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A Purified tonsil CD4 + T cells (~91% purity) or CD4 + CD25 + CD127 low T reg cells (>88% FOXP3 + ) were TCR activated and infected with HIV as described in “Methods.” GFP was assessed in FOXP3 + (left) or FOXP3 − (right; gated on Foxp3 neg cells) fractions 48 h post-infection. Representative flow cytometric data (left) and statistical analyses from three independent tonsil donors (right) are shown. Mean +/− SEM are shown; * P < 0.05; **<0.005, ****<0.00005; two-tailed; Mann–Whitney test. B – F TCR activated whole human tonsil cultures (HTC) were infected with HIV and allowed to expand with IL-2 for 6 days. Flow cytometric analyses of CD3 + FOXP3 + cells pre-gated on CD8 − cells ( B ), viability of CD3 + CD8 − FOXP3 + cells ( C <t>),</t> <t>PD-1</t> and IFN-γ expression in viable CD3 + FOXP3 + CD8 − cells ( D ), with respective statistical analyses (right) are shown. B – D n = 5; * P < 0.05; ** P = 0.007; two-tailed; Mann–Whitney test. E , F Flow cytometric plots showing the expression of the indicated proteins in PD-1 high and PD-1 low populations gated in D in HIV-infected HTC. B – F Five independent experiments showed similar results.
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A Purified tonsil CD4 + T cells (~91% purity) or CD4 + CD25 + CD127 low T reg cells (>88% FOXP3 + ) were TCR activated and infected with HIV as described in “Methods.” GFP was assessed in FOXP3 + (left) or FOXP3 − (right; gated on Foxp3 neg cells) fractions 48 h post-infection. Representative flow cytometric data (left) and statistical analyses from three independent tonsil donors (right) are shown. Mean +/− SEM are shown; * P < 0.05; **<0.005, ****<0.00005; two-tailed; Mann–Whitney test. B – F TCR activated whole human tonsil cultures (HTC) were infected with HIV and allowed to expand with IL-2 for 6 days. Flow cytometric analyses of CD3 + FOXP3 + cells pre-gated on CD8 − cells ( B ), viability of CD3 + CD8 − FOXP3 + cells ( C ), PD-1 and IFN-γ expression in viable CD3 + FOXP3 + CD8 − cells ( D ), with respective statistical analyses (right) are shown. B – D n = 5; * P < 0.05; ** P = 0.007; two-tailed; Mann–Whitney test. E , F Flow cytometric plots showing the expression of the indicated proteins in PD-1 high and PD-1 low populations gated in D in HIV-infected HTC. B – F Five independent experiments showed similar results.

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: A Purified tonsil CD4 + T cells (~91% purity) or CD4 + CD25 + CD127 low T reg cells (>88% FOXP3 + ) were TCR activated and infected with HIV as described in “Methods.” GFP was assessed in FOXP3 + (left) or FOXP3 − (right; gated on Foxp3 neg cells) fractions 48 h post-infection. Representative flow cytometric data (left) and statistical analyses from three independent tonsil donors (right) are shown. Mean +/− SEM are shown; * P < 0.05; **<0.005, ****<0.00005; two-tailed; Mann–Whitney test. B – F TCR activated whole human tonsil cultures (HTC) were infected with HIV and allowed to expand with IL-2 for 6 days. Flow cytometric analyses of CD3 + FOXP3 + cells pre-gated on CD8 − cells ( B ), viability of CD3 + CD8 − FOXP3 + cells ( C ), PD-1 and IFN-γ expression in viable CD3 + FOXP3 + CD8 − cells ( D ), with respective statistical analyses (right) are shown. B – D n = 5; * P < 0.05; ** P = 0.007; two-tailed; Mann–Whitney test. E , F Flow cytometric plots showing the expression of the indicated proteins in PD-1 high and PD-1 low populations gated in D in HIV-infected HTC. B – F Five independent experiments showed similar results.

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Purification, Infection, Two Tailed Test, MANN-WHITNEY, Expressing

Whole HTC was activated with TCR stimulation, infected with HIV, and allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days. Viral inhibitor Efavirenz (50 nM) or cell death/pan-caspase inhibitor z-VAD (10 μM) was added 28 h post-infection as described in “Methods.” Flow cytometry acquisition was done with constant time for all the samples. Percentage of CD3 + FOXP3 + cells pre-gated on CD8 − cells ( A ), viability of FOXP3 + cells pre-gated on CD3 + CD8 − cells ( B ), PD-1 and IFN-γ expression in viable CD3 + CD8 − FOXP3 + cells ( C ), Ki-67 expression in viable PD-1 high and PD-1 low FOXP3 + populations ( D ) are shown. A – D Representative contour plots (left), statistical analyses of proportions of the cells (middle), and statistical analyses of the absolute cell counts (right) are shown (ordinary one-way ANOVA alpha = 0.05* and multiple comparison t tests; * P < 0.05; **<0.005, ***<0.0005). Results are derived from three independent experiments ( n = 3) and are presented as mean value +/− SEM.

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: Whole HTC was activated with TCR stimulation, infected with HIV, and allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days. Viral inhibitor Efavirenz (50 nM) or cell death/pan-caspase inhibitor z-VAD (10 μM) was added 28 h post-infection as described in “Methods.” Flow cytometry acquisition was done with constant time for all the samples. Percentage of CD3 + FOXP3 + cells pre-gated on CD8 − cells ( A ), viability of FOXP3 + cells pre-gated on CD3 + CD8 − cells ( B ), PD-1 and IFN-γ expression in viable CD3 + CD8 − FOXP3 + cells ( C ), Ki-67 expression in viable PD-1 high and PD-1 low FOXP3 + populations ( D ) are shown. A – D Representative contour plots (left), statistical analyses of proportions of the cells (middle), and statistical analyses of the absolute cell counts (right) are shown (ordinary one-way ANOVA alpha = 0.05* and multiple comparison t tests; * P < 0.05; **<0.005, ***<0.0005). Results are derived from three independent experiments ( n = 3) and are presented as mean value +/− SEM.

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Infection, Flow Cytometry, Expressing, Derivative Assay

Purified tonsil CD4 + T cells (~93% purity) were TCR activated, infected with HIV, and allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days post-infection unless otherwise noted. Efavirenz (50 nM), LPS (10 μg/ml), PG-LPS (5 μg/ml), HKGT (10 6 /ml), IL-1β (20 ng/ml), IL-33 (20 ng/ml), Anakinra (10 μg/ml), LY294002 (10 μM), and Nigericin (10 nM) were added as indicated, 36 h post infection. A PD-1 and IFN-γ (left) and AREG (right) expression in FOXP3 + cells. B ELISA quantification of IL-1β (left) and AREG (right) in cell culture supernatants collected on day 3 post infection. p-Akt ( C ) and AREG ( D ) expression in FOXP3 + cells. E Percentage and absolute cell numbers of PD-1 hi IFN-γ + FOXP3 + cells in CD4 + population. F AREG expression in FOXP3 + cells (left) and ELISA quantification of AREG (right), 6 days post infection. A – F Data are representative of three independent experiments and are presented as mean value +/− SEM. (* P < 0.05; **<0.005, ***<0.0005, ****<0.00005; unpaired t tests). Source data are provided as a Source data file.

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: Purified tonsil CD4 + T cells (~93% purity) were TCR activated, infected with HIV, and allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days post-infection unless otherwise noted. Efavirenz (50 nM), LPS (10 μg/ml), PG-LPS (5 μg/ml), HKGT (10 6 /ml), IL-1β (20 ng/ml), IL-33 (20 ng/ml), Anakinra (10 μg/ml), LY294002 (10 μM), and Nigericin (10 nM) were added as indicated, 36 h post infection. A PD-1 and IFN-γ (left) and AREG (right) expression in FOXP3 + cells. B ELISA quantification of IL-1β (left) and AREG (right) in cell culture supernatants collected on day 3 post infection. p-Akt ( C ) and AREG ( D ) expression in FOXP3 + cells. E Percentage and absolute cell numbers of PD-1 hi IFN-γ + FOXP3 + cells in CD4 + population. F AREG expression in FOXP3 + cells (left) and ELISA quantification of AREG (right), 6 days post infection. A – F Data are representative of three independent experiments and are presented as mean value +/− SEM. (* P < 0.05; **<0.005, ***<0.0005, ****<0.00005; unpaired t tests). Source data are provided as a Source data file.

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Purification, Infection, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

CD4 + T cells were stimulated as in Fig. . A , B AEP, pAEP, FOXP3, BCL-2, and Ki-67 staining in PD-1 high FOXP3 + (blue) and PD-1 low FOXP3 + cells 6 days post-infection. Some CD4 + T cells stimulated and infected as above were moved to a plate coated with recombinant human PD-L1/B7-H1 Fc chimera (5 μg/ml) or treated with AEP inhibitor (10 μM) 36 h after infection. Percentage of FOXP3 + cells in CD4 + population and FOXP3 MFI on FOXP3 + gated cells ( C ), percentage and absolute cell numbers of PD-1 hi IFN-γ + cells in FOXP3 + population ( D ), and AREG expression in FOXP3 + cells ( E ), as determined by flow cytometric analyses. Results represent triplicate experiments with similar results and are presented as mean value +/− SEM (ordinary one-way ANOVA and multiple t tests were conducted; * P < 0.05; **<0.005, ***<0.0005).

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: CD4 + T cells were stimulated as in Fig. . A , B AEP, pAEP, FOXP3, BCL-2, and Ki-67 staining in PD-1 high FOXP3 + (blue) and PD-1 low FOXP3 + cells 6 days post-infection. Some CD4 + T cells stimulated and infected as above were moved to a plate coated with recombinant human PD-L1/B7-H1 Fc chimera (5 μg/ml) or treated with AEP inhibitor (10 μM) 36 h after infection. Percentage of FOXP3 + cells in CD4 + population and FOXP3 MFI on FOXP3 + gated cells ( C ), percentage and absolute cell numbers of PD-1 hi IFN-γ + cells in FOXP3 + population ( D ), and AREG expression in FOXP3 + cells ( E ), as determined by flow cytometric analyses. Results represent triplicate experiments with similar results and are presented as mean value +/− SEM (ordinary one-way ANOVA and multiple t tests were conducted; * P < 0.05; **<0.005, ***<0.0005).

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Staining, Infection, Recombinant, Expressing

Purified CD4 + T cells and T regs were stimulated and infected as in “Methods.” A CD25 and FOXP3 expression in all cells in the cultures (above) and PD-1 and IFN-γ expression in CD25 + FOXP3 + cells (below) at 96 h post infection. B Statistical analyses of PD-1 hi IFN-γ + cells in FOXP3 + population from these two cultures. C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1. As controls, T resp cells were cultured alone or co-cultured with purified naive CD127 low CD25 + T regs that were sequentially sorted to remove CD45RO + CD4 + cells using human CD45RO kit (Miltenyi) and purify CD25 high T reg cells using STEMCELL technology CD25 + T reg isolation kits. These control T regs were also previously stimulated and infected the same manner (purple) before co-culture with T resp . T resp proliferation was determined by cell-trace dye dilution in PD-1 hi CD25 + co-culture (blue), control T reg co-cultures (purple), or those cultured alone (green). D Statistical analyses of % T resp suppression mean values from three independent experiments showing similar results. B , D Mean +/− SEM are shown; * P < 0.05; **<0.005, ****<0.00005; two-tailed; Mann–Whitney test).

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: Purified CD4 + T cells and T regs were stimulated and infected as in “Methods.” A CD25 and FOXP3 expression in all cells in the cultures (above) and PD-1 and IFN-γ expression in CD25 + FOXP3 + cells (below) at 96 h post infection. B Statistical analyses of PD-1 hi IFN-γ + cells in FOXP3 + population from these two cultures. C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1. As controls, T resp cells were cultured alone or co-cultured with purified naive CD127 low CD25 + T regs that were sequentially sorted to remove CD45RO + CD4 + cells using human CD45RO kit (Miltenyi) and purify CD25 high T reg cells using STEMCELL technology CD25 + T reg isolation kits. These control T regs were also previously stimulated and infected the same manner (purple) before co-culture with T resp . T resp proliferation was determined by cell-trace dye dilution in PD-1 hi CD25 + co-culture (blue), control T reg co-cultures (purple), or those cultured alone (green). D Statistical analyses of % T resp suppression mean values from three independent experiments showing similar results. B , D Mean +/− SEM are shown; * P < 0.05; **<0.005, ****<0.00005; two-tailed; Mann–Whitney test).

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Purification, Infection, Expressing, Isolation, Labeling, Cell Culture, Co-Culture Assay, Two Tailed Test, MANN-WHITNEY

HOILs from gingival mucosa from healthy controls and HIV + patients on cART were processed for flow cytometry ex vivo. A FOXP3 expression in CD3 + CD4 + gated HOIL cells. PD-1 and IFN-γ ( B ), IL-10 ( C ), AREG ( D ), and BCL-2 ( E ) expression in FOXP3 + population. Statistical analyses and comparison between the groups for % PD-1 hi IFN-γ + cells ( n = 20; ** P = 0.0025) ( F ), % PD-1 hi cells ( n = 35; ** P = 0.005) ( G ), % IL-10 + cells ( n = 22; * P = 0.024) ( H ), AREG expression ( n = 22; *** P = 0.0002) ( I ), and BCL-2 expression ( n = 12; ** P = 0.0022) ( J ) in FOXP3 + population. K ELISA quantification of AREG levels in saliva ( n = 78; * P < 0.03). L , M Correlation of % PD-1 hi CD25 + cells in FOXP3 + population ( L ) and salivary AREG ( M ), with effector CD4 hyperactivation (% CD38 + HLADR + in FOXP3 neg CD4 + T cells in gingival mucosa; Fig. ; n = 20). F – K P values two-tailed; Mann–Whitney test; each data point represents a study participant, and the data are presented as mean value +/− SEM. Source data are provided as a Source data file.

Journal: Nature Communications

Article Title: Oral immune dysfunction is associated with the expansion of FOXP3 + PD-1 + Amphiregulin + T cells during HIV infection

doi: 10.1038/s41467-021-25340-w

Figure Lengend Snippet: HOILs from gingival mucosa from healthy controls and HIV + patients on cART were processed for flow cytometry ex vivo. A FOXP3 expression in CD3 + CD4 + gated HOIL cells. PD-1 and IFN-γ ( B ), IL-10 ( C ), AREG ( D ), and BCL-2 ( E ) expression in FOXP3 + population. Statistical analyses and comparison between the groups for % PD-1 hi IFN-γ + cells ( n = 20; ** P = 0.0025) ( F ), % PD-1 hi cells ( n = 35; ** P = 0.005) ( G ), % IL-10 + cells ( n = 22; * P = 0.024) ( H ), AREG expression ( n = 22; *** P = 0.0002) ( I ), and BCL-2 expression ( n = 12; ** P = 0.0022) ( J ) in FOXP3 + population. K ELISA quantification of AREG levels in saliva ( n = 78; * P < 0.03). L , M Correlation of % PD-1 hi CD25 + cells in FOXP3 + population ( L ) and salivary AREG ( M ), with effector CD4 hyperactivation (% CD38 + HLADR + in FOXP3 neg CD4 + T cells in gingival mucosa; Fig. ; n = 20). F – K P values two-tailed; Mann–Whitney test; each data point represents a study participant, and the data are presented as mean value +/− SEM. Source data are provided as a Source data file.

Article Snippet: C PD-1 hi CD25 + cells were purified from HIV-infected CD4 cultures using sequential sorting of PD-1-PE + cells and CD25 high T reg cells using STEMCELL technology PE isolation and CD25 + T reg isolation kits and were used in co-cultures with cell-trace violet-labeled responder T cells (T resp ) at ratio 1:1.

Techniques: Flow Cytometry, Ex Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY