CD4 T follicular helper cell dynamics during SIV infection

Abstract

CD4 T follicular helper (TFH) cells interact with and stimulate the generation of antigen-specific B cells. TFH cell interaction with B cells correlates with production of SIV-specific immunoglobulins. However, the fate of TFH cells and their participation in SIV-induced antibody production is not well understood. We investigated the phenotype, function, location, and molecular signature of TFH cells in rhesus macaques. Similar to their human counterparts, TFH cells in rhesus macaques represented a heterogeneous population with respect to cytokine function. In a highly differentiated subpopulation of TFH cells, characterized by CD150lo expression, production of Th1 cytokines was compromised while IL-4 production was augmented, and cells exhibited decreased survival, cycling, and trafficking capacity. TFH cells exhibited a distinct gene profile that was markedly altered by SIV infection. TFH cells were infected by SIV; yet, in some animals, these cells actually accumulated during chronic SIV infection. Generalized immune activation and increased IL-6 production helped drive TFH differentiation during SIV infection. Accumulation of TFH cells was associated with increased frequency of activated germinal center B cells and SIV-specific antibodies. Therefore, chronic SIV does not disturb the ability of TFH cells to help B cell maturation and production of SIV-specific immunoglobulins.

Figure 1. Characterization of TFH cells in RM LNs.

(A) Flow cytometry contour plots, showing the expression of BCL-6 against either PD-1 or CXCR5 in CD4 T cells from 2 chronic SIV-infected LNs (left panel). Histograms depicting the expression of CCR7 in bulk CM (solid gray), BCL-6^hiPD-1^hi, and BCL-6^hiCXCR5^dim/hi CD4 T cells (right panel). The frequency of the gated populations is shown. (B) The gating scheme used for identification of particular CD4 populations in LN preparation is shown. The naive and CM cells were analyzed with respect to ICOS and CD150 expression. The naive population was used as reference for setting the gates. The frequency of the gated populations is shown. (C) Confocal images were collected from LN and SP tissues from SIV-negative (n = 2) RMs. The CD4, CD20, PD-1, and Ki67 markers were simultaneously analyzed, and their relative localization in 1 SIV-negative LN is shown (left panel). The boxed area is shown at higher magnification in the top right and the bottom images (scale bars: 100 μm). The relative distance of CD4 T cells, expressing different amounts of PD-1, from the “center” of the B cell follicle was calculated, and pooled data obtained from 2 SIV-negative SPs are shown (right panel). Each dot represents a cell or a definable part of a CD4 T cell. Bars depict mean ± 95% confidence intervals.

Figure 2. TFH subpopulations are characterized by differential cytokine production.

(A) Cells from chronic SIV-infected SP tissues (n = 7) were sorted with respect to expression of CCR7, PD-1, ICOS, and CD150, and the mRNA expression for BCL6, MAF, IL21, and TBX21 was determined. The normalized CT value for CD28^hiCD95^hiCCR7^hiPD-1^lo cells was assigned a value of 1, and the fold change over this value for the populations tested is shown. ND, not detected. P values were calculated using the Mann-Whitney U test. (B) Cells (10⁵) were sorted from chronic SIV-infected SP tissues (n = 5), stimulated with PMA/ionomycin for 14 to 16 hours, and supernatants were analyzed for cytokines using Luminex technology. Signals from nonstimulated samples were below the detection limit. P values were calculated using the Mann-Whitney U test. (C) Sorted CD4 populations (n = 2 for naive and PD-1^lo and n = 5 for PD-1^dim and PD-1^hi) were cocultured (1:1) with autologous sorted B cells in the presence of SEB, and total IgG was analyzed in the supernatants on day 12. The titer for CCR7^hiPD-1^dim was assigned a value of 1, and the fold changes over this value for the population tested is shown.

Figure 3. RM TFH cells exhibit a distinct transcriptional profile compared with that of non-TFH cells.

(A) Heat map clustering analysis of gene expression in non-TFH (n = 5) compared with TFH (n = 5) cell populations from SIV-negative (n = 5) tissues. The top 200 genes, based on raw P value for the contrast, are shown. The relative expression (fold change) of selected genes (n = 15) between TFH and non-TFH populations is shown. Genes were significantly differentiated with fold change of more or less than 1.5 and raw P value of less than 0.05. (B) Heat map analysis of gene expression between TFH cells from SIV-negative (n = 5) and chronic SIV-infected (n = 6) tissues. The relative expression of selected genes (n = 15) is shown.

Figure 4. Accumulation of TFH cells during chronic SIV infection.

(A) Pooled data showing the frequency (%) of CCR7^hiPD-1^lo, CCR7^hi/loPD-1^dim, and CCR7^loPD-1^hi populations in the CM cellular compartment from LN tissues from noninfected (white; n = 10), acute SIV-infected (light gray; n = 11), and chronic SIV-infected RMs (dark gray: low percentage of TFH cells, n = 9; red: high percentage of TFH cells, n = 9). (B) Pooled data showing the levels (MFI) of BCL-6, expressed as fold change over the MFI of naive cells, in CD4 T cell subsets. (C) Pooled data showing the frequency (%) of total, naive, and CM CD4 T cells. (D) The frequency (%) of the CD150^lo and CD150^hi TFH cells is shown. (E) SIV-GAG DNA was measured by PCR. LN tissues from acute SIV-infected (samples collected on day 3, 7, 10, 14, 17, and 21 after infection) (n = 12) and chronic SIV-infected (n = 6) RMs were used. Each dot represents data for a single LN from an individual animal. Animals are represented only once in each plot. Horizontal lines depict either (A–D) mean or (E) median values. P values were calculated using the (A–D) Mann-Whitney U test or the (E) Wilcoxon matched-pairs signed-rank test.

Figure 5. TFH cells are characterized by low cell cycling capacity and increased sensitivity to in vitro cell death.

(A) The schedule of the in vivo administration of BrdU and collection of LN tissues from chronic SIV-infected RMs (n = 3) as well as the percentage of Ki67^hiBrdU^hi cells in naive, CM, and EM CD4 T cell populations are shown. The frequency (%) of Ki67^hiBrdU^hi cells in CM/CD4 T cell populations, based on their expression of CCR7, PD-1, and CD150, is also shown. (B) Pooled data showing the percentage of active caspase-3⁺ cells in the naive and CM subpopulations from noninfected (n = 7; white bars) and chronic SIV-infected RMs (n = 13; gray bars). (C) Pooled data showing the relative frequency (%) of PD-L1^lo, PD-L1^dim, and PD-L1^hiCD20^hiCD3^lo B cells in SIV^– (n = 10) and chronic SIV-infected LNs (n = 10 for low percentage of TFH cells and n = 4 for high percentage of TFH cells). (B and C) In the box-and-whiskers plot, box size represents the limits of data for the second and third quartiles, with medians shown as bars. Whiskers define the minimum and maximum of the data presented. P values were calculated using the Mann-Whitney U test. *P < 0.0001. (D) The relative expression of the S1PR1 mRNA in sorted cells from acute SIV-infected (n = 3; LN tissues) and chronic SIV-infected RMs, with low percentage of TFH cells (n = 3; LN tissues) and high percentage of TFH cells (n = 7; LN tissues), is shown. The normalized CT value for CD28^hiCD95^hiCCR7^hiPD-1^lo cells was assigned a value of 1, and the fold change over this value for the populations tested is shown.

Figure 6. TFH cell accumulation correlates with increased immune activation and skewed IL-6 signaling.

(A) Plasma sCD14 levels from noninfected (n = 6), acute (n = 8), and chronic SIV-infected RMs (dark gray: low TFH cells, n = 7; red: high TFH cells, n = 9). (B) Plasma IL-6 levels from noninfected (n = 6) and chronic SIV-infected (n = 12) RMs. (C) IL-6Ra expression on CD4 T cell subsets from noninfected (n = 10), acute (n = 11), and chronic SIV-infected (n = 18) LNs. (D) Linear regression analysis between IL-6Ra and frequency of TFH cells (chronic SIV-infected, n = 18). (E) IL-6–induced pSTAT-1 (3), after subtraction of basal (no stimulation) levels, in CD4 T cells from noninfected (n = 4), acute (n = 4), and chronic SIV-infected (n = 11) LNs. (F) Linear regression analysis between pSTAT-1 (pSTAT-1/pSTAT-3) in PD-1^hi CD4 T cells and frequency of TFH cells (chronic SIV-infected RMs, n = 11). (G) Viability and absolute cell numbers (counts after treatment minus initial counts) of sorted LN CD4 T cells (chronic SIV-infected, n = 4) stimulated with anti-CD3 with or without IL-6. (H) Pooled data showing levels of BCL-6 in LN (n = 5) CD4 T cell populations stimulated with anti-CD3 with or without IL-6. Box sizes represent limits of the second and third quartiles, with medians shown as bars. Whiskers define minimum and maximum of data. Each dot represents data for a single sample from an individual animal. Animals are represented once in each plot. Horizontal lines depict mean values. P values were calculated using the (A–C) Mann-Whitney U test or (E) Wilcoxon matched-pairs signed-rank test.

Figure 7. Accumulation of TFH cells in chronic SIV infection is associated with increased generation of GC B cells and SIV-specific antibodies.

(A) Flow cytometry plots showing the binding of PNA within the B cell compartment in the LNs and PBMCs from a chronic SIV-infected RM as well as pooled data showing the frequency (%) of PNA^hiIgG^hi and PNA^hiIgG^lo B cells in LNs from noninfected (n = 5) and chronic SIV-infected RMs (n = 5 for low percentage of TFH LNs and n = 4 for high percentage TFH LNs). (B) Contour plots showing the coexpression of BCL-6 and Ki67 or IgD or IgG in B cells from a chronic SIV-infected LN as well as pooled data showing the frequency (%) of BCL-6^hi B cells in LNs from noninfected (n = 5) and chronic SIV-infected RMs (n = 5 for low percentage of TFH LNs and n = 4 for high percentage TFH LNs). (C) Pooled data showing the IgG and IgA titers of SIV-specific antibodies in plasma from acute SIV-infected (n = 7; white symbols) and chronic SIV-infected RMs (n = 7 for low percentage of TFH cells, gray symbols; n = 9 for high percentage TFH cells, black symbols). (D) Pooled data showing the avidity score for plasma purified IgG from chronic SIV-infected RMs. The frequency of the gated populations is shown in the contour plots. Horizontal lines depict mean values. P values were calculated using Mann-Whitney U test.