Paradoxical aging in HIV: immune senescence of B Cells is most prominent in young age

Abstract

Combination antiretroviral therapies (cART)can lead to normal life expectancy in HIV-infected persons, and people aged >50 yrs represent the fastest growing HIV group. Although HIV and aging are independently associated with impaired humoral immunity, immune status in people aging with HIV is relatively unexplored. In this study influenza vaccination was used to probe age associated perturbations in the B cell compartment of HIV-negative "healthy controls" (HC) and virologically controlled HIV-infected participants on cART (HIV) (n=124), grouped by age as young (<40 yrs), middle-aged (40-59yrs) or old (>60 yrs). H1N1 antibody response at d21 post-vaccination correlated inversely with age in both HC and HIV. Immunophenotyping of cryopreserved PBMC demonstrated increased frequencies of double negative B cells and decreased plasmablasts in old compared to young HC. Remarkably, young HIV were different from young HC but similar to old HC in B cell phenotype, influenza specific spontaneous (d7) or memory (d21) antibody secreting cells. We conclude that B cell immune senescence is a prominent phenomenon in young HIV in comparison to young HC, but distinctions between old HIV and old HC are less evident though both groups manifest age-associated B cell dysfunction.

Keywords: B cells; HIV; PD1; aging; chronic infections; immunosenescence; influenza vaccination.

Figure 1. Serological response to H1N1 in HIV (Black) and HC (Grey)

(A) H1N1 specific HAI titer at T0, T1 and T2 between young and old HIV (black) and HC (grey) participants. Statistical significance (p value <0.05, Wilcoxon test) is depicted as * and †. (B) Mean ±SEM of fold change, (T2/T0) in H1N1-specific HAI titer; Mann-Whitney test did not show differences to be significant. (C) Correlation of biological age with HAI titer at T2 in HC (left) and HIV (right) by Spearman correlation test. Serological values are expressed in log2 scale. Age groups are depicted as squares (young, <40 years), triangles (middle, 40-60 years) and circles (old, ≥60 years).

Figure 2. Spontaneous H1N1-specific ASC at T1 in HIV (Black) and HC (Grey)

(A) Mean ±SEM of H1N1 specific spontaneous ASC was assessed by ELISpot using PBMC 7 days after vaccination any pre activation. Mann-Whitney test was performed; *, Statistical significance at p <0.05. (B) H1N1 specific spontaneous ASC assessed in 60 Healthy controls (Left) and 64 HIV infected individuals (Right). ELISpot values are expressed as log2 scale. Age groups depicted as squares (young, <40 years), triangles (middle, 40-59 years) and circles (old, ≥60 years). Spearman correlation was performed.

Figure 3. Correlation between age and H1N1-specific Memory B cells and plasmablasts response in HIV (Black) and HC (Grey)

(A) Evaluation of the H1N1 specific memory B cells at T0 and T2. Lines represent mean ±SEM. For comparisons between age groups at same time point, Mann-Whitney test was performed and for comparisons between time points of the same age group, Wilcoxon signed-rank test was performed; *, Statistical significance at p <0.05. (B) Age was correlated with (Top) H1N1 specific memory B cells at T0 assessed with ELISpot using PBMC pre vaccination stimulated for 5 days with H1N1 (5μg/ml) and (Bottom) H1N1 specific memory B cells at T2 assessed with ELISpot using PBMC 21 days after vaccination stimulated for 5 days with H1N1 (5μg/ml) in (Left) 60 Healthy controls and (Right) 64 HIV infected individuals. ELISpot values are expressed as log2 scale. Age groups depicted as squares (young, <40 years), triangles (middle, 40-59 years) and circles (old, ≥60 years). Spearman correlation was performed.

Figure 4. Evaluation of B cell subsets in HIV (Black) and HC (Grey)

B cells subsets are reported as frequencies of the CD20+CD3- B cells. Age groups are depicted as squares (young, <40 years) and circles (old, ≥60 years). *, Statistical significance at p <0.05 by Mann-Whitney test.

Figure 5. B Cells of young HIV exhibit markers of Immune activation and exhaustion

(A) Example of gating strategy for CD80+, PDL1+ and FcRL4+ B cells. (B) Frequencies of CD80+ B cell and (C) Frequencies of PDL1+ B cell in HC (Grey) and HIV (Black). Age groups are depicted as squares (young, <40 years) and circles (old, ≥60 years). *, Statistical significance at p <0.05 by Mann-Whitney test.

Figure 6. Plasmablasts at T0 are positively correlated with B cellular response to H1N1 in HIV (Black) and HC (Grey)

Frequencies of plasmablast before vaccination were correlated with the H1N1 specific spontaneous ASC by ELISpot in PBMC at 7 days after vaccination. ELISpot values are expressed as log2 scale. Age groups depicted as squares (young, <40 years), triangles (middle, 40-59 years) and circles (old, ≥60 years). Spearman correlation was performed.

Figure 7. Immune activation and immune regulation influence influenza Ab responses in HIV

(A) The frequency of the PDL1+ B cells before vaccination was correlated with the frequency of the plasmablasts before vaccination. (B) Frequencies of the CD80+ Naïve B cells at T0 were correlated with the H1N1 specific HAI titer fold change (T2/T0). Age groups are depicted as squares (young, <40 years), triangles (middle, 40-59 years) and circles (old, ≥60 years). Spearman correlation was performed.

Figure 8. PDL1+ B cells are related to immune aging and exhaustion of B cells

Frequencies of PDL1+ B cells at T0 were correlated with (A, B) frequencies of double negative B cells; (C, D) frequencies of exhausted B cells (DN/FcRL4+) and (E, F) with frequencies of exhausted T cells (CD4/PD1+) in 60 healthy controls (Left, Grey) and 64 HIV infected individuals (Right, Black). Age groups are depicted as squares (young, <40 years), triangles (middle, 40-59 years) and circles (old, ≥60 years). Spearman correlation was performed in all the correlation tests.

Figure 9. Time under cART reduces B cell immune activation and immune senescence but has no effect on PDL1 expression on B cells

(A) The frequency of the CD80+ B cells (Left) and CD80+ Naïve B cells (Right) before vaccination was correlated with the duration of cART. (B) Frequencies of the DN B cells were correlated with the years under cART. (C) Frequencies of the PDL1+ B cells at T0 were correlated with the years under cART. Age groups are depicted as squares (young, <40 years), triangles (middle, 40-59 years) and circles (old, ≥60 years). Spearman correlation was performed.