Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells

Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) alpha-chain, TRAV1-2-TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH2OH), specifically detect all human MAIT cells. Tetramer(+) MAIT subsets were predominantly CD8(+) or CD4(-)CD8(-), although a small subset of CD4(+) MAIT cells was also detected. Notably, most human CD8(+) MAIT cells were CD8 alpha(+)CD8 beta(-/lo), implying predominant expression of CD8 alpha alpha homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH2OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in V. 19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-beta repertoire, and although the majority of human MAIT cells expressed TRAV1-2-TRAJ33, some expressed TRAJ12 or TRAJ20 genes in conjunction with TRAV1-2. Accordingly, MR1 tetramers allow precise phenotypic characterization of human and mouse MAIT cells and revealed unanticipated TCR heterogeneity in this population

Poxvirus-Encoded Gamma Interferon Binding Protein Dampens the Host Immune Response to Infection

Ectromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-γ) receptor 1. We generated an IFN-γ binding protein (IFN-γbp) deletion mutant of ECTV to study the role of viral IFN-γbp (vIFN-γbp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-γbp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-γbp from ECTV allowed the generation of an effective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-γbp deletion mutant virus, designated ECTV-IFN-γbp(Δ), produced increased levels of IFN-γ and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-γ and the virus' ability to dampen its effects

Deficiency in Th2 Cytokine Responses Exacerbate Orthopoxvirus Infection

<div><p>Ectromelia virus (ECTV) causes mousepox in mice, a disease very similar to smallpox in humans. ECTV and variola virus (VARV), the agent of smallpox, are closely related orthopoxviruses. Mousepox is an excellent small animal model to study the genetic and immunologic basis for resistance and susceptibility of humans to smallpox. Resistance to mousepox is dependent on a strong polarized type 1 immune response, associated with robust natural killer (NK) cell, cytotoxic T lymphocyte (CTL) and gamma interferon (IFN-γ) responses. In contrast, ECTV-susceptible mice generate a type 2 response, associated with weak NK cell, CTL and IFN-γ responses but robust IL-4 responses. Nonetheless, susceptible strains infected with mutant ECTV lacking virus-encoded IFN-γ binding protein (vIFN-γbp) (ECTV-IFN-γbp^Δ) control virus replication through generation of type 1 response. Since the IL-4/IL-13/STAT-6 signaling pathways polarize type 2/T helper 2 (Th2) responses with a corresponding suppression of IFN-γ production, we investigated whether the combined absence of vIFN-γbp, and one or more host genes involved in Th2 response development, influence generation of protective immunity. Most mutant mouse strains infected with wild-type (WT) virus succumbed to disease more rapidly than WT animals. Conversely, the disease outcome was significantly improved in WT mice infected with ECTV-IFN-γbp^Δ but absence of IL-4/IL-13/STAT-6 signaling pathways did not provide any added advantage. Deficiency in IL-13 or STAT-6 resulted in defective CTL responses, higher mortality rates and accelerated deaths. Deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells, indicating an absence of a switch to a Th1-like response. Factors contributing to susceptibility or resistance to mousepox are far more complex than a balance between Th1 and Th2 responses.</p></div

Response of WT and GKO BALB/c strains to infection with ECTV-WT or ECTV-IFN-γbp^Δ.

<p>Groups of female mice were infected with ECTV-WT or ECTV-IFN-γbp^Δ. Mice were monitored daily for 21 days for disease signs. Data shown are combined results obtained from two separate experiments in which 5–15 mice per strain were used (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118685#pone.0118685.s008" target="_blank">S2 Table</a>). <i>P</i> values for survival proportions were obtained by using Kaplan-Meier Log rank statistical test: **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001; ****, <i>p</i> < 0.0001. The survival rates of WT mice compared to GKO mice infected with WT or mutant virus are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118685#pone.0118685.s005" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118685#pone.0118685.s006" target="_blank">S6</a> Figs., respectively and the statistical analysis presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118685#pone.0118685.s008" target="_blank">S2 Table</a>.</p

Numbers of CD4 T cells expressing IFN-γ, IL-4, T-bet or GATA-3.

<p>Mice (n = 5–10/group) were infected with ECTV-WT or ECTV-IFN-γbp^Δ, sacrificed on day 7 p.i. and splenocytes used for intracellular staining for IFN-γ, IL-4, T-bet or GATA-3 without stimulation (A and B) or with PMA + ionomycin stimulation (C and D). Shown are means of absolute numbers of IFN-γ⁺CD4 T cells with no stimulation (A) or PMA + ionomycin stimulation (C), IL-4⁺CD4 T cells with no stimulation (B) or PMA + ionomycin stimulation (D). Also shown are means of absolute numbers of T-bet⁺ and GATA-3⁺ CD4 T cells following ECTV-WT (E) or ECTV-IFN-γbp^Δ (F) infection. Two-way ANOVA followed by Fisher’s LSD test for significance was used. For panel A, numbers of IFN-γ⁺CD4 T cells in WT mice for both viruses were significantly higher (p<0.0001) compared to numbers in all GKO strains. In IL-4^−/− and STAT-6^−/− mice, IFN-γ⁺CD4 T cell numbers generated by ECTV-IFN-γbp^Δ infection were significantly higher (<i>p</i> = 0.0005 and <i>p</i> = 0.0013, respectively) compared to WT virus infection. In IL-13^−/− mice, IFN-γ⁺CD4 T cell numbers generated by WT virus infection were significantly higher (<i>p</i> = 0.0034) compared to ECTV-IFN-γbp^Δ infection. For panel B, numbers of IL-4⁺CD4 T cells in WT mice for both viruses were significantly higher (p<0.01) compared to numbers in all GKO strains. No other significant differences were found. For panel C, numbers of IFN-γ⁺CD4 T cells in WT mice for both viruses were significantly higher (p<0.0001 in ECTV-WT and <i>p</i> < 0.001 in ECTV-IFN-γbp^Δ) compared to numbers in all GKO strains. In IL-4^−/−, STAT-6^−/− IL-4Rα^−/− and IL-13^−/− / IL-4Rα^−/− mice, IFN-γ⁺CD4 T cell numbers generated by ECTV-IFN-γbp^Δ infection were significantly higher compared to WT virus infection. For panel D, numbers of IL-4⁺CD4 T cells in WT mice for ECTV-WT virus was significantly higher (p<0.05) compared to numbers in all GKO strains. In WT mice, IL-4⁺CD4 T cell numbers generated by WT virus infection were significantly higher (<i>p<</i>0.05) compared to ECTV-IFN-γbp^Δ infection. For panel E, T-bet⁺ CD4 T cell numbers were significantly increased (p<0.01) in STAT-6^−/− and IL-13^−/− mice compared to WT animals. For panel F, no significant differences were found between strains. Data shown for panels A and B are from one of two independent experiments with similar results. Data shown for panels C-F are from one experiment. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.0001.</p

Viral load in organs and blood of WT and GKO mice infected with ECTV-WT or ECTV-IFN-γbp^Δ.

<p>Groups of 5 mice were infected with either ECTV-WT or ECTV-IFN-γbp^Δ and sacrificed at day 7 p.i. Viral load was determined by viral plaque assay and expressed as Log₁₀ PFU virus/gram tissue. The dotted line indicates the limit of virus detection by viral plaque assay. Two-way ANOVA followed by Fisher’s LSD test for significance between (i) ECTV-WT titers in WT mice compared with all strains, (ii) ECTV-IFN-γbp^Δ load in WT mice compared with all strains, and (iii) ECTV-WT compared with ECTV-IFN-γbp^Δ load in individual strains (A) liver, (B) spleen, and (C) blood. The detailed statistical analysis of WT and mutant virus titers in each of the 5 GKO mouse strains is presented in S3 (Liver), S4 (Spleen) and S5 (Blood) Tables. Asterisks indicate significant differences in organ titers comparing ECTV-WT or ECTV-IFN-γbp^Δ. ****, p< 0.0001; ***, 0.0001< p <0.001; **, 0.001< p <0.01; * 0.01< p <0.05.</p

CD8 T cell responses to ECTV-WT and ECTV-IFN-γbp^Δ.

<p>Mice (n = 5–10/group) were infected with ECTV-WT or ECTV-IFN-γbp and sacrificed 7 days later to measure splenic CTL activity. Shown is % specific lysis of ⁵¹Cr-labelled, ECTV-infected P815 target cells by splenocytes from (A) ECTV-WT- or (B) ECTV-IFN-γbp^Δ-infected WT and GKO mice. (C) Percent specific lysis of targets by splenocytes at 75:1 effector-to-target ratio. Two-way ANOVA followed by Fisher’s LSD test for significance was used for statistical analysis: (i) CTL response to ECTV-WT vs. ECTV-IFN-γbp^Δ in IL-13^−/− (p < 0.0001) and STAT-6^−/− (p < 0.0001) (ii) CTL response to ECTV-WT in WT vs IL-4^−/− (p = 0.0083), vs STAT-6^−/− (p = 0.0003), vs IL-13^−/− (p = 0.0001), vs IL-13^−/−/IL-4Rα^−/− (p = 0.0037) mice; (iii) CTL response to ECTV-IFN-γbp in WT vs STAT-6^−/− (p = 0.0002); vs IL-13^−/− (p = 0.0478); vs IL-4Rα^−/− (p = 0.0174) and IL-13^−/−/IL-4Rα^−/− (p = 0.000046) mice. ****, p<0.0001. (D—F) Spleen cells from mice infected mice were re-stimulated for 5 h with ECTV-infected P815 cells or H-2^d 8 T cell peptide determinants prior to intracellular IFN-γ staining followed by flow cytometry. Data shown are means of absolute numbers of ECTV-specific IFN-γ⁺ 8 T cells in spleens of ECTV-WT- vs. ECTV-IFN-γbp^<b>Δ</b>-infected mice (D) and determinant-specific IFN-γ⁺ 8 T cells following infection with ECTV-WT (E) or ECTV-IFN-γbp (F) viruses. Two-way ANOVA followed by Fisher’s LSD test for significance was used for statistical analysis. For panels D, E and F, IFN-γ⁺ 8 T cell numbers in WT mice were significantly higher (p<0.0001) than numbers in all GKO strains. Data shown for D-F are representative of one of three independent experiments with similar results. *, <i>p</i>< 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.0001.</p

Poxvirus CD8(+) T-Cell Determinants and Cross-Reactivity in BALB/c Mice

Mouse models of orthopoxvirus disease provide great promise for probing basic questions regarding host responses to this group of pathogens, which includes the causative agents of monkeypox and smallpox. However, some essential tools for their study that are taken for granted with other mouse models are not available for these viruses. Here we map and characterize the initial CD8(+) T-cell determinants for poxviruses in H-2(d)-haplotype mice. CD8(+) T cells recognizing these three determinants make up around 40% of the total responses to vaccinia virus during and after resolution of infection. We then use these determinants to test if predicted conservation across orthopoxvirus species matches experimental observation and find an unexpectedly cross-reactive variant peptide encoded by ectromelia (mousepox) virus

Evidence for Persistence of Ectromelia Virus in Inbred Mice, Recrudescence Following Immunosuppression and Transmission to Naïve Mice

<div><p>Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status.</p></div