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. 2016 Dec 20;45(6):1270-1284.
doi: 10.1016/j.immuni.2016.10.018. Epub 2016 Dec 6.

The Chemokine Receptor CX3CR1 Defines Three Antigen-Experienced CD8 T Cell Subsets With Distinct Roles in Immune Surveillance and Homeostasis

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Free PMC article

The Chemokine Receptor CX3CR1 Defines Three Antigen-Experienced CD8 T Cell Subsets With Distinct Roles in Immune Surveillance and Homeostasis

Carmen Gerlach et al. Immunity. .
Free PMC article

Abstract

Infections induce pathogen-specific T cell differentiation into diverse effectors (Teff) that give rise to memory (Tmem) subsets. The cell-fate decisions and lineage relationships that underlie these transitions are poorly understood. Here, we found that the chemokine receptor CX3CR1 identifies three distinct CD8+ Teff and Tmem subsets. Classical central (Tcm) and effector memory (Tem) cells and their corresponding Teff precursors were CX3CR1- and CX3CR1high, respectively. Viral infection also induced a numerically stable CX3CR1int subset that represented ∼15% of blood-borne Tmem cells. CX3CR1int Tmem cells underwent more frequent homeostatic divisions than other Tmem subsets and not only self-renewed, but also contributed to the expanding CX3CR1- Tcm pool. Both Tcm and CX3CR1int cells homed to lymph nodes, but CX3CR1int cells, and not Tem cells, predominantly surveyed peripheral tissues. As CX3CR1int Tmem cells present unique phenotypic, homeostatic, and migratory properties, we designate this subset peripheral memory (tpm) cells and propose that tpm cells are chiefly responsible for the global surveillance of non-lymphoid tissues.

Figures

Figure 1
Figure 1. CX3CR1 expression levels identify three populations of pathogen-specific CD8+ TEff cells
(A) FACS analysis of CX3CR1-GFP induction by LCMV infection on PBMC (left: representative experiment; right: means of 3 experiments, n=3–5 mice/each) and (B) after gating on CD3+CD8+ or CD3+CD4+. (C) Staining of CD8 T cells by fractalkine fused to human IgG1 Fc (FKN-Ig) or CX3CR1 MAb. r: mean Pearson correlation ± SD. (D–E) Naïve Cx3cr1+/gfp CD45.1+ OT-I cells were transferred into C57BL/6 recipients followed by LCMV-ova or VSV-ova infection. (D) Gating strategy to identify TEff subsets. (E) Mean + SD. n = 2 experiments. All FACS plots are composite plots as described in Fig. S1A. See also Figs. S1–S3.
Figure 2
Figure 2. CX3CR1 is a differentiation marker for pathogen-specific CD8+ TEff cells
(A,B) Naïve Cx3cr1+/gfp CD45.1+ OT-I cells were transferred into C57BL/6 recipients followed by LCMV-ova or VSV-ova infection. Cytokine expression by splenic TEff subsets (day 10), gated as in Figure 1D (B: mean + SD) (C) Naïve p14 Cx3cr1+/gfp CD45.1+ [Tbx21+/+ or Tbx21−/−] cells were transferred into C57BL/6 followed by LCMV infection and analysis of splenic P14 cells. (D) Naïve p14 Cx3cr1+/gfp [Tbx21+/+ (CD45.1+CD45.2) or Tbx21−/− (CD45.1+CD45.2+)] were co-transferred to C57BL/6 mice followed by LCMV infection. Left: Composite plots of blood-derived P14 Right: mean + SD. (A–D) n=2 experiments pooled. ** p<0.01, *** p<0.001 by repeated measures one-way (A), repeated measures two-way (B) or regular one-way (C) ANOVA with Tukey’s (A,C) or Bonferroni (B) multiple comparisons test. See also Fig. S3–4A,B.
Figure 3
Figure 3. CX3CR1 expression levels identify three CD8+ TMem populations with distinct homeostatic properties
(A) Experimental protocol, absolute and relative numbers, and (B) phenotype of OT-I TMem cells recovered from spleen and LNs of recipients of sorted TEff subsets. (B) Mean+SD. (C) Cx3cr1+/gfp mice were infected with LCMV. Representative FACS plot, concentration and frequency of gp33-Dextramer+ CD8+ TMem subsets in blood. Mean±SD. (D) Experimental protocol and phenotype of OT-I TMem cells recovered from spleen and LNs. 1 mouse per time-point. (E) Frequency of Ki67+ cells among naïve (CD44CD62L+) CD8+ T cells and OT-I TMem subsets. Right: Fold-difference in %Ki67+ cells between CX3CR1int (int) and CX3CR1 (neg) or CX3CR1hi (hi) TMem. (F) Experimental protocol and phenotype of OT-I TMem cells recovered from spleen and LNs. (A–F) n=2 experiments pooled. * p<0.05, ** p<0.01, *** p<0.001 by regular (A) or repeated measures (E: Blood & Spleen) one-way ANOVA with Tukey’s multiple comparisons test or two-tailed T test (E: LN). See also Fig. S4C–E.
Figure 4
Figure 4. CX3CR1 levels on TMem cells distinguish TCM and TEM cells and a CX3CR1int TMem population that, unlike TEM cells, re-acquires CD62L
(A) Chemotactic response of OT-I TMem subsets to CCL19 in a Transwell assay. Chemotactic index: number of TMem that migrated towards CCL19 relative to medium alone. 4–5 wells/group/experiment. (B) OT-I TMem subset frequencies in lymphoid and non-lymphoid tissues. (C) Experimental protocol and frequency of OT-I TMem cells in blood after secondary infection. (D) Composite FACS plots and CD62L expression on OT-I TMem in blood. (E) Cx3cr1+/gfp mice were infected with LCMV. CD62L expression on blood circulating gp33-Dextramer+ CD8+ TMem cells (total) or subsets thereof. (F) Experimental protocol and frequency of CD62L+ cells among recovered splenic and LN resident OT-I TMem. (G) Homing efficiency of adoptively transferred TMem cells to peripheral LN (pLN), mesenteric LN (mLN) and spleen in 2h period. # recovered CX3CR1/# recovered CX3CR1int relative to input ratio. (A–E, G) n=2 and (D) n=3 experiments pooled. ** p<0.01, *** p<0.001 by one-way ANOVA with Tukey’s multiple comparisons test. Error bars indicate mean±SEM. See also Fig. S4C–S7.
Figure 5
Figure 5. Unidirectional differentiation from TCM to CX3CR1int TMem to TEM after re-challenge
(A) Experimental protocol and phenotype of OT-I TMem cells post 2° infection in blood. Mean + SD. (B) At day 55 post 2° infection, C57BL/6 recipient mice were reinfected with LM-ova (3° infection). Appearance of CD62L+ cells on transferred subsets over time. Mean ± SD. n=2 experiments pooled.
Figure 6
Figure 6. Peripheral tissues are largely devoid of TEM
Naïve OT-I Cx3cr1+/gfp CD45.1+ T cells were transferred into C57BL/6 followed by LCMV-ova or VSV-ova infection. (A) Gating strategy for intra- and extra-vascular OT-I cells. Composite FACS plot. (B) CD69 and CD103 expression on extra-vascular OT-I TMem cells and (C) frequency of CX3CR1 subsets among extra-vascular CD69+CD103+ OT-I TMem cells in indicated tissues. (B–C) Mean + SD. n=2 experiments. (D) Frequency of CX3CR1 subsets among OT-I TMem cells. Mean + SD. Left: n=4 experiments (Blood, Spleen, LN, SG) n=3 experiments (IEL, LPL, FRT). Right: 1 experiment.
Figure 7
Figure 7. CX3CR1int TMem cells, not TEM cells, are the major TMem subset circulating through peripheral tissues
(A) OT-I TMem subsets in blood (left) and TDL (right). FACS plots above were concatenated from 3 immunized mice. Data panels below show frequency of CX3CR1 (red), CX3CR1int (blue) and CX3CR1hi (green) TMem subsets among total (top), CD62L+ (middle) and CD62L (bottom) OT-I TMem cells. (B) Schematic for parabiosis experiments in panels (C–H). (C, F) FACS plots depicting blood- and lymph-borne OT-I TMem cells in naive WT (C) and Lta−/− (F) parabionts. Numbers show percentage of gated events. (D, G) Frequency of CD62L+ and CD62L OT-I TMem cells (mean + SD). (E) Frequency of TMem subsets among total (top), CD62L+ (middle) and CD62L (bottom) OT-I TMem cells in WT parabionts. (H) Frequency of TMem subsets among total OT-I TMem cells in Lta−/− parabionts. (I) Cell numbers and (J) phenotype of OT-I TMem cells in pooled axillary, brachial and inguinal LNs from control and anti-CD62L-treated naïve and immune parabiotic pairs (mean + SD). (A,I,J) n=2 (C–E) n=4 and (F–H) n=3 experiments pooled. * p<0.05, *** p<0.001 (one-way ANOVA with Tukey’s multiple comparisons test).

Comment in

  • Sifting through CD8+ T Cell Memory.
    Martin MD, Badovinac VP. Martin MD, et al. Immunity. 2016 Dec 20;45(6):1184-1186. doi: 10.1016/j.immuni.2016.12.005. Immunity. 2016. PMID: 28002725
  • T cells: Sorting memories.
    Bird L. Bird L. Nat Rev Immunol. 2016 Dec 23;17(1):2-3. doi: 10.1038/nri.2016.146. Nat Rev Immunol. 2016. PMID: 28008185 No abstract available.

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