1. Volume 44, Issue 1, Pages 167-178 (January 2016)
Circulating HIV-Specific Interleukin-21+CD4+ T Cells Represent Peripheral Tfh Cells with Antigen-Dependent Helper Functions 
Bruce T. Schultz, Jeffrey E. Teigler, Franco Pissani, Alexander F. Oster, Gregory Kranias, Galit Alter, Mary Marovich, Michael A. Eller, Ulf Dittmer, Merlin L. Robb, Jerome H. Kim, Nelson L. Michael, Diane Bolton, Hendrik Streeck 
Immunity 
Volume 44, Issue 1, Pages (January 2016)
DOI: /j.immuni
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1 IL-21 Secretion of CD4+ T Cells Defines Peripheral Tfh Cells
SEB-stimulated CD4+ T cells from four HIV-negative individuals were sorted in triplicate into 100-cell wells of IL-21+CD4+, IFN-γ+CD4+, or IL-21+IFN-γ+CD4+ T cells (Figure S1).
(A) Representative flow plot of the three sorted populations and box plots representing gene expression levels by Fluidigm BioMark (mRNA expression levels represented as Et values). Light gray depicts IL-21+CD4+ T cells, white depicts IFN-γ+CD4+ T cells, and dark gray depicts IL-21+IFN-γ+CD4+ T cells. Data are represented as median (min to max).
(B) Bivariate linear plot illustrating average mRNA expression level of genes predominantly found in IL-21+CD4+ versus IFN-γ+CD4+ T cells. Red dots depict genes defining Th1 cells, blue dots depict genes defining pTfh cells, and green dots depict genes defining Th2 cells. Black dots are genes not associated with any particular group. Higher expression values in IL-21+CD4+ T cells trend toward the y axis; higher expression in IFN-γ+CD4+ T cells trend toward the x axis.
(C) Radar plot displaying the T helper subset most closely associated with genes expressed in IL-21+CD4+, IFN-γ+CD4+, or IL-21+IFN-γ+CD4+ T cell subsets (Table S2). Higher defining values trend toward the periphery.
(D) Heatmap illustrating expression levels of Tfh-cell-associated genetic markers in various phenotypic subsets previously described as pTfh and IL-21+CD4+ T cells. See also Figure S4.
(E) Radar plot comparing peripheral Tfh cell phenotypes and IL-21+CD4+ T cells’ relative association for genes defining respective CD4+ T cell subsets as performed in (C).
Immunity  , DOI: ( /j.immuni )
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3. Figure 2
HIV-Specific IL-21+CD4+ T Cells Most Closely Resemble Phenotypic and Transcriptional Markers of Tfh Cells
HIV-positive CD4+ T cells were stimulated with Gag or Env overlapping peptide pools and sorted based on secretion of IL-21 or IFN-γ.
(A) PCA was performed on genes that were differentially expressed between IL-21+CD4+ and IFN-γ+CD4+ T cell populations (Table S3). Individual samples from respective populations were graphed and show distinct clustering of HIV-specific IL-21+CD4+ (blue) and HIV-specific IFN-γ+CD4+ (red) T cell populations, demonstrating almost no overlap between groups.
(B) Boxplots showing transcription factor expression levels of HIV-specific IL-21+CD4+ versus HIV-specific IFN-γ+CD4+ T cell responses in relationship to TBX21. See also Table S1 and Figure S3. Data are represented as median (Tukey).
(C) HIV epitope-specific IL-21+CD4+ or IFN-γ+CD4+ T cell lines from six individuals were generated and expanded in vitro with individual 15-mer OLP. Cells were then stimulated with their respective 15-mer and stained for IL-21, IFN-γ, CD3, CD4, CXCR5, CXCR3, ICOS, CCR7, IL-17, and PD-1 as described above, followed by flow cytometry. Overlaid representative flow plots show single-positive IL-21+CD4+ (blue) along and IFN-γ+CD4+ (red) T cells (left). Quantitative analysis for all HIV-specific CD4+ T cell lines generated reveal differences in marker expression in IL-21+CD4+ and IFN-γ+CD4+ T cells (right). Data are represented as mean ± SEM.
(D) CD4+ T cells isolated from the lymph node were stimulated with Gag or Env peptide pools and stained for CD3, CD4, CXCR5, PD-1, Bcl-6, and IL-21. Representative plots comparing IL-21+ (blue) and bulk CD4+ T cells (black) for both Gag- (top) and Env-specific (bottom) cells are shown with frequencies for IL-21+CD4+ T cells. Data are represented as mean ± SEM.
Immunity  , DOI: ( /j.immuni )
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4. Figure 3
Distinct Profiles in the Protein Specificity, Breadth, and Magnitude of HIV-Specific pTfh versus IFN-γ+CD4+ T Cell Responses
HIV-specific IL-21+CD4+ versus IFN-γ+CD4+ T cell responses from 26 treatment-naive chronically HIV-infected individuals were measured by ELISpot using PTE peptides spanning the entire Gag or Env proteome.
(A) Differences in the breadth (number of epitopes) and magnitude of HIV-specific IL-21+CD4+ or IFN-γ+CD4+ T cell responses. Magnitude is calculated as the sum of spot-forming cells (SFCs) that are both greater than 55 SFCs and more than three times the standard deviation. Data are represented as mean ± SEM.
(B) Relative distribution of protein specificity in HIV-specific IL-21+CD4+ pTfh cell responses versus HIV-specific IFN-γ+CD4+ T cell responses in a 10 × 10 dot plot with the total number of positive responses listed under each plot.
(C) Analysis of the Gag:Env response ratio revealed a significant difference between antigen-specific IL-21+CD4+ versus IFN-γ+CD4+ T cells. Data are represented as mean ± SEM.
Immunity  , DOI: ( /j.immuni )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 4
HIV-Specific pTfh Cells Facilitate HIV-Specific CD8+ T Cell Responses in an Antigen-Independent Manner
Influence of pTfh cells on CD8+ T cell function.
(A) Viral load measurements of 26 chronic, treatment-naive HIV-infected individuals demonstrates lower viral load set-points in individuals with measurable HIV-specific (as described in Figure 3A) IL-21+CD4+ T cell responses compared to individuals with no measurable HIV-specific IL-21+CD4+ T cell response. Data are represented as mean ± SEM.
(B) pTfh cell lines were established from sorted IL-21+CD4+ T cells, expanded in vitro, and co-cultured for 7 days with isolated, autologous CD8+ T cells at a 1:1 ratio and stimulated with respective peptide in the presence or absence of IL-21 nAb. HIV-specific IL-21+CD4+ T cell responses induced significantly higher Granzyme B and Perforin expression in HIV-specific CD8+ T cells, which was almost completely abrogated after neutralization of IL-21 in culture. There was no difference between Gag- and Env-specific Tfh cell responses in their ability to help CD8+ T cells. Data are represented as mean ± SEM.
Immunity  , DOI: ( /j.immuni )
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6. Figure 5
Gag- and Env-Specific pTfh Cells Provide Differential Helper Functions to Naive B Cells
pTfh cell lines were established with sorted IL-21+CD4+ T cells from 11 chronic, treatment-naive HIV-infected individuals, expanded in vitro, and co-cultured with CFSE-labeled autologous naive B cells 7 days as previously described and stimulated with Gag or Env PTE peptide pools.
(A) Flow cytometric analysis of B cells after co-culture revealed a divergent maturation marker profile in Gag- versus Env-specific pTfh cells including CD38 expression (both proliferated and nonproliferated cells), IgG, and IgM expression. Data are represented as median (min to max).
(B) Luminex analysis of immunoglobulins secreted into the co-culture supernatant collected on day 7 showed differences in the CSR induced by Gag- or Env-specific pTfh cells. Data are represented as mean ± SEM.
Immunity  , DOI: ( /j.immuni )
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7. Figure 6 Env-Specific pTfh Cells Share Features of Th2 Cells
Comparison of T helper program bias of pTfh cells.
(A) Boxplots comparing gene expression profiles in Gag- versus Env-specific pTfh cell responses assessed by Biomark Fluidigm. Data are represented as median (min to max).
(B) Env-specific pTfh cells have higher mRNA expression of the Th2 cell transcription factor GATA3 (left) and higher collective mRNA expression of Th2 cytokines (right). Data are represented as median (min to max) (left) and mean ± SEM (right).
(C) Luminex analysis of the supernatants collected from Env-stimulated PBMCs show higher Th2 cytokine secretion whereas Gag-stimulated cells have a higher frequency of Th1 cytokines. Data are represented as mean ± SEM.
Immunity  , DOI: ( /j.immuni )
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8. Figure 7
ALVAC Prime and ALVAC+AIDSVAX Boost Vaccine Regimen Induces Higher pTfh Cell Frequencies Compared to DNA Prime, Ad5 Boost, MVA-CMDR, and ALVAC Vector Regimens
Comparison of PBMCs isolated from vaccine recipients exposed to either an ALVAC prime alone, an ALVAC prime with ALVAC+AIDSVAX boost (ALVAC+AIDSVAX), a DNA prime with Ad5 boost (DNA+Ad5), or a DNA prime using a MVA vector (MVA) vaccine regimen at peak immunogenicity. Significantly higher frequencies of HIV-specific IL-21+ (top left) pTfh cell responses were observed in PBMCs exposed to ALVAC+AIDSVAX vaccines compared to the ALVAC alone and DNA+Ad5 vaccines. No differences were observed for both TNF-α+ (bottom right) and CXCR5+ (top right) CD4+ T cell responses whereas IFN-γ+CD4+ T cells displayed a comparatively lower response to the MVA+DNA regimen. Data shown for cytokine expression include responses to both Gag and Env immunogens. Data are represented as mean ± SEM.
Immunity  , DOI: ( /j.immuni )
Copyright © 2016 Elsevier Inc. Terms and Conditions