The Role of Naïve T-Cells in HIV-1 Pathogenesis: an Emerging Key Player Gabriela Khoury A, D, Reena Rajasuriar A, E, Paul U

Clinical Immunology (2011) 141, 253–267

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Clinical Immunology

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REVIEW The role of naïve T-cells in HIV-1 pathogenesis: An emerging key player Gabriela Khoury a, d, Reena Rajasuriar a, e, Paul U. Cameron a, b, c, d, Sharon R. Lewin a, c, d,⁎ a Department of Medicine, Monash University, 85 Commercial Road, Melbourne Victoria, 3004, Australia b Department of Immunology, Monash University, 85 Commercial Road, Melbourne Victoria, 3004, Australia c Infectious Diseases Unit, Alfred Hospital, 85 Commercial Road, Melbourne, Victoria, 3004, Australia d Centre for Virology, Burnet Institute, 85 Commercial Road Melbourne, Victoria 3004, Australia e Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

Received 8 July 2011; accepted with revision 8 September 2011 Available online 17 September 2011

KEYWORDS Abstract Functional naïve T-cells are critical for an effective immune response to multiple Naïve T-cells; pathogens. HIV leads to a significant reduction in CD4+ naïve T-cell number and impaired func- HIV-1; tion and there is incomplete recovery following combination antiretroviral therapy (cART). Here IL-7; we review the basic homeostatic mechanisms that maintain naïve CD4+ T-cells and discuss re- Immune reconstitution; cent developments in understanding the impact of HIV infection on naïve CD4+ T-cells. Finally Antiretroviral therapy; we review therapeutic interventions in HIV-infected individuals aimed at specifically enhancing Reservoirs recovery of naïve CD4+ T-cells. © 2011 Elsevier Inc. All rights reserved.

Contents

1. Introduction ...... 254 2. NaïveT-cellhomeostasis:insightsfrommurinemodels ...... 254 2.1. Homeostatic expansion ...... 254 2.2. Homeostatic proliferation ...... 255 3. NaïveT-cellhomeostasis,HIVinfectionandtheimpactofcART...... 256 3.1. Naïve T-cell number ...... 256 3.2. Naïve T-cell function ...... 257

⁎ Corresponding author at: Infectious Diseases Unit, Alfred Hospital, 85 Commercial Road, Melbourne, Victoria, 3004, Australia. Fax: +61 3 9076 2431. E-mail address: [email protected] (S.R. Lewin).

3.2.1. Response to IL-7 ...... 257 3.2.2. TCR diversity ...... 257 3.2.3. Response to neo-antigens ...... 257 4. HIV infection of naïve T-cells ...... 257 4.1. Infection of naïve T-cells in vitro ...... 257 4.1.1. Role of chemokines ...... 258 4.1.2. Role of IL-7 ...... 258 4.1.3. Tissue microenvironment and abortive infection ...... 258 4.1.4. DC-naïve T-cell interactions ...... 258 4.2. Infection of thymocyte subsets in vitro ...... 258 4.3. Infection of naïve T-cells in vivo ...... 258 4.3.1. Total and integrated HIV DNA ...... 258 4.3.2. Viral compartmentalization ...... 259 4.3.3. Viral co-receptor usage ...... 259 5. Therapeutic approaches to enhance naïve T-cell recovery ...... 260 5.1. Exogenous IL-2 therapy ...... 260 5.2. Exogenous IL-7 therapy ...... 260 5.2.1. Animal preclinical studies ...... 260 5.2.2. Human clinical trials: HIV-infected patients ...... 260 5.2.3. IL-7 Receptor polymorphisms ...... 260 5.3. Other novel therapeutic approaches ...... 261 6. Outstanding research questions ...... 261 7. Conclusion ...... 261 Role of the funding source ...... 261 Conflict of interest statement ...... 261 References ...... 261

1. Introduction decrease in CD31 expression and TREC concentration resulting in an increase in the proportion of CD31- naïve T-cells HIV infection is characterized by substantial depletion of CD4+ with aging [9,11]. The mechanisms which drive homeostatic T-cells including recent thymic emigrants, naïve T-cells and proliferation of naïve T-cells in humans are not fully under- memory T-cells. Following control of HIV replication with stood, however extensive studies in murine models have effective combination antiretroviral therapy (cART), CD4+ demonstrated multiple factors are involved in both post- T-cells recover to normal levels, in most but not all patients, thymic survival and proliferation of naïve T-cells. Exposure and impaired CD4+ T-cell recovery has been associated with to the cytokine IL-7 and contact with major histocompati- non-AIDS events including cardiovascular disease, liver disease bility complex (MHC) molecules presenting self-peptides and malignancy [1–4]. Therefore despite the substantial through the T-cell receptor (TCR) within secondary lym- reduction in morbidity and mortality from cART, life expec- phoid tissue are both critical for naïve T-cell homeostasis tancy has still not returned to normal [5,6]. A detailed under- [12–15].(SeeFig. 1). standing of naïve CD4 T-cell homeostasis is required in order to develop novel strategies to enhance the quality of immune reconstitution following cART. 2.1. Homeostatic expansion

In lymphopenic murine models, adoptive transfer of naïve T- 2. Naïve T-cell homeostasis: insights from murine cells results in a biphasic increase in naïve T-cells with both models rapid and slow proliferation phases. Rapid proliferation (or homeostatic expansion) was associated with conversion of naïve Naïve T-cells are characterized by the expression of surface T-cells into an effector/memory phenotype [16,17]. There are markers CD45RA, CD27, CD28, CD62L, CCR7 and the IL-7 diverse views on whether IL-7 is required for homeostatic ex- receptor [7,8]. Naïve T-cells exit the thymus following matu- pansion which is thought to be dependent on the strength of ration and are enriched for T-cell receptor excision circles binding between antigen and the TCR. Proliferative responses (TREC) and express the surface marker platelet endothelial following low-affinity binding, as seen with self-peptides, is cell adhesion molecule-1 (PECAM-1), also known as CD31 [9]. thought to be more dependent on IL-7 [12,18] compared to Naïve T-cells circulate between the blood and the lymphoid high-affinity cognate antigen binding (reviewed in [19]). tissue driven by cell surface markers CD62L and CCR7 [10]. Naïve T-cell homeostatic expansion has also been shown to The number of naïve T-cells in blood remains relatively be associated with exposure to microbial products. Following constant throughout adult life despite continuous stimulation adoptive transfer of T-cells from immunocompetent BALB/ with foreign antigens and a dramatic reduction in thymic out- cThy1.1 mice to mice with severe combined immunodeficien- put with age. Maintenance of naïve T-cells in adults is largely cy (SCID) that have been raised in a germ-free environment, dependent on homeostatic proliferation which leads to a there was little naïve T-cell proliferation, activation or The role of naïve T-cells in HIV-1 pathogenesis: An emerging key player 255

Figure 1 Naïve T-cell homeostasis. (A) Precursor T-cells from the bone marrow undergo maturation in the thymus through positive and negative selection. (B) Recent thymic emigrants (blue) leave the thymus and enter the periphery expressing CD45RA+CCR7+CD62L+CD31+ (blue square) and containing high concentrations of T-cell Receptor Excision Circles (TREC, blue circle). Naïve T-cells can then undergo different outcomes, including (C) homeostatic proliferation following contact with IL-7 and self peptide presented by antigen presenting cells resulting in the production of naïve T-cells with reduced concentration of TREC and decreased expression of CD31 (purple) or (D) conversion to memory/effector cells (orange and pink) which are CD45RO+CD45RA− CD31−, following presentation of foreign antigen by antigen presenting cells (green).

differentiation into a memory/effector phenotype suggesting proliferation) where the naïve phenotype is maintained and is that exposure to environmental pathogens my contribute to dependent on IL-7 [16,17,25,26]. When naïve T-cells were naïve T-cell expansion [20]. In addition, in mice who have adoptively transferred into IL-7-deficient mice, survival of had chemical ablation of the thymus, microbial products in these cells was significantly compromised [12]. Survival of plasma were elevated and there was an increase in chronic im- naïve T-cells was also dependent upon ongoing contact with mune activation thought to be secondary to a reduction in cir- self-peptide through presentation by either MHC Class I or II culating naïve T-cells and loss of gut integrity [21]. Therefore, and interaction with TCR of CD8+ and CD4+ naïve T-cells naïve T-cell homeostatic expansion is influenced by exposure respectively [14,15,27,28]. to environmental antigens and loss of naïve T-cells may in A recent study suggested that the source of IL-7 also plays turn indirectly influence gut integrity. Given HIV infection is an important role in homeostatic proliferation of CD4+ naïve characterized by high levels of circulating lipopolysaccharide T-cells [29]. Following adoptive transfer of T-cells into (LPS) and significant abnormalities in the gastrointestinal lymphopenic mice deficient in recombination–activation (GI) tract mucosa, these data may account for a significant gene-1 (Rag1−/−), CD8+ T-cells underwent proliferation in change in naïve T-cell homeostasis in HIV infection [22–24]. response to elevated peripheral IL-7 levels while proliferation of CD4+ naïve T-cells required the presence of dendritic cells (DCs) for both IL-7 and contact with MHC class II. The authors 2.2. Homeostatic proliferation demonstrated that during lymphopenia, when IL-7 levels were elevated, a subset of DCs that express the IL-7 receptor In addition to rapid proliferation, adoptive transfer of naïve α chain (CD127) down-regulate MHC class II by negative feed- T-cells also leads to slow proliferation (also called homeostatic back [29]. This reduced the ability of the DC to interact with 256 G. Khoury et al. the CD4+ naïve T-cell and therefore prevented proliferation. It depletion of naïve CD4+ T-cells and the FRC network in the set- is unknown whether similar mechanisms involving CD127-pos- ting of untreated HIV infection. itive DCs exist in humans. HIV infection leads to significant depletion of total muco- sal CD4+ T-cells in the GI tract which allow for migration of microbial products into the systemic circulation [22,24]. Mi- 3. Naïve T-cell homeostasis, HIV infection and the crobial products, specifically LPS, can drive DC activation via ligation of toll like receptors (TLR) leading to elevated pro- impact of cART inflammatory cytokines and enhanced T-cell proliferation [22,39,40]. Microbial products may also be responsible for 3.1. Naïve T-cell number enhanced naïve T-cell proliferation as demonstrated in mouse models but not yet confirmed in human studies Following HIV infection there is a significant decline in number [20,21]. More recently, the contribution of cytomegalovirus and function of naïve CD4+ T-cells in the blood and lymph (CMV) co-infection to naïve T-cell proliferation and deple- node compared to healthy individuals [30–34]. (summarized in tion has been explored. HIV-infected patients with strong Table 1) The reduction in naïve T-cells is likely multi-factorial CMV-specific T-cell responses had fewer naïve T-cells [41]. and secondary to reduced thymic function, increased naïve T- The authors suggested that the loss of naïve T cells could cell proliferation, enhanced immune activation and direct HIV be due to movement of naïve T-cells into the large pool of infection [30–32,35]. Collagen deposition in the paracortical CMV-specific memory T-cells leading to an adverse effect T-cell zones of the lymphoid tissue also plays a critical role in on total CD4+ T-cell immune recovery following cART. limiting naïve T-cell homeostasis [36,37]. Recent work in both Following initiation of cART, naïve T-cell proliferation de- SIV-infected macaques and HIV-infected humans demonstrated creases; thymic output increases and the total number of that following SIV/HIV infection, increased production of TGF-β naïve T-cells significantly increases [30,32,33,35].However, in lymphoid tissue resulted in procollagen production and depo- the numbers of naïve T-cells rarely reach normal levels in sition of collagen fibrils [38]. This resulted in restricted T-cell adults even after more than 7 years of cART [42].Incontrast, access to the fibroblastic reticular cell (FRC) network which is in HIV-infected children, naïve T-cells reached normal levels the major source of the survival factor IL7, resulting in apoptosis following cART, and recovery of thymic output was indepen- and depletion of naïve CD4+ T-cells. The loss of naïve T-cells in dent of age and time of initiation of cART [43]. A recent turn, removed a major source of lymphotoxin-β,asurvivalfac- study showed that naïve T-cell count at initiation of cART tor for FRCs [38]. The resulting loss of FRCs and the loss of IL-7 could predict successful immune reconstitution [44].Inthis produced by FRCs may thus perpetuate a vicious cycle of case, immune reconstitution was defined as an increase of

Table 1 Naïve T-cell abnormalities in the setting of HIV infection. Compartment assessed Abnormality in naïve T-cells in HIV (measurement) Reference Blood ↓ Numbers of naïve T-cells and RTE (CD45RA+, CD45RA+ CD62L+, TREC) [30–32,34] ↑ CMV responses associated with reduced number of naïve T-cells [41] ↑ Expansion proliferation (Ki67) [31] ↓ Cell cycle entry and proliferation (Ki67, CFSE) [48,49] ↓ Co-stimulatory response (CD27+ CD28) [49] ↑ Signaling (Basal STAT5 phosphorylation) [50,52] ↓ Survival (↓ Bcl-2) [50] ↑ Phosphorylated STAT5 in cytoplasm [52] ↓ Phosphorylated STAT5 nuclear localisation [52]

Lymph node ↓ Number of naïve T-cells (TREC) [32] ↑ Number of naïve T-cells (CD45RA+ CD62L+, TREC) [34] Collagen deposition in •Paracortical T zones [37] •High endothelial venules [36] •Fibroblastic reticular cell network [38] ↑ Distance between IL-7 source and naïve T-cells (CD45RA+) [38] ↓ Access to IL-7 [38] ↑ Naïve T-cell apoptosis (TUNEL+) [38]

Gastro-intestinal tract ↓ CD4+ T cells (naïve, effector and memory) [23,137,138] ↑ Microbial translocation (↑ LPS, 16s rDNA) [22,139] ↑ Fibrosis [140] The role of naïve T-cells in HIV-1 pathogenesis: An emerging key player 257

100–200 CD4+ T-cells after 2 years on therapy. There is a need higher functional response to IL-7 (measured by STAT5 phos- to further understand why recovery of naïve T-cells is im- phorylation), which correlated with increased total and paired following cART. The role of age, nadir CD4 T-cell naïve T-cell levels compared to patients with incomplete count or other biological factors that may enhance lymph responses (CD4 b500 cells/μl) [55]. Other studies have shown node fibrosis or impair thymic output remains to be further that defects in IL-7 responsiveness were only partially explored. corrected with cART while levels of IL-7 remained elevated and IL-7 receptor signaling defects were still present in many patients following cART [50,56–58]. 3.2. Naïve T-cell function 3.2.2. TCR diversity Abnormalities in naive. TCR diversity is also altered in patient with HIV infection. TCR diversity is quantified by the length of the hypervariable CDR3 3.2.1. Response to IL-7 Vβ regions of the TCR [59–62]. In healthy individuals the mul- The dramatic depletion of CD4+ T-cells in HIV infection is also tiple CDR3 lengths follow a Gaussian distribution but in HIV in- associated with significant functional defects in naïve T-cells fection this distribution is skewed and is associated with (summarised in Table 1). HIV infection is associated with in- disease progression and accelerated loss of total CD4+ T-cells creased systemic levels of IL-7, low surface expression of the in both adults and children [59–62]. Translocation of microbial α chain of the IL-7 receptor (lL7Rα), CD127, and impaired re- products as well as the HIV glycoprotein (gp)160 which can in- sponse to IL-7 [45–47]. Naïve T-cells from HIV-infected indi- duce superantigen responses in specific Vβ groups can also viduals had a reduced capacity to expand following lead to a skewed TCR repertoire [63]. In individuals who stimulation with TCR ligation and IL-7 [48].Inaddition, have a poor immunological response to cART (b20% increase CD31+ naïve T-cells, a subset of naïve T-cells that have re- in CD4+ T-cells from baseline or do not reach 200 cells/μl) cently left the thymus and not undergone proliferation there was an increased expansion in some Vβ groups and this [9,11], failed to express the co-stimulatory molecules CD28 was associated with reduced naïve T-cell number and thymic and CD27 after TCR stimulation [49]. Following TCR engage- output [58]. Two longitudinal studies have shown that despite ment of CD31- naïve T-cells, there was an increase in co-stim- an increase in total CD4+ T-cell count on long-term cART, ulatory molecules but the cells were unable to enter cell cycle CD4+ T-cell subsets remain skewed [64,65]. [49]. These data demonstrate discreet functional defects in response to IL-7 and TCR engagement in the setting of HIV in- 3.2.3. Response to neo-antigens fection, in addition to reduced total number of naïve T-cells. A reduction in functional responses to new antigens and vacci- IL-7 signaling which occurs through the STAT5 pathway has nations has also been associated with late initiation of cART also been shown to be altered during HIV infection. The basal and was most likely due to skewing of the proportion of CD4+ level of STAT5 phosphorylation was elevated in both naïve and T-cell populations and depletion of naïve T-cells [66].Other memory CD4+ T-cell subsets in patients receiving cART, who studies have shown that despite increases in CD4+ T-cells to were viremic and aviremic [50]. Expression of CD127 on levels >500 CD4+ T-cells with long-term cART, responses to naïve T-cells and the ability of IL-7 to bind to the IL-7R on neoantigens through vaccinations were lower in HIV-infected naïve T-cells in patients on cART were similar to healthy individuals compared to healthy controls [67]. Lack of cellular controls [50]. Recent analysis of signaling pathways in CD4+ response to neoantigens has also been observed in children T-cells from viremic and aviremic patients also suggested and adolescents vaccinated during their first year on cART that naïve CD4+ T-cells, in contrast to memory CD4+ T-cells, [68]. One possible explanation for these findings is a defect were able to sustain CD127-STAT5 signaling [51]. However, in naïve T-cell proliferation in response to neoantigen [69]. despite normal IL7 binding and sustained IL7R signaling in The implications of long-term impaired naïve T-cell recovery HIV-infected patients, the expression of the survival protein are unclear but it is highly likely that impaired naïve T-cell Bcl-2 was reduced in naïve T-cells from both viremic and number and function will contribute to ongoing immune dysre- cART-treated patients, consistent with a post-receptor block gulation in patients on cART. that prevented the induction of downstream pro-survival signals [50]. A study of viremic patients attributed this out- 4. HIV infection of naïve T-cells come to an increased accumulation of phosphorylated STAT5 within the cytoplasm due to the inability to enter the nucleus [52]. The multiple defects in signaling pathways important for One potential mechanism for depletion and impaired function survival of naïve and memory T-cells could therefore play an of naïve T-cells is that HIV can directly infect naïve T-cells important role in limiting the capacity for CD4+ T-cell recov- both in vivo and in vitro. However the extent of infection, ery following cART. the impact of HIV infection on function and the effect of Following long-term cART in HIV-infected adults [53],and cART on the reservoir of infected naïve T-cells is less clear. children [54], the concentration of IL-7 reduces to normal levels. One multivariate analysis showed that an increase in 4.1. Infection of naïve T-cells in vitro the mean CD4+ T-cell count in patients was associated with increased IL-7 responsiveness and this was independent of Naïve CD4+ T-cells are relatively resistant to HIV infection in T-cell activation, PD-1 expression, CCR5 expression and IL-7 vitro but in some conditions, direct infection can occur plasma levels [55]. In addition, patients on suppressive [70–72]. Infection of naïve T-cells with both CXCR4-using (X4) cART with a complete immunological response (≥500 CD4+ and CCR5-using (R5) viruses can lead to integration but the effi- T-cells/mm3 ≥5 years after initiation of ART), demonstrated ciency of infection of naïve CD4+ T-cells was significantly lower 258 G. Khoury et al. than in resting memory CD4+ T-cells [70]. In vitro infection of been described [83]. This newly described mechanism of accu- naïve T-cells was associated with accumulation of partial or in- mulation of incomplete reverse transcripts and abortive infec- complete viral transcripts and delayed viral integration but the tion could be an important contributing factor to naïve CD4+ major block to direct infection of naïve T-cells appears to be de- T-cell depletion in HIV-infected patients. creased efficiency of viral fusion [70,73]. Viral fusion in naïve T- cells can be significantly enhanced in viruses that co-express ve- 4.1.4. DC-naïve T-cell interactions sicular stomatitis virus (VSV) and R5 envelope protein, but fu- DCs located within the lymphoid tissue may play a role in en- sion was inefficient with VSV or R5 env protein alone [71].The hancing infection of naïve T-cells. One group has demonstrat- molecular mechanisms for this observation are currently ed that in vitro co-culture of naïve T-cells with DCs can unclear. significantly enhance infection of naïve CD4+ T-cells [84], while others have shown no effect of DCs [70]. A study using 4.1.1. Role of chemokines monocyte-derived DCs (MDDCs) explored the efficiency of Our laboratory recently demonstrated that direct infection of MDDCs to transmit HIV to different T-cell subsets including resting CD4+ T-cells can be established following incubation naïve, central and effector memory subsets [84], The study with chemokines that bind to chemokine receptors highly showed that MDDC-mediated transmission of virus was depen- expressed on the surface of resting T-cells, including CCL19 dent on virus co-receptor usage and co-receptor expression on and CCL21 which both bind to CCR7 which is highly expressed the target T-cell. MDDCs were only able to transfer X4 strains on both naïve and memory T-cells [74,75]. However, these to CD4+ naïve T-cells [84]. In other experiments, when resting chemokine mediated changes only facilitated infection of T-cells were infected in the presence of DCs and then sorted resting memory and not naïve T-cells, [75]. Taken together into naïve and memory cells, infection of naïve T-cells was these data demonstrate that there are clear differences in not enhanced by the presence of DCs [70]. Other groups have the ability of HIV to infect resting naïve and memory T-cells demonstrated that physical contact of total resting CD4+ T- and that the major block to infection of naïve T-cells occurs cells with uninfected autologous immature-MDDCs increased early in the viral life cycle, most likely at the level of viral infection of resting CD4+ T-cells [85]. This was not simply a re- fusion. sult of a mixed lymphocytic reaction as, an increase in activation markers was only observed when a higher ratio of DC:T- 4.1.2. Role of IL-7 cell was used [85]. The role of IL-7 in enhancing infection of naïve T-cells has also Combined, these studies demonstrate that HIV infection of been explored [76–79]. Naïve T-cells become more suscepti- naïve T-cells requires additional signals, mediated by either ble to infection following culture with IL-7 [79]. Following cytokines such as IL-7 or cell-cell contact via DC-T-cell con- treatment with IL-7, cord blood naïve T-cells and adult mem- tact, and that these signals are more likely to be present with- ory T-cells, but not adult naïve T-cells, enter G1b phase of in the endogenous microenvironment of lymphoid tissue. cell cycle and become susceptible to infection with both VSV-pseudotyped HIV and wild type HIV [76–78]. Total resting 4.2. Infection of thymocyte subsets in vitro CD4+ T-cells treated with IL-7 together with the phosphatidy- linositol 3-kinase (PI3K) inhibitor, wortmannin, led to no change in HIV infection suggesting that pathways other than HIV infection of thymocyte subsets could be the main source of PI3K may be involved [78]. HIV-infected naïve T-cells in the periphery. All thymocyte populations predominately express high levels of CXCR4 and 4.1.3. Tissue microenvironment and abortive infection low levels of CCR5 making these cells more susceptible to in- The microenvironment may also have an important role in fection with X4-using viruses [86,87]. However, HIV is able to susceptibility of naïve T-cells to HIV infection. HIV infection enter thymocytes and undergo reverse transcription after in- of resting naïve CD4+ T-cells was significantly enhanced fectionwitheitherX4orR5-usingviruses[88].Ourlabhas when infection was performed using tonsil lymphoid tissue demonstrated that thymic plasmacytoid dendritic cells play ex vivo [80,81]. In contrast, when naive CD4+ T-cells were an important role in the infection of thymocytes through purified from tonsil and cultured alone in vitro, HIV infection transfer of R5-using virus to both immature and mature thymo- did not occur [80]. Differences between susceptibility to HIV cytes [89]. Various cytokines have differential effects on thy- infection of naïve T-cells from lymphoid tissue and those iso- mocyte infection. IL-7 alone or in combination with IL-4 lated from the blood may be due to differences in the ex- increased expression of CXCR4 and enhanced infection with pression of active and inactive forms of the cellular protein X4-using viruses [87,90]. Thymocytes cultured with IL-2 and APOBEC3G [82]. IL-4 expanded, the expression of both co-receptors increased When human lymphoid aggregated cultures (HLACs) from and thymocytes were more permissive to X4 and R5-using vi- tonsil were infected with X4-using virus, incomplete reverse ruses [90]. In addition, mature thymocytes cultured with α transcription products accumulated within resting CD4+ TNF- and IL-7 were more likely to become infected as a result T-cells [72]. These incomplete transcripts triggered pro- of IL-7 enhancing cell survival of mature cells [91]. apoptotic and pro-inflammatory cellular responses resulting in the death of resting CD4+ T-cells and may potentially 4.3. Infection of naïve T-cells in vivo explain the significant CD4+ T-cell loss in HIV infection [72]. These experiments however didn't distinguish between memo- 4.3.1. Total and integrated HIV DNA ry and naïve T-cells, so it is currently unknown if this occurs in In contrast to findings in vitro, naïve T-cells are clearly infected both T-cell subsets [72]. Enhanced apoptosis of resting CD4+ with HIV in vivo, although at low levels compared with other T- T-cells isolated from the blood and infected in vitro has also cell subsets such as CD4+ memory T-cells [92–100]; The role of naïve T-cells in HIV-1 pathogenesis: An emerging key player 259

(summarized in Table 2). Accurate quantification of HIV in naïve A more recent cross-sectional study looking at both viremic T-cells in vivo can be complicated by the surface markers used and aviremic patients showed that both CD31+ and CD31- to define naïve T-cells. If insufficient surface markers are used naïve T-cell subsets were infected with HIV at similar frequen- to define a naïve T-cells (b3specificmarkers)[7] then it is pos- cies and in some patients, infected naïve T-cells made a sub- sible that memory T-cell contamination may be contributing to stantial contribution to the overall pool of infected T-cells the assessment of naïve T-cells [101–103].Inaddition,many [96]. This study also showed that in CD31+ naïve T-cells from studies only assess total HIV DNA rather than integrated DNA both viremic and aviremic patients, the concentration of HIV which again may provide an over estimation of the number of DNA positively correlated with IL-7 plasma levels, suggesting infected naïve T-cells [92–97,99,100]. However, to further a possible function of IL-7 in the infection of naïve T-cells. strengthen the case that naïve T-cells are indeed infected in vivo, replication competent virus has been detected in naïve 4.3.2. Viral compartmentalization T-cells from patients receiving cART [93,94,98,99]. In order to better understand the origin of infection of naïve There is conflicting data on whether specific naïve T-cell T-cells, several groups have examined the phylogenetic rela- subsets are preferentially infected. In one study, there was a tionship between virus isolated from naïve T-cell subsets, significantly higher concentration of HIV DNA in CD31- naïve memory cells and plasma. We recently showed that there T-cells compared with the CD31+ naive T-cells [92]. These was no compartmentalisation of virus envelope sequences in findings suggested that infection of naïve T-cells did not CD31+ and CD31- naïve and memory T-cells subsets from pa- occur in the thymus; but that infection potentially occurred tients prior to and following cART [95]. Similar findings have following naïve T-cell homeostatic proliferation in the pe- been described in another study using sorted samples from pa- riphery. However, the samples in this study were fixed tients naïve to cART [93]. In a smaller cohort of aviremic pa- prior to sorting and this could have had an effect on the ef- tients on cART for seven years, compartmentalization of ficiency of HIV DNA detection [92]. In a more recent longitu- virus in naïve T-cells was observed in two of the three patients dinal study of patients initiating cART, our group showed studied [100]. In order to better understand the origin of infec- that both CD31+ and CD31- naïve T-cell subsets were tion of naïve T-cells, these studies should be performed in infected at similar frequency [95]. In addition we showed larger prospective studies of patients on long term cART. that the contribution of naïve T-cells to the total reservoir increased following cART [95]. Overall, even though the ab- 4.3.3. Viral co-receptor usage solute number of infected naïve T-cells in patients on cART is Both R5-using and X4 using virus has been isolated from naïve T- significantly smaller than infected memory T-cells, naïve T- cells from HIV-infected patients [93–95,100].Inallofthese cells expand significantly following cART and therefore may studies naïve T-cells were predominantly infected with R5- represent a significant persistent reservoir in patients on using virus. However, two of the studies did detect some X4- cART. using viruses within the naïve T-cell compartment [94,100].It

Table 2 Detection of HIV infection of naïve T-cells in vivo. Studies that have assessed HIV infection of naïve T-cells in HIV-infected patients who are either on or off cART (upper rows without shading) or on cART (lower rows, gray shading) are shown.

Reference Patient CD4+ Plasma HIV Phenotype Total HIV Integrated Replication Compartment- Number cells/µl RNA copies/ml naïve T-cells DNA HIV DNA competent alisationa (naïve/cART) (Median) (Median) virus [92] 22 (11/11) 526 15,989 CD11a10 Yes (CD31 neg — — — CD45RO—CD57— >CD31pos) CD27+ CD31+/— [93] 13 (13/0) 610 17,090 CD27+CD45RO— Yes (total) — Yes No CD11a+CD57— [94] 11 (4/7) 547 1,085 CD45RA+CD62L+ Yes (total) Yes Yes — [95] 10 (10/10) 143 b 65,150 b CD45RO—CD28+ Yes (CD31+ = — — No CD31+/— CD31 neg) [96] 26 (11/15) 561 36,541(viremic) CD45RA+CCR7+ Yes (CD31+ = Yes CD31+ — — <40 (aviremic) CD31+/— CD31 neg) (10 cART— patients) [97] 102 (0/102) 271 6,908 CD45RA+CD62L+ Yes (total) — — — [98] 34 (0/34) 581 <50 CD45RA+ CCR7+ — Yes Yes — CD27+ [99] 11 (0/11) 582 <20 CD45RA+CD62L+ Yes (total) — Yes — [100] 3 (0/3) 305 <20 CD45RA+CD62L+ Yes (total) — — Yes (2/3)

–Not assessed. a Phylogenetic analysis. b Baseline sample at the beginning of longitudinal study. 260 G. Khoury et al. still remains unclear how naïve T-cells become infected with 5.2.2. Human clinical trials: HIV-infected patients R5-using viruses given the low expression of CCR5 on the surface There have been two completed trials of recombinant human of naïve T-cells. We have recently demonstrated in vitro that (rh) IL-7 in cART-treated HIV-infected patients. In one study, pa- thymic plasmacytoid DC are able to efficiently transfer R5- tients with CD4+ T-cell counts 100–400 cells/μlandHIVRNA using virus to both immature and mature thymocytes providing b50 copies/ml had eight subcutaneous injections three times a potential mechanism to explain R5-using infection of naïve per week over a 16 day period and two doses of rhIL-7 were ex- T-cells [89]. amined [121]. There was a preferential increase in both naïve and central memory T-cells following rhIL-7. Increases in CD4+ naïve T-cells peaked at day 14 and started to decline 5. Therapeutic approaches to enhance naïve 14 days after completion of rhIL-7. At the higher dose, T-cell recovery four patients also experienced a transient increase in plasma HIV RNA [121]. In addition to cART, there is now increasing interest to de- The second trial was performed in HIV-infected patients with velop immunotherapeutic agents that specifically enhance CD4+ T-cell counts >100 cells/μl, in patients with a viral load of CD4+ T-cell recovery, including naïve T-cells. b50 or 50–50,000 copies/ml HIV RNA [122]. A transient increase in HIV RNA was demonstrated in 6 out of 11 patients who had HIV b 5.1. Exogenous IL-2 therapy RNA 50 copies/ml at baseline. On day 1 following rhIL-7, there was a significant decrease in circulating CD4+ and CD8+ T-cells. By day 14, the levels had increased but mainly consisted of cen- Administration of IL-2 to lymphopenic mice increased total tral memory T-cells, with minimal increase in naïve T-cells. The CD4+ T-cells including naïve, resting memory and activated number of proliferating naïve T-cells increased compared to CD4+ T-cells [104]. Similar responses were seen in HIV- baseline, however these were mainly CD8+ T-cells [122]. infected patients on cART who received IL-2 [105–107]. Aside from the effects of IL-7 on CD4+ T-cell number, IL-7 Both thymic output and peripheral homeostatic proliferation can also have an impact on latently infected cells and possi- contributed to the increase in naïve CD4+ T-cells [104–109]. bly co-receptor usage of virus in vivo. In both clinical trials of In patients treated with cART and IL-2 there was a reduction IL-7, some patients experienced a transient increase in plas- in apoptosis of total and naïve CD4+ T-cells [106,110]. ma HIV RNA at the time of peak T-cell proliferation, which Two large, randomized international clinical trials of IL-2 eventually returned to b50 copies/ml [121,122]. There was in patients on cART, Evaluation of Subcutaneous Proleukin in no significant increase in the concentration of cell-associated a Randomized International Trial (ESPRIT) [111] and Subcu- HIV DNA in either PBMC or CD4+ T-cells following IL-7 [121]. taneous, Recombinant Human Interleukin-2 in HIV-Infected However, this study did not correct for the overall increase Patients With Low CD4+ Counts Receiving Active Antiretrovi- in total T-cell number following rhIL-7, meaning that the abso- ral Therapy (SILCAAT) [112] showed no additional clinical lute number of infected cells (expressed as HIV copies/ml benefit following administration of IL-2 to patients receiving blood) may have increased as a result of proliferation of la- cART. These studies demonstrated that IL-2 led to a clear in- tently infected naïve, central memory or transitional memory crease in total CD4+ T-cell counts, including naïve CD4+ T- T-cells, as has recently been demonstrated ex vivo [98]. cells, but this did not lead to a reduction in opportunistic Therefore, it is possible that IL-7 may induce proliferation of infections or mortality, showing that IL-2 therapy had no both uninfected and latently infected naïve and memory T- additional clinical benefit for patients on cART [111–113]. cells and it remains unclear if IL-7 will expand or activate la- Similar findings were observed in the Study of ALdesleukin tently infected cells. Clinical trials are currently underway to With and without AntiRetroviral Therapy, (STALWART), test this (ERAMUNE; www.clinicaltrials.gov). which tested intermittent IL-2 combined with peri-cycle IL-7 may also potentially lead to a change in HIV co-recep- cART in HIV-infected individuals who were cART-naïve and tor usage. Following long-term culture of HIV-infected PBMC asymptomatic [114]. These studies demonstrated that an with IL-7 in vitro, there was an increase in the expression of increase in CD4+ T-cell number secondary to IL-2 did not CXCR4 on CD4+ T-cells, which led to a change in co-receptor translate to effective function and improved clinical out- usagefromR5-usingtoX4-usingvirus[123]. The same group comes and further work is needed to understand the biolog- also suggested that increases in CXCR4 expression on CD4+ T- ical basis for these findings. cells from HIV-infected patients could lead to the emergence of R4-using viruses [124]. Using IL-7 as an immunotherapy 5.2. Exogenous IL-7 therapy could potentially have the same effect in vivo although to date this has not been demonstrated. 5.2.1. Animal preclinical studies Administration of IL-7 to SIV-infected and uninfected cynomo- 5.2.3. IL-7 Receptor polymorphisms logus monkeys, baboons and macaques, induced proliferation It is possible that response to endogenous and exogenous rhIL- of naïve CD4+ T-cells and a decline in the frequency of TREC 7 is dependent upon differences in the IL-7R which are deter- consistent with enhanced homeostatic proliferation [115– mined by genetic polymorphisms. The IL-7R is composed of an 119]. Administration of IL-7 induced migration of T-cells to α and common γ chain [125]. Four haplotypes of the gene cod- the lymph node where they entered cell cycle [120] and great- ing for IL-7Rα (CD127) have been described in Caucasians er TCR diversity was observed consistent with enhanced thy- [126]. These haplotypes are defined by single nucleotide poly- mic output [119]. Importantly, administration of IL-7 to SIV- morphisms located throughout the IL-7Rα gene and lead to al- infected macaques resulted in no increase in SIV viral load ternative splicing of mRNA resulting in varying ratios of soluble [116,117]. and membrane bound CD127 [127–129]. Using a multivariate The role of naïve T-cells in HIV-1 pathogenesis: An emerging key player 261 analysis, we have recently shown that the IL-7Rα haplotype 2 Table 3 Outstanding research questions on naïve CD4+ T- was significantly associated with more rapid recovery of CD4+ cells and HIV pathogenesis. T-cells >500 cells/μl following cART [53].Inaddition,soluble CD127 (sCD127) levels in cART-treated HIV-infected patients Outstanding research questions were significantly lower in individuals who were homozygous 1. Are naïve T-cells infected in all patients or only a select for haplotype 2 than non-haplotype 2 carriers. A potential ex- proportion? planation for these findings is that high levels of sCD127 2. At what stage of disease do naïve T-cells become infected? “mops” up circulating IL-7 allowing for less efficient IL-7- 3. Are naïve T-cells preferentially infected with X4-using or R5-using viruses? related cell signaling and impaired CD4+ T-cell recovery. Eval- 4. Does viral compartmentalization occur in naïve T-cell subsets? uation of these and other polymorphisms should be performed 5. How do R5-using viruses infect naïve T-cells? in other patient cohorts. In addition these polymorphisms may 6. How does self-peptide and microbial products drive naïve T- potentially be important in determining how effectively a pa- cell proliferation in humans? tient responds to exogenous IL-7, although these studies have 7. Which surrogate markers of immune function should be used not been performed to date. in future clinical trials of immune based therapies? 8. What genetic factors contribute to CD4 T-cell immune re- 5.3. Other novel therapeutic approaches constitution following cART?

Other immunotherapies like Growth Hormone (GH), Keratino- cyte Growth Factor (KGF), and androgen blockade which are known to improve thymopoiesis and thymic architecture regen- endpoints with better surrogate markers of immune function eration are also being considered for evaluation in HIV-infected other than the number of CD4+ T-cells alone will be needed. patients. Other approaches could also include anti-fibrotic The varying responses in immune reconstitution between indi- agents that could potentially reduce lymph node fibrosis [38]. viduals may potentially be predicted by genetic differences A randomized control trial of GH in a cohort of 46 HIV- and larger genome wide association studies similar to those re- infected patients receiving cART showed promising results cently conducted on HIV controllers [136] need be conducted with significant increases in thymopoiesis as measured by to assess the determinants of CD4+ T-cell reconstitution. TREC concentration [130]. Studies in macaques have shown These studies will be important in identifying novel therapeu- that KGF targets epithelial cells, improves both naïve T-cell tic targets to enhance immune recovery. numbers and TCR diversity [131] and also activates the production of IL-7 within the thymus [132]. At puberty when sex 7. Conclusion steroid levels increase, the thymus decreases in size and thymic output significantly reduces. Therefore androgen block- Successful immune reconstitution requires an increase in ade may improve thymic output of HIV patients as shown total CD4+ T-cells as well as the recovery of CD4+ naïve T- following castration in mouse models [133]. In addition, hypo- cell number and function. Understanding the multiple fac- gonadal men have been shown to have an increased number of tors involved in naïve T-cell homeostasis and proliferation RTE and following treatment with androgens, the levels of RTE will allow for the development of new strategies to enhance decline [134]. Sex steroid blockade using luteinizing-hormone immune reconstitution. releasing hormone antagonists is currently used in the treatment of prostate cancer [133] and has also been trialed in patients undergoing stem cell transplantations [135].Inbothof Role of the funding source these scenarios, thymic function and production of naïve T-cells were enhanced. No trials have yet been performed in G.K is a recipient of the National Health and Medical Research the setting of HIV infection. Council (NHMRC) biomedical postgraduate scholarship. S.R.L is funded by the NHMRC and Alfred Foundation and is an NHMRC Practitioner Fellow 6. Outstanding research questions P.U.C and S.R.L were funded by an NHMRC program grant for this work. Many outstanding research questions remain about naive T- R.R is a recipient of the King Scholarship from the Malaysian cells and HIV infection (Table 3). Most of our detailed under- government. standing of the mechanisms of naïve T-cell homeostasis is derived from mouse models. The effects of self-peptide and microbial translocation in driving enhanced naïve T-cell prolif- Conflict of interest statement eration should be further explored in ex vivo human models. New interventions targeting immune reconstitution like exog- The author(s) declare that there are no conflicts of interest. enous IL-7 have shown promise in clinical trials. However, a major challenge for the field is to determine how best to eval- References uate efficacy of immunomodulators such as IL-7 given the observation of enhanced CD4+ T-cell recovery but no [1] J.V.Baker,G.Peng,J.Rapkin,D.I.Abrams,M.J.Silverberg,R.D. improvement in clinical endpoints in the recent IL-2 studies. MacArthur, W.P. Cavert, W.K. Henry, J.D.f. Neaton, the Terry The efficacy of adjunct immunomodulatory therapy with Beirn Community Programs for Clinical Research on AIDS., CD4+ cART can no longer be evaluated on the basis of CD4+ T-cell count and risk of non-AIDS diseases following initial treatment number alone and large randomised trials with clinical for HIV infection, AIDS 22 (2008) 841–848. 262 G. Khoury et al.

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