IL-7 regulates basal homeostatic proliferation of antiviral CD4+T cell memory

Abstract

Heightened protection from infectious disease as conferred by vaccination or pathogen exposure relies on the effective generation and preservation of specific immunological memory. T cells are irreducibly required for the control of most viral infections, and maintenance of CD8(+)T cell memory is regulated by at least two cytokines, IL-7 and IL-15, which support survival (IL-7, IL-15) and basal homeostatic proliferation (IL-15) of specific CD8(+) memory T cells (T(M)). In contrast, the factors governing the homeostasis of pathogen-specific CD4(+)T(M) remain at present unknown. Here, we used a physiologic in vivo model system for viral infection to delineate homeostatic features and mechanisms of antiviral CD4(+)T(M) preservation in direct juxtaposition to CD8(+)T cell memory. Basal homeostatic proliferation is comparable between specific CD4(+) and CD8(+)T(M) and independent of immunodominant determinants and functional avidities but regulated in a tissue-specific fashion. IL-7, identified as the dominant cytokine, and IL-15, an accessory cytokine, regulate basal homeostatic proliferation and survival of antiviral CD4(+)T(M). Interestingly, a role for these cytokines in regulation of CD4(+)T cell memory is not readily discernible in the generic "memory-phenotype" population, apparently a consequence of its heterogeneous composition. We also describe a prominent, nonredundant role for IL-7 in supporting basal homeostatic proliferation of CD8(+)T(M). We propose that homeostatic control of antiviral CD4(+) and CD8(+) T cell memory is fundamentally similar and characterized by quantitative, rather than qualitative, differences.

Fig. 1.

Phenotypic and functional profiling of CD4⁺ and CD8⁺T_M.(A) Spleen cells from LCMV-immune mice (7–9 weeks after challenge) were restimulated for 5 h with the dominant MHC-I-restricted (GP₃₃) or MHC-II-restricted (GP₆₁) LCMV epitopes and stained for intracellular IFN-γ in combination with indicated surface or intracellular markers. (Upper) CD8⁺T cell gates. (Lower) CD4⁺T cell gates. Boxed numbers indicate frequencies of GP₃₃-specifc CD8⁺ and GP₆₁-specific CD4⁺T_M; numbers in upper right quadrants are percentages of all epitope-specific (IFN-γ⁺) T cells coexpressing indicated additional markers. Values are means of 6–10 mice analyzed. TNFα, tumor necrosis factor α. GM-CSF, granulocyte/macrophage colony-stimulating factor. (B) Cytokine receptor expression by splenic T_M. Black traces indicate staining for cytokine receptors; gray histograms are isotype control stains or polyclonal goat Ig stains (IL-15Rα histograms). (C) CD127/IL-7Rα, CD122/IL-2Rβ, and IL-15Rα expression by virus-specific CD8⁺ (black) and CD4⁺ (gray) T_M in different organs. Cytokine receptor expression levels were normalized by dividing the geometric mean of fluorescence intensity (GMFI) of experimental stains by that of the isotype control stains. CD127 expression by specific CD4⁺T_M was significantly higher as compared with specific CD8⁺T_M in spleen (P = 0.0367) and lower in the MLN (P = 0.0011). Comparative CD122 expression by specific CD4⁺T_M was reduced in all organs (P < 0.006), as was IL-15Rα expression (P < 0.02), with the exception of the peritoneal cavity.

Fig. 2.

Delineating basal homeostatic proliferation of CD4⁺ and CD8⁺T_M.(A) Basal homeostatic proliferation of T_M specific for MHC-I-restricted (GP₃₃, NP₃₉₆, GP₂₇₆, GP₁₁₈, NP₂₀₅, and GP₉₂) and MHC-II-restricted (GP₆₁ and NP₃₀₉) LCMV epitopes was evaluated ≈7 weeks after infection and 7-d BrdUrd administration before combined IFN-γ/BrdUrd staining. Bars (SEM) indicate the fraction of BrdUrd⁺T_M among respective epitope-specific populations that are displayed in order of immunodominance. Experiment (n = three mice per group) was performed twice with similar results. (B) Histograms gated on GP₃₃-specific CD8⁺T_M (Upper) and GP₆₁-specific CD4⁺T_M (Lower) show percentage of BrdUrd incorporation (7-d pulse) by T_M recovered from different tissues. (C) Summary of basal homeostatic proliferation (7-d BrdUrd pulse) as a function of anatomic location. Data (SEM; n = three mice per group) from one of three similar experiments. Statistical analyses for proliferative turnover in reference to spleen were performed by Student's t test; P values are indicated. (D) Ki-67 expression by virus-specific T_M ≈10 weeks after LCMV challenge in spleen (black) and peritoneal cavity (gray). Shown are the combined data from two of four separate experiments (n = three mice per group; differences between Ki-67⁺ splenic and peritoneal T_M, P < 0.001).

Fig. 3.

Cytokine-driven proliferation of T_M. (A) Memory mice (6–9 weeks after infection) were injected with PBS (control) or 2 μg of recombinant murine IL-2, IL-7, or IL-15 and pulsed with BrdUrd for 3 d. Plots are gated on CD8⁺ (Upper)orCD4⁺ (Lower) T cells. Virus-specific T_M are identified by IFN-γ stains. Additional controls included mice injected with mIL-17, which displayed BrdUrd uptake identical to PBS control mice. Boxed numbers indicate frequencies of GP₃₃-specifc CD8⁺ and GP₆₁-specific CD4⁺T_M. Bar diagrams display absolute numbers of specific T_M in spleen. Values indicate the factor by which cytokine administration increased specific T_M numbers. No significant changes were noted after IL-2 administration. (B) Cytokine-driven proliferation of memory-phenotype (CD44^hi) and specific T_M as induced by i.v. administration of 2 μg of IL-2, IL-7, or IL-15. P values were calculated in relation to PBS-injected control mice. (C) The “factor increased turnover” after cytokine injection was calculated only for specific and memory-phenotype (CD44^hi)T_M populations that demonstrated significantly altered proliferation and is compared to proliferation in control mice (PBS injected, given a value of 1). Data in B and C are combined from four separate experiments evaluating three to nine mice total.

Fig. 4.

Regulation of T_M proliferation by IL-7 and IL-15. (A) Enumeration of GP₃₃-specific CD8⁺ and GP₆₁-specific CD4⁺T_M (≈9 weeks after LCMV) in spleens of control mice, mice receiving four separate i.v. injections of 450 μg of IL-7Rα-blocking antibody over a period of 7 d, and IL-15^-/- mice. Shown are the representative data from one of four similar experiments, with error bars indicating SEM (n = three mice per group per experiment). (B) Basal homeostatic proliferation of GP₃₃-specific CD8⁺ and GP₆₁-specific CD4⁺T_M in spleen, MLN, peritoneal cavity, and bone marrow in control mice and under conditions of IL-7Rα-blockade (4 × 450 μg of IL-7Rα antibody i.v.) or IL-15-deficiency (7-d BrdUrd pulse). To directly compare the relative impact of receptor blockade or cytokine deficiency, proliferation of respective T_M populations in control mice (B6) was set to 100%; P values were calculated in comparison to respective T_M populations in B6 control mice. Data are from one of two experiments (n = three mice per group); similar results were obtained by employing 3 × 300 μg of IL-7Rα antibody in two additional experiments. (C) Proliferation of GP₃₃-specific CD8⁺ and GP₆₁-specific CD4⁺T_M under conditions of IL-7Rα-blockade (4 × 450 μg of IL-7Rα antibody i.v.) and/or IL-15-deficiency (7-d BrdUrd pulse). Values indicate the fraction of BrdUrd⁺ GP₃₃- or GP₆₁-specific T_M. (D) Proliferation of GP₃₃-specific CD8⁺ (Upper) and GP₆₁-specific CD4⁺ (Lower)T_M after treatment of B6 and B6.IL-15^-/- mice (≈12 weeks after LCMV) with 2 μg of mIL-7 (3-d BrdUrd pulse). Consistent with the data displayed in B, proliferation of specific CD8⁺ but not CD4⁺T_M was reduced in PBS-injected B6.IL-15^-/- as compared with B6 control mice (data not shown).

Fig. 5.

Regulation of T_M survival by IL-7 and IL-15. (A) Bcl-2 expression by specific CD8⁺ and CD4⁺T_M in peripheral blood as a function of time after virus challenge. Bcl-2 expression was normalized as indicated in the legend to Fig. 1C. (B) Up-regulation of Bcl-2 and Bcl-x_L expression by specific CD8⁺ and CD4⁺T_M (≈9 weeks after LCMV challenge) after 24 h in vitro treatment with 100 ng/ml mIL-7. Black traces represent data for mIL-7, and gray histograms represent data for PBS. (C) Summary of in vitro cytokine-induced Bcl-2 and Bcl-x_L up-regulation by specific T_M (100 ng/ml, 24-h stimulation); P values were calculated in comparison to respective T_M populations in PBS-treated control cultures. (D) Ex vivo Bcl-2 expression by virus-specific T_M 7 weeks after LCMV infection and under conditions of IL-7Rα-blockade (4 × 450 μg of antibody i.v. over 7 d) and/or IL-15-deficiency. Significant differences in Bcl-2 expression were also observed after administration of 4 × 300 μg of antibody (data not shown). Experiments (n = three mice per group) were performed four times; P values were calculated in comparison to respective T_M populations from PBS-injected or uninjected control mice (B6).