Mucosal HIV transmission is considered a rare event.1 The site of exposure (rectal or vaginal), the viral load (VL) of the infected partner and the presence of other sexually transmitted infections (STIs) constitute variables with major impact on the likelihood of transmission.2,3 However, there may be additional unidentified viral- and host-related characteristics that sway the chances of productive infection.
To proliferate and disseminate after exposure, HIV needs to evade numerous barriers in the mucosa and find its way to draining lymph nodes (LNs) and gut inductive sites.4 The availability of HIV targets cells at the site of exposure is influenced by several factors (eg, STIs) that impact the quality and quantity of cell subsets with differential ability to spread infection (CCR5+ and α4β7high CD4+ T cells).5–8
α4β7high CD4+ T cells constitute a major target of HIV/simian immunodeficiency virus (SIV) infection and are preferentially infected during the acute stages.5,6,8 It was reported that at day 2–4 postinfection α4β7high CD4+ T cells have 5 times more SIV-gag DNA than α4β7− CD4+ T cells,6 and their frequency in blood correlates with the loss of CD4+ T cells in the gut.9 Of note, blocking α4β7 by injecting a monoclonal antibody against α4β7 before intravenous SIV challenge reduces peak VL, decreasing proviral DNA in peripheral blood mononuclear cells (PBMCs) and jejunal and colorectal tissues.10
Once viral replication is underway at the site of exposure, the next critical stage in mucosal transmission is the ability of HIV infected cells to rapidly reach the mucosal draining LNs.4 Notably, α4β7 is responsible for trafficking and retention of lymphocytes in the gut-associated lymphoid tissue, Peyer patches, mesenteric lymph nodes (MLNs), and gut lamina propria.11 These are among the first sites reached by HIV and where the virus replicates at a high rate. Moreover, α4β7-expressing leukocytes play an essential role in mucosal immunity and are also important in the immune response of the female genital tract.12,13
Thus, the presence of α4β7high CD4+ T cells at the site of exposure can impact (1) the availability of good substrate and, therefore, the initial rate of viral replication, (2) the ability of infected cells to rapidly reach the mucosal draining LNs, and (3) the local immune response.
Additional evidence of the importance of α4β7high CD4+ T cells in HIV transmission and pathogenesis is reflected in the ability of HIV-gp120 to bind α4β7. Recent findings suggest that the ability of HIV to interact with α4β7 might be 1 of the viral features that help HIV to survive the mucosal barriers.5,6,9,10,14,15
The different lines of evidence noted above point to a role for α4β7high CD4+ T cells during the earliest stages of mucosal transmission. We postulated a link between the availability of these cells at the time of challenge and susceptibility to rectal SIV infection. Herein, we show that the frequency of α4β7high memory CD4+ T cells in blood before challenge correlates with the rate of acquisition of SIVmac239 infection. Moreover, we observed a strong direct correlation between the frequency of α4β7high CD4+ T cells in blood and rectal tissue. This indicates that the frequency of α4β7high CD4+ T cells in the rectal mucosa at the time of challenge correlated with susceptibility to infection. In addition, the frequency of α4β7+ CD11c+ myeloid DCs (mDCs) and α4β7+ CD80+ DCs correlated with infectious status and acute VL, whereas blood CCR5+ CD4+ T cells did not. Taken together, our results suggest that the availability of α4β7high CD4+ T cells play a critical role at a early stage of SIV transmission.
Macaque Treatments and Challenge
Adult male Indian rhesus macaques (RM, Macaca mulatta) were housed in compliance with the regulations under the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, at Tulane National Primate Research Center (TNPRC, Covington, LA). Monkeys were anesthetized with ketamine-HCl (10 mg/kg) or tiletamine/zolazepam (6 mg/kg) before all procedures. Preemptive and postprocedural analgesia (buprenorphine, 0.01 mg/kg) was required for procedures causing more than momentary pain or distress. Seventeen animals were challenged rectally with 3000 TCID50 of SIVmac239 in 1 mL of phosphate-buffered saline. Virus was propagated in PBMCs and titrated 174 × CEM cells. The SIV chronically infected RMs (n = 10, >1 year postinfection) used for the correlation of α4β7high CD4+ T cells between tissues (Fig. 1B) were healthy with CD4 count ranging from 364 to 785 cells/μL.
Determination of SIV Infection
Plasma VLs were measured by quantitative reverse transcriptase-polymerase chain reaction (PCR).16–18 Infection was confirmed by SIV-PCR on MLNs and iliac LNs at days 10 and 14. One animal was negative to SIV-PCR in plasma on day 10 and VL at day 14 could not be tested. This animal was positive by SIV-PCR in MLNs and iliac LNs both on days 10 and 14 and was considered infected. The animal is included in the graphs that show positive (POS) vs negative (NEG) animals, but not in the graphs that correlate VLs with the frequency of α4β7high or CCR5+ CD4+ T cells and α4β7+ DCs. For 1 animal, the data of the T cells panel at day 0 were not available; therefore, this animal is not represented in the graphs that show correlations with the frequency of the various T subsets at day 0 but included in the correlations at earlier time points.
Cell Isolation and Flow Cytometry
PBMCs were isolated using Ficoll-Hypaque density gradient centrifugation. Axillary (AX), inguinal (ING), iliac, and MLNs were cut in small pieces and passed through a 40-μm cell strainer. Rectal biopsies were incubated 1 hour in R10 with 1 mg/mL hyaluronidase, 0.5 mg/mL collagenase II (Sigma-Aldrich, St. Louis, MO), and 1 mg/mL DNAseI (Roche, Indianapolis, IN). The cell suspension was passed through a 40-μm nylon cell strainer. Cells were stained with the LIVE/DEAD Aqua dye (Invitrogen), anti-CD4-QDot605 (non-human primate repository; Beth Israel Medical Center, Boston, MA), anti-HLA-DR-QDot605 (Invitrogen, Grand Island, NY), and anti-dimeric α4β7 (NIH AIDS Research and Reference program) conjugated using PE-Conjugation Kit (AbD-Serotec). Also included in the T-cells panel are anti-: CD8-APC-Cy7, CD95-FITC, CD3-V450, CCR5-PeCy7, CD69-Alexa700, CD28-APC; DCs anti-: CD3-14-20(Lin)-V450, CD80-APC-H7, CD11c-Alexa700, CD1c-APC, CD123-PCPCy5.5 (BDBioscience). Greater than 200,000 events were acquired in the lymphocyte live-cells gate using the BD LSRII Flow Cytometer.
Mann–Whitney nonparametric test was used to compare variables between groups (SIV-POS vs SIV-NEG). Linear regression analysis was performed to determine the correlation between cell subsets in blood and other tissues and VLs. A 2-tailed P = α < 0.05 was considered significant. The analysis was performed using Prism5a (GraphPad Software, Inc).
The Frequency of α4β7high Memory CD4+ T Cells in Blood Correlates With Their Frequency in the Rectal Mucosa
On rectal challenge, the mucosa is the first site where the virus encounters susceptible target cells. Thus, its attributes are likely to have a strong impact on susceptibility to SIV infection. The α4β7+ T cells home to the gut and the gut inductive sites and are reportedly a major target population in acute infection.5,6,19 We postulated that there could be an association between the frequency of α4β7high CD4+ T cells in the colorectal area and in blood. If so, measuring the frequency of α4β7high CD4+ T cells in blood could provide a surrogate for their presence at the site of challenge.
Because the majority of α4β7high CD4+ T cells are CD95+ T cells (see Figure S1, Supplemental Digital Content, http://links.lww.com/QAI/A436;6,20), we measured the frequency of α4β7high within the CD95+ CD4+ T cells subset. This gating strategy allowed us to measure α4β7high cells independently of memory T cells (see Figure S1, Supplemental Digital Content, http://links.lww.com/QAI/A436). This gating strategy was used for blood, rectum, and LNs.
Indeed, the frequency of α4β7high CD4+ T cells in blood and colorectal tissues of 17 animals 8 weeks before challenge strongly correlated (Fig. 1A, left). In contrast, the frequency of α4β7high memory CD4+ T cells in AX and ING LNs biopsies 6 weeks before infection did not correlate with their frequency in blood on the same day (Fig. 1A, center and right). Because it was not possible to sample MLNs and iliac LNs from naive animals, we measured the frequency of this population in blood, rectal tissues, and AX, ING, MLNs, and iliac LNs of 10 healthy chronically infected animals (> 1 year postinfection) at the time of their killing. We found that, in chronically infected animals, the frequency of α4β7high memory CD4+ T cells significantly correlated between blood and rectum (Fig. 1B, left). Moreover, there was a significant correlation between blood and iliac LNs (Fig. 1B, center) but not with MLNs (Fig. 1B, right), AX, or ING LNs (not shown).
Higher Frequency of α4β7high CD4+ Memory T Cells in Blood Associates With Infection
We next asked whether the frequency of α4β7high memory CD4+ T cells in peripheral blood was related to susceptibility to infection through the rectal route. Ten naive RMs were challenged rectally with 3000 TCID50 of SIVmac239 (group 1), and 10 months later, an additional 7 animals were challenged rectally with 3000 TCID50 of the same stock of SIVmac239 (group 2). As determined by quantitative SIV-RNA PCR on plasma (VL) and SIV-DNA nested PCR on iliac LNs, 12 of 17 animals became infected.
We found that the animals that became infected (POS) had a significantly higher frequency of α4β7high memory CD4+ T cells in blood 21 days before challenge (Fig. 2, left) and at the time of challenge (Fig. 2, right) than the ones that remained uninfected (NEG). This was most significant if we separated the animals in positive (POS) and negative (NEG) by plasma VLs at day 7 postchallenge (see Figure S2, Supplemental Digital Content, http://links.lww.com/QAI/A436).
The acute VLs confirmed a positive association between the rate of viral replication and the frequency of α4β7high CD4+ T cells at the time of exposure. In fact, there was a strong direct correlation between the acute VLs and the frequency of this population in blood at the time of challenge (Fig. 3A; VLs at day 7 postchallenge, left; VLs at day 10 postchallenge, right). Interestingly, the frequency of these cells in the infected animals declined at a rate similar to the rate of increase in VL (Fig. 3B, left). This was in agreement with data from other groups.6 In contrast, there was little variation in the animals that remained uninfected (Fig. 3B, right).
Notably, the strong direct correlation between blood and rectal α4β7high CD4+ T cells (Fig. 1A) suggests that the animals with high levels of α4β7high CD4+ T cells in blood also had high levels in their rectal tissue. This indicates that the frequency of α4β7high memory CD4+ T cells in the rectal mucosa correlated with susceptibility to SIV infection.
It has been proposed that binding to α4β7 might constitute an advantage for the virus during transmission (increased transmission fitness15). Indeed, an interaction between the of SIVmac239 and α4β7 could help explain the correlation between infection and the frequency of α4β7high CD4+ T cells at the time of challenge. However, the strength of the gp120-α4β7 binding varies among gp120s from different viral isolates,15 and this prompted us to examine the binding of the gp120 of SIVmac239 to RM α4β7. The relative affinity of SIVmac239 gp120 for α4β7 is in the range of HIV-gp120s previously tested (see Figure S3, Supplemental Digital Content, http://links.lww.com/QAI/A436;5,15,19). If a role for this interaction in SIV/HIV transmission is confirmed, it is possible that the association of α4β7 with susceptibility to infection is lost when the challenging virus has low or no affinity for α4β7.
The Frequency of CCR5+ T Cells Does Not Associate With Infection
It has been reported that memory CD4+ T cells that express very high levels of α4β7 are highly susceptible to HIV/SIV infection.5,9 Thus, we investigated if the expression of other HIV/SIV receptors on highly susceptible cell subsets associated with rectal SIV infection. We found no association between higher frequency of CCR5+ or memory CCR5+ (frequency of CD95+ that are CCR5+) CD4+ T cells and infection status (Fig. 4A). Neither did we find a correlation with acute VL (Fig. 4B).
In the gut tissue, the majority of α4β7high CD4+ T cells expresses CCR5.5 However, in blood, only a fraction of α4β7high CD4+ T cells expresses CCR5 (mean, 13.8%; range, 13.2%–18.1% at the time of challenge), and the majority of CCR5+ CD4+ T cells did not express α4β7 (see gating strategy in Figure S4, Supplemental Digital Content, http://links.lww.com/QAI/A436). We found no correlation between the frequency of CCR5+ memory CD4+ T cells in blood and their frequency in the rectal tissue in the RM 8 weeks before infection (Fig. 4C). Finally, it has been shown that, when measured by flow cytometry, the expression of CCR5 may be underestimated and that reverse transcriptase-qPCR measurement of the CCR5 mRNA may be a more sensitive method.21,22 These observations indicate that we cannot rule out a correlation between the frequency of CCR5+ CD4+ T cells in the rectal tissue and the susceptibility to SIV infection.
It is also possible that high expression of α4β7 on lymphocytes is a characteristic of the activation state of the immune system, which favors virus survival and spread early after challenge. However, we found no association between infection status (or VL) and the frequencies of CD69+ CD4+ T cells or α4β7+ CD8+ T cells in blood at the time of challenge (not shown).
Higher Frequency of α4β7+ DCs in Blood Associates With Infection
DCs are among the first cell targets that HIV encounters and likely play a key role in driving viral expansion after transmission.23–26 Thus, we investigated if the expression of α4β7 on DCs at the time of challenge could be associated with infection and/or VL. We found that the majority of α4β7+ Lin- HLA-DR+ DCs in blood of RMs are CD11c− (mean, 77.4%; range, 61.9%–87.6%), whereas the CD11c+ α4β7+ mDCs express low/intermediate level of α4β7 (see Figure S5, Supplemental Digital Content, http://links.lww.com/QAI/A436). Interestingly, all the Lin− HLA-DR+ DCs that express intermediate/high levels α4β7 are CD80+, and there is a small population of DCs that expresses high levels of α4β7 and CD80 (α4β7high CD80+ DCs; see Figure S5, Supplemental Digital Content, http://links.lww.com/QAI/A436).
Indeed, we found that the frequency of α4β7+ Lin− HLA-DR+ DCs, α4β7+ mDCs, and α4β7high CD80+ DCs at the time of challenge was higher in the animals that were positive (POS) for SIV-RNA PCR on plasma at day 7 postinfection than those that were negative (NEG) (Fig. 5A; frequency of α4β7high CD80+ DCs was available only for group 1). Instead, when the animals were divided in positive and negative by VLs at day 10 or 14 (therefore, the final results of infected vs uninfected as in Figs. 2, right, and 4A), only the frequency of α4β7+ mDCs was significantly different. Higher frequency of α4β7+ mDCs and α4β7high CD80+ DCs in blood at the time of challenge also correlated with acute VL (Fig. 5B, center and right), whereas total α4β7+ Lin− HLA-DR+ DCs did not (Fig. 5B, left).
In contrast, the frequency of CD1c+ DCs and CD123high pDCs, which express low levels of α4β7 (see Figure S5, Supplemental Digital Content, http://links.lww.com/QAI/A436), were not associated with infection or VL (not shown).
Our results show that susceptibility to rectal SIV infection could be associated with the frequency of α4β7high memory CD4+ T cells. After a single rectal exposure to a high-dose of SIVmac239, the 12 (of 17) RM that became infected had a significantly higher frequency of α4β7high CD4+ T cells before challenge than did the animals that remained uninfected. Notably, the same significant difference was found when we compared the frequency of this cell subset in blood 3 weeks before challenge, suggesting that each RM had a relatively stable frequency of blood α4β7high memory CD4+ T cells. In contrast, this correlation was not present with CCR5+ memory CD4+ T cells. However, when we tried to associate the susceptibility to SIV with the frequencies of blood or rectal α4β7high CD4+ T cells (or blood/rectal CCR5+ CD4+ T cells) 8 weeks before infection, we found no correlation (although there was a tendency for a higher frequency of blood α4β7high CD4+ T cells for animals that become infected; data not shown). This may be explained by the several factors that can impact the frequency of these cells during the 8 weeks before challenge.
Moreover, we found a strong direct correlation between the frequency of α4β7high CD4+ T cells before challenge and acute VL. Importantly, higher acute VL is associated with CD4+ T-cell decline and disease progression.27 Therefore, although it is unclear if α4β7high CD4+ T cells are the main source of virus in plasma during the acute phase,8 our findings suggest a link between availability of α4β7high CD4+ T cells at the time of challenge and the magnitude of viral replication during acute infection. Of note, previous reports described the frequency of α4β7high cells within the CD4+ T cells population.6,9,19 However, having shown that the majority of α4β7high CD4+ T cells are CD95+ (see Figure S1, Supplemental Digital Content, http://links.lww.com/QAI/A436;6,20), we followed the α4β7high CD4+ T cells within the CD95+ cells, excluding the possibility that a higher frequency of α4β7high CD4+ T cells is a consequence of higher frequency of all memory cells.
The correlation between susceptibility to rectal SIV and α4β7high CD4+ T cells can be explained, at least, partly, by the role played by α4β7 in directing lymphocytes to gut inductive sites28 and by the high susceptibility of α4β7high CD4+ T cells.5,6,9 We propose that a higher frequency of α4β7high CD4+ T cells at the site of exposure increases the probability that SIV-infected cells reach MLNs and Peyer patches, where they encounter a large number of highly susceptible HIV cell targets. Therefore, the α4β7high CD4+ T cells could affect (1) the rate of viral replication at the site of exposure and (2) the rate with which the virus accesses mucosal draining LNs and gut-inductive sites. Both these factors constitute 2 major determinants of transmission and impact the chances of this rare event to occur.
By facilitating the rapid delivery of virus to inductive sites, infection of α4β7high CD4+ T cells may not only increase the likelihood of establishing infection but also influence the identity of the viral isolate that initiates infection. In fact, productive mucosal SIV/HIV infection, in most instances, reflects an expansion from a single viral isolate.29,30 It has been reported that the removal of transmission-linked putative N-linked glycosylation sites in HIV-gp120s can increase α4β7-affinity,15,30–32 and it was proposed that, in regard to transmission fitness, a strong α4β7 reactivity might constitute an advantage during mucosal exposure. We found that the relative affinity of SIVmac239 gp120 for α4β7 fell into in the range of other medium-reactive HIV-gp120s; however, gp120s from other SIVs might react with different strengths, and this could influence the correlation between α4β7high CD4+ T cells and infection.
We report that, in uninfected animals, the frequency of α4β7high CD4+ T cells was quite stable over time. However, STIs such as HSV-2, Chlamydia, and human papillomavirus modulate the frequency of α4β7 expressing T cells in the female genital tract and systemically,12,13,20,33 influencing in this way susceptibility to HIV mucosal infection. Moreover, other host-related factors, eg, hormonal environment, presence of other pathogens, and/or autoimmunity conditions, could impact the frequency of this cell subset and influence immune responses, in a way that also influences infection and could confound the correlation we are describing.
Of interest, not only to the HIV field is the finding that the frequency of α4β7high CD4+ memory T cells circulating in blood correlates with their frequency in the rectal mucosa and, perhaps, its draining LNs. This strong correlation suggests that, at the time of challenge, the animals that exhibited the highest frequency in blood also had the highest frequency in the rectal mucosa. Nevertheless, correlation does not prove causation and the link between the frequency of α4β7high CD4+ memory T cells and transmission might be indirect. More studies are needed to clarify the role of these cells in transmission.
We also found a correlation between the frequency of α4β7+ DCs in blood at the time of challenge and VL. It has been shown that α4β7+ pDCs are modulated by SIV infection and accumulate in the gut mucosa.34 However, we did not find a correlation between α4β7+ pDCs in blood and infection status. DCs at mucosal surfaces possess very different characteristics than circulating DCs; therefore, it might be inappropriate to draw a parallel between circulating DCs and the DCs present at the mucosal site of challenge.35,36 Nonetheless, similar to the α4β7high T cells, α4β7+ DCs traffic to the mucosal inductive sites. Here, the DCs acquire the ability to induce α4β7 on CD4+ T cells, which could be a way to influence the dynamics of viral replication during the acute phase of infection.
In conclusion, our results suggest that α4β7high CD4+ T cells and possibly α4β7+ DCs play an important role during transmission by the mucosal route and may constitute a determinant of susceptibility to infection. To the extent that these results can be extrapolated to mucosal transmission of HIV in humans, they present a novel and straightforward mean of identifying individuals at higher risk of rectal transmission within and across different groups of people.
The authors thank Nina Devine for her help editing the manuscript.
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