1. Differential Th17 CD4 T-cell depletion in pathogenic and nonpathogenic lentiviral infections
by Jason M. Brenchley, Mirko Paiardini, Kenneth S. Knox, Ava I. Asher, Barbara Cervasi, Tedi E. Asher, Phillip Scheinberg, David A. Price, Chadi A. Hage, Lisa M. Kholi, Alexander Khoruts, Ian Frank, James Else, Timothy Schacker, Guido Silvestri, and Daniel C. Douek
Blood
Volume 112(7):
October 1, 2008
©2008 by American Society of Hematology

2. Th17 cells are detected in PB and do not significantly overlap with Th1 CD4 T cells.
Th17 cells are detected in PB and do not significantly overlap with Th1 CD4 T cells. PB lymphocytes were stimulated with anti-CD3 overnight in the presence of brefeldin A. Cells were then stained and analyzed by flow cytometry as described in “Intracellular cytokine assay.” (A) Cells were gated based on characteristic light scatter properties, followed by positive staining for CD3 without binding to the dead cell dye, and then for CD4 staining without CD8 staining. Naive and memory CD4 T-cell subsets were gated based on characteristic expression patterns of CD45RO and CD27 (or CD28 and CD95 for studies with Sooty mangabeys, as shown in parentheses). (B) Individual subsets of CD4 T cells were analyzed for production of either IL-17 or IFN-γ. (C) The IL-17– and IFN-γ–producing memory subsets were further analyzed for the production of TNF and IL-2.
Jason M. Brenchley et al. Blood 2008;112:
©2008 by American Society of Hematology

3. Th17 cells respond to bacterial and fungal antigens.
Th17 cells respond to bacterial and fungal antigens. PB lymphocytes from cohorts of HIV-infected and uninfected persons were stimulated with a variety of viral (A) or bacterial antigens (B) overnight in the presence of brefeldin A and stained as in “Intracellular cytokine assay.” Production of IL-17 and IFN-γ by memory CD4 T cells in response to individual antigen-specific stimulation was measured by flow cytometry. (C) The frequencies of memory CD4 T cells from PB lymphocytes of several HIV+ (●) and HIV− patients (○) producing either IFN-γ or IL-17 in response to bacterial or fungal antigens are shown.
Jason M. Brenchley et al. Blood 2008;112:
©2008 by American Society of Hematology

4. Th17 cells are preferentially lost from the GI tracts of HIV-infected patients.
Th17 cells are preferentially lost from the GI tracts of HIV-infected patients. (A) GI tract and PB lymphocytes from HIV-uninfected persons (○) were stimulated with anti-CD3 in the presence of brefeldin A and stained as in “Intracellular cytokine assay.” GI tract lymphocytes from cohorts of HIV-infected (●) and uninfected persons (○) were stimulated with anti-CD3 overnight (B,D) or with PMA and ionomycin (C,E) for 4 hours in the presence of brefeldin A and stained as in Figure 1. Frequencies of GI tract memory CD4 T cells that produce either IL-17 (A-C) or IFN-γ (D,E) were then measured by flow cytometry. In panels C and E, red symbols signify chronically HIV-infected patients on HAART. (F) Expression of CCR5 by GI tract Th1 and Th17 cells from HIV-infected (●) and HIV-uninfected (○) patients. (G) The frequency of CD13+ myelomonocytic cells within total GI tract leukocytes was assessed after gating for CD45 expression with exclusion of dead cells in cohorts of HIV-infected (●) and uninfected persons (○). (H) Expression of IL-23R by memory CD4 T cells in the GI tracts of HIV-infected (●) and HIV-uninfected persons (○).
Jason M. Brenchley et al. Blood 2008;112:
©2008 by American Society of Hematology

5. Th17 cells are not preferentially lost from the BAL of HIV-infected patients.
Th17 cells are not preferentially lost from the BAL of HIV-infected patients. (A-F) GI tract, PB, and BAL lymphocytes from HIV-uninfected persons (○) and HIV-infected patients (●) were stimulated with anti-CD3 in the presence of brefeldin A and stained as in “Intracellular cytokine assay.” Frequencies of memory CD4 T cells that produce either IL-17 (A,C,E) or IFN-γ (B,D,F) were then measured by flow cytometry. (G) BAL lymphocytes from 4 HIV-infected patients were stimulated with CA antigen in the presence of brefeldin A and stained as in “Intracellular cytokine assay.” Production of either IL-17 or IFN-γ by memory CD4 T cells is shown.
Jason M. Brenchley et al. Blood 2008;112:
©2008 by American Society of Hematology

6. CD4 T cells in PB of HIV-infected patients are skewed toward a Th1 phenotype.
CD4 T cells in PB of HIV-infected patients are skewed toward a Th1 phenotype. PB lymphocytes from cohorts of HIV-infected (●) and uninfected persons (○) were stimulated with anti-CD3 overnight in the presence of brefeldin A and stained as in Figure 1. The frequency of memory CD27+ (A) and CD27− (B) CD4 T cells that produced IL-17 or IFN-γ was then measured by flow cytometry. The ratio of IL-17–producing cells to IFN-γ–producing cells was then compared within both memory CD27− and CD27+ CD4 T-cell subsets from HIV− (C, ○) and HIV+ (D, ○) patients. Statistical significance was determined by the Mann-Whitney test.
Jason M. Brenchley et al. Blood 2008;112:
©2008 by American Society of Hematology

7. Infection frequencies of Th17 cells in PB
Infection frequencies of Th17 cells in PB. PB lymphocytes from a cohort of HIV-infected patients were stimulated with anti-CD3 overnight in the presence of brefeldin A and stained as in “Intracellular cytokine assay.” CD27+ memory CD4 T cells that produced ...
Infection frequencies of Th17 cells in PB. PB lymphocytes from a cohort of HIV-infected patients were stimulated with anti-CD3 overnight in the presence of brefeldin A and stained as in “Intracellular cytokine assay.” CD27+ memory CD4 T cells that produced IFN-γ, IL-17, or neither were then sorted by flow cytometry, and the infection frequency was determined by quantitative PCR for viral DNA as described in “Quantitative PCR.”
Jason M. Brenchley et al. Blood 2008;112:
©2008 by American Society of Hematology

8. Th1 CD4 T cells are less functional than Th17 CD4 T cells in HIV-infected patients.
Th1 CD4 T cells are less functional than Th17 CD4 T cells in HIV-infected patients. PB lymphocytes from cohorts of HIV-uninfected and -infected patients were stimulated with anti-CD3 overnight in the presence of brefeldin A and stained as in “Intracellular cytokine assay.” The ability of IL-17–producing or IFN-γ–producing CD27+ (A,B) and CD27− (C,D) memory CD4 T cells to coproduce IL-2 and TNF was analyzed using SPICE as described in “Flow cytometric analysis.” Statistical significance was determined by the Mann-Whitney test.
Jason M. Brenchley et al. Blood 2008;112:
©2008 by American Society of Hematology

9. Th17 cells are not preferentially lost from the GI tracts of SIV-infected SMs. GI tract lymphocytes from cohorts of SIV-infected (●) and uninfected sooty mangabeys (○) were stimulated with PMA and ionomycin for 4 hours in the presence of brefeldin A and sta...
Th17 cells are not preferentially lost from the GI tracts of SIV-infected SMs. GI tract lymphocytes from cohorts of SIV-infected (●) and uninfected sooty mangabeys (○) were stimulated with PMA and ionomycin for 4 hours in the presence of brefeldin A and stained as in “Intracellular cytokine assay.” Frequencies of GI tract memory CD4 T cells that produce either IL-17 (A) or IFN-γ (B) were then measured by flow cytometry. (C) Expression of IL-23R by memory CD4 T cells in the GI tracts of SIV-infected (●) and SIV-uninfected sooty mangabeys (○). (D) Frequencies of PB memory T cells that produce IL-17 from SIV-infected (●) and uninfected (○) sooty mangabeys.
Jason M. Brenchley et al. Blood 2008;112:
©2008 by American Society of Hematology