Distinct APC subtypes drive spatially segregated CD4+ and CD8+ T-Cell effector activity during skin infection with HSV-1

Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4+ and CD8+ T-cells during skin infection with HSV-1. IFN-γ-producing CD4+ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8+ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4+ T cells, CD8+ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8+ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4+ and CD8+ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).The work was funded by grant (APP628423 and APP1059514) and fellowship support from the National Health and Medical Research Council Australia (NHMRC)and the Australian Research Council (ARC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Spatiotemporal dynamics of T Cell activation during localised HSV infection

© 2016 Dr. Jyh Liang HorThe induction of cell-mediated immune responses against localised infection requires complex orchestration involving multiple subsets of immune cells; from antigen acquisition by tissue-derived antigen-presenting cells (APC), to the priming of antigen-specific T lymphocytes in the secondary lymphoid tissues. The spatiotemporal coordination of T cell priming and the dynamic interplay between T cells and APC during localised viral infection, as well as how CD4+ T cells deliver help are poorly understood. Using a mouse localised cutaneous herpes simplex virus type 1 (HSV-1) infection model, this thesis investigated the intricate relationship of both innate and adaptive immune cells in shaping the optimal generation of cell-mediated immunity against localised viral infection. In light of recent studies demonstrating neutrophils involving in the crosstalk between innate and adaptive immunity, we examined the immunomodulatory roles of this innate immune cell during cutaneous HSV-1 infection. We identified an early transient wave of neutrophil infiltration to both the skin and draining lymph nodes (LN) triggered by dermal scarification. However, we found only minimal migration of neutrophils from skin to draining LN, and ablation of neutrophils did not affect T cell priming, or their migration to the infected skin, suggesting that neutrophils are dispensable for the generation of T cell responses against localised HSV-1 infection. To understand the spatiotemporal dynamics of T cell priming, the activation kinetics and cellular dynamics of CD4+ and CD8+ T cells was characterised in the draining LN through the combined use of flow cytometry, immunofluorescence confocal microscopy and intravital two-photon microscopy. The work here revealed that CD4+ and CD8+ T cells are activated at distinct times, facilitated by distinct dendritic cell (DC) subsets. CD4+ T cells were first activated at ~12 hr post-infection and preferentially clustered with migratory CD11b+ DC, whereas CD8+ T cells were activated at a later phase, ~36-48 hr post-infection and clustered with LN-resident XCR1+ DC. These findings highlight the complex interactions involving multiple DC and T cell subsets, and importantly the pivotal role of XCR1+ DC not only as the primary APC subset that primes CD8+ T cells, but also serving as the central platform for the delivery of CD4+ T cell help. Finally, we investigated the role of dedicator of cytokinesis 8 (DOCK8) proteins in the formation of CD8+ tissue-resident memory T cells (TRM) after skin HSV-1 infection. DOCK8-deficient CD8+ T cells exhibited severely impeded survival and TRM forming capacity, resulting in the loss of protection against subsequent HSV-1 challenge

Distinct APC Subtypes Drive Spatially Segregated CD4⁺ and CD8⁺ T-Cell Effector Activity during Skin Infection with HSV-1

<div><p>Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function <i>in situ</i> remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4⁺ and CD8⁺ T-cells during skin infection with HSV-1. IFN-γ-producing CD4⁺ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8⁺ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4⁺ T cells, CD8⁺ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8⁺ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4⁺ and CD8⁺ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).</p></div

Epidermal APCs trigger IFN-γ production by CD8⁺ T_EFF cells.

<p>(<b>A</b>,<b>B</b>) Analysis of IFN-γ⁺ <i>in vitro</i> activated gBT-I (<b>A</b>,<b>B</b>) and gDT-II (<b>B</b>) T_EFF cells cultured (as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004303#ppat-1004303-g005" target="_blank"><b>Figure 5</b></a>) with increasing numbers of CD45.2⁻ and CD45.2⁺ APCs sorted from epidermal sheets (pooled from 20 mice) 4 days after HSV-1 skin infection. Data representative (<b>A</b>) or pooled from 2–3 experiments (<b>B</b>). (<b>C</b>,<b>D</b>) Analysis of IFN-γ⁺ <i>in vitro</i> activated gBT-I cells cultured with sorted APCs from epidermal sheets (pooled from 20 mice) 4 days after infection. APC numbers were 5×10⁴ for keratinocytes (KC), CD11c⁺ and residual CD45.2⁺CD11c⁻Vγ3⁻ (CD45.2⁺) cells; 3×10⁴ for DETCs. Data representative (<b>C</b>) or pooled from 2 experiments (<b>D</b>). **, <i>P</i><0.01 by student t-test individually comparing specific APC subsets to control conditions (No APC).</p

Local proliferation maintains a stable pool of tissue-resident memory T cells after antiviral recall responses

Although tissue-resident memory T cells (TRM cells) are critical in fighting infection, their fate after local pathogen re-encounter is unknown. Here we found that skin TRM cells engaged virus-infected cells, proliferated in situ in response to local antigen encounter and did not migrate out of the epidermis, where they exclusively reside. As a consequence, secondary TRM cells formed from pre-existing TRM cells, as well as from precursors recruited from the circulation. Newly recruited antigen-specific or bystander TRM cells were generated in the skin without displacement of the pre-existing TRM cell pool. Thus, pre-existing skin TRM cell populations are not displaced after subsequent infections, which enables multiple TRM cell specificities to be stably maintained within the tissue.S.L.P. was supported by the University of Melbourne (Elizabeth and Vernon Puzey Postgraduate Scholarship). T.G. was supported by a fellowship from the Sylvia and Charles Viertel Charitable Foundation. This work was supported by the National Health and Medical Research Council of Australia (to S.N.M. and L.K.M.) and the Australian Research Council (to S.N.M.)

Localization of IFN-γ⁺ T_EFF cells in infected skin.

<p>IFM analysis of skin, 5 (<b>A</b>,<b>D</b>), 6 (<b>C</b>) and 8 (<b>B</b>) days post-infection, stained with anti-keratin and -IFN-γ antibodies, as indicated. (<b>C</b>,<b>D</b>) Detection of HSV-specific T_EFF cells after transfer of GFP⁺ naïve gBT-I (<b>C</b>) and gDT-II (<b>D</b>) cells prior to infection. Scale bars: <b>A-i</b>, 100 µm; <b>A-ii</b>, 2 µm; <b>B</b>, 200 µm; <b>C-i</b>, 70 µm; <b>C-ii</b>, 10 µm; <b>C-iii</b>, 10 µm; <b>D-i</b>, 200 µm; <b>D-ii</b>, 10 µm; <b>D-iii</b>, 10 µm. Photos representative of <i>n</i> = 4–6 mice/group.</p

CD11c⁺ DCs drive IFN-γ production by CD4⁺ T_EFF cells <i>in vivo</i>.

<p>(<b>A</b>) Mice received GFP⁺ gDT-II cells prior to HSV-1 skin infection. IFM analysis of skin 5 days post-infection after staining with anti-MHC-II and -IFN-γ antibodies. Arrows indicate IFN-γ⁺ gDT-II cells. Scale bars: <b>A-i</b>, 100 µm; <b>A-ii</b>, 20 µm; <b>A-iii</b>, 20 µm. (<b>B</b>,<b>C</b>) CD11c.DTR mice received naïve gDT-II cells prior to HSV-1 skin infection and 4 days post-infection were treated with diphtheria toxin (DT) or PBS (Ctrl). Analysis of IFN-γ⁺ gDT-II cells in skin (collagenase digestion). ***, <i>P</i><0.001 by Mann Whitney test; <i>n</i> = 11–15 mice/group from 3 experiments. (<b>D</b>) Wild-type mice received naïve gDT-II or gBT-II cells prior to infection and 4 days post-infection were treated with anti-CD80/86 (αCD80/86) or isotype control (Isotype) antibodies. Analysis of IFN-γ⁺ gDT-II cells in skin (collagenase digestion) and axillary LNs, and of IFN-γ⁺ gBT-I cells in epidermal sheets (dispase digestion) days 5 post-infection. *, <i>P</i><0.05; **, <i>P</i><0.01; ns, not significant by Mann Whitney test; <i>n</i> = 6–7 mice/group from 2–3 experiments.</p