Functional plasticity of an antigen-specific memory CD4 T cell population

Abstract

The protective nature of memory immune responses is attributed largely to terminally differentiated memory T cells that retain memory of the antigen via the antigen receptor and memory of the effector functions that initially cleared the pathogen. It is not known whether a given population of antigen-specific memory T cells is endowed with functional flexibility to provide protective responses against antigens reencountered in different immunological contexts. Here, we examine functional properties of influenza hemagglutinin (HA)-specific memory CD4 T cells recovered from adoptive hosts that received in vitro-activated HA-specific T cell receptor-transgenic CD4 T cells 2 months to 1 year previously. We demonstrate that this HA-specific memory CD4 T cell population bearing a clonal T cell receptor can produce predominantly T helper 1 or T helper 2 effector cytokines depending on the nature of the recall stimulus. Our findings reveal remarkable functional plasticity within an antigen-specific memory T cell population and have direct implications for modulating memory T cell function in vaccine design and treatments for autoimmune diseases.

Figure 1

Phenotype and cytokine profile of HA-TCR effector CD4 T cells. (A) CD25 expression profile (Left) and size (forward scatter, Right) of 6.5⁺ HA-TCR naive and effector CD4 T cells. (B) Production of IFN-γ and IL-4 during generation of HA-TCR effector CD4 T cells. Intracellular cytokine analysis of IFN-γ (Upper Left) and IL-4 production (Upper Right) by 6.5⁺ HA-TCR effector CD4 T cells during their generation in vitro is shown. Monensin was added 2–3 days after activation of HA-TCR CD4 T cells with HA peptide and APC. IFN-γ versus IL-4 production is gated on 6.5⁺ cells (Lower).

Figure 2

Functional responses of differentially stimulated HA-TCR memory CD4 T cells. HA-TCR memory CD4 T cells were recovered 10 weeks and 1 year posttransfer of HA-TCR effector cells into RAG2^−/− adoptive hosts. For controls, naive HA-TCR CD4 T cells were transferred into RAG2^−/− hosts in parallel. Memory and naive CD4 T cells were restimulated with 1 μg/ml HA peptide (black bars) or 1 μg/ml anti-CD3 antibody (gray bars) in the presence of mitomycin C-treated APC. Freshly purified HA-TCR CD4 T cells (marked Fresh Tg or FTg) were cultured in parallel. IFN-γ and IL-4 content in 48-h culture supernatants was measured by specific ELISA, and proliferation was assessed by measuring [³H]thymidine incorporation after 72 h (see Materials and Methods). These results are representative of five different experiments.

Figure 3

Cytokine production from HA-TCR memory CD4 T cells in response to increasing antigen doses. Memory CD4 T cells were isolated from adoptive hosts 6 months posttransfer and activated with the indicated doses of HA peptide or 1 μg/ml anti-CD3 antibody in the presence of APC. IFN-γ and IL-4 content in culture supernatants was quantitated by specific ELISA as described for Fig. 2.

Figure 4

ICS analysis of differentially stimulated naive and memory HA-TCR CD4 T cells. HA-TCR memory CD4 T cells isolated from adoptive hosts 3 months previously were reactivated with HA peptide (antigen) or anti-CD3 in the presence of APC followed by incubation with monensin, surface staining for 6.5 and CD25, and intracellular staining for IFN-γ, IL-4, and IL-2. (A) Production of IFN-γ from 6.5 TCR-expressing and CD25⁺ memory T cells gated on large, activated cells. Unactivated T cells showed no IFN-γ production, and quadrants are designated based on staining with isotype-matched control antibodies for each cytokine. Numbers in quadrants refer to the percentage of total activated cells. (B) Production of IL-4 from 6.5⁺ and CD25⁺ HA-TCR memory CD4 T cells. The numbers in parentheses refer to the absolute number of cells in each quadrant. Activation of naive HA-TCR CD4 T cells yielded negligible numbers of IL-4-producing cells (data not shown). (C) IL-4⁺ and IFN-γ⁺ memory T cells shown together. The numbers in parentheses indicates absolute cell number. (D) IL-2 and IFN-γ production from 6.5⁺ activated naive and memory CD4 T cells. The numbers in parentheses refer to the absolute numbers of 6.5⁺ T cells, and data shown are gated on 6.5⁺ CD4⁺ T cells.