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1.
Fig. 6

Fig. 6. IL-22 expands memory CD4+ cells after BCG vaccination and M. tb H37Rv infection. From: NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis.

A. C57BL/6 mice (5 mice per group) were unimmunized or immunized subcutaneously with 106 CFU of BCG. Some BCG-vaccinated mice were treated with anti-NK1.1 or isotype control Ab (0.3 mg per mouse 0, 24 and 48 h after vaccination). Some anti-NK1.1-treated mice received recombinant IL-22 (2 ng) at the same time points. Sixty days after BCG vaccination, mice were infected with 50–100 CFU of M. tb H37Rv by aerosol, and 30 days later, the number of CD4+CD44+CD127+CD117+ cells in the lungs was measured by flow cytometry. Means ±SEs are shown. B. A representative flow cytometry plot of CD4+CD44+CD127+CD117+ lung cells is shown. We gated on CD4+CD44+ cells, and then gated on CD127+ and CD117+ cells.

Rohan Dhiman, et al. J Immunol. ;189(2):897-905.
2.
Fig. 1

Fig. 1. BCG vaccination enhances NK cell number and production of IFN-γ and IL-22. From: NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis.

C57BL/6 mice (5 mice per group) were unimmunized or immunized subcutaneously with 106 CFU of BCG in 100 μl of PBS. After 24, 48 and 72 h, spleen and peripheral lymph node cells were isolated. A. The percentages of CD3+NKp46+ and CD3-NKp46+ cells were measured by flow cytometry. B. The frequency of IFN-γ-producing NK cells was determined by ELISPOT. Purified NK cells were obtained from spleen and peripheral lymph node cells by negative selection, and incubated overnight in triplicate wells on an ELISPOT plate to determine the frequency of IFN-γ-producing NK cells. C. Intracellular staining was performed to detect IL-22-producing CD3+NKp46+ and CD3-NKp46+ spleen cells. Surface staining was performed with anti-CD3 and anti-NKp46, and intracellular staining was performed with isotype control or anti-IL22. Mean values and SEs are shown. D. A representative flow cytometry result of staining for IL-22 and NKp46, after gating on CD3- and CD3+ spleen cells is shown.

Rohan Dhiman, et al. J Immunol. ;189(2):897-905.
3.
Fig. 5

Fig. 5. NK1.1+ cells and IL-22 enhance the protective efficacy of BCG vaccination. From: NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis.

C57BL/6 mice were unimmunized or immunized subcutaneously with 106 CFU of BCG. Some BCG-vaccinated mice were treated with anti-NK1.1, or isotype control Ab (0.3 mg per mouse 0, 24 and 48 h after vaccination). Some anti-NK1.1-treated mice received recombinant IL-22 (2 ng) at the same time points. Sixty days after BCG vaccination, mice were challenged with 50–100 CFU of M. tb H37Rv by aerosol. A. Thirty days post-infection, CD4+CD25+FoxP3+ T-cells in lungs were measured by flow cytometry. B. A representative flow cytometry plot of CD4+CD25+FoxP3+ T-cells is shown. Gating was performed as in Fig.3B. C and D. Thirty days post-infection, lung cells were isolated and stimulated with γ-irradiated M. tb (C) or Ag85a (D). After 72 h, IFN-γ levels were measured by ELISA. Values shown are those obtained after subtracting IFN-γ levels from wells containing medium alone. E. Sixty days post-infection, lungs were homogenized and plated on 7H11 agar with THC, and CFU per lung were counted after 3 weeks.

Rohan Dhiman, et al. J Immunol. ;189(2):897-905.
4.
Fig. 2

Fig. 2. NK1.1+ cells reduce expansion of CD4+CD25+FoxP3+ T-cells after BCG immunization. From: NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis.

C57BL/6 mice (3-6 mice per group) were unimmunized or immunized subcutaneously with 106 CFU of BCG in 100 μl of PBS. Some BCG-vaccinated mice were treated with anti-NK1.1, or isotype control Ab (0.3 mg per mouse 0, 24 and 48 h after vaccination). A. One week after vaccination, spleen and peripheral lymph node cells were isolated and CD4+CD25+FoxP3+ T-cells were measured by flow cytometry. B. One week after vaccination, CD4+ and CD11b+ cells from spleens and peripheral lymph nodes were isolated and cultured, with or without γ-irradiated M. tb H37Rv. After 5 days, CD4+CD25+FoxP3+ T-cells were measured by flow cytometry. C. A representative flow cytometry result of in vitro expanded CD4+CD25+FoxP3+ T-cells is shown. CD25+ and Foxp3+ cells were shown on gated CD4+ cells. D. One week after vaccination, CD4+ and CD11b+ cells from spleens and peripheral lymph nodes were isolated and cultured, with or without γ-irradiated M. tb H37Rv. After 5 days, supernatants were collected and IFN-γ levels were measured by ELISA. Pooled cells from 3 to 5 mice were used and the experiment was performed twice. Mean values and SEs are shown.

Rohan Dhiman, et al. J Immunol. ;189(2):897-905.
5.
Fig. 4

Fig. 4. NK cells lyse expanded Tregs during the response to BCG vaccination in vivo. From: NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis.

A. C57BL/6 mice (5 mice per group) were unimmunized or immunized subcutaneously with 106 CFU of BCG in 100 μl of PBS. After 72 h, CD4+CD25hi cells were isolated from pooled spleen and lymph node cells, using the Treg isolation kit. CD4+CD25hi cells from BCG-vaccinated mice and control unvaccinated mice were designated as expanded and natural Tregs, respectively. Expanded and natural Tregs were labeled with 5 μM (CFSEhigh) and 0.5 μM (CFSElow) of CFSE, respectively, mixed 1:1 and inoculated intravenously (6 × 106 cells/mouse) into recipient C57BL/6 mice, 72 h after BCG vaccination. Recipient mice were treated with anti-NK1.1 or isotype control Abs, 0, 1 and 2 days after vaccination. Eighteen h after transfer of Tregs, CFSElow and CFSEhigh populations were detected by flow cytometry of single cell suspensions from spleens and peripheral lymph nodes draining the vaccination site. The percent of in vivo lysis of BCG-expanded Tregs was calculated, using the formula given in the methods. Mean values and SEs of five independent experiments are shown. B. A representative flow cytometry result of cells from a BCG-vaccinated mouse is shown. CFSEhi cells are CD4+CD25hi BCG-expanded Tregs and CFSElow cells are CD4+CD25hi natural Tregs.

Rohan Dhiman, et al. J Immunol. ;189(2):897-905.
6.
Fig. 3

Fig. 3. NK1.1+ cells inhibit expansion of immunosuppressive Tregs after BCG vaccination and challenge with M. tb H37Rv. From: NK1.1+ cells and IL-22 regulate vaccine-induced protective immunity against challenge with Mycobacterium tuberculosis.

C57BL/6 mice (3-8 mice per group) were unimmunized or immunized subcutaneously with 106 CFU of BCG in 100 μl of PBS. Some BCG-vaccinated mice were treated with anti-NK1.1, or isotype control Ab (0.3 mg per mouse 0, 24 and 48 h after vaccination). Sixty days after BCG vaccination, mice were challenged with 50–100 CFU of M. tb H37Rv by aerosol. A. Thirty days post-infection, CD4+CD25+FoxP3+ T-cells in the lungs were measured by flow cytometry. B. A representative flow cytometry result of lung cells is shown. We gated on lung CD4+ cells and then gated on CD25+ and FoxP3+ expressing cells. C. M. tb H37Rv-expanded CD4+CD25hi cells are immunosuppressive. Three C57BL/6 mice were immunized subcutaneously with 106 CFU of BCG. One week later, CD4+ and CD11b+ cells from spleens and peripheral lymph nodes were isolated and cultured with γ-irradiated M. tb H37Rv. After 72 h, CD4+CD25hi cells and CD4+CD25- cells were isolated and cultured in Transwells, within large wells containing CD8+ and CD11b+ cells obtained from mice one week after BCG vaccination and γ-irradiated M. tb H37Rv. After 72 h, the Transwells were removed, and IFN-γ mRNA was quantified in the cells in the large wells by real-time PCR. D. C57BL/6 mice (3 mice per group) were immunized subcutaneously with 106 CFU of BCG. Some BCG-vaccinated mice were treated with anti-NK1.1, or isotype control Ab (0.3 mg per mouse 0, 24 and 48 h after vaccination). Sixty days after BCG vaccination, mice were challenged with 50–100 CFU of M. tb H37Rv by aerosol. Thirty days post-infection, CD4+CD25hi Tregs from pooled lung, spleen and mediastian lymph nodes were isolated and cultured in Transwells, within large wells containing CD8+ and CD11b+ cells from BCG-vaccinated mice and γ-irradiated M. tb H37Rv. After 72 h, the Transwells were removed, and IFN-γ mRNA was quantified in the cells in the large wells by real-time PCR. Mean values and SEs are shown. E. C57BL/6 mice (8 mice per group) were unimmunized or immunized subcutaneously with 106 CFU of BCG. Some BCG-vaccinated mice were treated with anti-NK1.1, or isotype control Ab (0.3 mg per mouse 0, 24 and 48 h after vaccination). Sixty days after BCG vaccination, mice were challenged with 50–100 CFU of M. tb H37Rv by aerosol. Thirty days post-infection, lungs were homogenized and plated on 7H11 agar with THC, and CFU per lung were counted after 3 weeks.

Rohan Dhiman, et al. J Immunol. ;189(2):897-905.

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