The role of follicular helper T cells and the germinal center in HIV-1 gp120 DNA prime and gp120 protein boost vaccination

Abstract

The importance of follicular T helper (TFH) cells and the germinal center (GC) reaction in the humoral immune response has become clear in recent years, however the role of TFH cells and the GC in an HIV vaccine strategy remains unclear. In this study, we primed mice with gp120-encoding DNA and boosted with gp120 protein, a regimen previously shown to induce high titers of high affinity and cross-reactive anti-gp120 Abs. Priming with gp120 DNA caused increased TFH cell differentiation, GC B cells, and antigen-specific antibody titers, compared with priming with gp120 protein. Priming with DNA also caused more activated CD4(+) T cells to become TFH cells and more GC B cells to become memory cells. Deletion of BCL6 midway through the vaccine regimen resulted in loss of TFH cells and GCs, and, unexpectedly, increased anti-gp120 IgG titers and avidity. Our data suggests vaccination with gp120-encoding DNA elicits a stronger and more rapid TFH and GC response than gp120 protein. Furthermore, we demonstrate that the GC reaction may actually limit antigen-specific IgG secretion in the context of repeated immunizations.

Keywords: BCL6; HIV vaccine; follicular helper T cells; germinal center; prime-boost.

Figure 1. Experimental design for testing DNA vs. protein priming. C57BL/6 mice were primed i.m. with either gp120-encoding DNA, gp120 protein, or empty vector, 3 times, 2 wk apart. Some mice were sacrificed (S) 3 and 7 d after final priming injections. The remaining mice were rested for 4 wk, then all groups were given 2 booster injections of gp120 protein, 2 wk apart. Mice were then sacrificed 3 and 7 d after the final boosters.

Figure 2. Increased germinal center activity after gp120 DNA priming. Mice were immunized according to Figure 1 and sacrificed 3 and 7 d after final priming immunizations. (A) TFH and GC B cell populations after priming only. TFH cells gated on CD4⁺ CXCR5⁺ ICOS⁺ PD-1^hi. GC B cells gated on B220⁺ Fas⁺ PNA⁺ GL7⁺. n = 3. Percent of total spleen; mean ± SE. (B) Representative flow plots of TFH cells and GC B cells in (A) from day 3 after final immunization. (C) Serum anti-gp120-specific IgG titers after prime only. n = 3. D) Avidity of gp120-specific IgG antibodies after priming only. n = 3; mean ± SE *P < 0.05, **P < 0.01, ***P < 0.001. Representative of 2 experiments.

Figure 3. Enhanced GC B cells and TFH cell proportions with gp120 DNA priming. Mice were immunized as in Figure 1 and sacrificed 3 (day 73) and 7 (day 77) d after final gp120 protein booster injections (day 70). (A) TFH and GC B cell populations after prime-boost regimen. Cells gated as in Figure 1. Percent of total spleen. n = 3 – 6; mean ± SE **P < 0.01, ***P < 0.001 (B) Representative flow plots of TFH cells and GC B cells in (A) from day 3 after final protein booster. (C) Percent effector memory T cells (CD3⁺ CD4⁺ CD44^hi CD62L^-) in gp120 DNA and protein primed mice after protein boosters. Percent of T_h cells (CD3⁺ CD4⁺). n = 5 – 6; mean ± SE. (D) Ratio of TFH to effector memory cells. n = 5 – 6; mean ± SE ***P < 0.001 by t test. Representative of 3 experiments.

Figure 4. Priming with gp120-encoding DNA improves germinal center activity. Mice were immunized and sacrificed as in Figure 3. (A) Memory B cells (gated as CD19⁺ IgD^- GL7^- CD38⁺) in mice primed with gp120-encoding DNA or gp120 protein. Percent of total spleen. n = 4 – 6; mean ± SE (B) Memory B cells originating from the GC. Gated as CD19+ IgD^- GL7^- CD38⁺ CD73⁺. The CD73+ B cells are shown as a percentage of the memory B cell population. n = 4 – 6; mean ± SE *P < 0.05, **P < 0.01 by t test. (C) Transitional B cells (gated on CD19+ IgD^- GL7⁺ CD38⁺); percent of total spleen. n = 4 – 6; mean ± SE. Representative flow plots of day 3 and day 7 from mice primed with gp120 DNA. Quadrants show memory B cells (MB; top left) and transitional B cells (TB; top right). (D) Serum anti-gp120-specific IgG titers after boosting. mean ± SE. E) Avidity of serum anti-gp120-specific antibodies after boosting. mean ± SE n = 4 – 6; *P < 0.05, ***P < 0.001. Representative of 3 experiments.

Figure 5. Experimental design for deletion of BCL6. Cre-ERT2 BCL6^fl/fl mice were primed with either gp120-encoding DNA or gp120 protein, followed by 2 boosters of gp120 protein, as was done for Figures 3 – 4. Some Cre-ERT2 BCL6^fl/fl mice also received 5 i.p. injections of tamoxifen (T) between the prime and boost injections to delete BCL6. Mice were sacrificed 3 d after final injections.

Figure 6. Germinal centers are lost with deletion of BCL6, yet antibody titers increase. Cre-ERT2 BCL6^fl/fl mice were immunized as in Figure 5 and sacrificed 3 d after final protein booster immunizations. Some mice were given tamoxifen injections between prime and boost immunizations to delete BCL6. (A) TFH cells (gated as in Fig. 2) in spleen of mice either untreated or given i.p. tamoxifen injections before boosting. Percent of total spleen. n = 3 – 4; mean ± SE. Representative flow plots of each treatment condition are also shown. (B) GC B cells (gated as in Fig. 2) in spleen after prime-boost regimen with representative flow plots of each treatment condition. Percent of total spleen. n = 3 – 4; mean ± SE. (C) Serum anti-gp120-specific IgG titers and avidity of anti-gp120-specific antibodies after boosting. mean ± SE n = 3 – 4; *P < 0.05, **P < 0.01, ***P < 0.001