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

Human immunodeficiency virus type I-specific CD8+ T cell subset abnormalities in chronic infection persist through effective antiretroviral therapy

Background: Effective highly active antiretroviral therapy (HAART) reduces human immunodeficiency virus (HIV) replication, restores CD4 +T lymphocyte counts and greatly reduces the incidence of opportunistic infections. While this demonstrates improved generalized immune function, rapid rebound to pre-treatment viral replication levels following treatment interruption indicates little improvement in immune control of HIV replication. The extent to which HAART can normalize HIV-specific CD8 +T cell function over time in individuals with chronic infection remains an important unresolved issue. In this study, we evaluated the magnitude, general specificity and character of HIV specific CD8 +T cell responses at four time points across 2-9 years in 2 groups of chronically infected individuals separated on the basis of either effective antiretroviral suppression or ongoing replication of HIV.Methods: Peripheral blood mononuclear cells (PBMC) were stimulated with overlapping 15mer peptides spanning HIV Gag, Pol, Env and Nef proteins. Cells producing interferon-γ (IFN-γ) or interleukin-2 (IL-2) were enumerated by ELISPOT and phenotyped by flow cytometry.Results and Conclusions: The magnitude of the HIV-specific CD8 +T cell response ranged from < .01 to approximately 1.0% of PBMC and was significantly greater in the group with detectable viral replication. Stronger responses reflected higher numbers of CD8 +CD45RA -effector memory cells producing IFN-γ, but not IL-2. Magnitude, general specificity and character of the HIV-specific CD8 +T cell response changed little over the study period. While antiretroviral suppression of HIV in chronic infection reduces HIV-specific CD8 +T cell response magnitude in the short term, it had no significant effect on response character over periods up to 9 years

Linear Fidelity in Quantification of Anti-Viral CD8+ T Cells

Enumeration of anti-viral CD8+ T cells to make comparisons between mice, viruses and vaccines is a frequently used approach, but controversy persists as to the most appropriate methods. Use of peptide-MHC tetramers (or variants) and intracellular staining for cytokines, in particular IFNγ, after a short ex vivo stimulation are now common, as are a variety of cytotoxicity assays, but few direct comparisons have been made. It has been argued that use of tetramers leads to the counting of non-functional T cells and that measurement of single cytokines will fail to identify cells with alternative functions. Further, the linear range of these methods has not been tested and this is required to give confidence that relative quantifications can be compared across samples. Here we show for two acute virus infections and CD8+ T cells activated in vitro that DimerX (a tetramer variant) and intracellular staining for IFNγ, alone or in combination with CD107 to detect degranulation, gave comparable results at the peak of the response. Importantly, these methods were highly linear over nearly two orders of magnitude. In contrast, in vitro and in vivo assays for cytotoxicity were not linear, suffering from high background killing, plateaus in maximal killing and substantial underestimation of differences in magnitude of responses

Analysis of A47, an Immunoprevalent Protein of Vaccinia Virus, Leads to a Reevaluation of the Total Antiviral CD8+ T Cell Response▿

Vaccinia virus (VACV) is the prototypic orthopoxvirus and was the live vaccine used to eradicate smallpox. In addition, VACV is a possible vector for recombinant vaccines. Despite these reasons for study, the roles of many VACV genes are unknown, and some fundamental aspects, such as the total size of immune responses, remain poorly characterized. VACV gene A47L is of interest because it is highly transcribed, has no sequence similarity to any nonpoxvirus gene, and contains a larger-than-expected number of CD8+ T cell epitopes. Here it is shown that A47L is not required for growth in vitro and does not contribute to virulence in mice. However, we confirmed that this one protein primes CD8+ T cells to three different epitopes in C57BL/6 mice. In the process, one of these epitopes was redefined and shown to be the most dominant in A47 and one of the more highly ranked in VACV as a whole. The relatively high immunogenicity of this epitope led to a reevaluation of the total CD8+ T cell response to VACV. By the use of two methods, the true size of the response was found to be around double previous estimates and at its peak is on the order of 60% of all CD8+ T cells. We speculate that more CD8+ T cell epitopes remain to be mapped for VACV and that underestimation of responses is unlikely to be unique to VACV, so there would be merit in revisiting this issue for other viruses

Direct quantification of HSV-specific CD8⁺ T cells.

<p>Splenocytes from mice infected with HSV KOS were serially diluted in splenocytes from mice infected with HSV K.L8A and CD8⁺ T cell responses to the peptides gB₄₉₈ and RR1₉₈₂ were measured using three methods. (A) Data from DimerX, IFNγ-ICS and IFNγ-ICS/CD107 methods as indicated on each graph. Backgrounds determined using irrelevant peptides were subtracted from the values presented. (B) Linear regression analysis and r² statistics for quantification of gB₄₉₈–specific CD8⁺ T cells by each method.</p

Measuring in vitro cytotoxicity of VACV-specific CD8⁺ T cells.

<p>The same set of serial dilutions of splenocytes from VACV WR and VACV ΔB8R mice as made for the experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039533#pone-0039533-g002" target="_blank">figure 2</a> were used as a source of effectors in an <i>in vitro</i> cytotoxicity assay. Target cells were RMA cells coated with B8₂₀ or A3₂₇₀ and these were incubated with effectors at the E:T ratios shown. (A) The percent loss of B8₂₀ or A3₂₇₀ (as shown on graphs) coated cells as compared with non-peptide-coated control targets. (B) Linear regression analysis and r² statistics derived from the B8₂₀–specific killing data shown in panel A. Data are representative of two independent experiments.</p

Comparison of DimerX, IFNγ-ICS and an in vivo cytotoxicity assay.

<p>OVA₂₅₇-specific CD8⁺ T cells were generated from OT-I mice and increasing numbers were injected i.v. into B6 mice. After 24 hours, naïve B6.SJL splenocytes coated with OVA₂₅₇ (labeled CFSE^high) mixed with uncoated splenocytes (labeled CFSE^low) were injected i.v. into the OT-I T cell-treated B6 mice. After 4 hours, splenocytes were prepared and the percentage of OVA₂₅₇-specific CD8⁺ T cells was determined by DimerX and/or IFNγ-ICS and OVA₂₅₇–specific killing measured by comparing recovery of CD45.1⁺ CFSE^high and CFSE^low cells. Two experiments are shown (A and C) and graphs are labeled with the method used. (B and D) Linear regression analysis and r² statistics for the experiments shown in panels A and C, respectively.</p

Vaccinia virus F5 is required for normal plaque morphology in multiple cell lines but not replication in culture or virulence in mice

AbstractVaccinia virus (VACV) gene F5L was recently identified as a determinant of plaque morphology that is truncated in Modified Vaccinia virus Ankara (MVA). Here we show that F5L also affects plaque morphology of the virulent VACV strain Western Reserve (WR) in some, but not all cell lines, and not via previously described mechanisms. Further, despite a reduction in plaque size for VACV WR lacking F5L there was no evidence of reduced virus replication or spread in vitro or in vivo. In vivo we examined two mouse models, each with more than one dose and measured signs of disease and virus burden. These data provide an initial characterization of VACV F5L in a virulent strain of VACV. Further they show the necessity of testing plaque phenotypes in more than one cell type and provide an example of a VACV gene required for normal plaque morphology but not replication and spread