Chronic LCMV infection regulates the effector T cell response by inducing the generation of less immunogenic dendritic cells

Chronic viral infection impairs systemic immunity in the host; however, the mechanism underlying the dysfunction of immune cells in chronic viral infection is incompletely understood. In this study, we studied the lineage differentiation of hematopoietic stem cells (HSCs) during chronic viral infection to elucidate the changes in dendritic cell (DC) differentiation and subsequent impact on T cell functionality using a chronic lymphocytic choriomeningitis virus (LCMV) infection model. We first investigated the lineage differentiation of HSCs in the bone marrow (BM) to elucidate the modulation of immune cell differentiation and found that the populations highly restrained in their differentiation were common myeloid progenitors (CMPs) and common dendritic cell progenitors (CDPs). Of interest, the main immune cells infected with LCMV Clone 13 (CL13) in the BM were CD11b/c+ myeloid DCs. We next characterized CD11b+ DCs that differentiated during chronic LCMV infection. These DCs displayed a less immunogenic phenotype than DCs in naive or acutely infected mice, showing low expression of CD80 but high expression of PD-L1, B7-H4, IDO, TGF-β, and IL-10. Consequently, these CD11b+ DCs induced less effective CD8+ T cells and more Foxp3+ regulatory T (Treg) cells. Furthermore, CD11b+ DCs generated during CL13 infection could not induce effective CD8+ T cells specific to the antigens of newly invading pathogens. Our findings demonstrate that DCs generated from the BM during chronic viral infection cannot activate fully functional effector CD8+ T cells specific to newly incoming antigens as well as persistent antigens themselves, suggesting a potential cause of the functional alterations in the T cell immune response during chronic viral infection.

The cytokine-secreting potential of adoptively transferred OT-I T cells was analyzed by intracellular cytokine staining and CellTrace dye staining. The kinetics of effector cytokine expression during OT-I T cell proliferation were analyzed (top three FACS plots and bar graphs). The expression levels of IFN-γ, IL-2, and TNF-α in activated CD44 hi proliferating OT-I T cells were investigated (bottom three FACS plots and bar graphs). The bar graphs show the means ± SDs (6 mice in each group). Each experiment was repeated twice. The p values in the figures indicate the following: *P < 0.05; **P < 0.01; **P < 0.01; ***P < 0.001  Supplementary Fig. 1

. Analysis of HSC and progenitor populations in the BM on Days 3 and 5 after LCMV infection.
HSCs and progenitor cells were identified based on surface molecule expression, as described in the gating strategy for the lineage marker-negative population. On Days 3 and 5 after LCMV infection, BM cells were isolated, and the percentage of each HSC and progenitor population was analyzed (5 mice in each group).

Supplementary Fig. 2. Phenotypic analysis of BMDCs differentiated from ARM-infected
mice and ex vivo splenic CD11b + DCs.
[a] The LCMV CL13 viral titer in the BM was analyzed by a plaque formation assay.
[b] The frequency of the LCMV NP + CD11b + population in the BM was analyzed at 0, 10 and 30 days after LCMV CL13 infection.
[c] Number of CD11b/c + cells on Days 6 and 9 of BMDC differentiation.  [h] Expression of surface molecules on CD11b/c + cells on Day 4 of BMDC differentiation.
[i] The bar graph presents the MFI values of each molecule on CD11b/c + cells on Day 4 of BMDC differentiation.

[j] The concentrations of inflammatory cytokines secreted by UN-BMDCs or ARM-infected
BMDCs were measured by ELISA.
[k] The histograms present the expression of each surface molecule on splenic CD11b + DCs after 10 days of LCMV infection.
[l] The bar plots present the MFI values of each surface molecule on CD11b + splenic DCs.
The bar graphs show the means ± SDs (n = 3 samples). Each experiment was repeated 3 times.
The p values in the figures indicate the following: *P < 0.05; **P < 0.01; **P < 0.01; ***P < 0.001. [c] The total amount of each cytokine expressed in the supernatant of P14-BMDC cocultures was measured by a multiplex bead assay.
[d] Conversion ratio of Foxp3 + Treg cells in a coculture containing CD4 + T cells with UN-BMDCs or ARM-infected BMDCs in each condition.
[e] The proliferation and division of P14 T cells cocultured with ex vivo SPDCs from uninfected or ARM-infected mice were analyzed. The bar graph presents the percentages of the cell populations in each division number.
[f] The cytokine-secreting potential of P14 T cells was analyzed. The kinetics of effector cytokine expression during P14 T cell proliferation were analyzed (top two FACS plots and bar graph). The expression levels of IFN-γ, IL-2, TNF-α and GzmB in activated CD44 hi proliferating P14 T cells were investigated (bottom two FACS plots and bar graph).
[g] The total amount of each cytokine expressed in the supernatant of P14-SPDC cocultures was measured by a multiplex bead assay.
[h] Conversion ratio of Foxp3 + Treg cells derived from CD4 + T cells cocultured with SPDCs from uninfected or ARM-infected mice in each condition.
[a] Experimental scheme for manipulating CD11b + DCs in the spleen with GM-CSF expressing B16F10 cells.
[b] Expansion of each subset of SPDCs with B16F10 cells engineered to express GM-CSF or [c] Proliferation and effector cytokine expression analysis of P14 cells primed with CD11b + SPDCs expanded by injection with GM-CSF-expressing B16F10 cells.
The bar graphs show the means ± SDs (3 mice in each group). Each experiment was repeated twice. The p values in the figures indicate the following: *P < 0.05; **P < 0.01; **P < 0.01; ***P < 0.001. [a] The blockade of PD-L1 on the surface of BMDCs after treatment with a purified anti-PD-L1 antibody was verified by flow cytometry analysis.
[b] The kinetics of the expression of various molecules, such as PD-1, IFN-γ, IL-2 and TNFα, in P14 T cells cocultured with BMDCs were investigated.
[c] Upon coculture with BMDCs, the frequency of each effector cytokine-expressing CD44 hi activated P14 T cell population was analyzed in each condition.
[d] The total amount of each effector cytokine in the supernatant of P14-BMDC cocultures was estimated by a multiplex bead assay.
[e] The blockade of PD-L1 on the surface of SPDCs after treatment with purified anti-PD-L1 antibody was verified by flow cytometry analysis.
[f] The kinetics of the expression of various molecules, such as PD-1, IFN-γ, IL-2 and TNF-α, in P14 T cells cocultured with SPDCs were assessed.
[g] Upon coculture with SPDCs, the frequency of each effector cytokine-expressing CD44 hi activated P14 T cell population was analyzed in each condition.
[h] The total amount of each effector cytokine in the supernatant of P14-SPDC cocultures was estimated by a multiplex bead assay.
[a] The in vitro proliferation and division of OT-I T cells cocultured with OVA-pulsed naïve or ARM-infected SPDCs were analyzed by CellTrace dye staining. The bar graph presents the percentages of the cell populations in each division number.
[b] The potential cytokine secretion of OT-I T cells cocultured with OVA-pulsed naïve or ARM-infected SPDCs was analyzed by intracellular cytokine staining and CellTrace dye staining. The kinetics of effector cytokine expression during OT-I T cell proliferation were analyzed (top two FACS plots and bar graph). The expression levels of IFN-γ, IL-2, and TNFα in activated CD44 hi proliferating OT-I T cells were investigated (bottom two FACS plots and bar graph).
[c] Analysis of the proliferation and division of adoptively transferred OT-I T cells in the spleen of uninfected or ARM-infected mice after VV-OVA coinfection. The bar graph presents the percentages of the cell populations in each division number.
[d] The cytokine-secreting potential of adoptively transferred OT-I T cells was analyzed by intracellular cytokine staining and CellTrace dye staining. The kinetics of effector cytokine expression during OT-I T cell proliferation were analyzed (top three FACS plots and bar graphs). The expression levels of IFN-γ, IL-2, and TNF-α in activated CD44 hi proliferating OT-I T cells were investigated (bottom three FACS plots and bar graphs).