Inflammatory monocytes contribute to the persistence of CXCR3hi CX3CR1lo circulating and lung-resident memory CD8+ T cells following respiratory virus infection

Abstract

Phenotypically diverse memory CD8⁺ T cells are present in the lungs that either re-circulate or reside within the tissue. Understanding the key cellular interactions that regulate the generation and then persistence of these different subsets is of great interest. Recently, DNGR-1⁺ dendritic cell (DC) mediated priming was reported to control the generation of lung-resident but not circulating memory cells following respiratory viral infection. Here, we report an important role for Ly6C⁺ inflammatory monocytes (IMs) in contributing to the persistence of memory CD8⁺ T cells but not their generation. Effector CD8⁺ T cells expanded and contracted normally in the absence of IMs, but the memory compartment declined significantly over time. Quite unexpectedly, this defect was confined to tissue resident and circulating CXCR3^hi CX3CR1^lo memory cells but not CXCR3^hi CX3CR1^int and CXCR3^lo CX3CR1^hi subsets. Thus, two developmentally distinct innate cells orchestrate the generation and persistence of memory T cell subsets following a respiratory virus infection. See also: News and Commentary by Lafouresse & Groom.

Keywords: CD8+ T cells; lung; monocytes; resident memory T cells; vaccinia.

Figure 1. Expansion of inflammatory monocytes in response to respiratory vaccinia virus infection

WT BL/6 mice were infected with VACV-WR (i.n) and lung and spleen cells were harvested. (a) Gating strategy for identifying IMs includes exclusion of other prominent cell types. Cells were stained with live/dead dye followed by CD3, B220, CD11b, NK1.1, Ly6G and Ly6C. (b) Total number of LY6C⁺ cells were calculated at days 0, 2, 4, 6 and 8 post-infection. (c & d) Lungs were harvested from BL/6 mice and CCR2^−/− recipient mice at day 8 post-infection with rVacV-WR-OVA. Cells were stained as described in (a) and total cell numbers of each innate cell type was determined. *, P < 0.05; **, P < 0.01 and results are a representative of one of the three repeats with mean ± SEM (n = 3 mice/group).

Figure 2. Inflammatory monocytes do not affec expansion and accumulation of antigen specific CD8 T cells

Equal numbers (5 × 10⁴) of WT naïve (CD44^lo) OT-I (Vα2⁺Vβ5⁺) transgenic CD8 T cells were adoptively transferred into BL/6 mice and CCR2^−/− mice, and infected with rVacV-WR-OVA (2 × 10⁴ PFU i.n) the following day. (a) Lungs, (b) spleen were harvested at days 8 post-infection and stained for CD8, CD44, Vα2, Vβ5 extracellularly and Ki67 intra-nuclearly and frequencies and cell numbers of OT-I CD8 T cells determined. Lung cells pre-gated on CD8, CD44, Vα2, and Vβ5 were stained for (c) KLRG1 & CD127, (d) CD27 & CD43 (e) KLRG1 & CXCR3 (g) Tbet & Eomes. (f) Lungs cells were re-stimulated in vitro with OVA and OT-I cells were stained for IFN-γ. *, P < 0.05; **, P < 0.01 and results are the mean ± SEM (n = 3 mice/group). Similar results were obtained in two independent experiments.

Figure 3. Generation of tissue resident memory cells in CCR2 deficient mice

(a–e) At day 20, recipient mice were injected with anti-CD45.2 antibody intravenously, three minutes before euthanizing and lungs were harvested. Frequencies and total number of TRM (CD45.2⁻) cells were determined after pre-gating on CD8⁺CD44⁺ OT-I (Vα2⁺Vβ5⁺) cells followed by staining with CXCR3. (b) The gated CD45.2 negative cells were analyzed for the expression of CD103 and CD69 using flow cytometry at respective days and absolute numbers of cells calculated. (c) Cells were stained with CXCR3 and absolute numbers of CD45.2⁺ cells (non-TRM cells) both CXCR3^lo and CXCR3^hi was quantified. (d) The gated CD45.2 negative cells were analyzed for the expression of CD103 and CD69 using flow cytometry at respective days and absolute numbers of cells calculated. (e) After extracellular staining, cells were stained with Ki-67 intra-nuclearly without peptide stimulation. (f–g) At day 50, recipient mice were injected with anti-CD45.2 antibody intravenously, three minutes before euthanizing and lungs were harvested. Frequencies and total number of TRM (CD45.2⁻) cells were determined after pre-gating on CD8⁺CD44⁺OT-I (Vα2⁺Vβ5⁺) cells followed by staining with CXCR3. (g) Cells were stained with CD69 and CD103 and total cell numbers in each gate were calculated. These experiments were performed twice with (n = 3 or 4 mice/group) and results are the mean ± SEM; with significance value determined by *, P < 0.05.

Figure 4. Long-term maintenance of tissue resident memory cell subset segregated based on CXCR3 and CX3CR1 expression

(a–e) Cells were also stained with CX3CR1 and CXCR3 and (a) frequencies and (b) total cell numbers of were calculated. (c) CX3CR1^neg (d) CX3CR1^int (e) CX3CR1^hi were also analyzed for TRM cells after recipient mice were injected with anti-CD45.2 antibody intravenously. Total number of circulating/vascular (CD45.2⁺) and resident (CD45.2⁻) cells were calculated for each of the CX3CR1 subsets. These experiments were performed twice with (n = 3 or 4 mice/group) and results are the mean ± SEM; with significance value determined by *, P < 0.05;