The chemokine receptor CXCR6 is required for the maintenance of liver memory CD8⁺ T cells specific for infectious pathogens

Abstract

It is well established that immunization with attenuated malaria sporozoites induces CD8(+) T cells that eliminate parasite-infected hepatocytes. Liver memory CD8(+) T cells induced by immunization with parasites undergo a unique differentiation program and have enhanced expression of CXCR6. Following immunization with malaria parasites, CXCR6-deficient memory CD8(+) T cells recovered from the liver display altered cell-surface expression markers as compared to their wild-type counterparts, but they exhibit normal cytokine secretion and expression of cytotoxic mediators on a per-cell basis. Most importantly, CXCR6-deficient CD8(+) T cells migrate to the liver normally after immunization with Plasmodium sporozoites or vaccinia virus, but a few weeks later their numbers severely decrease in this organ, losing their capacity to inhibit malaria parasite development in the liver. These studies are the first to show that CXCR6 is critical for the development and maintenance of protective memory CD8(+) T cells in the liver.

Keywords: Plasmodium; chemokine receptor; liver; malaria; memory CD8+ T cells.

Figure 1.

Expression of chemokine receptors on antigen-specific memory CD8⁺ T cells after irradiated sporozoite immunization. A, Naive CD45.1⁺ OT-I were adoptively transferred to C57BL/6 recipient mice 1 day prior to immunization with γ-spz. Representative histograms showing the expression of CXCR3, CCR5, and CXCR6 gated on memory OT-I cells 1 month after immunization. B–D, Ccr5^−/− (B), Cxcr3^−/− (C), and Cxcr6^−/− (D) mice were immunized, and total numbers of tetramer⁺ cells were determined 1 month after immunization. E, Number of interferon γ–expressing cells determined by ex vivo stimulation with SIINFEKL peptide (10 μg/mL) in spleen and liver of immunized Cxcr6^+/− and Cxcr6^−/− mice. Data shown are results from 1 experiment, which are representative of 2 independent experiments (n = 3–4 per group). Error bars show mean ± standard error of the mean. **P < .01. Abbreviations: IFN-γ, interferon γ; NS, not significant; WT, wild type.

Figure 2.

Expression of CXCR6 on CD8⁺ T cells is critical for the establishment of a memory population in the liver. A, Schematics of the experimental design, of which naive CXCR6⁺ or CXCR6⁻ CD45.1⁺ OT-I cells were adoptively transferred to Cxcr6^+/− C57BL/6 recipients prior to immunization with γ-spz. B and C, Representative fluorescence-activated cell sorting plots gated on total lymphocytes (B) and total numbers of OT-I cells in spleen, lungs, and liver of mice receiving CXCR6⁺ OT-I or CXCR6-OT-I cells on day 25 after immunization (C). Data shown are results from 1 experiment, which are representative of 6 independent experiments with 3–4 mice per group. Data are mean ± standard error of the mean. **P < .01. Abbreviation: NS, not significant.

Figure 3.

CXCR6 is not required during priming, homing, or homeostatic proliferation of CD8⁺ T cells. A and B, Representative fluorescence-activated cell sorting plot gated on total lymphocytes and total numbers of OT-I cells recovered from spleen, lung, and liver of immunized mice on day 3 (A) and day 9 (B) after immunization. C, For the in vivo homing assay, equal number of effector CXCR6⁺ and CXCR6⁻ OT-I cells were adoptively transferred to naive wild-type recipients. Twenty hours after adoptive transfer, total numbers of donor cells in spleen and liver in recipient mice were recovered. D, To measure homeostatic proliferation, mice receiving CXCR6⁺ or CXCR6⁻ OT-I were immunized with γ-spz. One month later, mice were administered BrdU for 10 days. Percentages are of BrdU⁺ among memory OT-I in spleen and liver. The data are results from 1 experiment, which are representative of 2 (A, C, and D) and 4 (B) independent experiments with 3–4 mice per group. Data are mean ± standard error of the mean. Abbreviations: dLN, draining lymph node; NS, not significant; P. berghei, Plasmodium berghei.

Figure 4.

Functional and phenotypical characterization of CD8⁺ T cells in the absence of CXCR6. A, Cytokine production in memory CXCR6⁺ and CXCR6⁻ OT-I was evaluated after 4-hour ex vivo stimulation with cognate peptide. Data are pooled results of 2 independent experiments. B, Expression of CD62L, CD127, CD27, KLRG1, and CXCR3 among OT-I in spleen and liver on day 30 after immunization. Data shown are results from 1 experiment, which are representative of 2 independent experiments. C, Day 9 after immunization with γ-spz, the expression of BCL2 was evaluated among CXCR6⁺ or CXCR6⁻ effector OT-I cells, shown as representative histograms (Ci; left) and percentage of BCL2⁺ (Cii; right) among gated OT-I . Data shown are results from 1 experiment, which are representative of 2 independent experiments with 3–4 mice per group. Data are mean ± standard error of the mean. *P < .05. Abbreviations: IFN-γ, interferon γ; IL-2, interleukin 2; TNF-α, tumor necrosis factor α.

Figure 5.

Immunized mice harboring Cxcr6^−/− memory CD8⁺ T cells do not inhibit the development of Plasmodium liver stages. Total numbers of OT-I cells in spleen and liver in mice on day 25 after immunization with recombinant vaccinia virus (rVV). A, Naive Cxcr6^+/− or Cxcr6^−/− OT-I were adoptively transferred to recipient mice, which were later immunized with rVV. B, Percentage of interferon γ (IFN-γ)–expressing cells among gated CD45.1⁺ OT-I. C, Seven days or 21 days after rVV immunization, mice were challenged with live Plasmodium berghei CS^5M. Liver parasite burden was determined by reverse-transcription polymerase chain reaction 40 hours later. Data shown are results from 1 experiment, which are representative of 2 independent experiments with 3–5 mice per group. Data are mean ± standard error of the mean. *P < .05 and **P < .01. Abbreviations: NS, not significant; rRNA, ribosomal RNA.