Secondary Logo

Journal Logo

HIV RESERVOIR: Edited by Matthieu Perreau and Constantinos Petrovas

The active human immunodeficiency virus reservoir during antiretroviral therapy: emerging players in viral persistence

Astorga-Gamaza, Antonio; Buzon, Maria J.

Author Information
Current Opinion in HIV and AIDS: July 2021 - Volume 16 - Issue 4 - p 193-199
doi: 10.1097/COH.0000000000000685
  • Open

Abstract

INTRODUCTION

Human immunodeficiency virus (HIV) establishes a persistent infection for which nowadays there is not an available cure. Combined antiretroviral therapy (ART) strongly inhibits viral replication and stops disease progression but is unable to fully eliminate the virus, which accumulates and persists long-term in cell reservoirs [1]. The classical view of the HIV reservoir consists of resting memory CD4+ T cells harboring replication-competent virus, located in different cellular and anatomical compartments, that could reignite systemic infection when therapy is interrupted [2,3]. The frequency of such cells is very low; first approximations estimated that only ∼1 out of a million infected CD4+ T cells contain replicative viral forms [4,5]. Initially, this low proportion of cells was considered one of the main obstacles to cure the infection, but soon after it was recognized that this number, most likely, underestimated the real frequency of cells with potential to produce infectious virus [6,7]. Additionally, a bigger fraction of cells, roughly 2–18% of all infected cells have been detected to transcribe HIV and produce viral proteins during ART [8▪▪,9,10]. Transcriptionally and translationally competent HIV-infected cells are considered the active viral reservoir. Importantly, the extent to which some of these cells contribute to long-term HIV persistence has been poorly defined. In the past, cells with HIV proviral activity were systematically excluded from reservoir studies. This fact was due to several assumptions; (i) activated cells, usually linked to HIV expression, are thought to be short-lived and therefore, will not represent a long-term reservoir for HIV, and (ii) transcriptionally active cells producing viral particles will be eliminated by viral cytotoxicity and/or immune recognition [11]. However, investigations indicate that cells with resting phenotypes can also transcribe HIV [12,13], that transcriptionally active HIV-infected cell clones can be maintained for years [8▪▪,9,14,15▪▪,16], and that active reservoirs might be the source of residual viremia [14]. These studies uncover the active reservoir as an important component of the long-term reservoir, and therefore, convene the study of this particular cell reservoir for comprehensive approaches directed to target viral persistence. Here, we will review new technological advances that have been instrumental to study the active HIV reservoir, the cell sources and markers of active HIV-infected cells, and the potential role of the active reservoir in long-term viral persistence. 

Box 1
Box 1:
no caption available

IDENTIFICATION OF ACTIVE HUMAN IMMUNODEFICIENCY VIRUS-INFECTED CELLS

A detailed review of methods used to quantify the different forms of the HIV reservoir can be found elsewhere [17]. In this review, we will focus on approaches used for the identification and characterization of the active HIV reservoir, meaning cells with transcriptionally and/or translationally active HIV.

The understanding of the composition and location of active viral reservoirs during ART has been possible by the development of more specific and sensitive novel assays [9,18–21]. The traditional polymerase chain reaction (PCR)-based assay, and more recently the droplet digital PCR approach, allow quantifying different cell-associated HIV viral transcripts, including spliced and unspliced ribonucleic acid (RNA) molecules, in bulk cell populations. This methodology can be combined with the quantification of total viral HIV deoxyribonucleic acid (DNA) to obtain a relative measure of the transcriptional activity of HIV in infected cells [22–24]. This method per se does not allow the identification of cells producing HIV-RNA neither the quantification of the transcriptional activity in individual cells; however, it could be used to assess the transcriptional activity of specific cell subpopulations after cell isolation. Notably, in recent years, novel in situ hybridization (ISH)-based techniques have allowed the detection of intracellular HIV RNA molecules at the single-cell level with high sensitivity and specificity. The Prime Flow RNA assay (fluorescence in situ hybridization-flow) and the HIV-RNA scope ISH technologies have permitted the identification of different subpopulations carrying transcriptionally active provirus in blood and tissue specimens in patients on ART [19,25▪▪,26]. Additionally, these techniques have been used to measure the frequency of cells with active HIV upon exogenous cell stimulation or addition of latency reversal agents [20,25▪▪,27,28]. Of relevance, these approaches have also provided valuable insights into the variable response of different cell subpopulations to drugs intended to stimulate HIV expression [20,27,28]. Other induction-based viral RNA reactivation assays, including the tat/rev induced limiting dilution assay [29,30], have been used to quantify cells that can reactivate HIV transcription upon cell stimulation. However, recent efforts have been directed to develop new methodologies to provide information on the proviral sequences in cells with transcriptional viral activity. In this sense, the cell-associated HIV RNA and DNA single-genome sequencing method was developed to investigate the fraction of proviral expression [9]. This method revealed that identical HIV RNA from infected cell clones could arise from multiple single cells and across multiple times. Currently, new approaches comprising technological advances and aimed at assessing the intactness of proviral DNA, integration sites, viral expression, and the biology of cells harboring HIV at the single-cell level, are being developed [16,31,32▪] and will certainly provide relevant information about the nature and significance of the active HIV reservoir.

CELL RESERVOIRS FOR ACTIVE HUMAN IMMUNODEFICIENCY VIRUS

Curing HIV most likely will require the elimination of all infected cells with potential to initiate new rounds of infection, thus the identification and characterization of reservoir cells in patients on ART have represented an important goal during the last decade. The main cell type supporting long-term HIV persistence are CD4+ T cells; although macrophages containing integrated HIV DNA, RNA, viral proteins, and intact virions in virus-containing compartment-like structures have also been described in tissues [33]. Resting memory phenotypes have been the most studied subpopulations due to their higher intrinsic capacity to harbor silent HIV and therefore, their likelihood to constitute a long-term niche for the virus through the evasion of the immune system and the action of antiretroviral drugs [5,34]. However, CD4+ T cells are intrinsically a heterogeneous population and are defined by differential expression of cell surface receptors associated with different stages of cell maturation, activation, differentiation, function, and cell turnover. In blood, subpopulations of CD4+ T cells showing central memory (TCM), transitional memory (TTM), and effector memory (TEM) phenotypes comprise the largest proportion of HIV proviruses [35,36]. A detailed review of the major cell subpopulations that compose the total HIV reservoir can be found here [37]. From the total pool of reservoir cells, only a small fraction of cells contain intact viral forms [6,7,38,39]; higher proportions of intact viral regions seem to be located, in some studies, in the TEM subset [7,15▪▪,38,40], but naïve CD4+ T cells could also contain intact HIV [41,42]. Importantly, only a small fraction of infected cells contain active HIV without previous cell activation [8▪▪,9]. In blood, TEM and TTM, cells with relatively short half-lives, are the main contributors to HIV transcription [19,43,44▪]. Grau-Expósito et al. found TEM CD4+ T cells as a major niche for HIV transcription in ART-treated patients [19], whereas Yukl et al. found enrichment in HIV transcriptional activity in TEM but also in TTM cells [43]. Similarly, TEM cells which is the subpopulation with the fastest replacement rates were found highly enriched for HIV-RNA and contained the most clonal proviral expansion [44▪]. However, viral burden in several tissues, including lymph nodes (LN), cervix, and the gut-associated lymphoid tissues is systematically higher than in blood [26,43,45–47]. Indeed, CD4+ T-follicular helper (TFH) cells found in LN are highly enriched in viral RNA and replication-competent HIV [47,48]. Similarly, in the female genital tract, tissue-resident memory CD4+ T cells (TRM) contain transcriptionally active HIV and are the highest contributors to the total pool of HIV-infected cells [46]. Some of these cellular subsets and anatomical compartments might constitute sanctuaries for HIV persistence under ART [46,48,49] and might promote viral rebound upon treatment interruption [40]. Collectively, active cellular reservoirs for HIV are widely distributed in different anatomical and cellular compartments, which might vary inter-patient, and represent potential sources of replication-competent virus that, eventually, might fuel systemic viral replication after ART discontinuation.

Given the highly diverse sources of persistent cell reservoirs, finding a unique, or a set of markers, able to identify cells carrying replication-competent virus could significantly facilitate the development of new strategies directed to cure HIV [50]. Several surface molecules, such as CD2high, CXCR3, CD32a, and CD161 [51–54] or immune exhaustion markers like PD-1, TIGIT, CTLA-4, and TIM-3 [55,56], have been reported to identify latent cells enriched in inducible proviruses. Recently, cells expressing the activation marker human leukocyte antigen – DR were shown to contain high levels of intact HIV [57,58]. Importantly, similar viral transcriptional levels are found in these cells and their negative counterparts, suggesting that phenotypic markers of cell activation are not necessarily surrogates for cells with a transcriptionally active viral status [58]. Besides, other molecules, such as CD30 and CD20 have been able to identify transcriptionally active cells, and the targeting of these active reservoirs with specific antibodies ex vivo and in vivo have showed reduction in the HIV cellular burden [25▪▪,59]. Among cell markers, CD32a probably remains one of the most promising candidates, since it was shown to contain latent HIV [52,60] and to be enriched in transcriptionally active HIV in several compartments; CD32a was detected in infected cells in blood and main tissue reservoirs like the LN and the gastrointestinal tract in people on ART [61–63]. Although the value of CD32a as a marker of the HIV reservoir was challenged by early investigations denoting technical artifacts [64,65], the isolation of bona fide CD32+ CD4+ T cells has pointed out the usefulness of CD32a as a relevant marker of HIV reservoir cells [46,60,61]. Moreover, CD32a is also expressed on cells enriched in molecules related to HIV susceptibility and long-term maintenance [46,61], and importantly, the possible interaction of CD32a with immune complexes might confer to the infected cell an escaping mechanism from immune surveillance [66,67]. Together, up to date, several markers have been reported to identify active HIV in CD4+ T cells. To what extend the combination of these markers or their expression kinetics during the HIV infection cycle might represent a more accurate measure to identify active reservoir cells warrants further investigation. A summary of the main cell markers proposed for the study of the active HIV reservoir, as well as the main cell subpopulations and anatomical compartments showed to contribute to HIV expression in subjects on ART, is illustrated in Fig. 1.

FIGURE 1
FIGURE 1:
Active HIV reservoirs identified in individuals on ART. Illustration representing the relative contributions to the total HIV reservoir of both, the silent and the active viral fractions, highlighting the main different cell markers, cell subsets and anatomical compartments that might support HIV transcription during ART.

THE ACTIVE HUMAN IMMUNODEFICIENCY VIRUS CELL RESERVOIR AND LONG-TERM VIRAL PERSISTENCE

The mechanisms driving HIV latency in vivo are currently not well defined; the existence of a pool of cells with different blocks in several steps of the HIV replication process precedes the high heterogeneity in viral silencing observed in cell reservoirs [24,68,69]. Although HIV transcription and production of viral particles do not necessarily mean infectious competence, here we summarize some data supporting the importance of characterizing the active cell reservoirs during ART.

In viremic patients, the clinical significance of measuring the intracellular levels of viral RNA soon became apparent by the direct association between intracellular HIV-RNA quantification and CD4+ T cell counts [70–72]. Moreover, the level of viral transcription has been associated with virological failure in patients on ART, suggesting the frequency of HIV RNA+ cells as a potential biomarker of therapy success [22]. Importantly, in several investigations, viral expression predicted time to viral rebound after treatment interruption [73▪▪,74]. During ART, however, only a small fraction of the total pool of reservoir cells are able to transcribe HIV [8▪▪,9,75]. The fraction of these cells containing replication-competent HIV and therefore, representing a potential target for cure approaches, has not been very well established. The disconnection between the assays used to detect different forms of the viral reservoir has not allowed to estimate the fraction of transcriptionally active cells with potential to produce infectious viral particles on a per cell level basis. Recently, however, several investigations found the existence in some ART-treated individuals, of persisting cell clones expressing HIV that represented the source of residual viremia, being closer to a replication-competent state [14]. Furthermore, new methodologies based on the simultaneous genome DNA and RNA viral sequencing in single cells have demonstrated that intact genomes could also express viral RNA [15▪▪,16], and importantly, that intact HIV located in cell clones could transcribe HIV during long periods of time [8▪▪,9,14,15▪▪]. Whether or not these specific cell clones have a constitutive or intermittent expression of viral RNA, and/or are properly targeted by the immune system, are still open questions. Likewise, further research on T cell clones and residual viremia in patients on ART might be very informative [76], since viral blips and low-level viremia might affect long-term dynamics of the HIV reservoir, slowing down the reduction of its size [77].

Besides, recent studies have also demonstrated the presence of defective provirus encoding translationally competent HIV-RNA transcripts [78–80]. These transcriptionally and translationally competent cell reservoirs could be responsible for the persistent immune activation and cell exhaustion evidenced in ART-treated and some Elite controllers patients [81–83]. The direct relationship between the expression of viral products and viral-driven pathogenesis during ART evidences the need to pay more attention to the existence and the impact of the active reservoir. It is tempting to speculate that the elimination of RNA-expressing cells or the acceleration of the active viral reservoir decay, could have a positive effect not only on systemic immune activation, but also on shifting the reservoir toward a profound dormant state as recently shown in elite controllers [32▪,84]. However, to accomplish this goal will likely require the restoration of relevant antiviral immune responses and revert possible immune resistant mechanisms raised in reservoir cells, as recently suggested [67,85].

CONCLUSION

The role of the active viral reservoir on HIV persistence has not been completely considered until recently. During ART, HIV can be found in a transcriptionally and translationally active state, comprising a small proportion of the total reservoir cells. Recently, new technological advances have allowed characterizing the reservoir landscape in depth, and have identified the presence of active reservoir cells with intact genomes with potential to survive long-term. Based on these recent findings, we believe that a more profound study of the active reservoir might lead to the development of new targeted strategies for the continuous elimination of relevant reservoir cells over time. Elimination of cells with viral transcriptional activity could significantly impact viral persistence.

Acknowledgements

We would like to thank Dr Meritxell Genescà for a thoughtful review of the manuscript.

Financial support and sponsorship

This work was supported by the Spanish Secretariat of Science and Innovation and FEDER funds (grants SAF2015-67334-R and RTI2018-101082-B-I00 [MINECO/FEDER]), and the Fundació La Marató TV3 (grant 201805-10FMTV3). M.J.B. is supported by the Miguel Servet program funded by the Spanish Health Institute Carlos III (CP17/00179). A.A.-G. is supported by the Spanish Secretariat of Science and Innovation Ph.D. fellowship (BES-2016-076382). The funders had no role in the decision to publish or the preparation of the manuscript.

Conflicts of interest

There are no conflicts of interest.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

REFERENCES

1. Perelson AS, Essunger P, Cao Y, et al. Decay characteristics of HIV-1-infected compartments during combination therapy. Nature 1997; 387:188–191.
2. Blankson JN, Persaud D, Siliciano RF. The challenge of viral reservoirs in HIV-1 infection. Annu Rev Med 2002; 53:557–593.
3. Eisele E, Siliciano RF. Redefining the viral reservoirs that prevent HIV-1 eradication. Immunity 2012; 37:377–388.
4. Eriksson S, Graf EH, Dahl V, et al. Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PLoS Pathog 2013; 9:e1003174.
5. Finzi D, Hermankova M, Pierson T, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 1997; 278:1295–1300.
6. Ho YC, Shan L, Hosmane NN, et al. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 2013; 155:540–551.
7. Bruner KM, Wang Z, Simonetti FR, et al. A quantitative approach for measuring the reservoir of latent HIV-1 proviruses. Nature 2019; 566:120–125.
8▪▪. McManus WR, Bale MJ, Spindler J, et al. HIV-1 in lymph nodes is maintained by cellular proliferation during antiretroviral therapy. J Clin Investig 2019; 129:4629–4642.
9. Wiegand A, Spindler J, Hong FF, et al. Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART. Proc Natl Acad Sci USA 2017; 114:E3659–E3668.
10. DeMaster LK, Liu X, VanBelzen DJ, et al. A subset of CD4/CD8 double-negative T cells expresses HIV proteins in patients on antiretroviral therapy. J Virol 2015; 90:2165–2179.
11. Chun TW, Finzi D, Margolick J, et al. In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency. Nat Med 1995; 1:1284–1290.
12. Hermankova M, Siliciano JD, Zhou Y, et al. Analysis of human immunodeficiency virus type 1 gene expression in latently infected resting CD4+ T lymphocytes in vivo. J Virol 2003; 77:7383–7392.
13. Jennifer L., Groebner LP, Rachel Sklutuis, et al. Resting and activated CD4+ T cells both have silent and active HIV proviruses in vivo. Conference on Retroviruses and Opportunistic Infections. June 3–November 2021. Abstract number 304. Spotlight-E4.
14. Kearney MF, Wiegand A, Shao W, et al. Origin of rebound plasma HIV includes cells with identical proviruses that are transcriptionally active before stopping of antiretroviral therapy. J Virol 2016; 90:1369–1376.
15▪▪. Musick A, Spindler J, Boritz E, et al. HIV infected T cells can proliferate in vivo without inducing expression of the integrated provirus. Front Microbiol 2019; 10:2204.
16. Kevin B., Einkauf MO, Ce Gao, et al. Evolutionary dynamics of HIV reservoir cells via a novel single-cell multiomics assay. Conference on Retroviruses and Opportunistic Infections. 202. 2021. Abstract number 155. Oral-10.
17. Abdel-Mohsen M, Richman D, Siliciano RF, et al. Recommendations for measuring HIV reservoir size in cure-directed clinical trials. Nat Med 2020; 26:1339–1350.
18. Baxter AE, O’Doherty U, Kaufmann DE. Beyond the replication-competent HIV reservoir: transcription and translation-competent reservoirs. Retrovirology 2018; 15:18.
19. Grau-Exposito J, Serra-Peinado C, Miguel L, et al. A novel single-cell FISH-flow assay identifies effector memory CD4 (+) T cells as a major niche for HIV-1 transcription in HIV-infected patients. mBio 2017; 8:e00876-17.
20. Pardons M, Fromentin R, Pagliuzza A, et al. Latency-reversing agents induce differential responses in distinct memory CD4 T cell subsets in individuals on antiretroviral therapy. Cell Rep 2019; 29:2783–2395 e5.
21. Deleage C, Chan CN, Busman-Sahay K, Estes JD. Next-generation in situ hybridization approaches to define and quantify HIV and SIV reservoirs in tissue microenvironments. Retrovirology 2018; 15:4.
22. Pasternak AO, Lukashov VV, Berkhout B. Cell-associated HIV RNA: a dynamic biomarker of viral persistence. Retrovirology 2013; 10:41.
23. Pasternak AO, Adema KW, Bakker M, et al. Highly sensitive methods based on seminested real-time reverse transcription-PCR for quantitation of human immunodeficiency virus type 1 unspliced and multiply spliced RNA and proviral DNA. J Clin Microbiol 2008; 46:2206–2211.
24. Yukl SA, Kaiser P, Kim P, et al. HIV latency in isolated patient CD4(+) T cells may be due to blocks in HIV transcriptional elongation, completion, and splicing. Sci Transl Med 2018; 10:eaap9927.
25▪▪. Serra-Peinado C, Grau-Exposito J, Luque-Ballesteros L, et al. Expression of CD20 after viral reactivation renders HIV-reservoir cells susceptible to Rituximab. Nat Commun 2019; 10:3705.
26. Estes JD, Kityo C, Ssali F, et al. Defining total-body AIDS-virus burden with implications for curative strategies. Nat Med 2017; 23:1271–1276.
27. Grau-Exposito J, Luque-Ballesteros L, Navarro J, et al. Latency reversal agents affect differently the latent reservoir present in distinct CD4+ T subpopulations. PLoS Pathog 2019; 15:e1007991.
28. Baxter AE, Niessl J, Fromentin R, et al. Single-cell characterization of viral translation-competent reservoirs in HIV-infected individuals. Cell Host Microbe 2016; 20:368–380.
29. Procopio FA, Fromentin R, Kulpa DA, et al. A novel assay to measure the magnitude of the inducible viral reservoir in HIV-infected individuals. EBioMedicine 2015; 2:874–883.
30. Cillo AR, Sobolewski MD, Bosch RJ, et al. Quantification of HIV-1 latency reversal in resting CD4+ T cells from patients on suppressive antiretroviral therapy. Proc Natl Acad Sci USA 2014; 111:7078–7083.
31. Gantner P, Pagliuzza A, Pardons M, et al. Single-cell TCR sequencing reveals phenotypically diverse clonally expanded cells harboring inducible HIV proviruses during ART. Nat Commun 2020; 11:4089.
32▪. Jiang C, Lian X, Gao C, et al. Distinct viral reservoirs in individuals with spontaneous control of HIV-1. Nature 2020; 585:261–267.
33. Ganor Y, Real F, Sennepin A, et al. HIV-1 reservoirs in urethral macrophages of patients under suppressive antiretroviral therapy. Nat Microbiol 2019; 4:633–644.
34. Siliciano RF, Greene WC. HIV latency. Cold Spring Harb Perspect Med 2011; 1:a007096.
35. Chomont N, El-Far M, Ancuta P, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med 2009; 15:893–900.
36. Buzon MJ, Sun H, Li C, et al. HIV-1 persistence in CD4+ T cells with stem cell-like properties. Nat Med 2014; 20:139–142.
37. Fromentin R, Chomont N. HIV persistence in subsets of CD4+ T cells: 50 shades of reservoirs. Semin Immunol 2020; Nov 30:101438.
38. Hiener B, Horsburgh BA, Eden JS, et al. Identification of genetically intact HIV-1 proviruses in specific CD4(+) T cells from effectively treated participants. Cell Rep 2017; 21:813–822.
39. Bruner KM, Murray AJ, Pollack RA, et al. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med 2016; 22:1043–1049.
40. De Scheerder MA, Vrancken B, Dellicour S, et al. HIV rebound is predominantly fueled by genetically identical viral expansions from diverse reservoirs. Cell Host Microbe 2019; 26:347–58 e7.
41. Zerbato JM, McMahon DK, Sobolewski MD, et al. Naive CD4+ T cells harbor a large inducible reservoir of latent, replication-competent human immunodeficiency virus Type 1. Clin Infect Dis 2019; 69:1919–1925.
42. Venanzi Rullo E, Pinzone MR, Cannon L, et al. Persistence of an intact HIV reservoir in phenotypically naive T cells. JCI insight 2020; 5:e133157.
43. Yukl SA, Shergill AK, Ho T, et al. The distribution of HIV DNA and RNA in cell subsets differs in gut and blood of HIV-positive patients on ART: implications for viral persistence. J Infect Dis 2013; 208:1212–1220.
44▪. Bacchus-Souffan C, Fitch M, Symons J, et al. Relationship between CD4 T cell turnover, cellular differentiation and HIV persistence during ART. PLoS Pathog 2021; 17:e1009214.
45. Yukl SA, Gianella S, Sinclair E, et al. Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. J Infect Dis 2010; 202:1553–1561.
46. Cantero-Perez J, Grau-Exposito J, Serra-Peinado C, et al. Resident memory T cells are a cellular reservoir for HIV in the cervical mucosa. Nat Commun 2019; 10:4739.
47. Perreau M, Savoye AL, De Crignis E, et al. Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production. J Exp Med 2013; 210:143–156.
48. Banga R, Procopio FA, Noto A, et al. PD-1(+) and follicular helper T cells are responsible for persistent HIV-1 transcription in treated aviremic individuals. Nat Med 2016; 22:754–761.
49. Chaillon A, Gianella S, Dellicour S, et al. HIV persists throughout deep tissues with repopulation from multiple anatomical sources. J Clin Invest 2020; 130:1699–1712.
50. Neidleman J, Luo X, Frouard J, et al. Phenotypic analysis of the unstimulated in vivo HIV CD4 T cell reservoir. eLife 2020; 9:e60933.
51. Banga R, Procopio FA, Ruggiero A, et al. Blood CXCR3(+) CD4 T cells are enriched in inducible replication competent HIV in aviremic antiretroviral therapy-treated individuals. Front Immunol 2018; 9:144.
52. Descours B, Petitjean G, Lopez-Zaragoza JL, et al. CD32a is a marker of a CD4 T-cell HIV reservoir harbouring replication-competent proviruses. Nature 2017; 543:564–567.
53. Li X, Liu Z, Li Q, et al. CD161(+) CD4(+) T cells harbor clonally expanded replication-competent HIV-1 in antiretroviral therapy-suppressed individuals. mBio 2019; 10:e02121–19.
54. Iglesias-Ussel M, Vandergeeten C, Marchionni L, et al. High levels of CD2 expression identify HIV-1 latently infected resting memory CD4+ T cells in virally suppressed subjects. J Virol 2013; 87:9148–9158.
55. Darcis G, Berkhout B, Pasternak AO. The quest for cellular markers of HIV reservoirs: any color you like. Front Immunol 2019; 10:2251.
56. Fromentin R, Bakeman W, Lawani MB, et al. CD4+ T cells expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART. PLoS Pathog 2016; 12:e1005761.
57. Lee E, Bacchetti P, Milush J, et al. Memory CD4 + T-cells expressing HLA-DR contribute to HIV persistence during prolonged antiretroviral therapy. Front Microbiol 2019; 10:2214.
58. Horsburgh BA, Lee E, Hiener B, et al. High levels of genetically intact HIV in HLA-DR+ memory T cells indicates their value for reservoir studies. Aids 2020; 34:659–668.
59. Hogan LE, Vasquez J, Hobbs KS, et al. Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30. PLoS Pathog 2018; 14:e1006856.
60. Darcis G, Kootstra NA, Hooibrink B, et al. CD32(+)CD4(+) T cells are highly enriched for HIV DNA and can support transcriptional latency. Cell Rep 2020; 30:2284–96 e3.
61. Abdel-Mohsen M, Kuri-Cervantes L, Grau-Exposito J, et al. CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells. Sci Transl Med 2018; 10:eaar6759.
62. Noto A, Procopio FA, Banga R, et al. CD32(+) and PD-1(+) lymph node CD4 T cells support persistent HIV-1 transcription in treated aviremic individuals. J Virol 2018; 92:e00901-18.
63. Vasquez JJ, Aguilar-Rodriguez BL, Rodriguez L, et al. CD32-RNA co-localizes with HIV-RNA in CD3+ cells found within gut tissues from viremic and ART-suppressed individuals. Pathog Immun 2019; 4:147–160.
64. Bertagnolli LN, White JA, Simonetti FR, et al. The role of CD32 during HIV-1 infection. Nature 2018; 561:E17–E19.
65. Perez L, Anderson J, Chipman J, et al. Conflicting evidence for HIV enrichment in CD32(+) CD4 T cells. Nature 2018; 561:E9–E16.
66. Holgado MP, Sananez I, Raiden S, et al. CD32 ligation promotes the activation of CD4(+) T Cells. Front Immunol 2018; 9:2814.
67. Antonio Astorga-Gamaza, Judith Grau-Expósito, Joaquin Burgos-Cibrian, et al. Expression of CD32 in HIV-Reservoir cells confers resistance to natural killer cells. Conference on Retroviruses and Opportunistic Infections. 2021. June 3-November 2021. Abstract number 306. Spotlight-E4.
68. Moron-Lopez S, Telwatte S, Sarabia I, et al. Human splice factors contribute to latent HIV infection in primary cell models and blood CD4+ T cells from ART-treated individuals. PLoS Pathog 2020; 16:e1009060.
69. Lindqvist B, Svensson Akusjarvi S, Sonnerborg A, et al. Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells. PLoS Pathog 2020; 16:e1008264.
70. Saksela K, Stevens CE, Rubinstein P, et al. HIV-1 messenger RNA in peripheral blood mononuclear cells as an early marker of risk for progression to AIDS. Ann Intern Med 1995; 123:641–648.
71. Furtado MR, Kingsley LA, Wolinsky SM. Changes in the viral mRNA expression pattern correlate with a rapid rate of CD4+ T-cell number decline in human immunodeficiency virus type 1-infected individuals. J Virol 1995; 69:2092–2100.
72. Pasternak AO, Jurriaans S, Bakker M, et al. Steady increase in cellular HIV-1 load during the asymptomatic phase of untreated infection despite stable plasma viremia. Aids 2010; 24:1641–1649.
73▪▪. Pasternak AO, Grijsen ML, Wit FW, et al. Cell-associated HIV-1 RNA predicts viral rebound and disease progression after discontinuation of temporary early ART. JCI insight 2020; 5:e134196.
74. Moron-Lopez S, Kim P, Sogaard OS, et al. Characterization of the HIV-1 transcription profile after romidepsin administration in ART-suppressed individuals. Aids 2019; 33:425–431.
75. Pasternak AO, Berkhout B. What do we measure when we measure cell-associated HIV RNA. Retrovirology 2018; 15:13.
76. Halvas EK, Joseph KW, Brandt LD, et al. HIV-1 viremia not suppressible by antiretroviral therapy can originate from large T cell clones producing infectious virus. J Clin Investig 2020; 130:5847–5857.
77. Bachmann N, von Siebenthal C, Vongrad V, et al. Determinants of HIV-1 reservoir size and long-term dynamics during suppressive ART. Nat Commun 2019; 10:3193.
78. Ishizaka A, Sato H, Nakamura H, et al. Short intracellular HIV-1 transcripts as biomarkers of residual immune activation in patients on antiretroviral therapy. J Virol 2016; 90:5665–5676.
79. Imamichi H, Dewar RL, Adelsberger JW, et al. Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy. Proc Natl Acad Sci USA 2016; 113:8783–8788.
80. Pollack RA, Jones RB, Pertea M, et al. Defective HIV-1 proviruses are expressed and can be recognized by cytotoxic T lymphocytes, which shape the proviral landscape. Cell Host Microbe 2017; 21:494–506 e4.
81. Lichtfuss GF, Cheng WJ, Farsakoglu Y, et al. Virologically suppressed HIV patients show activation of NK cells and persistent innate immune activation. J Immunol 2012; 189:1491–1499.
82. Nabatanzi R, Bayigga L, Cose S, et al. Aberrant natural killer (NK) cell activation and dysfunction among ART-treated HIV-infected adults in an African cohort. Clin Immunol 2019; 201:55–60.
83. Fenwick C, Joo V, Jacquier P, et al. T-cell exhaustion in HIV infection. Immunol Rev 2019; 292:149–163.
84. Einkauf KB, Lee GQ, Gao C, et al. Intact HIV-1 proviruses accumulate at distinct chromosomal positions during prolonged antiretroviral therapy. J Clin Investig 2019; 129:988–998.
85. Ren Y, Huang SH, Patel S, et al. BCL-2 antagonism sensitizes cytotoxic T cell-resistant HIV reservoirs to elimination ex vivo. J Clin Investig 2020; 130:2542–2559.
Keywords:

active reservoir; cell markers; human immunodeficiency virus; latency; viral persistence

Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.