative of two independent experiments. The * indicates p<0.05 by ANOVA (Kruskal-Wallis, Tukey's post-test).

Figure 4

CD8 depletion enhances OT-IIT accumulation. 10³ (A) or 10⁵ (B) OT-IIT cells were transferred into naive mice, which were primed (H1ova), rested, and challenged (H3ova) as in Figure 3. Three days prior to secondary challenge, mice were inject interperitoneally with either an anti-CD8 monoclonal antibody(2.43) or vehicle control. Injections continued on alternating days thereafter. On day 8 after infection, OT-IIT cells were enumerated from the spleen, BAL and MLN. Data represent averages of 3–4 mice/group and two independent experiments. The * indicates p<0.05 by Student's t-test between control and depleted animals.

Figure 5

Limited clonal expansion by endogenous OT-IIe specific cells in wt mice. (a) The wt B6 mice were infected i.n. with H3ova (primary) or primed i.p. with H1ova, rested for at least 30d, then challenged i.n. with H3ova (secondary). Spleens were harvested on day 10 (primary) or day 8 (secondary) and assayed by ELISPOT as described in the Methods. PA and PB1 refer to the D^bPA₂₂₄, and K^bPB1₇₀₃ CD8⁺ T cell responses, respectively. (b) The wt B6 mice were primed i.p. with H1wt or H1sly, rested for at least 30d, then challenged i.n. with H3wt (which contains the SLY1e). Spleens were harvested on day 8 and assayed by ELISPOT as described in the Methods. Data are representative of at least two independent experiments. The * indicates p<0.05 by ANOVA (Kruskal-Wallis, Tukey's post-test).