cHG+abx interference of bacterial biofilms.

(a) Visualization of bacterial biofilms after 12 hours of topical treatment, compared to a no-treatment control. (b) Quantification of bacterial density stained with SYTO-61 in developed biofilms over 12 hours of topical treatment. (c) Absorbance (595 nm) of crystal violet solution from biofilms treated with cHG+abx pre-eluted for various timepoints, compared to control. One-way ANOVA followed by Tukey’s test was used to determine significance for both (b) and (c). * indicates p-value <0.05, ** indicates p-value < 0.01.</p

Tregs limit T-lymphocyte activation within MCMV-infected brains.

<p>Activation of CD8⁺ and CD4⁺ T-cells was evaluated based on flow cytometric analysis using PE-Cy5-labeled inducible co-stimulatory molecule (ICOS) Abs. (A) Representative contour plots show the percentage of CD8⁺ T-cells expressing the activation marker ICOS from infected, untreated, as well as DTx-treated animals at the indicated time points. (B) Data presented show the mean fluorescent intensity (MFI) of ICOS expression on CD8⁺ T-cells (mean ±SD) between groups. **p <0.001 DTx- versus DTx+ MCMV-infected at 7 & 30 dpi and ***p<0.0002 at 14 dpi. (C) Pooled data presents the mean fluorescent intensity (MFI) of ICOS expression on CD4⁺ T-cells (mean ±SD) between groups. **p <0.001 DTx- versus DTx+ MCMV-infected at 14 & 30 dpi. (D) The number of ICOS⁺CD8⁺ T-cells in infected brains with and without DTx treatment at the indicated time points is shown. (E) The number of ICOS⁺CD4⁺ T-cells in infected brains with and without DTx treatment at the indicated time points is shown. **p < 0.001 DTx- versus DTx+ animals</p

cHG+abx efficacy in an ex vivo fibroblast (FB) scratch model.

(a) MRSA-colonized FB scratch assay imaged over 48 hours. Non-infected FBs treated with cHG alone (2nd row) show similar growth compared to control (1st row, untreated, non-infected FBs). FBs infected with MRSA without treatment (4th row) showed significant FB death compared to infected FBs treated with cHG + abx (3rd row). (b) Quantification of cell density within scratches of the modified scratch assay over 48 hours of migration. Two-way ANOVA with Tukey’s post-hoc test was used to establish statistical significance between experimental groups. * indicates p-value < 0.05, ** indicates p-value < 0.01, **** indicates p-values < 0.0001.</p

Tregs limit IFN-γ production by T lymphocytes within MCMV-infected brains.

<p>Single cell suspension of brain tissue obtained from MCMV-infected DTx-treated and untreated mice (2–4 animals per time point) were banded on a 70% percoll cushion. Brain leukocytes at the 30–70% percoll interface were collected. For intracellular IFN-γ staining, brain leukocytes (2 x 10⁶ cells/ml) were pulsed with either anti-CD3/CD28 antibodies or with a MCMV-specific, MHC class 1-restricted M45 peptide (6 h at 37°C) and treated with Brefeldin A. After incubation, cells were washed in FACS buffer and stained for the surface molecules CD45, CD8, and for intracellular IFN-γ using a Cytofix/Cytoperm kit (BD Pharmingen), before flow cytometry. (<b>A</b>) Representative plots shows the ratio of CD8⁺ T lymphocytes producing IFN-γ among the DTx-treated (+DTx) and untreated (-DTx) groups at 7,14, & 30 dpi in response to peptide treatment. (B) Pooled data show the (mean ±SD) of CD8⁺ T-cells producing IFN-γ at the indicated time points from 2 independent experiments. **p < 0.001 DTx- versus DTx+ MCMV-infected at 7, 14, & 30 dpi.</p

Tregs attenuate TNF-α production by T lymphocytes within MCMV-infected brains.

<p>For intracellular staining of TNF-α, brain leukocytes (2 x 10⁶ cells/ml) were pulsed with either anti-CD3/CD28 antibodies or with an MCMV-specific, MHC class 1-restricted M45 peptide (6 h at 37°C) and treated with Brefeldin A. After incubation, cells were washed in FACS buffer and stained for the surface molecules CD45, CD8, and for intracellular staining of TNF-α using a Cytofix/Cytoperm kit (BD Pharmingen), before flow cytometry. (A) Representative plots show the ratio of CD8⁺ T lymphocytes producing TNF-α among the DTx-treated (+DTx) and untreated (-DTx) groups at 7,14, & 30 dpi in response to peptide treatment. (B) Pooled data show the (mean ±SD) of CD8⁺ T-cells producing TNF-α at the indicated time points from 2 independent experiments. **p < 0.001 DTx- versus DTx+ MCMV-infected at 14 dpi and 30 dpi.</p

Treg depletion results in an increased number of CD8⁺T-cells displaying dual cytokine production.

<p>Pooled data show absolute numbers of CD8⁺ T-cells producing both IFN-γ and TNF-α among the DTx-treated (+DTx) and untreated (-DTx) groups at 7,14,and 30 dpi in response to peptide stimulation. Data are from 2 independent experiments using 2–4 animals per treatment group/time points. **p < 0.001 DTx- versus DTx+ MCMV-infected at 7, 14, & 30dpi</p

Modified Kirby-Bauer assay.

(a) Representative images of the zone of inhibition (ZOI) after indicated hours of pre-elution. (b) Quantification of bacterial ZOI post-treatment with cHG+abx pre-eluted for up to 60 hours, compared to a no-treatment control. One-way ANOVA followed by Tukey’s test was used to establish significance. * indicates p-value < 0.05, ** indicates p-value < 0.01.</p

Discrimination between brain parenchyma-localized and vasculature-localized lymphocyte proliferation.

<p>Foxp3-DTR, MCMV-infected, DTx-treated and untreated mice at 14 dpi were injected intravenous with anti-CD8α-PE and anti-CD4-FITC mAb. Lymphocytes were isolated and stained <i>ex-vivo</i> for the anti-CD8 β-AF647 and anti-CD4-AF700 using different clones, as described in the methods. Plots are representative of two experiments using three animals per groups. (A) Contour plots show CD8⁺ T-cells in the vasculature that stained both for anti-CD8α-PE and anti-CD8β-AF647; while tissue lymphocytes were stained by anti-CD8β-AF647 alone in both DTx-treated and untreated groups. (B) Contour plots show proliferation of CD8⁺ T-cells both within the tissue and in the vasculature. <b>C.</b> Contour plots represent proliferation of CD4⁺ T-cells both in tissue and vasculature. (D) The number of parenchyma-localized CD8⁺ T-cells within MCMV-infected brains of animals with and without DTx treatment is shown. (E) The number of parenchymal CD4⁺ T-cells with and without DTx treatment is shown. **p < 0.001 DTx- versus DTx+ animals</p

<i>In vitro</i> blocking effect of PD-1-PD-L1 interactions on the effector T cell functions and survival among PTB patients.

<p>PBMCs from tuberculosis patients were stimulated with <i>Mtb.</i> antigen (WCL) in the presence or absence of anti-PD-1 mAb, anti-PD-L1 and anti-PD-1+PD-L1 mAb for 72 hrs followed by intracellular detection of IFN-γ by flow cytometry. Lymphocytes were gated based on their scatter (FSC/SSC) profile and further gated on CD3⁺ T cells. (<b>A</b>) Representative FACS plots shows production of IFN-γ by CD3⁺ T cells which markedly increases upon blocking PD-1-PD-L1 interaction (<b>B</b>) Each histogram depicts the effect of PD-1, PD-L1 blocking on the percentage of IFN-γ⁺ CD3⁺ T cells (mean ± S.E.M. for n = 8). (<b>C</b>) Enriched Treg cells (Foxp3⁺) were obtained by magnetic sorting of CD25⁺ cells. These cells were treated with anti-PD-1+PD-L1 blocking antibody and subsequently added to T cells enriched population in ratio of 1∶2 (Treg Vs Teff) with or without <i>Mtb.</i> antigen (WCL). Blocking PD-1 and PD-L1 markedly increases the frequency of IFN-γ and IL-2 producing T cells (I, II and III). Data is representative of three individual experiments. (<b>D</b>) <i>In vitro</i> blocking effect of PD-1-PD-L1 interactions on survival of effector T cells. PBMCs from PTB patients were stimulated with <i>Mtb.</i> antigen (WCL) for 72 hrs in the presence or absence of anti-PD-1 mAb, anti-PD-L1 and anti-PD-1+PD-L1 mAb and intracellular IFN-γ expression was determined by flow cytometry. Further annexin V and caspase-3 positivity was determined on effector T cells (IFN-γ⁺ CD3⁺). Blocking PD-1-PD-L1 interactions significantly decreases the apoptosis of <i>Mtb.</i> specific IFN-γ producing T cells as determined by expression of annexinV and caspase-3. (<b>E</b>) The pooled data represents the mean ± S.D. performed on 3 PTB patients in duplicates.** P≥0.001 and *** P≥0.0001.</p

Overview of human-derived collagen hydrogel preparation and properties.

(a) Complete process of cHG+abx fabrication. (b) SEM image of the surface of crosslinked hydrogel. (c) Pharmacodynamics of clindamycin elution from cHG over a 72-hour period measured by LCMS. Panel A created with BioRender.com.</p