Memory-like CD8+ T cells generated during homeostatic proliferation defer to antigen-experienced memory cells

Abstract

Naive T cells proliferate in response to lymphopenia and acquire the phenotypic and functional qualities of memory T cells, providing enhanced protection against infection. How well memory-like T cells generated during lymphopenia-induced homeostatic proliferation (HP)-memory differentiate into secondary memory cells and compete with Ag-experienced true-memory cells is unknown. We found that CD8(+) HP-memory T cells generated robust responses upon infection and produced a secondary memory population comparable to true-memory cells in the absence of competition. However, when true-memory and HP-memory T cells competed during infection, HP-memory cells contributed less to the effector population, contracted earlier, and formed fewer secondary memory cells. Furthermore, HP- and true-memory cells demonstrated distinct chemokine receptor expression and localization within the spleen during infection, indicating differential access to signals necessary for secondary memory formation. Thus, HP-memory T cells provide protection without compromising the true-memory population. Differences in HP- and true-memory T cells may reveal the basis of competition for limited resources within the memory-T cell compartment.

FIGURE 1

True-memory CD8⁺ T cells outcompete HP-memory cells during secondary infection. A, Generation of memory T cell subsets and experimental design. Immune response of the donor memory subsets was measured as a percentage of the total CD8⁺ cells in the indicated tissue. B, Co-transfer: HP- and true-memory, PBL. C, Single transfer: HP- and true-memory, PBL. D, Co-transfer: HP- and true-memory, spleen. E, Single transfer: HP- and true-memory, spleen. F, Total cell numbers recovered from spleen for indicated transfer conditions. Co-transfer: naive OT-I and HP-memory, PBL. G, Single transfer: naive OT-I and HP-memory, PBL. H, Co-transfer: naive and HP-memory. Representative of >3 experiments (n = 3). Error bars indicate SD. I, Co-transfer: naive and true-memory. Mem, memory; transf, transfer.

FIGURE 2

Defect in HP-memory cell accumulation is not due to peripheral localization. The ratios of co-transferred memory subsets were monitored in the indicated tissues after Lm.OVA infection. A, Relative accumulation of the donor (CD45.1⁺ CD8⁺) subsets in the spleen (SPL), lymph nodes (LN), bone marrow (BM), liver, lung, and gut following infection. Memory subsets in the spleen and lymph nodes after infection: (B) percent of each donor memory subset (HP, solid line; true-memory, dotted line), (C) CD62L expression on co-transferred memory subsets (HP, gray; true-memory, white). D, Average total cell numbers from pooled spleen and all lymph nodes (HP, black; true-memory, white). E, Total cell numbers for donor HP- or true-memory cells from the spleen (left) and all recovered lymph nodes (right) following treatment with anti-CD62L or PBS (similar results obtained with isotype control). Host naive CD8⁺ cell numbers indicated in separate graphs (right). Representative of >3 experiments (n = 3). Error bars indicate SD. Mem, memory; Inf, infection; d, day; H, HP; M, true-memory.

FIGURE 3

Localization of HP- and true-memory cells relative to CD11c⁺ cells. Confocal images of serial spleen sections from co-transfer recipients (day 2 of infection). Sections were stained for CD11c, B220, or CD4, and congenic markers for HP-memory (A), true-memory (B), and HP- and true-memory (C). D, Spleen (day 5) was stained for CD11c, both memory subsets, and B220 (10× magnification). Images are taken at 20× magnification unless noted. Representative of three experiments (n = 2–3) per time point.

FIGURE 4

HP- and true-memory cells display distinct localization and clustering in the spleen. A, Spleen sections of co-transfer recipients on days 5, 6, and 10. Stained for both memory subsets and B220. B, Panels with the indicated stains and merged image of spleen (day 5). Images are taken at 10× magnification. Representative of three experiments (n = 3) per time point.

FIGURE 5

mRNA levels of chemokines and chemokine receptors by HP- and true-memory cells. cDNA was generated from co-transferred HP- and true-memory cells sorted from pooled spleen (day 6). Relative (Rel.) mRNA levels for indicated genes were determined with a qPCR array and normalized to GAPDH. A, The transcripts displaying a 2-fold or greater difference in expression were listed and further divided into receptors (left) and chemokines (right). B, qPCR verification of CXCR5 (left) and CCR7 (right) mRNA levels. mRNA from total spleen used as a reference. Representative of two co-transfers tested >3 independent times.

FIGURE 6

The role of Ag and cytokines in the defective competition by HP-memory cells. A, Percentage of donor cells among total CD8⁺ cells, PBL: OVAp, and LPS treated. B, Ratio of the percent of true-memory to HP-memory cells in spleen on day 10 of infection, recipients treated with PBS, IL-7/anti-IL-7 mAb, or IL-15/IL-15Rα complexes. C, Total cell number of each memory subset (day 10 Lm.OVA) treated with PBS (D), IL-7/anti-IL-7 mAb (E), and IL-15/IL-15Rα complexes (F). Representative of at least three experiments (n = 3). Error bars represent SD.

FIGURE 7

True-memory cells accumulate and show increased basal homeostatic proliferation compared with HP-memory cells in the absence of infection. HP and true-memory subsets were monitored after transfer into naive B6 hosts without infection. A, Percentage of donor cells in PBL after co-transfer. B, CFSE detection of the co-transferred memory cells in the spleen and lymph nodes at 120 days post transfer. Percentage of the indicated subset among total CD8⁺ T cells: (C) co-transfer, HP-memory; (D) co-transfer, true-memory; (E) single transfer, HP-memory; and (F) Single transfer, true-memory. Representative of two experiments (n = 5). Error bars indicate SD.