Inadequate T follicular cell help impairs B cell immunity during HIV infection

Abstract

The majority of HIV-infected individuals fail to produce protective antibodies and have diminished responses to new immunizations. We report here that even though there is an expansion of follicular helper T (TFH) cells in HIV-infected individuals, the cells are unable to provide adequate B cell help. We found a higher frequency of programmed cell death ligand 1 (PD-L1)(+) germinal center B cells from lymph nodes of HIV-infected individuals suggesting a potential role for PD-1-PD-L1 interaction in regulating TFH cell function. In fact, we show that engagement of PD-1 on TFH cells leads to a reduction in cell proliferation, activation, inducible T-cell co-stimulator (ICOS) expression and interleukin-21 (IL-21) cytokine secretion. Blocking PD-1 signaling enhances HIV-specific immunoglobulin production in vitro. We further show that at least part of this defect involves IL-21, as addition of this cytokine rescues antibody responses and plasma cell generation in vitro. Our results suggest that deregulation of TFH cell-mediated B cell help diminishes B cell responses during HIV infection and may be related to PD-1 triggering on TFH cells. These results demonstrate a role for TFH cell impairment in HIV pathogenesis and suggest that enhancing their function could have a major impact on the outcome and control of HIV infection, preventing future infections and improving immune responses to vaccinations.

Figure 1

Tfh cells from HIV-infected subjects are unable to provide appropriate B cell help. (a) Frequency of T cell and B cell subsets in LNs from HIV⁻ and HIV⁺ subjects. T cell subsets were defined as: naïve (CD3⁺CD4⁺CD45RA⁺CD27⁺), central memory (CD3⁺CD4⁺CD45RA⁻CD27⁺), effector cells (CD3⁺CD4⁺CD45RA⁻CD27⁻) and Tfh cells (CD3⁺CD4⁺CD45RA⁻CXCR5^hi). (b) B cell subsets were defined as: naïve (CD3⁻CD19⁺CD38⁻IgD⁺), GC (CD3⁻CD19⁺CD38⁺⁺IgD⁻) and total memory B cells (CD3⁻CD19⁺CD38^+/−IgD⁻). For T cell subsets (HIV⁻ n=9; HIV⁺ n=9) for Tfh cell subset (HIV⁻n=10; HIV⁺ n=13) for B cell subsets (HIV⁻ n=11; HIV⁺ n=12). (c) IgG production (ng ml⁻¹) in cocultures from LNMCs of HIV⁻ and HIV⁺ subjects after 7 d (HIV⁻ n=6; HIV⁺ n=6) as measured by ELISA. (d) Percent difference in the levels of secreted IgG from cocultures of Tfh cells and GC-enriched B cells from HIV⁺ LNMCs when compared to uninfected controls (HIV⁻ n=6; HIV⁺ n=6). (e) Absolute number of B cells in coculture with Tfh and non-Tfh cells (HIV⁻ n=6; HIV⁺ n=6) (f) Absolute number of Tfh cells after 7 d (HIV⁻ n=6; HIV⁺ n=6) in coculture.

Figure 2

Ex-vivo characterization of Tfh cells and B cells in LNs from HIV-infected and uninfected individuals. (a) Enrichment of Tfh cells in the CXCR5^hi population of both HIV⁻ and HIV⁺ LNMCs as determined by Bcl-6, ICOS and PD-1 staining. (b) Expression levels of PD-1 on Tfh cells from HIV⁻ and HIV⁺ LNMCs as measured by mean fluorescence intensity (MFI). (c) Frequency of PD-L1 expression on B cell subsets from infected and uninfected LNMCs. Subsets were defined as naïve (CD3⁻CD19⁺CD38⁻IgD⁺), GC (CD3⁻CD19⁺CD38^hiIgD⁻), early memory (CD3⁻CD19⁺CD38⁺IgD⁻) and late memory (CD3⁻CD19⁺CD38⁻IgD⁻) (HIV⁻ n=6; HIV⁺ n=6). (d) Frequency of PD-L2 expression on B cell subsets (HIV⁻ n=5; HIV⁺ n=5). (e) Representative images for PD-L1 staining on LN sections from HIV-uninfected (n=6) and infected (n=5) subjects as well as SIV-uninfected (n=4) and infected (n=2) macaques. Scale bar, 50 μm.

Figure 3

Triggering PD-1 on Tfh cells leads to a decrease in cell proliferation, activation and cytokine secretion. Tfh cells from tonsil mononuclear cells (TMNCs) of uninfected individuals were cultured for 3 d in the presence of anti-CD3, anti-CD28 and isotype coated beads (Stim), anti-CD3, anti-CD28 and PD-L1 coated beads (Stim + PD-L1) or left unstimulated (Unstim). (a) Frequency of Ki-67 and CD38 expression on Bcl-6⁺ Tfh cells. The dot plots depict the percent of Ki-67⁺ and CD38⁺ Tfh cells after stimulation with the beads (n=10). (b) Effect of PD-1 triggering on the frequency and absolute number of live Tfh cells (n=10) (c) Percent of Ki-67⁺ Bcl-6⁺ Tfh cells at different time points after stimulation with the beads (n=3; *P < 0.01; **P < 0.001 between control and PD-L1 beads). (d) Effect of PD-1 triggering on the absolute number of ICOS⁺ Tfh cells and ICOS MFI (n=5). Results are representative of two independent experiments (e) Effect of PD-1 triggering on IL-21 secretion as measured by ELISA on day 3 (n=4).

Figure 4

Supplementation with IL-21 restores IgG production in co-cultures from HIV-infected LNs. (a) Representative graph depicting IgG levels in co-cultures of GC-enriched B cells with CXCR5^hi, CXCR5⁻ or CD45RA⁺ T cell subsets in the presence of SEB with or without recombinant human IL-21 (10 ng ml⁻¹) after 7 d. (b) Increase in IgG production and CD27 MFI on B cells when cocultures of Tfh and GC-enriched B cells from HIV⁺ LNMCs are supplemented with IL-21 (n=5). (c) Dose dependent effect of IL-21 on IgG production and CD27 MFI on B cells (n=4). (d) Dose dependent effect of IL-21 supplementation on the absolute number of B cells and IgG production in co-cultures of Tfh and GC-enriched B cells from HIV⁺ LNMCs (n=4) after 7 d. (e) Effect of IL-21 supplementation on total IgG levels following antigen specific stimulation by culturing Tfh cells and GC-enriched B cells in the presence of primed monocytes after 7 d. (n=2). (f) Effect of PD-L1/L2 blocking on IgG production in cocultures of Tfh cells and GC-enriched B cells in the presence of primed monocytes after 7 d (n=2).