Monocyte-Derived Dendritic Cells Dictate the Memory Differentiation of CD8+ T Cells During Acute Infection

Abstract

Monocyte-derived dendritic cells (moDCs) have been shown to robustly expand during infection; however, their roles in anti-infectious immunity remain unclear. Here, we found that moDCs were dramatically increased in the secondary lymphoid organs during acute LCMV infection in an interferon-γ (IFN-γ)-dependent manner. We also found that priming by moDCs enhanced the differentiation of memory CD8⁺ T cells compared to differentiation primed by conventional dendritic cells (cDCs) through upregulation of Eomesodermin (Eomes) and T cell factor-1 (TCF-1) expression in CD8⁺ T cells. Consequently, impaired memory formation of CD8⁺ T cells in mice that had reduced numbers of moDCs led to defective clearance of pathogens upon rechallenge. Mechanistically, attenuated interleukin-2 (IL-2) signaling in CD8⁺ T cells primed by moDCs was responsible for the enhanced memory programming of CD8⁺ T cells. Therefore, our findings unveil a specialization of the antigen-presenting cell subsets in the fate determination of CD8⁺ T cells during infection and pave the way for the development of a novel therapeutic intervention on infection.

Keywords: IFN-γ; LCMV; acute infection; memory CD8+ T cells; monocyte-derived dendritic cells.

Figure 1

IFN-γ-dependent expansion of monocyte-derived dendritic cells during acute infection. (A) Gating strategies of cDCs and moDCs in the spleen of naïve or LCMV-Arm-infected mice. Numbers indicate the percentages within the gates. (B) Cell numbers and frequencies of cDCs and moDCs in the spleen during LCMV-Arm infection. (C) Expression patterns of indicated surface molecules on cDCs and moDCs in LCMV-Arm-infected mice at day 4 p.i., Numbers indicate the MFI values of each molecule. (D) Kinetics of IFN-γ levels in the serum of LCMV-Arm-infected mice. (E,F) LCMV-Arm-infected mice were treated with IFN-γ-neutralizing Ab. (E) Cell numbers (left) and frequencies (right) of cDCs and moDCs were measured in the indicated organs on day 8 p.i., and are shown as graph plots. (F) Frequency of moDCs in the BM. Data are representative of three independent experiments and are shown as the mean ± SEM. n = 5 per group at each time point. ^**p < 0.01;^***p < 0.001.

Figure 2

IFN-γ acts directly on common monocyte progenitor cells and promotes the differentiation of moDCs. (A) Differentiation patterns of total BMPs at day 5 under different stimuli (GM-CSF alone, GM-CSF + IL-4, and GM-CSF + IFN-γ are shown as flow cytometry plots (left) and graphs (right). Numbers in the plots indicate the percentages within the gates. (B) IFN-γR (CD119) expression levels of the BMP subsets. Numbers indicate the MFI values of each subset. (C,D) Daily differentiation of the subdivided BMP subsets into moDCs under GM-CSF and IFN-γ stimulation. (C) Representative flow cytometry plots of sorted BMP subsets after a day of culture (D) Graph shows the proportions of cells that differentiate into moDCs in each BMP subset. (E–G) Sorted cMoPs or non-cMoPs were transferred into LCMV-Arm-infected mice on day 5 p.i., and their differentiation into moDCs was analyzed 3 days later. (E) Experimental schedule. (F–G) Differentiation patterns of donor cells into moDCs in the indicated organs are shown as flow cytometry plots (F) and graphs (G). Numbers in the plots indicate the percentages within the gates. Data are representative of two or three independent experiments and are shown as the mean ± SEM. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 3

CD8⁺ T cells primed by moDCs have reduced effector function than those primed by cDCs. (A,B) Representative histograms (A) and graph (B) of CTV dilutions in P14 cells primed by cDCs or moDCs in the presence of different doses of GP₃₃₋₄₁ peptide for 3 days. Numbers in the histograms indicate the percentage of cells that were divided at least once. (C–F) P14 cells were activated by cDCs or moDCs in the presence of 0.2 μg/ml GP₃₃₋₄₁ peptide for 3 days. (C) Expression levels of indicated surface molecules are shown as histograms (right) and a graph plot (left). Numbers in the histograms indicate the percentages of positive cells for each molecule. (D) Coexpression of CD25 and CD62L on P14 cells primed by cDCs or moDCs are shown as flow cytometry plots (upper) and graph (lower). Numbers in the plots indicate the percentages within each gate. (E) Secretion levels of the indicated effector molecules in P14 cells that were primed by cDCs or moDCs are shown as histograms (upper) and graph (lower). Numbers in the histograms indicate the percentages of positive cells for each molecule. (F) in vitro target killing ability of P14 cells primed by cDCs or moDCs. Cr⁵¹-labeled GP₃₃₋₄₁-loaded EL4 tumor cells were used as the target cells. Data are representative of three independent experiments and are shown as the mean ± SEM. ^*p < 0.05;^**p < 0.01; ^***p < 0.001.

Figure 4

Stimulation by moDCs dictates the developmental program of memory CD8⁺ T cells by transcriptional regulation. (A) Expression levels of the indicated transcription factors in P14 cells primed by cDCs or moDCs are shown as histograms (upper) and graph (lower). Numbers in the histograms indicate the percentages of positive cells for each molecule. (B) Coexpressions of T-bet and Eomes in P14 cells primed by cDCs or moDCs are shown as flow cytometry plots (upper) and graph (lower). Numbers in the plots indicate the percentages of the cells in each quadrant. (C) P14 cells primed by cDCs or moDCs were gated by their cell division (left). TCF1 expression levels of each gate are shown as histograms (center) and graphs (right). Numbers in the histograms indicate the MFI values of TCF1 expression in each gate. (D–G) CD45.1⁺ P14 cells were primed in vitro by cDCs or moDCs, transferred to infected recipient mice on day 8 p.i., and analyzed on day 28 post transfer. (D) Experimental schedule. (E) Representative flow cytometry plots of donor cells in the spleens of recipient mice. (F) Graphs show the number of donor P14 cells in the indicated organs. (G) Graphs show the coexpressions of CD127 and CD62L of the donor cells in the spleen of the recipient mice. Data are representative of two or three independent experiments and are shown as the mean ± SEM. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 5

CD8⁺ T cells fail to differentiate into MPECs in CCR2-deficient mice. (A,B) Coexpressions of KLRG1 and CD127 of CD45.1⁺ P14 cells in the spleen and LN of LCMV-Arm-infected WT and Ccr2^−/− mice on day 8 p.i., are shown as flow cytometry plots (left) and graph (right). Numbers in the plots indicate the percentage of cells within each quadrant. (C) Secretion levels of IFN-γ and TNF-α in CD45.1⁺ P14 cells in the spleen of WT and Ccr2^−/− mice on day 8 p.i., are shown as histograms (left) and graph (right). (D) In vitro target killing ability of CD45.1⁺ P14 cells from WT and Ccr2^−/− splenocytes on day 8 p.i., Cr⁵¹-labeled GP₃₃₋₄₁-loaded EL4 tumor cells were used as the target cells. (E,F) Expression levels of T-bet, Eomes and TCF1 in SLECs and MPECs of CD45.1⁺ P14 cells from WT and Ccr2^−/− splenocytes and LN cells on day 8 p.i., are shown as histograms (upper) and graph (lower). Numbers in the histograms indicate the percentage of positive cells for each molecule. (G) Gene expression levels of Tbx21, Prdm1, Eomes, and Tcf7 in CD45.1⁺ P14 cells from WT and Ccr2^−/− splenocytes on day 8 p.i., were measured by real-time PCR. Expression levels were normalized to mHprt. Data are representative of three independent experiments and are shown as the mean ± SEM. n = 5 per group. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 6

CD8⁺ T cells primed in CCR2-deficient mice cannot respond to reinfection. Effector P14 cells of WT and Ccr2^−/− mice at day 8 p.i., were sorted and equivalent numbers of the cells were transferred into naïve mice. Recipient mice were analyzed at least 20 days after transfer. (A) Experimental schedule. (B) Temporally enumerated CD45.1⁺ P14 cells in blood PBMCs of recipient mice after the transfer of effector P14 cells from WT and Ccr2^−/− mice. (C) The frequencies of CD45.1⁺ P14 cells in the spleen (left) and liver (right) of recipient mice on day 24 post transfer of P14 cells from WT and Ccr2^−/− mice. (D) The memory phenotypes of CD45.1⁺ P14 cells in the spleen of recipient mice on day 24 post transfer of P14 cells from WT and Ccr2^−/− mice. (E) Recipient mice were challenged with Lm-GP33 at day 26 post transfer. Graph shows the bacterial titers in the spleen of recipient mice at day 3 after rechallenge. Data are representative of two independent experiments and are shown as the mean ± SEM. n = 3–4 per group. ^*p < 0.05; ^**p < 0.01.

Figure 7

Defective IL-2 signaling grants moDCs an ability to induce memory CD8⁺ T cells. (A) IL-2 concentrations in the supernatant from the cultures of P14 cells with cDCs or moDCs. (B) Gene expression levels of Il2 in cDCs and moDCs isolated from LCMV-Arm-infected splenocytes. (C–F) Recombinant IL-2 or anti-IL-2 mAbs were added to the cultures of P14 cells with moDCs or cDCs. (C,D) Coexpressions of CD25 and CD62L under each condition are shown as flow cytometry plots (C) and graphs (D). (E,F) Expression levels of TCF1 are shown as flow cytometry plots (E) and graph (F). Numbers in the plots indicate the percentage within each gate. Data are representative of two independent experiments and are shown as the mean ± SEM. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.