HIV-1 causes chronic disease because of its persistence in CD4+ T cells that contain replication-competent provirus but exhibit little or no active viral gene expression and effectively resist antiretroviral therapy (ART) because they are not recognized and eliminated by virus-specific T cells.1–3
These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells with remarkable long-term stability. Different particular T cell subpopulations have previously been reported to be important HIV reservoirs, among them are resting memory CD4+ T lymphocytes, ie, central memory T cells (TCM), and transitional memory CD4+ T cells (TTM).4
Recently, Descours et al5 discovered that CD32a can serve as a marker of latently HIV-infected CD4+ T cells. In that study, virtually no expression of CD32a on bystander cells was detectable, and infection frequencies seemed to correlate with the expression of CD32a.5 These findings have sparked a lot of interest since CD32(a) might be a potential target for future HIV cure strategies.6 However, some of the findings of this study were based on in vitro experiments and solely HLA-DR–negative cells were studied.5
CD32 is a low-affinity receptor for the immunoglobulin G (IgG)Fc fragment and mostly expressed on B cells, monocytes, neutrophils, and eosinophils.7 Of note, several isoforms of CD32 (a, b, and c, activating and inhibitory) have been described,8,9 and there also exists a polymorphism of this gene that is correlated with faster HIV progression.10
So far, there are few data about the surface expression pattern of CD32 on human T cells in healthy individuals or in other diseases.11
The aim of our study was to further characterize the distribution of CD32 expression on CD4+ memory subpopulations in peripheral blood and lymph nodes of HIV-infected patients with different disease status.
MATERIALS AND METHODS
Study Subjects and Samples
At the University Medical Center Hamburg-Eppendorf, written informed consent was obtained from all participants who were recruited for this study, which was approved by the local Institutional Review Board of the Ärztekammer Hamburg. CD4+ T cell counts and plasma viral loads were extracted from the clinical database.
Immune Phenotypic Analysis for Surface Markers
Cryoconserved peripheral blood mononuclear cells (PBMC) or lymph nodal mononuclear cells (LNMC) were isolated and used for immunophenotypic staining as previously described.12,13 Briefly, biopsied lymph nodes were disintegrated with a scalpel. LNMC were isolated from the tissue by gentle squeezing and flushing with medium before cryoconservation. Cells were stained with Zombie NIR fixable viability stain (BioLegend, San Diego, CA) and the following fluorochrome-conjugated surface antibodies: anti-CCR5, anti-CD27 (BD Biosciences, Heidelberg, Germany), anti-CD8, anti–HLA-DR, anti-TIGIT, anti-CD45RA, anti-CD127, anti-CD19, anti-CCR7, anti-PD-1, anti-CD4, anti-CCR6, anti-CXCR4, anti-CD25, anti-CD3, anti-CD32, and anti-CD14 (all BioLegend). All samples were run on a on a Becton Dickinson LSR Fortessa flow cytometer with FACS Diva version 8 (BD Biosciences). Cytometric data were analyzed using FlowJo version 10.3 (FlowJo LLC., Ashland, OR).
Data Analysis and Statistics
Statistical analysis was performed using GraphPad Prism version 7.0c (GraphPad Software, Inc., La Jolla, CA). For multiple comparisons, Kruskal–Wallis and Dunn's post-test with an alpha value of 0.05 were performed. All reported P values were multiplicity adjusted according to Dunn. To compare ranks, 2-tailed Mann–Whitney tests were performed. Pearson correlation and Spearman rank correlation coefficient were applied for bivariate correlation analysis. Data are expressed as mean with SD. A P value of less than 0.05 was considered significant.
Expression Pattern of CD32 on Different T Cell Subsets of PBMC and LNMC of HIV Patients and Healthy Controls
In this study, we present data on the ex vivo CD32 expression of T cells in a cohort of viremic and HIV patients on ART in comparison with healthy individuals (see Table 1 for cohort characteristics). In line with previous reports, although CD32 was detectable on most CD3-negative cells (data not shown),14,15 overall, only a small minority of CD4+ T cells expressed CD32.
Next, we compared the expression of CD32 on total CD4+ T cells between viremic, ART-treated, and healthy individuals. A representative plot of CD32 expression and applied gating strategy is shown in Supplemental Digital Content Figure 1, http://links.lww.com/QAI/B104. We only detected slightly, but significantly higher frequencies of CD32+ CD4+ cells in viremic HIV patients (mean 1.50%, range 0.05%–9.04%) than in healthy controls (0.23%, range 4.94 × 10−5 to 0.78%, P = 0.0266) (Supplemental Digital Content Figure 2A, http://links.lww.com/QAI/B104). We found no significant difference between viremic and ART-treated patients nor between ART-treated (0.28%, range 0.02%–1.30%) and healthy individuals. Likewise, CD32 was more frequently expressed on CD8+ T cells of viremic than ART-treated patients or healthy controls (mean values 1.07%, 0.69%, and 0.84%, respectively) (Supplemental Digital Content Figure 3, http://links.lww.com/QAI/B104). Since previous studies either used fresh or thawed PBMC,5,16 we also compared CD32 stainings in fresh versus thawed PBMC of healthy controls side by side. CD32 expression was slightly lower on thawed PBMC, but this difference did not reach statistical significance (Supplemental Digital Content Figures 2A, B, http://links.lww.com/QAI/B104).
In a next step, we examined the distribution of CD32 on naive and memory CD4+ T cell subsets in more detail, ie, naive T cells (Tnaive) (CD45RA+/CCR7+), TCM (CD45RA−/CCR7+), effector memory T cells (TEM) (CD45RA−/CCR7−/CD27+), and TTM (CD45RA−/CCR7−/CD27−) (Fig. 1A).
In the memory compartment, we found that TCM, TEM, and TTM of viremic HIV patients had a significantly higher expression of CD32 than the respective memory subsets in ART-treated patients or healthy controls (mean values TCM: viremic: 1.32%, ART: 0.06%, healthy: 0.05%; TEM viremic: 2.35%, ART: 0.08%, healthy: 0.07%; and TTM: viremic: 1.86%, ART: 0.1%, healthy: 0.09%). However, there was no significant difference between CD32 expression of naive CD4+ T cells of viremic, ART-treated, or healthy individuals (viremic: 0.94%, ART: 0.42% and healthy: 0.36%).
Next, we also analyzed CD32+ expression of lymph nodal T cells from biopsies of 5 uninfected and 4 HIV patients on ART, as well as 4 viremic HIV patients. On LNMC, we saw a lower frequency of CD32 on CD4+ T cell subsets than on the respective peripheral mononuclear cells (Fig. 1B). The mean frequency of CD32+ CD4+ T cells in HIV-infected individuals ranged from 0.25% (TCM) to 0.41% (TEM). CD32 expression was highest on TEM and TTM cells of lymph nodal CD4+ T cells. There was a trend of higher CD32 expression on all CD4+ T cell subsets of HIV patients compared with uninfected patients; however, this difference did not reach statistical significance.
CD32 Expression on CD4+ T Cell Subsets Correlates With General T Cell Activation Regardless of Disease Status
The frequency of CD32 on CD4+ T cells of viremic patients and, in particular, on CD4+ TTM correlated with activation, defined as percentage of HLA-DR+ expression on CD8+ T cells (Spearman ρ = 0.5706 and 0.5813, respectively) (Fig. 1C). Also, there was a trend towards a correlation of HLA-DR+ on CD4+ T cells and CD32 expression; however, this trend did not reach statistical significance (data not shown).
Previously, it has been described that CD4+ T cells upregulate CD32 after in vitro activation.10,17 In this current cohort, ex vivo stained CD32+ CD4+ T cells showed significantly higher expression of HLA-DR than their CD32− counterparts regardless of the disease status (Fig. 1C).
Of note, Descours et al5 only analyzed CD32 on HLA-DR− CD4+ T cells and excluded HLA-DR+ cells. We additionally compared the HLA-DR+ versus the HLA-DR− CD4+ subpopulation. The level of CD32 expression was slightly yet significantly higher when gating on HLA-DR+ cells throughout all patient groups (Supplemental Digital Content Figure 4, http://links.lww.com/QAI/B104).
Mirroring the lower level of CD32 seen in CD4+ T cells in the lymph node, LNMC showed markedly lower HLA-DR expression compared with peripheral T cells (Supplemental Digital Content Figure 5, http://links.lww.com/QAI/B104).
Importantly, in our relatively small cohort, no correlation was found between CD32 expression on CD4+ T cell populations and the CD4+ T cell count or HIV plasma viral load (data not shown). Also, no correlation was found between activated TTM and CD32 expression on TTM (Spearman ρ = 0.2875, Fig. 1C).
CD32 was expressed on regulatory T cells at similar ranges as on T effector cells, and expression of CD32 was again slightly higher in viremic patients (Fig. 1D).
When we looked at the expression of the HIV coreceptors, CCR5 and CXCR4, we found that the percentage of CXCR4+ cells was higher in the CD32+ CD4+ population than in CD32− CD4+ T cells regardless of study population. Interestingly, only CD32+ CD4+ T cells of viremic HIV patients showed significantly higher levels of CCR5, than their CD32− counterparts (Fig. 1E). In addition, we assessed the expression of TIGIT and PD-1 on CD32+ and CD32− CD4+ T cells in a small study population of viremic and patients on ART (n = 5 each). We found elevated levels of TIGIT and PD-1 on CD32+ cells. However, the difference was not statistically significant (data not shown).
To address the possibility of a longer life span of CD32+ CD4+ T cells, we measured their comparative expression of CD127, a marker known to be associated with longer cellular half-life. However, differences were only seen according to the infection status and not according to CD32 expression (Fig. 1F).
Recently, Descours et al5 have postulated that CD32a might be a specific marker for resting, latently HIV-infected CD4+ T cells. Here, we present one of the first studies examining the CD32 expression in different memory T cell subsets of peripheral blood and lymph node.
Compared to ART-treated or healthy individuals we found that a significantly higher proportion of CD4+ T cells, and in particular CD4+ memory subpopulations, of viremic HIV patients expressed CD32. Surprisingly, CD32+ levels of total CD4+ T cells or naive and memory subpopulations did not significantly differ between patients on ART and healthy, uninfected controls.
Indeed, a recently published preliminary report showed that, although HIV DNA was enriched 16–333 times in CD32+ versus CD32− CD4+ T cells,16 this group of researchers could not detect a direct correlation between integrated HIV DNA in CD4+ T cells and percentage of CD32+ CD4+ T cells in ART-treated or therapy naive patients. The authors argue that the fold enrichment varies widely among different patients.16
We performed a preliminary post hoc analysis in a small cohort of patients with primary HIV infection: total HIV DNA as measured by droplet digital polymerase chain reaction in PBMC was compared with CD32 expression (previously described in Ref. 18). No significant correlation was detectable between total HIV DNA in PBMC and CD32 expression on CD4+ T cell subsets (data not shown).
In the current cohort, CD32 expression correlated with the general immune activation (HLA-DR expression of CD8+ T cells) and was significantly higher in memory T cell subsets of viremic HIV patients in comparison with ART-treated and healthy individuals. Previously, it could be shown that CD32 was upregulated by immune activation in vitro.9,16 Furthermore, CD32+ CD4+ T cells showed a higher expression of HLA-DR than CD32− cells regardless of the HIV-infection status. CD32 expression within different CD4+ T cell subpopulations did not, however, correlate with expression of HLA-DR within the same subpopulation (eg, activation of TTM did not correlate with CD32 expression on the same population), thus suggesting that CD32 is not a marker of T cell activation in the classical sense. The relationship between CD32 and the reservoir of latently infected T cells seems to be more complex and other factors such as active infection, activation, and differentiation status of the cell have to be taken into account. It has to be noted that Grau-Expósito et al report a 2-fold upregulation of both HLA-DR and CD32 expression after ex vivo infection with HIV-1.6,19 It cannot be ruled out that CD32+ is to some extent also expressed on actively HIV-infected T cells, for example, in our viremic patients.
So, the jury is still out on whether a) infection leads to upregulation of CD32 in latently infected T cells or b) as Martin et al hypothesize, “preferential infection or survival, rather than upregulation” are the reason for the enriched proviral HIV DNA detectable in CD32+ CD4+ T cells. Our data which showed increased CXCR4 and CCR5 expression on CD32+ CD4+ T cells seems to support the latter model. Also, both effects could play an equal role.
It has to be noted that the results of Martin et al16 and the results of this study are based on the analysis of cryoconserved cells, whereas Descours et al5 analyzed fresh PBMC. Although only a small, non-significant difference between CD32 expression of fresh versus cryopreserved PBMC could be detected in our hands (Supplemental Digital Content Figures 2A, B, http://links.lww.com/QAI/B104), the effect of freezing-thawing on CD32 expression has to be considered as a possible variable in future studies.
It also has to be taken into consideration that the gating strategy applied in this study differed from the one used by Descours et al5 because we used fluorescence minus one instead of isotype controls. Our approach could only distinguish between negative and positive events, and the clear separation of CD32 intermediate versus CD32 high events was not possible.
Future studies have to assess the exact relationship between CD32 expression and integrated HIV DNA for each patient group and each peripheral (and tissue-resident) CD4+ T cell subpopulation, thereby also differentiate between active infection versus latently infected cells. Buzón et al described so-called episomal cDNAs forming on treatment with an integrase inhibitor, which could enable discrimination between actively and latently infected cells.20,21
Furthermore, the individual role of different CD32 isotypes has to be assessed using isotype-specific antibodies. Also of importance is the possible influence of a previously described polymorphism of the FcyRII gene (which affects the IgG binding affinity) on the reservoir size or the number of integrated HIV DNA copies.10
In addition, since we observed a high background expression of CD32 in healthy HIV-negative controls, the combination of different putative markers of latently HIV-infected CD4+ T cells should be tested to increase the specificity of CD32.5 For example, PD-1 has recently been reported as a marker for latently infected cells.21
In summary, we find low-level expression of CD32 on CD4+ T cells regardless of the infection status that correlates with immune activation. Follow-up studies will have to re-evaluate CD32 as marker of latently HIV-infected cells in different CD4+ T cell subsets and different patient groups as other factors such as immune activation seem to influence CD32 expression on different T cell subsets.
The authors thank the patients who participated in this study and Alexandra Rickert for technical help.
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