Journal: Mucosal Immunology

Article Title: Vaccine protection by Cryptococcus neoformans Δ sgl1 is mediated by γδ T cells via TLR2 signaling

doi: 10.1038/s41385-022-00570-3

Figure Lengend Snippet: CBA/J mice ( n = 6 mice/group/timepoint) were administered isotype ( a – d ), anti-CD8 ( e , f ), or anti-CD4 ( g , h ) antibodies. Mice then received either C. neoformans Δ sgl1 (white symbols) or PBS (black symbols). After 30 days, mice were challenged with C. neoformans WT (day 0). On days −15, −1, 7, 15, and 24, mice were assessed for T cell-derived cytokines (IFNγ, IL-17A) via intracellular cytokine staining. Graphed data represents the mean ± SD from 2 independent experiments ( n = 3 mice/group/timepoint for each biological replicate). Representative flow cytometry plots for days −1 and 7 for each group are shown, which were gated from live, CD45 + singlets (gating scheme shown in Supplementary Fig. ). Significance was determined by a two-way ANOVA using Šídák’s multiple comparisons test for P value adjustment, and significance is denoted as * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.001.

Article Snippet: All antibodies for depletion of specific cell populations or neutralization of specific cytokines were purchased from BioXCell.

Techniques: Derivative Assay, Staining, Flow Cytometry

Journal: Mucosal Immunology

Article Title: Vaccine protection by Cryptococcus neoformans Δ sgl1 is mediated by γδ T cells via TLR2 signaling

doi: 10.1038/s41385-022-00570-3

Figure Lengend Snippet: CBA/J mice ( n = 8–10 mice/group) were neutralized of specific cytokines and/or cell types (as indicated in the figure legends) prior to vaccination with C. neoformans Δ sgl1 (white symbols) or PBS controls (black symbols), and neutralizations continued for the entirety of the experiment at noted intervals. a , b . Vaccinated and unvaccinated mice were challenged with C. neoformans WT strain and assessed for survival during specific cytokine neutralization ( a ) or cytokine neutralization during either CD4 or CD8 T cell deficiency ( b ). Survival significance was determined by the Mantel–Cox log-rank test, and denoted on each graph: a . # P < 0.001 for Δ sgl1 /Isotype → WT vs. Δ sgl1 /anti-IL-17A → WT; b . % P < 0.001 for Δ sgl1 /Isotype → WT vs. Δ sgl1 /anti-CD4 + anti-IL-17A → WT or Δ sgl1 /anti-CD8 + anti-IL-17A → WT.

Article Snippet: All antibodies for depletion of specific cell populations or neutralization of specific cytokines were purchased from BioXCell.

Techniques: Neutralization

Journal: Mucosal Immunology

Article Title: Vaccine protection by Cryptococcus neoformans Δ sgl1 is mediated by γδ T cells via TLR2 signaling

doi: 10.1038/s41385-022-00570-3

Figure Lengend Snippet: C57BL/6 (black symbols) or TLR2 −/− (white symbols) were administered C. neoformans Δ sgl1 and assessed for T cell-derived cytokines via intracellular cytokine stimulation on days −30 (uninfected; a ), −27 ( b ), and −23 ( c ). 30 days after C. neoformans Δ sgl1 administration, mice were challenged with C. neoformans WT and assessed for T cell-derived cytokines via intracellular cytokine stimulation on days −1 (unchallenged; d ), 3 ( e ), and 7 ( f ). At all timepoints, mice were assessed for the total number of γδ T, CD4 + , and CD8 + T cells as well as the number of IFNγ- or IL-17A-producing subsets of these cells. Graphed data represent the mean ± SD and are representative of 1–2 independent experiments ( n = 3–6 mice/timepoint/group). Significance was determined by a two-way ANOVA using Šídák’s multiple comparisons test for P value adjustment, and denoted as * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001.

Article Snippet: All antibodies for depletion of specific cell populations or neutralization of specific cytokines were purchased from BioXCell.

Techniques: Derivative Assay

Journal: Mucosal Immunology

Article Title: Vaccine protection by Cryptococcus neoformans Δ sgl1 is mediated by γδ T cells via TLR2 signaling

doi: 10.1038/s41385-022-00570-3

Figure Lengend Snippet: Upon intranasal administration of C. neoformans Δ sgl1 , the yeast cells travel down the bronchioles into the alveoli (1) encountering resident alveolar macrophages (AM), dendritic cells (DCs), and γδ T cells. Both sterylglucosides (SGs) and the glucuronoxylomannan (GXM)-rich capsule of C. neoformans Δ sgl1 can be found on the surface of yeast cells or/and in the extracellular environment. γδ T cells recognize and respond directly to C. neoformans Δ sgl1 via a toll-like receptor 2 (TLR2)-directed mechanism (2) producing the host-protective cytokines IFNγ and IL-17A leading to a pro-inflammatory lung cytokine environment with augmented leukocyte recruitment to the lungs (3) including neutrophils and monocytes that mediate early host control by limiting C. neoformans Δ sgl1 replication. Antigen-presenting cells (APCs) become activated, phagocytose the yeast, and travel to the lung-draining lymph node to prime naive CD4 + or CD8 + T cells when CD4 + T cells are absent (4). The naive T cells differentiate into protectively polarized IFNγ- and IL-17A-producing T cells and travel to the lungs (5) resulting in the pulmonary clearance of the mutant. While most recruited leukocytes die upon resolution of the inflammatory response, a small percentage of αβ and γδ T cells become host-protective memory T cells (6) that rapidly respond upon a subsequent WT challenge, promptly producing both IFNγ and IL-17A and conferring strong vaccine protection. Graphical illustration created with BioRender.com.

Article Snippet: All antibodies for depletion of specific cell populations or neutralization of specific cytokines were purchased from BioXCell.

Techniques: Mutagenesis

Journal:

Article Title: Dose-dependent synergy of Th1-stimulating cytokines on bacille Calmette-Gu?rin-induced interferon-? production by human mononuclear cells

doi: 10.1111/j.1365-2249.2007.03413.x

Figure Lengend Snippet: (a) Bacille Calmette–Guérin (BCG) dose-dependent interferon (IFN)-γ production by human peripheral blood mononuclear cells (PBMCs). PBMCs were incubated with medium or five different doses of BCG ranging from 2·5 × 103 to 2·5 × 106 colony-forming units (CFU)/ml for 3 days and then assayed for IFN-γ production in the conditioned media. (b) The roles of endogenous T helper 1 (Th1) and Th2 cytokines in BCG-induced IFN-γ production by human PBMCs. PBMCs were incubated with 2·5 × 105 CFU/ml of BCG in the presence or absence of indicated cytokine-neutralizing antibodies (3 µg/ml) for 3 days and then assayed for IFN-γ production in the conditioned media. Species and isotype-matched control antibodies had no effect on IFN-γ production (data not shown). *Significantly decreased.

Article Snippet: Human cytokine-neutralizing antibodies were obtained from Schering for IFN-α (sheep polyclone) and from R&D Systems (Minneapolis, MN, USA) for IL-2 (clone 5334·21, mouse IgG1), IL-10 (clone 23738·111, mouse IgG2b) and IL-12 (goat polyclone).

Techniques: Incubation