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. 2018 Apr 15;200(8):2714-2726.
doi: 10.4049/jimmunol.1701403. Epub 2018 Mar 5.

Associations of Simian Immunodeficiency Virus (SIV)-Specific Follicular CD8 + T Cells With Other Follicular T Cells Suggest Complex Contributions to SIV Viremia Control

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Associations of Simian Immunodeficiency Virus (SIV)-Specific Follicular CD8 + T Cells With Other Follicular T Cells Suggest Complex Contributions to SIV Viremia Control

Mohammad Arif Rahman et al. J Immunol. .
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Abstract

Follicular CD8+ T (fCD8) cells reside within B cell follicles and are thought to be immune-privileged sites of HIV/SIV infection. We have observed comparable levels of fCD8 cells between chronically SIV-infected rhesus macaques with low viral loads (LVL) and high viral loads (HVL), raising the question concerning their contribution to viremia control. In this study, we sought to clarify the role of SIV-specific fCD8 cells in lymph nodes during the course of SIV infection in rhesus macaques. We observed that fCD8 cells, T follicular helper (Tfh) cells, and T follicular regulatory cells (Tfreg) were all elevated in chronic SIV infection. fCD8 cells of LVL animals tended to express more Gag-specific granzyme B and exhibited significantly greater killing than did HVL animals, and their cell frequencies were negatively correlated with viremia, suggesting a role in viremia control. Env- and Gag-specific IL-21+ Tfh of LVL but not HVL macaques negatively correlated with viral load, suggesting better provision of T cell help to fCD8 cells. Tfreg positively correlated with fCD8 cells in LVL animals and negatively correlated with viremia, suggesting a potential benefit of Tfreg via suppression of chronic inflammation. In contrast, in HVL macaques, Tfreg and fCD8 cell frequencies tended to be negatively correlated, and a positive correlation was seen between Tfreg number and viremia, suggesting possible dysfunction and suppression of an effective fCD8 cell immune response. Our data suggest that control of virus-infected cells in B cell follicles not only depends on fCD8 cell cytotoxicity but also on complex fCD8 cell associations with Tfh cells and Tfreg.

Figures

Figure 1
Figure 1
Virological and immunological status of the study animals. (A–B) Viral load of HVL and LVL animals, respectively, over the course of infection. (C) Median viral load of the HVL and LVL groups post-infection. (D–E) CD4 counts of HVL and LVL animals, respectively, post infection. (F) Median CD4 counts of the HVL and LVL groups post-infection. (G) LN viral loads of the macaques. LN cells from 8 LVL and 11 HVL macaques were available for analysis. (H) Correlation between LN viral loads and median plasma viral load. (I) Correlation between LN viral loads and plasma viral loads at the time of LN collection. Viral loads at time of LN collection for 2 HVL macaques were not determined. Data of panel G were analyzed by the Mann-Whitney test. Data of panels H–I were analyzed by the Spearman correlation test. * * * P < 0.001.
Figure 2
Figure 2
Localization of CD4 and CD8 cells in GCs of rhesus macaques. (A) Representative imaging of GCs for CD4+ cells. Immunofluorescence staining for Ki67 (purple), CD4 (red), PD-1 (green) and nuclei (blue) of a LN section from an acutely infected macaque (top panels). Representative staining of GCs from SIV-infected macaques (acutely-infected and LVL and HVL chronically-infected macaques) and a naïve macaque as indicated (bottom panels). (B) Representative imaging of GCs for CD8+ cells. Immunofluorescence staining for Ki67 (red), CD8 (purple), PD-1 (green) and nuclei (blue) of a LN section from an acutely-infected macaque (top panels). Representative staining of GCs from SIV-infected macaques (acutely-infected and LVL and HVL chronically-infected macaques) and a naïve macaque as indicated (bottom panels).
Figure 3
Figure 3
Quantitation of CD4+ and CD8+ cells in GCs of rhesus macaques. (A) Frequency of CD8+ cells in GCs of naïve and acutely and chronically SIV infected macaques. (B) CD4+ cell frequency in GCs of all groups of macaques. (C–D) Correlation between the number of CD8+ and CD4+ cells in acutely infected and LVL animals, respectively. Data analyzed by repeated measures ANOVA (A, B) and the Spearman correlation test (C–D). In (A) and (B), p values for multiple pairwise comparisons of the groups were corrected using Tukey’s method. Horizontal and vertical bars denote mean and standard deviation. * P < 0.05; * * P < 0.01; * * * P < 0.001, * * * * P < 0.0001.
Figure 4
Figure 4
fCD8 cells during SIV infection: frequency, functionality and association with viremia control. (A) Representative gating of fCD8 cells. A representative CXCR5 FMO control is shown. (B) Frequency of fCD8 cells in LN of naïve and acutely and chronically SIV infected macaques. (C–D) Frequency of SIV Gag- and Env-specific fCD8 cells determined by ELISPOT in acutely and chronically infected macaques. (E–H) Frequency of Env-and Gag-specific Granzyme B+ and perforin+ fCD8 cells in all macaque groups following stimulation with Env and Gag peptides. (I) SIV Gag-specific Granzyme B expression on fCD8 cells of LVL and HVL macaques, shown by MFI. Frequencies and MFI were calculated as the peptide-stimulated response minus the unstimulated response. MFI calculated as 0 were assigned a value of 1 for plotting. (J) Correlation between median chronic viral load and percentage of fCD8 cells among T cells (CD3+CXCR5+) in B cell follicles of LVL macaques. (K) Gag-specific killing by fCD8 cells of SIV-infected macaques. Data of panels B–I and K analyzed by the Mann-Whitney test; data of panel J were analyzed by the Spearman correlation test. Horizontal and vertical bars denote mean and standard deviation. * P < 0.05; * * P < 0.01; * * * P < 0.001, * * * * P < 0.0001.
Figure 5
Figure 5
Association of Tfh and fCD8 cells in B cell follicles. (A) Representative gating of Tfh cells. (B) Frequency of Tfh cells among CD4+ cells in LN of the four macaque groups (C–D) Percentage of IL-21+ Tfh cells in response to Gag (C) and Env (D) peptide stimulation. (E–F) IL-21 expression on Env- (E) and Gag- (F) specific Tfh cells determined by MFI in HVL and LVL animals. Frequencies and MFI were calculated as the peptide-stimulated response minus the unstimulated response. MFI calculated as 0 were assigned a value 0.1 for plotting. (G) Correlation between Tfh and fCD8 cell frequencies among T cells (CD3+CXCR5+) in B cell follicles of LVL macaques. (H–I) Correlation between Env- (H) and Gag-specific (I) IL-21+ Tfh cells and median chronic viral load of LVL animals. The data of (B–F) were analyzed by Mann-Whitney test and (G–I) were analyzed by Spearman correlation test. Horizontal and vertical bars denote mean and standard deviation. * P < 0.05; * * P < 0.01; * * * P < 0.001, * * * * P < 0.0001.
Figure 6
Figure 6
Association of Tfreg with fCD8 and Tfh cells in B cell follicles. (A) Representative gating of Tfreg cells. (B) Frequency of Tfreg cells among CD4+ cells in LN of four animal groups. (C–D) Correlation between Tfreg and fCD8 cell frequencies of LVL (C) or HVL (D) animals. (E) Correlation between Tfreg and Tfh frequencies in acutely infected macaques. (F–G) Correlation between Tfreg number and Tfh number (F) or IL-21+ Tfh number (G) in acutely infected macaques. (H–I) Correlation between the frequencies of Tfreg in LVL (H) or HVL (I) macaques and median chronic viral loads. (J) Correlation between Tfreg number and median chronic viral load in HVL macaques. The percentages in panels C–E and H–I were taken among T cells (CD3+CXCR5+) in B cell follicles of the macaques. Numbers of Tfreg, Tfh, and IL-21+Tfh cells in panels F, G, and J were normalized to one million CD3+ cells. The data of (B) analyzed by Mann-Whitney test; (C–J) were analyzed by Spearman correlation test. Horizontal and vertical bars denote mean and standard deviation. * P < 0.05; * * P < 0.01; * * * P < 0.001, * * * * P < 0.0001.
Figure 7
Figure 7
Comparison of SIV-specific frequency and cytokine producing cells in fCD8 and non-fCD8 cells. (A–B) Frequency of SIV-Gag and Env-specific fCD8 and non-fCD8 cells in LN of SIV-infected macaques. (C–F) Percentage of SIV-specific perforin+ (C, D) and Granzyme B+ (E, F) fCD8 and non-fCD8 cells in response to Gag (C, E) or Env (D, F) peptide stimulation. The data of (A–F) were analyzed with ANOVA. Horizontal and vertical bars denote mean and standard deviation. * P < 0.05; * * P < 0.01; * * * P < 0.001, * * * * P < 0.0001.
Figure 8
Figure 8
Comparison of phenotypic and functional properties of fCD8 and non-fCD8 cells. (A) Percentage of PD-1High fCD8 cells among four groups of macaques. (B) PD-1High fCD8 and non-fCD8 cells in naive and SIV-infected animals. (C) Specific killing by fCD8 and non-fCD8 cells of SIV-infected macaques. (D–E) Frequencies of SIV Env- and Gag-specific non-fCD8 cells in acutely and chronically SIV infected macaques, determined by ELISPOT. (F) Gag-specific killing by non-fCD8 cells among acutely and chronically-infected macaques. The data of (A, D–F) analyzed by Mann-Whitney test and (B–C) analyzed by ANOVA. Horizontal and vertical bars denote mean and standard deviation. * P < 0.05; * * P < 0.01; * * * P < 0.001, * * * * P < 0.0001.

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