Impaired peripheral blood T-follicular helper cell function in HIV-infected nonresponders to the 2009 H1N1/09 vaccine

Abstract

The generation of Ab-secreting plasma cells depends critically on CD4 T-follicular helper (TFH) cells during the germinal center reaction. Germinal center TFH cells share functional properties with circulating CXCR5(+) CD4 T cells, referred to herein as peripheral TFH (pTFH) cells. Because deficient Ab production and CD4 T-cell loss are recognized features of HIV infection, in the present study, we investigated pTFH cells in 25 HIV-infected patients on antiretroviral therapy. pTFH frequency was equivalent in patients and healthy controls (HCs), and these cells displayed a central memory phenotype. Sixteen patients and 8 HCs in this group were given a single dose of H1N1/09 influenza vaccine during the 2009 H1N1 influenza outbreak. In the vaccine responders (n = 8) and HCs, pTFH cells underwent expansion with increased IL-21 and CXCL13 secretion in H1N1-stimulated PBMC culture supernatants at week 4 (T2). These changes were not seen in vaccine nonresponders (n = 8). In coculture experiments, sorted pTFH cells supported HIN1-stimulated IgG production by autologous B cells only in vaccine responders. At T2, frequencies of pTFH were correlated with memory B cells, serum H1N1 Ab titers, and Ag-induced IL-21 secretion. Characterization of pTFH cells may provide additional insight into cellular determinants of vaccine-induced Ab response, which may have relevance for vaccine design.

Figure 1

Characterization of peripheral TFH cells in HCs and HIV-infected patients. (A) Representative dot plots show gating strategy for pTFH cells, defined as CD4⁺CXCR5⁺ cells in an HIV-infected patient. Isotype control Ab was used to set the gate for CXCR5. T-cell differentiation subsets in the CD4⁺CXCR5⁺ and CD4⁺CXCR5⁻ subsets were based on the surface expression of CD45RO, CD27, and CD57 as T_N (CD45RO⁻CD27⁺), T_CM: (CD45RO⁺CD27⁺), T_EM (CD45RO⁺CD27⁻), T_E (CD45RO⁺CD57⁺), and T_TE (CD45RO⁻CD57⁺). (B) Frequency of pTFH (CD4⁺CXCR5⁺) cells are indicated as a percentage of total CD4 T cells in HCs and HIV-infected patients. (C-D) Distribution of CD4⁺CXCR5⁺ (C) and CD4⁺CXCR5⁻ (D) cells within the differentiation subsets. Box plots represent median with 25th and 75th percentile borders. Error bars represent 10th and 90th percentile means from 17 HCs and 25 HIV-infected aviremic patients. (E) Phenotyping for differentiation subsets in IL-21⁺ pTFH cells obtained after 5 hours of stimulation with 50 ng/mL of PMA, 1 μg/mL of ionomycin, and 10 μg/mL of brefeldin A.

Figure 2

pTFH cell expansion in vaccine responders is correlated with Ab production and B-cell differentiation. (A) Frequency of pTFH (CD4⁺CXCR5⁺) cells indicated as a percentage of total CD4 T cells in HCs and HIV-infected patients who were responders (R) or nonresponders (NR) to H1N1/09 influenza vaccine. Peripheral TFH cell frequencies were measured prevaccination (T0) and 4 weeks after vaccination (T2). (B-C) Frequency of Ki67⁺ pTFH (B) and non-pTFH (CD4⁺CXCR5⁻; C) cells at T0 and at T2. Box plots represent median with 25th and 75th percentile borders. Error bars represent the 10th and 90th percentile means from 8 HCs, 8 HIV⁺ responders, and 8 HIV⁺ nonresponders. (D-E) Linear correlation between frequency of pTFH cells with frequency of memory B cells (CD20⁺ CD21^high CD10⁻ CD27⁺) at T2 (D) and with H1N1 serum Ab titer at T2 (E).

Figure 3

pTFH cells promote B-cell Ab production and differentiation on H1N1-specific stimulation. Cryopreserved PBMCs obtained 4 weeks after H1N1/09 vaccination were thawed and rested overnight. B cells (CD20⁺) were isolated with magnetic beads and pTFH (CD3⁺CD4⁺CD45RA⁻CXCR5⁺) and non-pTFH (CD3⁺CD4⁺CD45RA⁻CXCR5⁻) cells were purified by cell sorting. B cells were cocultured with either pTFH or non-pTFH cells at a 1:1 ratio in medium alone or in the presence of 5 μg/mL of H1N1 vaccine Ag or 1 μg/mL of staphylococcal enterotoxin B for 7 days. (A,C) Culture supernatants from the pTFH + B-cell cocultures (A) or non-pTFH + B-cell cocultures (C) were harvested and IgG production was measured by ELISA. (B,D) Cells were harvested and frequency of plasmablasts (defined as CD20^lowCD21^low/−CD27⁺CD10⁻Ki67⁺ events) within the B cells was evaluated by flow cytometry in the pTFH + B-cell cocultures (B) or the non-pTFH + B-cell cocultures (D). Bars represent the means and error bars indicate the SD of 3 HCs, 3 HIV⁺ H1N1/09 vaccine responders (R), and 3 HIV⁺ vaccine nonresponders (NR).

Figure 4

H1N1 Ag promotes IL-21 production by PBMCs from vaccine responders on in vitro stimulation. (A) Cryopreserved PBMCs obtained at T0 and 4 weeks after H1N1/09 vaccination (T2) were thawed, rested overnight, and cultured at 1 × 10⁶ cells/mL for 5 days at 37°C in the presence of 5 μg/mL of H1N1 vaccine Ag. On day 5, supernatants were harvested and IL-21 levels were determined by ELISA. (B) Graph shows the linear correlation between the frequency of peripheral TFH cells at T2 with IL-21 levels in H1N1-stimulated PBMC culture supernatants. Bars represent the means and error bars indicate the SD of 5 HCs, 5 HIV⁺ responders (R), and 4 HIV⁺ nonresponders (NR). *P < .05.

Figure 5

ICOS expression in pTFH cells is correlated with IL-21 production and Ab response to H1N1/09 vaccine. (A) Representative dot plots showing the gating strategy for IL-21 and ICOS expression in pTFH cells, defined as CD4⁺CXCR5⁺ events, using appropriate isotype control Abs. Freshly isolated PBMCs at T2 were cultured in complete medium alone or in the presence of PMA, ionomycin, and brefeldin A for 5 hours and stained for intracellular IL-21. (B) Linear correlation of frequency of ICOS⁺ pTFH cells with IL-21–producing pTFH cells at T2 of 8 HCs, 7 HIV⁺ responders (R), and 7 HIV⁺ nonresponders (NR). (C) Linear correlation of frequency of ICOS⁺ pTFH cells with H1N1 serum Ab titer at T2 for 8 HCs, 7 HIV⁺ responders, and 7 HIV⁺ nonresponders. (D) Maturation subsets within ICOS⁺ pTFH cells. (E) Cryopreserved PBMCs were thawed, rested overnight, and cultured at 1 × 10⁶ cells/mL for 5 days at 37°C with H1N1 vaccine Ag or medium as described. Supernatants were harvested and CXCL13 levels were determined by ELISA. Bar graphs show CXCL13 levels at T0 and T2 after vaccination in 5 HCs, 6 HIV⁺ responders, and 6 HIV⁺ nonresponders. Box plots represent median with 25th and 75th percentile borders. Error bars represent the 10th and 90th percentiles.