Sex differences in HIV-1 reservoir cell selection are linked to altered innate immune profiles

Abstract

HIV-1 persistence despite suppressive antiretroviral therapy (ART) is primarily because of infected memory CD4 T cells, so-called viral reservoir cells, that harbor chromosomally integrated viral DNA as a "provirus" and resist clearance by the human immune system. Biological sex affects host immune responses and may influence selection and evolution of HIV-1 reservoir cells during long-term ART for HIV infection. We assessed more than 4073 individual proviruses through single-molecule amplification from 30 females and 35 males living with HIV-1 and treated with ART for a median of 20 years. We observed that the HIV-1 reservoir profile in females was characterized by lower proviral phylogenetic complexity, an increased proportion of clonally expanded intact proviruses, and a higher proportion of intact proviruses integrated into repressive heterochromatin locations of the human genome. The evolution of this distinct viral reservoir profile in females was associated with an improved signature of innate immune responses, specifically those of NK cells. On the contrary, signs of viral sequence adaptation to adaptive T cell immune responses were more pronounced in intact HIV-1 proviruses from males. Collectively, these data suggest a stronger ability of the female immune system to drive immune selection of HIV-1 reservoir cells during ART, putatively because of improved innate immune function.

Fig. 1.. Proviral reservoir size and clonality in people living with HIV-1 on long-term suppressive ART.

(A-C) Shown are the frequencies of total (A), intact (B), and defective (C) proviruses in individuals on moderate durations of antiretroviral treatment (mART) and individuals on long-term suppressive ART (LT-ART). Open circles represent data at the limit of detection. Horizontal lines reflect the median. (D) Shown are proportions of intact or defective proviruses among all proviral genomes. Psi, packaging signal. (E) Shown are proportions of clonal and non-clonal intact proviruses. Participants with at least two detectable genome-intact proviruses are included. Mann-Whitney U nonparametric tests were used in A-C; two-tailed χ² tests were used in D-E. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Number of participants in panels A-C is represented by n; number of viral sequences in panels D-E is represented by n.

Fig. 2. Sex-based differences in reservoir size and clonality in people living with HIV-1 on long-term suppressive ART.

(A-C) Shown are frequencies of total (A), intact (B), defective (C) proviruses in individuals on moderate ART (mART) or long-term suppressive ART (LT-ART), segregated based on sex. Open circles represent data at the limit of detection. (D) Shown are proportions of intact or defective proviruses. Psi, packaging signal. (E, F) Shown are frequencies (E) and proportions (F) of clonally-expanded intact proviruses in indicated cohorts stratified based on sex. Participants with at least two detectable intact proviruses are included. (G) Circular maximum-likelihood phylogenetic trees for all genome-intact proviral sequences from LT-ART males and females. HXB2, reference HIV-1 sequence. Symbols indicate sequences generated by FLIP-seq (circle) or MIP-seq (square). Genome-intact proviral sequences from a given study participant are depicted in the same color. Grey circles reflect detection of a sole single genome-intact sequence in a given study participant. Clonal sequences, defined by complete sequence identity, are indicated by grey arches. Open symbols represent non-clonal genome-intact sequences in participants with at least 2 genome-intact sequences detected. The maximum-likelihood phylogenetic trees were inferenced using PhyML. (H) Stacked bar charts reflect the clonal composition of HIV-1 reservoir cells in LT-ART males and females. Each color represents an individual clone of sequence-identical genome-intact proviruses, whereas grey-colored sub-bars represent proviruses detected only once. Sequences derived from participants with at least 2 genome-intact sequences are included. Horizontal lines reflect the median (A-C, E). Mann-Whitney U nonparametric tests were used in panels A-C and E; two-tailed χ² tests were used in panels D, F and H. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. n, indicates the number of participants in panels A-C, E and G or the number of viral sequences in panels D, F and H.

Fig. 3.. Chromosomal positioning of intact HIV-1 proviruses in females on long-term suppressive ART.

Maximum-likelihood phylogenetic trees of intact proviruses from females on LT-ART (LT-F01 – LT-F014). Coordinates of chromosomal integration sites and corresponding gene names (if applicable) are indicated. Symbols indicate sequences generated by FLIP-seq, MIP-seq, or from quantitative viral outgrowth assays (qVOAs). *Sequences generated by FLIP-seq or MIP-seq that may be part of adjacent clonal clusters but differ by 1–2 base pairs, possibly due to PCR amplification or sequencing errors. ^#(n)Sequences derived from replication-competent proviral genomes retrieved in qVOAs that differ by n number of base pairs from adjacent clonal sequences; these sequence variations likely reflect mutations originating from viral sequence diversification during in vitro culture in qVOA assays.

Fig. 4.. Chromosomal positioning of intact HIV-1 proviruses in males on long-term suppressive ART.

Maximum-likelihood phylogenetic trees of intact proviruses from males on LT-ART (LT-M01 – LT-M017). Coordinates of chromosomal integration sites and corresponding gene names (if applicable) are indicated. Symbols indicate sequences generated by FLIP-seq, MIP-seq, or from quantitative viral outgrowth assays (qVOAs). *Sequences generated by FLIP-seq or MIP-seq that may be part of adjacent clonal clusters but differ by 1–2 base pairs, possibly due to PCR amplification or sequencing errors. ^#(n)Sequences derived from replication-competent proviral genomes retrieved in qVOAs that differ by n number of base pairs from adjacent clonal sequences; these sequence variations likely reflect mutations originating from viral sequence diversification during in vitro culture in qVOA assays.

Fig. 5.. Integration site profile of intact and defective HIV-1 proviruses in people living with HIV-1 on long-term suppressive ART.

(A, B) Proportions of intact and defective proviruses in defined genomic regions in indicated study cohorts (A), and in LT-ART males and females (B). (C, D) Proportions of proviral integration sites located in Hi-C genomic compartments A and B (and associated subcompartments) in mART and LT-ART individuals (C), and in LT-ART males and females (D). (E, F) Chromosomal distance of proviral integration sites to most proximal host transcriptional start sites (TSS), as determined by RNA-seq in CD4 T cells from reference datasets in indicated study cohorts (E) or in LT-ART males and females (F). Horizontal lines reflect the median. (G, H) Box and whisker plots reflect activating histone protein modifications in proximity to proviral integration sites inferred from reference datasets of the ROADMAP consortium (68). The median, the 25% and 75% percentile and the minimum and maximum of H3K27ac and H3K4me1ChIP-seq reads within ± 2500 basepairs of the proviral integration site are shown in indicated study cohorts (G), and in LT-ART males and females (H). Integration sites not covered in the reference dataset were excluded in panels C-H. Kruskal-Wallis nonparametric tests were used in panels E-H, two-tailed χ² tests were used in panels A-D. Nominal P values are shown in panels E-H. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Clonal integration sites are counted once. n, indicates the number of proviral integration sites.

Fig. 6.. Footprints of viral immune escape from host T and B cell immune responses.

(A, B) Average genetic distance, determined by pairwise comparisons of nucleotide differences, between all intact proviruses from a given study participant, in indicated study cohorts (A), and in males and females on LT-ART (B). Participants with at least two intact proviruses detected are included. (C, D) Number of signature sites associated with resistance to broadly neutralizing antibodies (bNAbs) in intact proviral sequences of indicated study cohorts (C), and in males and females on LT-ART (D). Signature sites associated with resistance to four classes of bNAbs were determined as described before (66). (E-H) Proportions of CTL epitopes (restricted by autologous HLA class I alleles) within intact proviruses that harbor the clade B consensus wild-type sequences (E-F) or previously described CTL escape variants (G-H). Defined escape mutations listed in the LANL HIV Immunology Database (www.hiv.lanl.gov) were considered. Horizontal lines reflect the median. Mann-Whitney U non-parametric tests were used. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Clonal sequences are counted once. n, indicates the number of participants in panels A and B or the number of viral sequences in panels C-H.

Fig. 7.. Innate immune responses in people living with HIV-1 on long-term suppressive ART.

(A, D) Linear discriminant analysis of total innate immune cells (A) and NK cells (D) in indicated study subgroups of LT-ART participants is shown. The phenotypic profile of total innate immune cells and NK cells in PBMC samples was characterized by the expression of 25 (A) or 15 (D) relevant surface markers. (B, C) Proportion of CD64⁺PD-L1⁺ mDCs in indicated study subgroups (B) and their associations with frequencies of clonal intact proviruses and proportions of intact proviruses in heterochromatin regions among total proviruses (C). (E-L) SPICE diagrams highlighting proportions of NK cells with zero, one or more inhibitory (E-H) or activating (I-L) phenotypic markers. Pie chart colors reflect the proportions of cells expressing the indicated numbers of markers; arcs reflect the proportion of cells expressing a given marker. Proportions of NK cells with at least one feature (G, K), and their associations with frequencies of clonal intact proviruses and proportions of intact proviruses in heterochromatin regions among total proviruses (H, L) are shown. (M, N) Ratio of NKG2A-expressing versus NKG2C-expressing NK cells in indicated study subgroups (M) and their associations with frequencies of clonal intact proviruses and proportions of intact proviruses in heterochromatin regions among total proviruses (N). The study subgroups are segregated based on sex (F, female; M, male) and the proportion of intact HIV-1 reservoir in heterochromatin locations (HTC^high or HTC^low/−). Horizontal bars reflect the median. Mann-Whitney U tests or Kruskal-Wallis nonparametric tests were used in panels B, G, K and M, permutation tests were used in panels E, F, I, and J, and linear regressions were used in panels C, H, L, and N. Linear regression coefficients (r²) and nominal P values are shown. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

Fig. 8.. T cell immune responses in people living with HIV-1 on long-term suppressive ART.

(A, J) Linear discriminant analysis of CD4 (A) and CD8 (H) T cells in indicated study subgroups in participants on LT-ART. The phenotypic and functional profile was characterized using the expression of 10 shared surface and intracellular markers following stimulation with HIV-1 Gag peptide pool. (B-E, K-N) Proportions of HIV-1-Gag specific CD4 (B, D) and CD8 (K, M) T cells in total CD4 or CD8 T cells, identified based on upregulation of any of the activation-induced markers (CD25, OX40, CD69 and CD40L) (B, K) or secretion of cytokines (IFN-γ, IL-2, TNF-α) (D, M) in indicated study subgroups. Associations with frequencies of clonal intact proviruses and proportions of intact proviruses in heterochromatin regions among total proviruses are indicated (C, E, L, N). (F-G, O-P) SPICE diagrams highlighting proportions of HIV-1-specific CD4 (F, G) and CD8 (O, P) T cells with one or more of the indicated functions. Pie chart colors reflect the number of effector functions, whereas proportions of cells expressing a specific effector function are indicated by an arc. (H-L, Q-R) Proportions of HIV-1-specific CD4 (H) and CD8 (Q) T cells with at least two functions and their associations with frequencies of clonal intact proviruses and proportions of intact proviruses in heterochromatin regions among total proviruses (I, R) are shown. The study subgroups are segregated based on sex (F, female; M, male) and the proportion of intact HIV-1 reservoir in heterochromatin locations (HTC^high or HTC^low/−). Horizontal bars in dot plots represent the median. Mann-Whitney U tests or Kruskal-Wallis nonparametric tests were used in panels B, D, H, K, M and Q, permutation tests were used in panels F-G and O-P, and linear regressions were used in panels C, E, I, L, N and R. Linear regression coefficients (r²) and nominal P values are shown. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.