Increased competition for antigen during priming negatively impacts the generation of memory CD4 T cells

Abstract

The factors involved in the differentiation of memory CD4 T cells from naïve precursors are poorly understood. We developed a system to examine the effect of increased competition for antigen by CD4 T cells on the generation of memory in response to infection with a recombinant vesicular stomatitis virus. Competition was initially regulated by increasing the precursor frequency of adoptively transferred naïve T cell antigen receptor transgenic CD4 T cells. Despite robust proliferation at high precursor frequencies, memory CD4 T cells did not develop, whereas decreasing the input number of naïve CD4 T cells promoted memory development after infection. The lack of memory development was linked to reduced blastogenesis and poor effector cell induction, but not to initial recruitment or proliferation of antigen-specific CD4 T cells. To prove that availability of antigen alone could regulate memory CD4 T cell development, we used treatment with an mAb specific for the epitope recognized by the transferred CD4 T cells. At high doses, this mAb effectively inhibited the antigen-specific CD4 T cell response. However, at a very low dose of mAb, primary CD4 T cell expansion was unaffected, although memory development was dramatically reduced. Moreover, the induction of effector function was concomitantly inhibited. Thus, competition for antigen during CD4 T cell priming is a major contributing factor to the development of the memory CD4 T cell pool.

Fig. 1.

CD4 T cell precursor frequency affects memory T cell development in response to virus infection. CD45.2 TEa CD4 T cells (1 × 10⁶, 1 × 10⁵, or 1 × 10⁴ cells) were transferred into CD45.1 mice before infection with VSV-SED. (A) The TEa response was assessed at days 7, 16, and 30 after infection. (The asterisk indicates that mice from the 1 × 10⁶ transfers were bled at day 28 after infection.) Numbers indicate the percentage of donor CD4 T cells (CD45.2⁺) relative to the total CD4 population in the blood. (B) Comparison of the percentage of donor cells within the entire CD4 population in response to VSV-SED when either 1 × 10⁶ or 1 × 10⁵ TEa cells were initially transferred. Days 3, 4, and 5 after infection represent the lymphocyte population from the spleen, whereas days 7, 16, and 30 represent donor CD4 T cells from the blood. (C) The presence of TEa memory cells was determined in the indicated tissues 58 days after infection. (D) The production of IL-2 and IFN-γ by TEa cells (1 × 10⁵ cells transferred) isolated from the spleen 56 days after infection was determined by intracellular cytokine staining after in vitro stimulation with the Eα peptide. The values in the upper right quadrants represent the percentages of positive CD4 cells.

Fig. 2.

Effect of initial precursor frequency on early activation phenotype and IL7R expression during the primary response. (A) Either 1 × 10⁶ (solid line) or 1 × 10⁵ (dashed line) CD45.2 TEa cells were transferred to CD45.1 mice that were infected 1 day later with VSV-SED. Cells were isolated 24 h later from spleen (Upper) and peripheral lymph nodes (LNs) (Lower), and donor cells were analyzed for expression of the indicated proteins. Shaded histograms represent naïve transferred TEa cells. (B) Either 1 × 10⁶ (solid line) or 1 × 10⁵ (dashed line) CD45.2 TEa cells were transferred as above. Mice were infected with 1 × 10⁵ pfu of VSV-SED, and spleens were harvested on days 3, 4, 5, and 10 after infection. CD4⁺ CD45.2⁺ TEa cells were analyzed for IL-7Rα expression by flow cytometry. Shaded histograms represent naïve TEa cells.

Fig. 3.

Increased precursor frequency limits blastogenesis but not early division in response to infection. Naïve CD45.2 TEa cells were CFSE-labeled and transferred into CD45.1 mice that were then infected with VSV-SED 1 day later. Spleen cells were analyzed for the presence of CD45.2 TEa cells on the indicated days, and donor cells were analyzed for CFSE dilution (A) and forward light scatter (FSC) properties (B) by flow cytometry. Solid line, 1 × 10⁶ TEa cells transferred; dashed line, 1 × 10⁵ TEa cells transferred. Shaded histograms represent naïve CFSE-labeled TEa cells.

Fig. 4.

Naïve CD4 T cell precursor frequency modulates induction of effector function. Splenocytes from mice receiving 1 × 10⁶ or 1 × 10⁵ TEa cells were analyzed 5 days after VSV-SED infection for the production of IL-2 and IFN-γ by intracellular cytokine staining after Eα peptide stimulation in vitro. Numbers indicate the percentage of TEa cells producing IL-2 (Left) or IFN-γ (Right).

Fig. 5.

IL-2 production early after infection is regulated by CD4 T cell precursor frequency. The indicated number of CD45.2 TEa cells was transferred to CD45.1 mice that were infected 24 h later with VSV-SED. The mice were injected i.v. with 250 μg of BFA 18 h later. TEa cells in the spleen were analyzed 6 h later by intracellular cytokine staining directly ex vivo. Numbers represent the percentage of TEa CD4 T cells producing IL-2 (Left) or IFN-γ (Right).

Fig. 6.

Y-Ae mAb limits CD4 T cell expansion in vivo in a dose-dependent and antigen-specific manner. (A Upper) Mice that received 1 × 10⁵ TEa cells were treated with 100 μg of control mouse IgG2b or 1, 10, or 100 μg of the Y-Ae mAb immediately before VSV-SED infection. Donor T cell expansion in the spleen was assessed 6 days after infection. Numbers indicate the percentage of donor T cells of the total CD4 population. (Lower) Graphical representation of the flow cytometry data shown in Upper. Error bars indicate the standard error. (B) Y-Ae specifically blocks antigen presentation to Eα-specific CD4 T cells. TEa cells (1 × 10⁵) and OT-II CD4 T cells (1 × 10⁵) were cotransferred into CD45.1 C57BL/6 mice. Mice were then treated with 250 μg of the Y-Ae or control mouse IgG2b Ab before coinfection with VSV-SED and VSV-OVA (1 × 10⁵ pfu). At day 6 after infection, expansion of TEa and OT-II cells was assessed via flow cytometry. Differentiation of TEa (CD45.2⁺ Vα2⁺Vβ6⁺) from OT-II (CD45.2⁺ Vα2⁺ Vβ6⁻) cells was attained by differential expression of the Vα and Vβ TCR chains.

Fig. 7.

Y-Ae treatment at the time of infection inhibits generation of memory TEa cells. (A) Recipient mice that received 1 × 10⁵ TEa cells were treated i.p. with 1 μg of Y-Ae or 1 μg of mouse IgG2b before VSV-SED infection. Cells from the spleen and lung were analyzed for the presence of TEa cells at day 5 (primary) or day 23–24 (memory) after infection. Numbers indicate the percentage of the donor TEa population of the total CD4 population (values represent the mean of three mice for primary and eight mice for memory experiments). (B) Graphical representation of data presented in A. *, P < 0.05. (C) Cytokine production by TEa cells in Y-Ae-treated mice. TEa T cells (1 × 10⁵) were transferred into congenic recipients. Before infection with VSV-SED, mice were treated with 1–2 μg of Y-Ae mAb (n = 6) or IgG2b control Ab (n = 3). TEa cells were analyzed 5 days later for intracellular cytokine production after in vitro peptide stimulation.