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Peripheral blood CD4+CCR6+ compartment differentiates HIV-1 infected or seropositive elite controllers from long-term successfully treated individuals

HIV-1 infection induces a chronic inflammatory environment not restored by suppressive antiretroviral therapy (ART). As of today, the effect of viral suppression and immune reconstitution in people living with HIV-1 (PLWH) has been well described but not completely understood. Herein, we show how PLWH who naturally control the virus (PLWHEC) have a reduced proportion of CD4+CCR6+ and CD8+CCR6+ cells compared to PLWH on suppressive ART (PLWHART) and HIV-1 negative controls (HC). Expression of CCR2 was reduced on both CD4+, CD8+ and classical monocytes in PLWHEC compared to PLWHART and HC. Longer suppressive therapy, measured in the same patients, decreased number of cells expressing CCR2 on all monocytic cell populations while expression on CD8+ T cells increased. Furthermore, the CD4+CCR6+/CCR6− cells exhibited a unique proteomic profile with a modulated energy metabolism in PLWHEC compared to PLWHART independent of CCR6 status. The CD4+CCR6+ cells also showed an enrichment in proteins involved in apoptosis and p53 signalling in PLWHEC compared to PLWHART, indicative of increased sensitivity towards cell death mechanisms. Collectively, this data shows how PLWHEC have a unique chemokine receptor profile that may aid in facilitating natural control of HIV-1 infection.


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Alterations in chemokine receptor expression and chemokine levels modulate the activity of the immune 51 response and inflammatory levels during HIV-1 infection 12 . In people living with HIV-1 (PLWH) on 52 suppressive ART, these aberrations may contribute to i) higher risk of age associated co-morbidities 53 together with elevated immune activation ii) microbiome dysbiosis leading to microbial translocation 54 and iii) dysregulated immune cell activation and function 13,14,15,16 . Consequently, HIV-1 pathogenesis 55 and immune dysfunction promote chronic immune activation. This results in a vicious cycle as immune 56 activation likely drives HIV-1 disease progression and inflammaging, defined as an age-related increase 57 of inflammatory markers, that could possibly lead to earlier onset of age-related diseases. In our earlier 58 study on a small group of 10 PLWH on ART for two decades, we reported normalisation of most of the 59 plasma proinflammatory cytokines and chemokines to the level of HIV-negative controls 17 . Therefore, 60 although there are several studies on immune reconstitution and viral suppression during short-term 61 ART, the long-term effect of ART on the immune system is not known.

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Eradication of latently infected cells, i.e. a sterilising cure, has proven to be a major hurdle. An 64 alternative approach is a functional cure aimed at suppressing viral replication without ART. A model markers compared to PLWH who progress in their disease 24 . However, as no consensus exists on how 72 the PLWHEC phenotype is maintained, further studies comparing variability of immune function 73 between PLWHEC and PLWH on suppressive ART (herein PLWHART) are required to understand the 74 mechanisms of natural control of HIV-1.

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In our recent study 25 , we hypothesised that a modulated CCR6/CCL20 chemokine axis and CCR2-77 CCL7-CCL2 signalling may play a protective role in the PLWHEC phenotype. We identified a 78 downregulation of CCR2 and CCR6 receptors on CD4 + and CD8 + lymphocytes and higher plasma 79 abundance of CCL4, CCL7 and CCL20 in PLWHEC compared to the HIV-1-negative controls. This can 80 provide natural resistance to HIV-1 infection although the expression profile during long-term 81 suppressive ART is not known. In the present study, we extended our analysis to evaluate the expression  Fig 1). The clinical and demographic data are presented in Table 1 97 and Supplementary Table 1. All the PLWHART were successfully treated at the time of sample 98 collection and plasma viral load was below the detection level (<40 copies/mL). Initial discrimination 99 between CD4 + T cells (CD3 + CD4 + ) and CD8 + T cells (CD3 + CD8 + ) showed that both PLWHEC and 100 PLWHART exhibited a reduced proportion of CD4 + T cells and elevated proportion of CD8 + T cells 101 compared to HC (Fig. 1a). Single receptor expression in PLWHEC showed a distinct profile compared 102 to both PLWHART and HC (Fig. 1b). Expression of CCR2 and CCR6 was reduced on both CD4 + and 103 CD8 + T cells together with a decreased proportion of CD8 + CCR2 + , CD4 + CCR6 + , and CD8 + CCR6 + in 104 PLWHEC compared to both PLWHART and HC (Fig. 1c, d and Supplementary Fig. 2a and b). The 105 expression levels of CCR2 were also reduced on CD8 + T cells in PLWHART compared to HC, as well as 106 the proportion of CD8 + CCR6 + cells (Fig. 1c, and Supplementary Fig. 2b). Even as the total frequency 107 of CD4 + CCR3 + was low in all three groups, the expression of CCR3 was high in both PLWHEC and lower on CD8 + T cells in PLWHEC compared to PLWHART (Supplementary Fig. 2d), while no 110 difference was detected on CD4 + T cells. Collectively, these data show how PLWHEC have a unique 111 expression profile of CCR2 and CCR6 while the expression signature of CCR3 is more similar to 112 PLWHART on lymphocytic cells (Fig. 1e). Continuing, co-expression analysis on CD4 + cells showed 113 that CCR2 + CCR5 + CCR6 + , CCR2 + CCR6 + , and CCR5 + CCR6 + were reduced in PLWHEC compared to 114 both PLWHART and HC, while no differences were detected between PLWHART and HC (Fig. 1f).

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Furthermore, single receptor expression of CCR2 was reduced in PLWHEC compared to PLWHART and 116 CCR6 expression was reduced in PLWHEC compared to both PLWHART and HC (Fig. 1f). Within the 117 CD8 + T cell population, co-receptor expression of CCR2 + CCR5 + CCR6 + was reduced in both PLWHART 118 and PLWHEC compared to HC while CCR2 + CCR5 + was reduced in PLWHEC compared to HC and 119 PLWHART (Fig. 1g). Single receptor expression analysis also showed that CCR5 was increased in 120 PLWHEC compared to HC and CCR6 expression increased in PLWHEC compared to both HC and 121 PLWHART (Fig. 1g). As the major differences were detected on CCR2 and CCR6 expression, we 122 measured the levels of their ligands, namely monocyte chemoattractant protein 1, MCP1 (CCL2) and 123 macrophage inflammatory protein 3, MIP-3 (CCL20) respectively, in plasma from PLWHART (n=51), 124 PLWHEC (n=12), and HC (n=17). Plasma levels of CCL20 were increased in both PLWHEC and 125 PLWHART compared to HC, while no significant difference was detected for CCL2 (Supplementary

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Generally, the receptor expression of CCR3 and CCR6 was low on all three monocytic cell subsets ( Fig.   140 2c). The major difference was a reduced expression of CCR2 on CM and IM in PLWHEC compared to 141 HC and on CM compared to PLWHART ( Fig. 2d and Supplementary Fig. 3b). In PLWHART, both 142 frequency of IM-CCR2 + cells and expression of CCR2 on IM cells were reduced compared to HC ( Fig.   143 2d and Supplementary Fig. 3b). Although the proportion of CM-CCR6 + cells was decreased in 144 PLWHEC compared to PLWHART, CCR6 expression was increased in PLWHEC compared to HC and Furthermore, NCM-CCR6 + cells were lower in PLWHEC compared to PLWHART and HC, and the 147 frequency of CM-CCR3 + cells was decreased in PLWHEC compared to PLWHART (Supplementary Fig.   148 3c and d). The receptor expression of CCR5 was reduced on IM in PLWHART compared to HC and on 149 NCM in PLWHEC compared to both PLWHART and HC (Supplementary Fig. 3e). This data indicates 150 that the main difference on the monocytic cell subsets is a reduced expression of CCR2 in PLWHEC 151 compared to the other groups (Fig. 2e). The IM were the only category of cells which exhibited 152 differences in co-receptor expression. For these cells, the largest variation was a reduction of 153 CCR2 + CCR5 + expressing cells in both PLWHEC and PLWHART compared to HC. An increase in cells 154 lacking all four receptors was found in both PLWHEC and PLWHART compared to HC (Fig. 2f).

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Furthermore, PLWHART exhibited reduced numbers of cells expressing only CCR2 compared to HC 156 ( Fig. 2f). Collectively, this data shows that the largest variation occurred on IM where PLWHART and 157 PLWHEC showed a similar trend compared to HC.

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Longer suppressive therapy reduces expression of CCR2 on monocytes

160
The chronic inflammatory condition due to HIV-1 persistence and an activated immune response 161 persists during suppressive ART. Therefore, to evaluate if longer treatment duration affects the receptor 162 expression, we included longitudinal samples (timepoint 1 (T1) n=10 and timepoint 2 (T2) n=10) where 163 the same patients were followed-up after three years of additional suppressive therapy (clinical 164 parameters in Supplementary Table 2). Treatment duration did not affect frequencies of CD4 + , CD8 +

165
T cells or monocytic cell populations (Supplementary Fig. 4a and b). For the detected receptor 166 expressions, most of the differences were detected in the monocytic cell populations while no significant 167 variations were seen on CD4 + T cells (Fig. 3a). Longer treatment duration increased the frequency of 168 CD8 + CCR2 + cells, while all three monocytic cell populations exhibited reduced numbers of cells 169 expressing CCR2 in T2 ( Fig. 3b and c). Following a similar trend, CCR2 expression was increased on 170 CD8 + T cells and IM exhibited reduced expression of CCR2 in T2 while CM and NCM showed no 171 difference (Fig. 3d). The proportion of CD8 + CCR6 + cells was increased in T2, without showing any 172 significant effect of CCR2 expression levels ( Fig. 3e-g). Furthermore, the proportions of IM-CCR3 + and 173 IM-CCR5 + cells decreased over time while only expression levels of CCR5 decreased in T2 ( Fig. 3h-174 m). Investigations of co-receptor expression on the lymphocytic cell populations showed that the only 175 difference was an increase in CCR2 + CCR5 + in T2 on CD8 + T cells (Fig. 3n). Within the monocytic cell 176 populations, the only differences of co-expression of the receptors were seen on IM where T2 exhibited 177 higher percentage of cells negative for all four receptors while CCR2 + CCR3 + CCR5 + , CCR2 + CCR3 + , 178 and CCR2 + CCR5 + were decreased in T2 (Fig. 3o). Conclusively, the effect induced by longer 179 suppressive treatment mainly reduces expression levels of these chemokine receptors on IM.

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The HIV-1 reservoir is lower in PLWHEC compared to PLWHART To evaluate the proportion of HIV-1 infected cells in our cohort, we quantified total HIV-1 DNA in the 183 PLWHEC (n=14) and PLWHART (n=54). Herein, we observed a higher level of total HIV-1 DNA in 184 PLWHART compared to PLWHEC (Fig. 4a) despite a median of eight years of successful treatment. No 185 significant difference was observed between long term ART (lnART; >10yrs (median 20yrs,

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The subpopulation of CD4 + CCR6 + cells has been described as more permissive towards HIV-1 and 211 targets for productive infection 31, 32 . To further characterise the CD4 + T cells that express or lack the 212 CCR6 receptor we sorted out these cell populations using fluorescence activated cell sorting (FACS) 213 from HC (n=3), PLWHEC (n=6) and PLWHART (n=6) (Fig. 5a). The PLWHART samples were selected 214 from PLWH on long-term suppressive ART (median 20 years, IQR; 17.5-22.25). First, as the HIV-1 215 reservoir has been proposed to reside in CD4 + CCR6 + T cells, we measured the amount of integrated 216 HIV-1 in PLWHEC (n=5) and PLWHART (n=3). However, the variation within the data and the small 217 sample size made the comparison inconclusive (Supplementary Fig. 6a). To evaluate the specific 218 proteomic profile of CD4 + CCR6and CD4 + CCR6 + cell populations we isolated these cell populations by FACS (15,000-30,000 cells) and performed mass spectrometry-based proteomics using a quantitative 220 tandem mass tags (TMT) labelling approach. Due to low protein detection by mass spectrometry four 221 PLWHEC samples, two samples out of each of the CCR6 + and CCR6cell populations were excluded 222 from the analysis. The principal component analysis (PCA) identified heterogeneity among the samples 223 ( Supplementary Fig. 6b). Differential expression analysis was unable to find statistically significant 224 regulated proteins due to heterogeneity among samples and low sample size. Therefore, functional 225 analysis was performed using a consensus scoring approach based on multiple protein set analysis (PSA) 226 run by incorporating the directionality of protein abundance. Using the group specific consensus scores 227 (PLWHEC vs PLWHART) and directionality classes, we identified distinct upregulation of oxidative 228 phosphorylation (OXPHOS) (adj p<0.05) and interferon-a response (adj p<0.05) and distinct 229 downregulation of glycolysis (adj p<0.05) in both CD4 + CCR6 + and CD4 + CCR6cells while using the 230 MSigDB hallmark gene-set (Fig. 5b). Similarly, the KEGG gene-set also identified a distinct 231 upregulation of OXPHOS (adj p<0.1), and a distinct downregulation of other pathways of amino acid 232 and carbohydrate metabolism (Supplementary Fig. 6c). This data indicates that the metabolic trade-off 233 of increased OXPHOS and reduced glycolysis is elevated in PLWHEC compared to PLWHART (Fig. 5c).

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The relative abundance of the OXPHOS proteins were higher in PLWHEC compared to HC while lower 235 in PLWHART compared to HC (Supplementary Fig. 7a and b). Interestingly, only p53 signalling and 236 apoptosis were distinctly upregulated in CD4 + CCR6 + (adj p <0.05) (Fig. 5b) and both pathways had 237 unique features in PLWHART (Fig. 5d and e). The majority of the PLWHART clustered together. There 238 were two downregulated proteins in PLWHEC (ISCU and LDHB) that were common between OXPHOS 239 and p53 signalling. Furthermore, four proteins (CASP7, VDAC2, RHOT2 and GPX4) were common 240 between OXPHOS and apoptosis, while seven proteins (FAS, TAP1, FDXR, TXNIP, CASP1, APP and 241 HMOX1) were common between p53 signalling and apoptosis among the proteins upregulated in 242 PLWHEC compared to PLWHART (Fig. 5f). Taken together, this data shows how the CD4 + CCR6 + cell 243 population have a unique profile in PLWHEC. The co-regulation of OXPHOS, p53-signalling and 244 apoptosis may be a contributing factor to control of HIV-1 infection.

Discussion 247
In our study, a unique cell type expression profile of CCR2 and CCR6 was identified in PLWHEC, while 248 PLWHART exhibited an expression profile more like HC. CCR6 + cells were thus decreased on both CD4 + 249 and CD8 + T cells in PLWHEC but not in PLWHART. Furthermore, the CD4 + T cells exhibited a protein

268
We have earlier showen that low expression of CCR6 and high levels of its ligand, CCL20, may result 269 in low levels of inflammation in PLWHEC 25 . Herein, we detected increased plasma levels of CCL20 in 270 PLWH, independent of HIV-1 status compared to HC, confirming earlier observations 42 . CCL20 has 271 previously been described to have an effect on the infection step of HIV-1, migration of immune cells 272 and immune cell function. However, we did not see any difference in CCL20 levels between PLWHEC 273 and PLWHART. Therefore, most likely it is the CD4 + CCR6 + cells, rather than levels of CCL20, that in 274 PLWHEC could contribute to natural control of HIV-1.

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Furthermore, CD4 + CCR6 + and CD8 + CCR6 + cells decline in untreated patients with HIV-1 43 since the 276 CCR6 + cells are more susceptible towards apoptosis and therefore involved in the depletion of memory 277 T cells. However, we did not detect any alterations in CD4 + CCR6 + compartments in PLWHART 278 compared to HC, although for CD8 + CCR6 + cells there was a reduction in both patient-groups. We

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Earlier studies have shown that 3' deletion clones decline during suppressive therapy 53 . These specific 308 mutated proviruses may be under negative selection due to their capacity to stimulate the immune system

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For the quantification of intact, 5' deleted, and 3' deleted / hypermutated proviruses, the Intact Proviral 395 DNA Assay was performed as previously described 28 . Briefly, a first ddPCR was performed as 396 described above with primer/probe combinations for the packaging signal (Ψ), which frequently shows 397 small mutations. The second reaction targets the HIV-1 Rev response element, present in the Env coding region. Two hydrolysis probes were used in this reaction: a HEX-labeled probe specific for the intact 399 proviral sequence and an unlabeled probe specific for the APOBEC-3G hypermutation, which competes 400 with the HEX-labeled probe and results in no signal in the case of APOBEC hypermutations. Duplicate 401 wells for IPDA were merged during analysis. All primer and probe details can be viewed in

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Gene set enrichment analysis was performed using R/Bioconductor package piano v2.2.0. Gene level t-459 statistics given by Limma, and hallmark gene set downloaded from MSigDB were used to find 460 significantly enriched gene-sets.