Journal:

Article Title: In Vitro-Generated Antigen-Specific CD4 + CD25 + Foxp3 + Regulatory T Cells Control the Severity of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions ▿

doi: 10.1128/JVI.00697-08

Figure Lengend Snippet: In vitro generation of Foxp3+ CD4+ T cells from OVA-specific precursor Foxp3− CD4+ T cells. (A) Splenocytes from DO11.10 RAG2−/− mice were cultured in the presence of 0.3 μg/ml of anti-CD3 antibody, 25 U of recombinant IL-2, and the indicated concentrations of TGF-β. More than 99% of CD4+ T cells were KJ1.26 positive (gated on CD4+ T cells). After 5 days of culture, cells were analyzed for the expression of CD4 and Foxp3. Under these conditions, approximately 75% of CD4+ T cells became Foxp3+. (B) A dose-response curve for Foxp3 induction with various concentrations of anti-CD3 antibody, 10 ng/ml of TGF-β, and 25 U of IL-2 is shown. A dose of 0.125 μg/ml of anti-CD3 antibody (plate bound) was found to be optimal. (C) A dose-response histogram for Foxp3 induction with various concentrations of TGF-β, 25 U of IL-2, and 0.125 μg/ml of anti-CD3 is shown. At a dose of 10 ng/ml of TGF-β, 80 to 90% of CD4+ T cells converted to become Foxp3+. (D) Representative histograms for gated CD4+ T cells are shown to show the Foxp3 induction under optimal conditions (0.125 μg/ml of plate-bound anti-CD3 antibody, 25 U of IL-2, 10 ng/ml of TGF-β). (E) SPCs from DO11.10 RAG2−/− animals were CFSE labeled and cultured with plate-bound anti-CD3, IL-2, and TGF-β for 5 days. After 5 days, cells were stained with CD4 and Foxp3. CFSE dilution and Foxp3 expression were shown in gated CD4+ T cells. TGF-β induced CD4+ CD25+ Foxp3+ T cells to proliferate extensively.

Article Snippet: Female 6- to 8-week-old BALB/c DO11.10 RAG2 −/− mice were purchased from Taconic, Thy1.2 + BALB/c and CB.17 SCID mice were purchased from Charles River, and Thy1.1 + BALB/c mice were a kind gift from D. Woodland (Trudeau Institute, Saranac Lake, NY).

Techniques: In Vitro, Cell Culture, Recombinant, Expressing, Labeling, Staining

Journal:

Article Title: In Vitro-Generated Antigen-Specific CD4 + CD25 + Foxp3 + Regulatory T Cells Control the Severity of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions ▿

doi: 10.1128/JVI.00697-08

Figure Lengend Snippet: Splenic CD11c+ DCs are intricately involved in causing conversion of Foxp3− T cells into Foxp3+ CD4+ T cells. The kinetics of CD103 expression on CD4+ Foxp3+ T cells (A) and CD11c+ DCs (B) in an in vitro conversion culture is shown. The expression of CD103 was observed earlier on DCs than on CD4+ Foxp3+ T cells. (C) CD11c+ DCs were purified from 48-h SPC cultures in the presence of anti-CD3, IL-2, and either without TGF-β as used in panel C(a) or with TGF-β as used in panels C(b) and C(d) or with TGF-β plus anti-TGF-β as used in panel C(c). These DCs were then cocultured in 1:5 ratios with anti-CD3-stimulated purified CD4+ CD25− Foxp3− T cells isolated from naïve DO11.10 RAG2−/− animals in the presence of IL-2 alone or with anti-TGF-β antibody. Shown is a representative FACS plot illustrating Foxp3 induction when the DCs were isolated from primary culture with no TGF-β [C(a), primary culture in presence of TGF-β [C(b)], or primary cultures in presence of TGF-β and anti-TGF-β and when DCs were from primary culture with TGF-β but anti-TGF-β antibody was added in secondary cultures.

Article Snippet: Female 6- to 8-week-old BALB/c DO11.10 RAG2 −/− mice were purchased from Taconic, Thy1.2 + BALB/c and CB.17 SCID mice were purchased from Charles River, and Thy1.1 + BALB/c mice were a kind gift from D. Woodland (Trudeau Institute, Saranac Lake, NY).

Techniques: Expressing, In Vitro, Purification, Isolation

Journal:

Article Title: In Vitro-Generated Antigen-Specific CD4 + CD25 + Foxp3 + Regulatory T Cells Control the Severity of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions ▿

doi: 10.1128/JVI.00697-08

Figure Lengend Snippet: In vitro-generated Tregs inhibit the proliferation of antigen-specific and polyspecific CD4+ CD25− T cells. CD25+ CD4+ T cells were isolated from both cultures stimulated with the conversion medium (Tregs) as well as cultures that were stimulated in the presence of IL-2 only (“control cells”). Additionally, CD4+ CD25+ T cells were also isolated from pooled spleens and LNs of BALB/c animals. These cells, in twofold serial dilutions, were used in suppression assays against the cultures of anti-CD3 antibody-stimulated CFSE-labeled CD4+ CD25− T cells (1 × 105) from naïve DO11.10 RAG2−/− mice (i.e., to measure antigen-specific effect) and Thy1.1 BALB/c animals (i.e., to measure polyspecific effect) with irradiated T-cell-depleted SPCs (2 × 105) from a homologous system as described in Materials and Methods. (A) The extent of CFSE dilution as an indication of suppressive activity of in vitro-generated CD4+ CD25+ regulatory T cells against anti-CD3-stimulated labeled CD4+ CD25− T cells from naïve DO.11.10 RAG2−/− animals is shown. Of the CD4+ CD25+ T cells, about 90% and 3% were Foxp3+ from cultures in the presence or absence of TGF-β, respectively. A first gate was applied on CD4+ T cells, and then the extent of CFSE dilution in CD4+ CFSE+ T cells was analyzed. (B) Blocking antibodies (Ab) to either TGF-β (10 μg/ml) or IL-10 (10 μg/ml) or PD-1 (10 μg/ml) or ICOS (10 μg/ml) were added to the suppression cultures, and the extent of division of CFSE in labeled cells was determined. Representative FACS plots at two dilutions of Tregs to T effectors are shown for PD-1- and ICOS-neutralized cultures. A dashed line represents dilution of CFSE when no Tregs were added, a solid line represents dilution of CFSE when Tregs were added, and a solid boldface line represents dilution of CFSE when, along with Tregs, either anti-PD1 or anti-ICOS antibodies were added. (C) Representative histograms indicating CFSE dilution in the Thy1.1 gated population is shown. CD4+ CD25+ T cells were isolated from the iTreg culture, control cells, and splenic nTregs from BALB/c animals and were used against anti-CD3-stimulated labeled CD4+ CD25− T cells from Thy1.1 animals. The markers show the percentages of cells that underwent less than two divisions.

Article Snippet: Female 6- to 8-week-old BALB/c DO11.10 RAG2 −/− mice were purchased from Taconic, Thy1.2 + BALB/c and CB.17 SCID mice were purchased from Charles River, and Thy1.1 + BALB/c mice were a kind gift from D. Woodland (Trudeau Institute, Saranac Lake, NY).

Techniques: In Vitro, Generated, Isolation, Labeling, Irradiation, Activity Assay, Blocking Assay

Journal:

Article Title: In Vitro-Generated Antigen-Specific CD4 + CD25 + Foxp3 + Regulatory T Cells Control the Severity of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions ▿

doi: 10.1128/JVI.00697-08

Figure Lengend Snippet: In vitro-generated OVA Tregs control SK severity in a bystander disease model. Foxp3+ CD4+ T cells (5 × 105) were adoptively transferred to DO11.10 RAG2−/− animals 24 h before or 6 days postocular HSV-1 infection. The disease severity and angiogenesis were recorded. (A) Gross eye pictures of control and transfer recipient animals from a representative experiment when 5 × 105 Foxp3+ cells were transferred 24 h before infection are shown. (B) Cumulative data on SK severity and angiogenesis from different experiments at 11 days p.i. are shown. Foxp3+ T cells (5 × 105) were transferred 24 h before or 6 days p.i. P values were calculated with one-way ANOVA using Dunnett's post-test settings. (C) Distribution of adoptively transferred Foxp3+ T cells in lymphoid organs (spleen and draining cervical LN) and ocular tissues at 11 days p.i. is shown. (D) Representative FACS plots demonstrating the infiltration of neutrophils (CD11b+ Gr1+) in collagenase-digested cornea from control and transfer recipient animals given 5 × 105 Foxp3+ cells transferred 24 h before infection are shown. Absolute numbers of neutrophils/cornea are shown in parentheses.

Article Snippet: Female 6- to 8-week-old BALB/c DO11.10 RAG2 −/− mice were purchased from Taconic, Thy1.2 + BALB/c and CB.17 SCID mice were purchased from Charles River, and Thy1.1 + BALB/c mice were a kind gift from D. Woodland (Trudeau Institute, Saranac Lake, NY).

Techniques: In Vitro, Generated, Infection

Journal: The Journal of Experimental Medicine

Article Title: CCR7 is required for the in vivo function of CD4 + CD25 + regulatory T cells

doi: 10.1084/jem.20061405

Figure Lengend Snippet: CCR7 KO mice show enhanced CHS reaction to oxazolone. (A) The ear thickness before and after five repeated oxazolone challenges in sensitized mice; data are expressed as mean differences between the hapten- and vehicle-challenged ears. From the third challenge onwards, CCR7 KO mice (white symbols; n = 19) have an enhanced ear swelling compared with WT mice (black symbols; n = 10). The augmented ear swelling in CCR7 KO was reversed by the transfer of 5 × 10 5 naive WT T reg cells (dotted line; n = 7). Inset table shows the area under the curve values and their statistical analysis (Student's t test). Error bars represent SEM. (B) After the last challenge, the difference between WT and CCR7 KO mice can be observed as an enhanced flare. (C and D) Microscopically, the CHS lesions contain a massive mononuclear infiltrate in CCR7 KO mice (C) in comparison with a moderate infiltrate in WT mice (D). (E and F) CD3 immunostaining of the CHS lesions in CCR7 KO (F) and WT mice (E) revealed that the infiltrate consists mostly of CD3 + cells. Bars, 50 μm.

Article Snippet: The CCR7 KO, DO11.10, and DO11.10 × CCR7 KO mice were bred and housed under specific pathogen-free conditions at the animal facility of the Novartis Institutes for BioMedical Research (NIBR) and were used between the ages of 8 and 14 wk.

Techniques: Comparison, Immunostaining

Journal: The Journal of Experimental Medicine

Article Title: CCR7 is required for the in vivo function of CD4 + CD25 + regulatory T cells

doi: 10.1084/jem.20061405

Figure Lengend Snippet: CCR7 is expressed by FoxP3 + CD4 + CD25 + T reg cells. Flow cytometric analysis of peripheral blood and single-cell suspensions of spleen and inguinal LNs of WT mice revealed that CD4 + CD25 + T reg cells coexpress FoxP3 and CCR7. Dot plots of FoxP3 and CCR7 staining (bottom) are gated on the CD4 + CD25 + T cells shown in the top panel. Numbers indicate the percentage of cells in each quadrant.

Article Snippet: The CCR7 KO, DO11.10, and DO11.10 × CCR7 KO mice were bred and housed under specific pathogen-free conditions at the animal facility of the Novartis Institutes for BioMedical Research (NIBR) and were used between the ages of 8 and 14 wk.

Techniques: Staining

Journal: The Journal of Experimental Medicine

Article Title: CCR7 is required for the in vivo function of CD4 + CD25 + regulatory T cells

doi: 10.1084/jem.20061405

Figure Lengend Snippet: Numbers of CD4 + and FoxP3 + cells in the peripheral LN, blood, and spleen of WT and CCR7 KO mice. (A) Few CD4 + and FoxP3 + cells can be found in the peripheral LNs of CCR7 KO mice (white circles) compared with WT mice (black circles). (B and C) In contrast, both populations are increased in the spleen (B), whereas in blood, only the CD4 + cells are increased, and FoxP3 + cells are reduced ( n = 4; C). Horizontal lines represent the mean values. (D) CD3 + and FoxP3 + T cells show altered distribution in retroauricular LNs of CCR7 KO mice compared with normal architecture in WT mice. Organized T cell zones of WT LNs are shown. This microarchitecture cannot be found in CCR7 KO mice, where diffuse T cell distribution is observed. Bars, 200 μm.

Article Snippet: The CCR7 KO, DO11.10, and DO11.10 × CCR7 KO mice were bred and housed under specific pathogen-free conditions at the animal facility of the Novartis Institutes for BioMedical Research (NIBR) and were used between the ages of 8 and 14 wk.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: CCR7 is required for the in vivo function of CD4 + CD25 + regulatory T cells

doi: 10.1084/jem.20061405

Figure Lengend Snippet: Adoptively transferred CCR7 KO T reg cells cannot suppress the antigen-induced expansion of TCR transgenic T cells. (A) 5 × 10 5 transferred CFSE-labeled T reg cells from DO11.10 mice enter LNs and, after antigen stimulation, expand in draining popliteal LNs and not in counterlateral LNs. Only a few T reg cells from DO11.10 × CCR7 KO can be detected in the LNs after their transfer. (B) Antigen-induced expansion of 5 × 10 5 CFSE-labeled DO11.10 CD4 + Th cells; their concurrent CD25 up-regulation is suppressed by the cotransfer of DO11.10 T reg cells but not DO11.10 × CCR7 KO T reg cells. Th cells regulated by WT T reg cells divide approximately one generation less in comparison with two other groups (dot plots). In unregulated and CCR7 KO T reg cell–regulated groups, the majority of Th cells are in generations three, four, and five, whereas the majority of Th cells regulated by WT T reg cells are in generations two, three, and four; their overall numbers are also reduced (histograms). (C) Statistical evaluation of the data in B ( n = 4). The cotransfer of T reg cells from DO11.10 mice reduced (**, P < 0.01) the mean total number of CFSE + T cells in the draining popliteal LNs, whereas the cotransferred DO11.10 × CCR7 KO T reg cells showed no effect. Error bars represent SEM.

Article Snippet: The CCR7 KO, DO11.10, and DO11.10 × CCR7 KO mice were bred and housed under specific pathogen-free conditions at the animal facility of the Novartis Institutes for BioMedical Research (NIBR) and were used between the ages of 8 and 14 wk.

Techniques: Transgenic Assay, Labeling, Comparison

Journal: The Journal of Experimental Medicine

Article Title: CCR7 is required for the in vivo function of CD4 + CD25 + regulatory T cells

doi: 10.1084/jem.20061405

Figure Lengend Snippet: In vitro function of CD4 + CD25 + T reg cells is not dependent on CCR7 expression. Polyclonal CD4 + T cells were stained with CFSE and stimulated with anti-CD3 mAb for 72 h in the presence of CD4 + CD25 + T reg cells from either WT or CCR7 KO mice. Both T reg cell populations show similar in vitro suppression of T cell proliferation in a dose-dependent manner (a 1:1 ratio is shown).

Article Snippet: The CCR7 KO, DO11.10, and DO11.10 × CCR7 KO mice were bred and housed under specific pathogen-free conditions at the animal facility of the Novartis Institutes for BioMedical Research (NIBR) and were used between the ages of 8 and 14 wk.

Techniques: In Vitro, Expressing, Staining

Journal: The Journal of Experimental Medicine

Article Title: CCR7 is required for the in vivo function of CD4 + CD25 + regulatory T cells

doi: 10.1084/jem.20061405

Figure Lengend Snippet: FoxP3 + T reg cells enter the CHS lesions of CCR7 KO mice. Immunofluorescence in the unchallenged (left) ears and oxazolon-challenged (right) ears of WT and CCR7 KO mice. The challenged ears of CCR7 KO mice contain an elevated number of CD3 + FoxP3 + T reg cells in comparison with WT mice. One representative ear sample out of 10 mice per group is shown. Bars, 200 μm.

Article Snippet: The CCR7 KO, DO11.10, and DO11.10 × CCR7 KO mice were bred and housed under specific pathogen-free conditions at the animal facility of the Novartis Institutes for BioMedical Research (NIBR) and were used between the ages of 8 and 14 wk.

Techniques: Immunofluorescence, Comparison

Journal: The Journal of Experimental Medicine

Article Title: CCR7 is required for the in vivo function of CD4 + CD25 + regulatory T cells

doi: 10.1084/jem.20061405

Figure Lengend Snippet: FoxP3 + T reg cells in CCR7 KO mice have a higher expression of CD44 and an increased frequency of CD103 + and CCR2 + subpopulations. Histograms of data acquired from the blood, spleen, and peripheral LNs are shown. 10 4 FoxP3 + T cells are depicted per histogram. In comparison with CCR7 KO mice (white histograms), WT mice (black histograms) have comparatively smaller subpopulations of CD103 + and CCR2 + T reg cells. CCR7 KO T reg cells also express higher levels of CD44. One out of four measurements on cells from different mice are shown. Numbers indicate the percentages of positive cells.

Article Snippet: The CCR7 KO, DO11.10, and DO11.10 × CCR7 KO mice were bred and housed under specific pathogen-free conditions at the animal facility of the Novartis Institutes for BioMedical Research (NIBR) and were used between the ages of 8 and 14 wk.

Techniques: Expressing, Comparison

Journal: The Journal of Experimental Medicine

Article Title: CCR7 is required for the in vivo function of CD4 + CD25 + regulatory T cells

doi: 10.1084/jem.20061405

Figure Lengend Snippet: Ineffective control of experimental IBD by CD4 + CD25 + CCR7 KO T reg cells. (A and B) The transfer of WT (A) or CCR7 KO (B) CD4 Th cells (black circles) into SCID mice induces weight loss, albeit with different kinetics. Disease induced by WT Th cells is prevented by the cotransfer of WT T reg cells in both ratios of 1:2 (black squares) and 1:1 (black triangles), whereas CCR7 KO T reg cells in ratios 1:2 (white squares) and 1:1 (white triangles) were less effective than their WT counterparts. The regulatory effect for both WT and CCR7 KO T reg cells was cell dose dependent (A). IBD induced by CCR7 KO Th cells developed with a 1-wk delay and was completely prevented by WT T reg cells and to a lesser extent by CCR7 KO T reg cells. In the latter case, no cell dose dependency could be observed (B). The last data points of the curves were analyzed with one-way analysis of variance and Dunnett's correction in comparison with untransferred control mice (**, P < 0.01). Error bars represent SEM.

Article Snippet: The CCR7 KO, DO11.10, and DO11.10 × CCR7 KO mice were bred and housed under specific pathogen-free conditions at the animal facility of the Novartis Institutes for BioMedical Research (NIBR) and were used between the ages of 8 and 14 wk.

Techniques: Control, Comparison