Epigenetic Features of HIV-Induced T-Cell Exhaustion Persist Despite Early Antiretroviral Therapy

T cell dysfunction occurs early following HIV infection, impacting the emergence of non-AIDS morbidities and limiting curative efforts. ART initiated during primary HIV infection (PHI) can reverse this dysfunction, but the extent of recovery is unknown. We studied 66 HIV-infected individuals treated from early PHI with up to three years of ART. Compared with HIV-uninfected controls, CD4 and CD8 T cells from early HIV infection were characterised by T cell activation and increased expression of the immune checkpoint receptors (ICRs) PD1, Tim-3 and TIGIT. Three years of ART lead to partial – but not complete – normalisation of ICR expression, the dynamics of which varied for individual ICRs. For HIV-specific cells, epigenetic profiling of tetramer-sorted CD8 T cells revealed that epigenetic features of exhaustion typically seen in chronic HIV infection were already present early in PHI, and that ART initiation during PHI resulted in only a partial shift of the epigenome to one with more favourable memory characteristics. These findings suggest that although ART initiation during PHI results in significant immune reconstitution, there may be only partial resolution of HIV-related phenotypic and epigenetic changes.


Fig. S2 -PD-1 expression on CD8 T cell subsets defined by T-bet and Eomes expression
(A) The percentage of each subset (T-bet neg Eomes neg , T-bet high Eomes low and Tbet low Eomes high ) expressing PD-1 is shown during PHI (red, left panel, n=54) and following 1 year of ART (blue, right panel, n=60). Groups were compared using a Friedman test (overall p for all tests <0.001) with subsequent pairwise comparisons performed with Dunn's test. Bars indicate median and interquartile range. ** indicates p<0.01; **** indicates p<0.0001. (B) Representative flow plots from each subset are overlaid from one donor during PHI.

Fig. S3 -Immune checkpoint receptor expression on CD8 T cell memory subsets during primary HIV infection
The expression of PD-1 (A-B), Tim-3 (C-D) and TIGIT (E-F) on naïve, central memory (CM), transitional memory (TM), effector memory (EM) and TEMRA cells is shown for HIVinfected individuals during PHI (n=60). Panels A, C, and E show ICR expression on CD8 T cells and panels B, D, and F show the same on CD4 T cells. Groups were compared using a Friedman test (overall p<0.05 for all cases), with subsequent pairwise comparison with Dunn's test corrected for multiple comparisons. Bars indicate median and interquartile range. * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.01; **** indicates p<0.001.

Fig. S4 -Increases in immune checkpoint receptor expression during primary HIV infection
Increases in ICR expression during PHI. Summary of comparisons of ICR expression between healthy controls (n=10) and individuals during PHI (n=60). Blocks, each corresponding to an ICR measured on a CD4 or CD8 T cell subset, are shaded according to the fold change in PHI relative to healthy controls. Groups were compared using Mann-Whitney tests and adjustment of p-values for multiple comparisons was performed across all comparisons presented here to control the false discovery rate. Asterisks and listed p-values correspond to the adjusted p-value. * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

Fig. S5 -Viral load and sampling timing for individuals used for epigenetic characterisation
Viral load dynamics and sampling timing for individuals used for epigenetic characterisation are shown (n=9). Individuals who received ART are shown in red, and those that were untreated are shown in green. VL measurements below the limit of detection are shown as open circles. Shading indicates the window during which samples were taken, with the individual timing indicated at the top.

Yes D Yes
A -RLRPGGKKR is an escape mutation which is poorly recognised by T cells which respond to RLRPGGKKK during primary HIV infection 1 . CD8 T cells which respond to RLRPGGKKK show decreased avidity, improved functionality and decreased PD-1 expression 2 . In this individual the wild type RLRPGGKKK is present as a variant at both time point, and there is ELISpot evidence of robust T cell response to this epitope.
B -GPGHKARIL is not a described escape mutation and there is ELISpot evidence of T cell responses to this epitope.
C -TSNLQEQIAW is a well-characterised early escape mutation which is not cross-recognised by wild-type T cells 3 . Similarly, TSNLQEQIQW is also escaped, with the Q at position 9 acting as compensatory mutation for the fitness cost associated with position 3 N substitution 4 . The binding position is given relative to a subtype B reference viral genome (HXB2). Abbreviations: cDNA, complementary DNA.