The Latent Human Immunodeficiency Virus (HIV) Reservoir Resides Primarily in CD32⁻CD4⁺ T Cells in Perinatally HIV-Infected Adolescents With Long-Term Virologic Suppression

Abstract

Background

High-level expression of the Fcγ receptor, CD32^hi, on CD4⁺ T cells was associated with enhanced human immunodeficiency virus (HIV) infection of the latent reservoir in a study of adults receiving antiretroviral therapy. We tested the hypothesis that CD32 was the preferential marker of the latent HIV reservoir in virally suppressed, perinatally HIV-infected adolescents.

Methods

The frequency of CD32^hiCD4⁺ T cells was determined by flow cytometry (N = 5) and the inducible HIV reservoir in both CD32^hi and CD32⁻CD4⁺ T cells was quantified (N = 4) with a quantitative viral outgrowth assay. Viral outgrowth was measured by the standard p24 enzyme-linked immunosorbent assay and an ultrasensitive p24 assay (Simoa; Quanterix) with lower limits of quantitation.

Results

We found a 59.55-fold enrichment in the absolute number of infectious cells in the CD32⁻ population compared with CD32^hi cells. Exponential HIV replication occurred exclusively in CD32⁻CD4⁺ T cells (mean change, 17.46 pg/mL; P = .04). Induced provirus in CD32^hiCD4⁺ T cells replicated to substantially lower levels, which did not increase significantly over time (mean change, 0.026 pg/mL; P = .23) and were detected only with the Simoa assay.

Conclusions

Our data suggests that the latent HIV reservoir resides mainly in CD32⁻CD4⁺ T cells in virally suppressed, perinatally HIV-infected adolescents, which has implications for reservoir elimination strategies.

The infectious latent human immunodeficiency virus (HIV) reservoir resides predominantly in CD32⁻CD4⁺ T cells in perinatally HIV-infected adolescents. Using ultrasensitive p24 methods, inducible virus was detected in CD32^hiCD4⁺ T-cell cocultures, with limited outgrowth. This has implications for reservoir elimination strategies.

Despite the advent of combination antiretroviral therapy (ART) that controls viral replication to clinically undetectable levels [1–3], long-term control of human immunodeficiency virus (HIV) replication without ART (state of viral remission) remains elusive owing to the persistence of latent, inducible, replication-competent provirus in long-lived, resting memory CD4⁺ T cells (the latent reservoir) [4–6]. Eradication of the latent reservoir is an intense area of research, because this may achieve ART-free remission for the millions of individuals living with HIV [7–10].

Eradicating the latent reservoir is challenging: the lack of viral gene expression enables immune and antiretroviral escape [11–13]. The recent identification of CD32 as a putative marker for nearly 50% of latent reservoir cells in a study of ART-treated, HIV-infected adults is important because it may render the reservoir amenable to elimination through Fc receptor–targeted approaches [14, 15]. However, additional studies in adults have failed to confirm CD32 as a unique marker for the latent reservoir based on infected cell concentrations and expression of immune activation markers, along with CD14 and CD19 expression [16–18]. We aimed to determine whether CD32 is a marker of the latent reservoir in CD4⁺ T cells of virally suppressed, perinatally HIV-infected adolescents.

MATERIALS AND METHODS

Study Population

Participants were recruited from the Johns Hopkins Pediatric and Adolescent HIV/AIDS Program, which provides care for HIV-infected individuals from infancy through 25 years. Inclusion criteria for this study were 13 years of age or older with confirmed perinatal HIV infection, known adherence to ART, and virologic suppression for ≥1 year. Virologic suppression was defined as ≤2 successive low-level (<450) detectable viral load measures, with no more than a half-log increase in the second measure (Supplementary Table 1).

Peripheral Blood Mononuclear Cell Collection and Isolation

Blood was collected in ethylenediaminetetraacetic acid–containing tubes and processed within 24 hours. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Hypaque gradient centrifugation (GE Healthcare) and cryopreserved in 90% fetal calf bovine serum containing 10% dimethyl sulfoxide and in liquid nitrogen until further processing. For flow cytometry analysis and cell sorting, 10 and 100 million PBMCs, respectively, were thawed, washed, and then rested overnight. Cells for downstream analyses were sorted using 2 different methods and analyzed in coculture accordingly (Figure 1A and 1B).

Figure 1.

Experimental workflow and number of wells tested by all methods. A, Processing of peripheral blood mononuclear cells (PBMCs) by 2 methods to yield populations of CD32^hi and CD32⁻CD4⁺ T cells for quantitative viral outgrowth assay (QVOA) (method 1, n = 3; method 2, n = 3) and additional surface marker analyses for CD14, CD19, and HLA-DR (method 2 only). B, Number of CD32^hi and CD32⁻ cocultured wells analyzed by standard enzyme-linked immunosorbent assay (ELISA) and ultrasensitive Simoa assay.

Cell Sorting: Method 1 With Negative Enrichment of CD4⁺ T Cells

Total CD4⁺ T cells from 100 million rested PBMCs per participant were isolated using a CD4 negative enrichment kit (Miltenyi Biotec), which depletes CD8, CD14, CD15, CD16, CD19, CD36, CD56, CD123, T-cell receptor γ/δ, and CD235 before cell sorting [4, 19] (Figure 1A). Total CD4⁺ T cells were subsequently incubated with Fc block (BD Biosciences) for 10 minutes to reduce nonspecific antibody binding on the CD32 epitope, after which the CD4⁺ enriched T cells were stained for 30 minutes with an antibody panel containing CD4–phycoerythrin (PE) (lone RPA-T4; BD Biosciences), CD3–fluorescein isothiocyanate (FITC) (Clone UCHT1; BD Biosciences) and CD32–allophycocyanin (APC) (Clone FUN-2; Sony Biotechnology) before cell sorting with a MoFlo Cell Sorter (Beckman Coulter). Dead cells were excluded using a propidium iodide viability marker. Cells were then gated for singlets, because the doublet population is enriched with nonspecific fluorescence (Supplementary Table 2). CD4⁺ T cells were selected using gating for highly fluorescent CD3⁺CD4⁺ T-cell markers (Supplementary Figure 1A–1C). To sort for the CD32⁻ population, the gate was set based on an unstained control, and any cells below this threshold were determined to be CD32⁻ (Figure 2B). For the sorting of the CD32^hi population, the gate selected was well above the background fluorescence of the CD32 isotype control (Figure 2C).

Figure 2.

Representative staining and cell sorting (method 2; sample 302V2). A, Gating for high-fluorescence CD3⁺ and CD4⁺ T cells. B, Sorting of CD32^hi and CD32⁻CD4⁺ T cells into distinct populations. C, CD32 isotype control showing low nonspecific fluorescence in the CD32^hi gate. (Similar data obtained with method 1 are presented in Supplementary Figure 1.) Abbreviations: APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Cell Sorting: Method 2 With No Enrichment

Method 2 employed direct cell sorting of total PBMCs because CD32^hiCD4⁺ T cells may express surface markers that would be removed during the negative bead enrichment procedure, such as CD14 and CD19. First, 100 million PBMCs per participant were incubated with Fc block for 10 minutes, before staining for 30 minutes with the following antibody panel: HLA-DR-BV605 (Clone G46-6; BD Biosciences), CD14-BV421 (Clone MφP9; BD Biosciences), CD19-PE-Cy7 (Clone SJ25C1; BD Biosciences), CD4-PE (Clone RPA-T4; BD Biosciences), CD3-FITC (Clone UCHT1; BD Biosciences), and CD32-APC (Clone FUN-2; Sony Biotechnology). The CD32⁻ and CD32^hi populations were sorted using the same equipment and gating strategy as described for method 1 (Figure 2A–2C).

Cell Staining Analysis Without Sorting

To determine the effect of negative enrichment on the presence of CD14 and CD19, CD4⁺ T cells negatively selected from 10 million PBMCs from participants 0301V1, 0302V2, and 0116V2 were incubated with Fc block for 10 minutes and stained with CD4-PE, CD3-FITC, CD32-APC, CD14-BV421, and CD19-PE-Cy7 for 30 minutes. Cells were analyzed with a Becton Dickinson LSRII (Becton Dickinson). An additional 10 million PBMCs (participants 0300V2, 0301V1, 0302V2, and 0116V2) were stained using the same protocol as in method 2 and analyzed for the presence of HLA-DR, CD14, and CD19.

Quantitative Viral Outgrowth Assay

CD32⁻CD4⁺ T cells were assayed with a standard quantitative viral outgrowth assay, as described elsewhere, which has been used to quantify latent reservoirs in perinatal and adult HIV infection [20]. Owing to low cell frequency, CD32^hi cells were cocultured in replicate dilutions based on cell yields. Additional CD32⁻ cocultures matching the cell inputs of the CD32^hi cultures were assayed in parallel. Viral outgrowth is defined as the presence of HIV p24 at day 14 in the supernatant measured with the ultrasensitive Simoa assay (mean limit of detection, 0.003 pg/mL; limit of quantitation [LOQ], 0.01 pg/mL) (Quanterix), and at day 21 for the standard enzyme-linked immunosorbent assay (ELISA) (limit of detection, 4.3 pg/mL [21]; LOQ, 6.25 pg/mL) (PerkinElmer). The HIV reservoir size was expressed as infectious units per million cells (IUPM) [22]. For the cultured wells testing negative for viral outgrowth, maximum likelihood estimates were used. In addition, in calculating an average IUPM value, repeated measurements for individual participants were first averaged.

A qualitative assay was performed to examine for the presence of multiply-spliced HIV RNA. A small fraction (1 μL) of cultured cells from days 7 and 14, which were also assayed with the Simoa assay, were tested using a modification of the Tat/rev-induced limiting dilution assay (TILDA) that detects HIV transcription events in cells bearing proviral genomes [23]. RNA for the housekeeping glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) was used as positive control for the reverse-transcription step.

HIV DNA Quantitation in Peripheral Blood

Total DNA was extracted from PBMCs and quantified by means of droplet digital polymerase chain reaction, using methods published elsewhere, with modified primers to target the HIV Long Terminal Repeat-Gag (LTR-Gag) region [24]. Proviral load is expressed as LTR-Gag copies per million PBMCs, with RPP30 as the housekeeping gene to determine input number of cells assayed, and is subsequently corrected to a million cell equivalents.

Absolute Infected Cell Fold Enhancement Calculation

Absolute infected cells were calculated by first multiplying the IUPM for each population (CD32^hi and CD32⁻) by the respective proportion of total CD4⁺ T cells per participant. Fold enhancement was defined as the ratio of absolute infected CD32⁻ cells to infected CD32^hi cells. In calculating average fold enhancement across study participants, an average of fold enhancement was taken for the 2 participants with repeated time points.

Statistics

Data were analyzed using Student paired t tests. Statistical significance was established at P = .05.

Study Approval

This study was approved by the Johns Hopkins Medicine Office of Human Subjects Research institutional review boards (study No, NA_00087629). Written informed consent was received from all participants before inclusion in the study.

RESULTS

Five perinatally HIV-infected adolescents were included in this study, with 2 participants (paticipants 0301 and 0302) evaluated at 2 separate time points (Supplementary Table 1). At study entry, participants had a mean age of 17.4 years (range, 15–21 years) and an average duration of virologic suppression of 10.62 years (range, 4.83–17.32 years). Four of the 5 study participants were female, and 4 were African American (Supplementary Table 1). At all time points studied, study participants had undetectable viral loads (<20 copies/mL) (Supplementary Table 1). Across all 5 study participants, including 2 time points for participants 0301 and 0302 (7 unique measurements), HIV DNA was detected in PBMCs at concentrations ranging from 2.2 to 206.2 copies per million cells (mean, 101.45 copies per million cells) at the time of analysis (Table 1).

Table 1.

Immunologic and Virologic Profiles of Study Cohort

Participant	Visit	CD4+ T Cells in Total PBMCs at Sample Collection, % (Absolute Count)	HIV-1 DNA, Copies per Million Cells	CD32hiCD4+ T Cells, %	Reservoir Size, IUPM (95% CI)
					Standard p24 ELISA		Ultrasensitive p24 Simoa Assay
					CD32hi	CD32−	CD32hi	CD32−
Method 1
0301	1	29.4 (777)	132.1	0.312	<28.88b	1.10 (.44–2.76)	137.33 (32.00–589.37)	3.85 (1.31–11.32)
0302	1	47 (810)a	106.7	0.055	<721.02b	0.25 (.04–1.79)	1267.08 (176.09–9117.24)	0.69 (.17–2.85)
0116	2	37.1 (1092)a	2.2	0.022	<721.02b	<0.13b	<721.02b	< 0.14b
Method 2
0300	2	37.3 (617)	53.92	0.073	NAc	NAc	NAc	NAc
0301	2	27.9 (777)a	81.1	0.207	<51.34b	<0.16b	<51.34	0.20 (.03–1.42)
0302	2	41 (810)a	127.9	0.068	<86.63b	<0.09b	143.84 (20.12–1028.11)	1.94 (.76–4.92)
0304	1	43 (885)	206.2	0.058	<69.31b	0.47 (.15–1.48)	<69.31	6.55 (.33–130.28)

Abbreviations: CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; IUPM, infectious units per million cells; NA, not applicable; PBMCs, peripheral blood mononuclear cells.

^aCD4 measurements closest to the sample collection visit, when measurements were not obtained (all measurements within 8 months of visit).

^bNo detected virus; values represent the posterior median estimate, the median of the Bayesian posterior distribution, and numbers in parenthesis represent lower and upper bounds of 95% CIs.

^cNA because insufficient cells were obtained.

As reported in HIV-infected adults, a population of CD4⁺ T cells expressing CD32 of a high fluorescent intensity (well above isotypic control; CD32^hi) (Figure 2A–C, Supplementary Figure 1A–1I) was present in all 5 individuals, at proportions ranging from 0.022% to 0.312% (mean, 0.130%) with method 1 and from 0.058% to 0.207% (mean, 0.102%) with method 2 (overall mean, 0.11%) (Table 1) [14]. No significant differences in the proportion of CD32^hi-expressing cells were seen between study methods (P = .76). The mean numbers of CD32⁻ and CD32^hiCD4⁺ T cells sorted were 6.33 million and 9563 cells, respectively.

A mean of 17.88%, 0.82%, and 1.61% of CD3⁺CD4⁺ T cells analyzed by the direct sort method (method 2) also expressed HLA-DR, CD14, and CD19, respectively (Figure 3A). However, when stratified by CD32 expression, a significantly higher proportion of CD32^hi cells expressed HLA-DR compared with CD32⁻ cells (P < .001) (Figure 3B). In addition, higher levels of CD19 (mean, 16.56%; range, 5.93%–23.51%) and CD14 (mean, 15.57%; range, 7.06%–29.13%) were found in the CD32^hi population than in the CD32⁻ population (mean for CD19, 1.70% [range, 0.45%–3.80%]; mean for CD14, 0.76% [0.38%–1.56%]; P = .03 and P = .001, respectively) (Figure 3C and 3D). Significantly higher levels of HLA-DR, CD14, and CD19 were also observed even when the analyzed CD3⁺CD4⁺ T cells analyzed were derived from the negative enrichment procedure (method 1) (Supplementary Table 3).

Figure 3.

Surface marker analysis for HLA-DR, CD14, CD19, and CD32 directly sorted from peripheral blood mononuclear cells (method 2). A, Distribution of HLA-DR, CD14, CD19, and CD32 expression in CD3⁺CD4⁺ T cells. B, Proportion of HLA-DR expression in CD32^hi and CD32⁻CD4⁺ T cells. C, Proportion of CD19 expression in CD32^hi and CD32⁻CD4⁺ T cells. D, Proportion of CD14 expression in CD32^hi and CD32⁻CD4⁺ T cells. Data derived from sorted and cultured cells are represented in gray and data from staining analysis only is represented in black. Mean and 95% confidence intervals are presented. Significance was calculated using Student paired t tests. Similar data from method 1 are presented in Supplementary Table 3.

To quantify and characterize the inducible HIV reservoir in CD32^hiCD4⁺ and CD32⁻CD4⁺ T cells from perinatally infected participants, we also performed quantitative viral outgrowth assays on the sorted cell populations (CD32^hi vs CD32⁻CD4⁺ T cells) from 4 study participants with sufficient cell counts (100 million PBMCs) (Table 1). Infectious provirus is determined by the presence of p24 antigen in the culture supernatant on day 21 of coculture for the standard p24 ELISA and day 14 for the ultrasensitive p24 Simoa assay (Figure 4) [14].

Figure 4.

Human immunodeficiency virus (HIV) reservoir size, measured in infectious units per million cells (IUPM) with standard enzyme-linked immunosorbent assay (ELISA) and Simoa assay in CD32^hi and CD32⁻CD4⁺ T Cells. Quantitative viral outgrowth assay was used to quantify IUPM by both p24 ELISA and Simoa assay. Black and dark-gray symbols represent estimated reservoir size with cells cultured after purification with method 1 and method 2, respectively. Different shapes represent different study participants. Undetectable data are represented in light gray and have been set to 0.01 IUPM; maximum likelihood estimates of these data are shown in Table 1.

A total of 125 individual cocultures (104 CD32⁻ and 21 CD32^hi wells) were obtained from the 4 study participants at 6 independent sample time points (Figure 1B and Supplementary Table 4). Using the standard p24 ELISA, virus production after T-cell activation was detected in the CD32⁻ population in 3 of 4 study participants (3 of 6 independent measures), with a mean reservoir size of 0.35 IUPM (range, 0.13–0.63), using maximum likelihood estimates (Figure 4). Replication-competent virus was not detected in the participant with a low proviral load, at 2.2 copies per million PBMCs (participant 0116; Table 1). In the CD32^hi population, no participant had inducible provirus detectable by the standard p24 assay.

The ultrasensitive Simoa assay was performed on 76 wells collected at day 14 (21 CD32^hi and 55 CD32⁻ wells; Supplementary Table 4). Nine CD32⁻ wells that were identified by standard ELISA as harboring inducible infectious provirus were also positive with the ultrasensitive Simoa assay. However, another 16 CD32⁻ culture wells (negative by standard ELISA) from these 3 study participants were found to be positive for infectious provirus with the Simoa assay (mean concentration, 1.91 pg/mL). This led to an estimated 7.16-fold increase in the size of the CD32⁻ reservoir (mean reservoir size, 2.51 IUPM; range, 0.14–6.55 IUPM; Figure 4). In the CD32^hi population, induced provirus was detected in 5 of the 21 cultured wells in the same 3 study participants (3 of 6 independent measures) to yield a mean reservoir size of 397.53 IUPM, using maximum likelihood estimates (range, 69.31–721.02). However, the p24 concentration (mean, 0.07 pg/mL) at day 14 of coculture in the CD32^hi inducible reservoir was substantially lower than that of the CD32⁻ inducible provirus (mean, 32.53 pg/mL) in the cultured CD32⁻ cells. These positive wells in the CD32^hi cocultures were above the LOQ of the Simoa assay and the measured background signal from negative control culture wells, devoid of cells from the donor participant and performed at the same time of coculture. The participant with the lowest proviral load in PBMCs had no detectable induced provirus, in either CD32⁻ or CD32^hiCD4⁺ T cells, as determined with the Simoa assay (participant 0116; Table 1).

To quantify the absolute number of infectious provirus in CD32^hi and CD32⁻CD4⁺ T cells, the ratio of cells with induced provirus was calculated using the reservoir size in IUPM, as determined with the Simoa assay and normalized to the proportion of their respective cell frequencies. For participants with repeated measurements, we calculated the average fold enhancement. Overall, we found enhancement of cells with induced provirus in the CD32⁻ population, compared with the CD32^hi population (Figure 5). Of the 2 participants (3 independent measures) with detectable induced proviruses in both cell populations, as determined with the Simoa assay, there was, on average, a 9.68-fold enhancement of absolute infected cells in the CD32⁻ population, compared with the CD32^hi population (Figure 5). When using maximum likelihood estimates in the CD32^hi IUPM value for the 2 study participants with detectable induced provirus in the CD32⁻ but not the CD32^hi population (participants 0301V2 and 0304V1), we found on average, overall, a 59.55-fold enhancement of infected cells in the CD32⁻ population, compared with the CD32^hi population (range, 5.42–162.84-fold).

Figure 5.

Fold enhancement of absolute infected cells in the CD32⁻CD4⁺ compared with the CD32^hiCD4⁺ T-cell population. Gray bars represent the 2 participants with proviral genomes undetectable by the Simoa assay in the CD32^hi population; maximum likelihood estimates are used.

To assess differences in replication kinetics of the induced proviruses residing in CD32^hi and CD32⁻CD4⁺ T cells, we analyzed the change in p24 concentration from day 7 to day 14 using the ultrasensitive Simoa assay (Figure 6A and 6B). Wells were included in the analysis if the concentration exceeded the LOQ at either day 7 or day 14. Of all CD32^hi wells, 47.6% (10 of 21) had detectable p24 concentrations with the Simoa assay, with average p24 concentrations of 0.028 and 0.054 pg/mL for days 7 and 14, respectively. For the CD32⁻ population, 61.8% of cultured wells (34 of 55) had induced provirus by either day 7 or day 14, as shown by the Simoa assay. Of the 34 cultured wells with detectable p24 in the CD32⁻ population, 20 (58.8%) increased from day 7 (mean p24 concentration, 5.51 pg/mL) to 14 (22.97 pg/mL). Overall, there was a significant increase in p24 antigen concentration from day 7 to day 14 in the CD32⁻ induced population (mean change, 17.46 pg/mL; P = .04). Conversely, in the CD32^hi induced population, p24 antigen concentration increased in only 3 of the 10 cultured wells (30.0%), with an overall increase that was not statistically significant (mean change, 0.026 pg/mL; P = .23) (Figure 6).

Figure 6.

Change in p24 concentrations from day 7 to day 14 in wells with inducible virus, as detected with the Simoa assay in CD32⁻CD4⁺ (A) and CD32^hiCD4⁺ (B) T-cell cocultures.

To assess the transcriptional state of HIV in the cultured cells, all 76 wells that tested with the Simoa assay were also qualitatively assayed for ms-HIV RNA. Of these, 44 wells (57.9%) had induced provirus detectable with the Simoa assay (34 CD32⁻ wells and 10 CD32^hi wells) (Supplementary Figure 2A and 2B). Nine wells, all of which were CD32⁻, had evidence of induced provirus by all 3 measures, with detectable p24 by standard ELISA, Simoa assay, and ms-HIV RNA. In addition, 3 of the 25 CD32⁻ wells with induced provirus detectable with the Simoa assay but not with standard ELISA also had detectable ms-HIV RNA. None of the 10 CD32^hiCD4⁺ T-cell cultured wells with detectable p24 had detectable ms-HIV-RNA.

DISCUSSION

In this first study of the CD32^hiCD4⁺ T-cell HIV reservoir in perinatally infected individuals, a population of CD32^hiCD4⁺ T cells was readily detected in the PBMCs of all study participants, as observed in the first study to report this finding in adults [14]. CD32^hiCD4⁺ T cells were detected even with very low concentrations of HIV DNA (2.2 copies per million PBMCs). These results agree with those of previously published studies, showing that in HIV-infected persons, a small fraction of CD4⁺ T cells express CD32, with an average of 0.11% by our gating strategy compared with approximately 0.012%0.279% in studies of ART-treated adults [14, 18].

In the study by Descours et al [14], there was up to a 3000-fold enrichment of cells containing inducible provirus in CD4⁺ T cells that coexpress CD32^hi. In our study, we were able to detect induced provirus after T-cell activation in these CD32^hi cells in nearly 50% of cultured wells (10 of 21 wells), but with very low levels of p24 detectable only with the Simoa assay. In contrast, after T-cell activation, exponential viral outgrowth was exclusively detected in CD32⁻ wells, with 9 wells at a high enough p24 concentration to be detected by means of standard p24 ELISA. Notably, cultured wells with low levels of p24 antigen were also identified in the CD32⁻ T-cell population. This suggests the presence of provirus in both populations that are not efficiently induced after a single round of T-cell activation, as reported by Hosmane et al [25]

Owing to the small proportion of CD32^hi cells in the total CD4⁺ T-cell pool, and the likelihood of overestimation of the inducible reservoir, we calculated the ratio of infectious cells in the CD32^hi and CD32⁻ population and found on average a 59.55-fold higher number of cells with infectious provirus in the CD32⁻ population than in the CD32^hi population, This confirms that, even taking into account the low p24-producing provirus in CD32 ^hi cells, the inducible HIV reservoir resides predominantly in the CD32⁻ population.

We observed marked differences in growth kinetics between induced proviruses harbored in CD32⁻ and CD32^hiCD4⁺ T cells. Although low p24 producers were present in the CD32⁻ population, there was nevertheless overall a significant increase in p24 from day 7 to 14 only in the CD32⁻ population (P = .04), whereas p24 levels in CD32^hi wells did not increase significantly (P = .23). Altogether, these findings support a difference in the induced provirus from the 2 cell populations. However, it is important to recognize that a similar phenotype of induced provirus exhibiting nonexponential increase in p24 over the time course of the culture was also identified in the CD32⁻ population. The contribution of these low p24-antigen producing variants in the latent T-cell reservoir warrants further study in the context of HIV reservoir eradication efforts.

The quantitative difference in p24 production in the CD32^hi wells is also corroborated by the qualitative assay for detection of HIV transcripts (ms-HIV RNA) in cultured wells positive with the Simoa assay, because none of these wells had ms-HIV RNA detected. In contrast, all CD32⁻ cultured wells that were positive by standard ELISA had detectable ms-HIV RNA, suggesting associations between detection of ms-HIV RNA by this assay and exponential viral replication to levels detectable by standard ELISA. Notably, the finding that 12% (3 of 25) of the CD32⁻ wells were positive with Simoa and ms-HIV RNA assays, but not with standard ELISA, supports low-level viral spread in culture, warranting consideration of the use of more sensitive p24 detection methods to quantify viral reservoirs.

Importantly, substantial phenotypic differences were found between CD32^hi and CD32⁻CD4⁺ T-cell reservoirs, irrespective of the methods used to select the CD3⁺CD4⁺ T cells before cell sorting. The CD32^hiCD4⁺ T-cell reservoir represented a largely activated T-cell phenotype with substantially higher concentrations of HLA-DR, and therefore would not be considered a classically latent reservoir. This is in agreement with recent findings of an association between CD32 expression and immune activation [16–18]. It is possible that these cells represent a partially activated T-cell reservoir, with a likely shorter lifespan than the latent reservoir. The interaction between the CD32⁻ latent and the CD32^hiCD4⁺ T-cell reservoir will need to be further defined with respect to the similarities and differences in their genotypic landscape. We also found that the CD32^hiCD4⁺ T-cell reservoir expressed non–T-cell surface markers, such as CD14 and CD19. Whereas these markers may indicate contamination by B cells or monocytes, though this less likely given the sorting technique used in our study, the possibility of trogocytosis during immunologic synapses, as reported by Whitney [26], is plausible. This would suggest that CD32^hi expression on CD4⁺ T cells might represent an immunologic rather than a purely virologic process [27, 28].

This study is limited by its small sample size, as well as the limited cell yields after cell sorting from allowable blood volumes in our cohort, which also precluded analyses of other markers of HIV persistence (HIV DNA quantitation in the CD32a^hiCD4⁺ T cells) or in the total CD4⁺ T-cell population, as was done in the study in adults [14]. In addition, the analyses for p24 and ms-HIV RNA in the 2 cell populations were limited by the number of cells sampled from the CD32^hiCD4⁺ T cells. Nevertheless, we have identified quantitative differences in induced provirus and activation states between the CD32^hi and CD32⁻CD4⁺ T-cell reservoirs in perinatally HIV-infected adolescents.

In conclusion, the inducible HIV reservoir exhibiting high p24-producing viral replication kinetics in perinatally HIV-infected adolescents was mainly present in the CD32⁻CD4⁺ T-cell population. However, inducible provirus was detected in CD32^hi cells; it remains to be determined whether provirus harbored in the CD32^hi compartment requires more than a single round of T-cell activation for amplification, as previously reported by Ho et al [29] and Hosmane et al [25] Nevertheless, based on our findings, we conclude that, though CD32 is not a unique and specific marker of the latent reservoir, it may indicate potentially infected T cells that have experienced an immunologic response.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Acknowledgments. We thank the study participants and families for contributing to this study. We also thank Quanterix for the use of their Simoa platform and for assistance with data collection and analysis.

Financial support. This research was supported by the National Institutes of Health (NIH) (grants R01 HD080474 and P01 1P01AI131365 to D. P.); the NIH-funded BELIEVE Collaboratory (grant 1UM1AI26617 to D. P); the Johns Hopkins University Center for AIDS Research, an NIH-funded program (grant P30 AI094189 to D. P.), and the EPIICAL Consortium (D. P.).

Potential conflicts of interest. P. P. is an employee and has ownership stock at Quanterix Corporation and has patents pertaining to the Simoa technology. All other authors report no potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Presented in part: Conference on Retroviruses and Opportunistic Infections, March 4–7, 2018, Boston, Massachusetts.