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Breast Milk CD4+ T Cells Express High Levels of C Chemokine Receptor 5 and CXC Chemokine Receptor 4 and Are Preserved in HIV-Infected Mothers Receiving Highly Active Antiretroviral Therapy

  1. Athena P. Kourtis1,5,
  2. Chris C. Ibegbu3,
  3. Regan Theiler4,
  4. Yong-Xian Xu3,
  5. Pooja Bansil1,
  6. Denise J. Jamieson1,4,
  7. Michael Lindsay4,
  8. Salvatore Butera2 and
  9. Ann Duer1
  1. 1 Division of Reproductive Health, National Center for Chronic Disease Prevention and Health Promotion, Emory University School of Medicine, Atlanta, Georgia
  2. 2 Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV, STD and TB Prevention, Centers for Disease Control and Prevention, Emory University School of Medicine, Atlanta, Georgia
  3. 3 Center for AIDS Research Immunology Core Laboratory, Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia
  4. 4 Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, Georgia
  5. 5 Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk
  1. Reprints or correspondence: Dr. Athena P. Kourtis, DRH/NCCDPHP/CDC, MSK34, 2900 Woodcock Blvd., Atlanta, GA 30341 (apk3{at}cdc.gov).
 
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Abstract

Background. Transmission of human immunodeficiency virus (HIV) to the infant through breast-feeding is a major problem worldwide; however, the biological circumstances of such transmission remain unclear. Little characterization of breast milk CD4+ T lymphocytes has been done so far.

Methods. We performed a detailed immunophenotypic analysis of T lymphocytes in the breast milk, compared with the blood, of HIV-uninfected (n = 9) and HIV-infected (n = 10) women receiving highly active antiretroviral therapy, by use of multiparameter flow cytometry. Descriptive statistics and nonparametric comparisons were performed using SAS software (version 9.1; SAS Institute).

Results. In uninfected women, 44%–78% of breast milk CD4+ T cells expressed the C chemokine receptor 5 (CCR5), whereas 26%–73% of cells coexpressed CCR5 and CXC chemokine receptor 4 (CXCR4). In contrast, only 7%–20% of peripheral blood CD4+ T cells expressed CCR5 and 1%–20% coexpressed CCR5 and CXCR4. The level of CCR5 expression in CD4+ T cells in breast milk was higher than in blood. In HIV-infected women, the high frequency of CD4+CCR5+ T cells in breast milk was preserved.

Conclusions. A majority of CD4+ T cells in breast milk express high levels of CCR5 and CXCR4. Unlike other mucosal immune sites, in which CD4+CCR5+ T cells are rapidly eliminated by HIV, these cells are preserved in breast milk during HIV infection.

Transmission of HIV from mother to infant through breast-feeding causes one-third to one-half of new HIV infant infections worldwide (reviewed in [1]); however, over half of all breast-fed infants remain uninfected even after prolonged exposure to breast milk. Breast milk is known to contain a multitude of immune factors that promote the infant's immune defenses, such as antimicrobial factors, immunoglobulins, and inflammatory substances, as well as immune cells [2–5]. The highest concentration of breast milk cells occurs in the first few days of lactation and is in the magnitude of 1×106–3×106 cells/mL [2, 3]. The cells present in breast milk are mainly neutrophils, macrophages, and mammary epithelial cells, with a small percentage of lymphocytes [1–6]. The study of lymphocyte subsets in breast milk—their activation status, chemokine receptor expression, and functional responses—is important in furthering our understanding of the immunological aspects of normal breast milk and how they are altered during HIV infection. To our knowledge, no studies to date have directly compared T cell homeostasis, activation, or phenotypic characteristics of the CD4+ T cell compartment in peripheral blood and breast milk from HIV-infected and HIV-uninfected women.

The objectives of this study were (1) to immunophenotype in detail the T lymphocytes present in breast milk of healthy HIV-uninfected mothers early in lactation (colostrum); (2) to study the expression of trafficking markers on such cells, including chemokine and lymph node— and mucosa-homing receptors; and (3) to compare these findings with those of the breast milk of HIV-infected women. We recruited 10 HIVinfected women and 9 HIV-uninfected women and sampled their breast milk and venous blood. From each subject and within each fluid, we examined (1) CD4+ T cell homeostasis; (2) T cell activation based on CCR5, HLA-DR, and Ki67 expression; (3) relative levels of naive, effector-memory, and terminally differentiated T lymphocytes; and (4) mucosal homing receptor expression. Our data reveal high activation in breast milk lymphocytes and, quite unexpectedly, preservation of CD4+CCR5+ T cells in the breast milk of HIV-infected women. Collectively, these data shed light on mechanisms underlying mucosal immune activation, lymphocyte recruitment, and homeostasis in the mammary gland and on the relationship of the dynamics of HIV infection in this mucosal site, compared with those in peripheral blood.

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Patients and Methods

Patients. We sequentially enrolled 10 adult mothers with established HIV infection had been delivered of their infants at Grady Memorial Hospital in Atlanta, Georgia. We also sequentially enrolled 9 HIV-uninfected women who had been delivered of their infants at the same hospital. The exclusion criteria were as follows: having a chronic illness such as diabetes mellitus, having chronic liver or renal disease, having other immunodeficiency, receiving immunosuppressive or immunomodulatory drugs, having a history of neoplastic disease, or having a history of illicit drug abuse within a year of sampling. For HIV-infected women, we obtained clinical information about their recent CD4+ T cell counts, viral load measurements, and current highly-active antiretroviral therapy (HAART). All women had been delivered of their infants full term. A small amount of fresh breast milk was obtained at one time from these women during their stay in the hospital for the birth of their infants (in the first 3 days after delivery). The milk was manually expressed in a sterile container; a venous blood sample was also collected from the women.

The women gave informed consent for the study, which was approved by the Centers for Disease Control and Prevention's and Emory University's ethics boards. The breast milk samples were transported to the laboratory on ice and processed within 6 h of collection. A portion of the sample was frozen at −70°C.

Isolation of blood and breast milk mononuclear cells and flow cytometry. Breast milk mononuclear cells and peripheral blood mononuclear cells were isolated by density gradent centrifugation (Ficoll-Paque; Pharmacia). The cells were washed in PBS and resuspended in fluorescence-activated cell sorter (FACS) buffer (PBS containing 2% bovine serum albumin and 0.1% NaN3) at 3×106 cells/mL. An amount of 100 μL was stained at room temperature for 20 min and then was washed with FACS buffer before fixing in 300 μL of 1% parafolmadehyde. The Ki67 tubes were permeabilized with 500 μL of Perm-2 (BD Biosciences) for 10 min at room temperature and then were washed before staining with Ki67 and fixed, as described above. The panel of markers used included the following: T lymphocytes (CD3, CD4, CD8), B lymphocytes (CD19), NK cells (CD16, CD56), chemokine receptors (CCR5, CXCR4), activation markers (HLA-DR), proliferation markers (Ki67), and naive (CD45RA), memory (CD45RO), lymph node—homing (CCR7), and mucosa-homing (CD103) receptors, as well as the senescence marker CD57. The complete staining panel of fluorochrome combinations used in the lymphocyte gate follows: Formula. The monoclonal antibodies are commercially available and directly conjugated with fluorescein isothiocyanate, phyco-eerythrin, PerCP (peridinin-chlorophyll-protein complex), or allophycocyanin (BD-Biosciences). Acquisition of data and cell subset analysis were performed with a FACSCalibur flow cytometer (BD Biosciences) using FlowJo software (Treestar).

Statistical analysis. Descriptive statistics (means, SEs, medians, and ranges) were used for the different lymphocyte subpopulations in the blood and breast milk of HIV-infected and HIV-uninfected women. Nonparametric tests (Wilcoxon Mann-Whitney U test) were used to compare each lymphocyte subpopulation in the blood or breast milk of HIV-infected with that of uninfected women. A paired t test was used to compare the distribution of each lymphocyte subpopulation in breast milk with that in their respective blood specimens for the 2 groups of women. All statistical analyses were performed using the SAS statistical package (version 9.1; SAS Institute).

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Results

Study population. Breast milk was obtained from HIV-positive women who were delivered of their infants at Grady Memorial Hospital, an inner-city hospital in Atlanta, Georgia (n = 10) and from HIV-uninfected women of similar sociodemographic status who were delivered of their infants at the same hospital (n = 9). All HIV-infected women were receiving combination HAART at the time of sampling. The median absolute CD4+ T cell count in the HIV-infected subjects was 312 cells/mm3 (range, 167–799 cells/mm3). The median level of HIV RNA in plasma was 365 copies/mL (range, undetectable to 13,700 copies/mL). For the women with virus load undetectable by the standard assay, a value of 200 copies/mL was assigned for computing the median. All women were free of opportunistic infections or other conditions at the time of sampling, and none had clinical evidence of mastitis. Breast milk was collected 1–3 days postpartum.

Percentages of CD4+ and CD8+ T cells in breast milk and blood of HIV-uninfected women. Gating on the lymphocytes and CD3+ T cells in the colostrum of uninfected, healthy mothers revealed that 4.4%–53.5% (median, 15.3%) of lymphocytes were CD8−, whereas 2.4%–27.1% (median, 9.1%) expressed CD8 (table 1 and figure 1). This compared with 42.3%–69.5% (median, 52%) for CD4+ and 17.2%–37.5% (median, 20.3%) for CD8+, respectively, in peripheral blood. Expression of the lymph-node homing marker CCR7 was significantly lower in both CD4+ and CD8+ breast milk T cells, compared with that in peripheral blood (table 1). In contrast, expression of the mucosal homing marker CD103 was significantly higher in breast milk CD4+ and CD8+ T cells, compared with that in peripheral blood (table 1).

Figure 1.
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Figure 1.

CD4+ and CD8+ T cell percentages of lymphocytes in breast milk and peripheral blood. Lymphocytes gated from first-week breast milk and simultaneously obtained blood from HIV-negative women (top) and HIV-positive women (bottom) who were delivered of their infants at Grady Memorial Hospital, Atlanta, Georgia, were stained with anti-CD3, anti-CD4, and anti-CD8 antibodies. The percentage of lymphocytes that express CD4 represents lymphocyte-gated events that stain positively with anti-CD3 and negatively with anti-CD8.

Activated status of CD4+ T lymphocytes in the breast milk of HIV-uninfected women. Many CD4+ T lymphocytes in the colostrum of HIV-uninfected women exhibited an activated phenotype (30.2%–60.1% [median, 39%] of CD4+ T cell expressed HLA-DR) (figure 2). In contrast, only 2.7%–10% (median, 3.9%) of peripheral blood CD4+ T cells expressed HLADR ( P < .01) (figure 2). Only 0.3%–15.5% (median, 2.8%) of CD4 cells from the colostrum had a naive (CD45RA+CCR7+) phenotype, compared with 13%–57.2% (median, 49.5%) of peripheral blood CD4+ T cells (Pp.003) (table 1). Moreover, breast milk CD4+ T cells expressed the proliferation marker Ki67 at a higher percentage than peripheral blood (0.8%–10.2% [median, 2.6%] vs. 0.9%–3.5% [median, 1.2%]; P = .05).

Figure 2.
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Figure 2.

HLA-DR expression by CD4+ or CD8+ T lymphocytes in breast milk and peripheral blood. Lymphocytes in peripheral blood and first-week breast milk obtained from HIV-negative women (top) and HIV-positive women (bottom) who were delivered of their infants at Grady Memorial Hospital, Atlanta, Georgia, were stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, followed by staining with anti—HLA-DR.

Chemokine receptor expression was also markedly different between CD4+ T cells in the colostrum, compared with CD4+ T cells from the peripheral blood. Forty-four percent to 77.7% (median, 61.2%), of CD4+ T cells in the breast milk of healthy women expressed CCR5 (figure 3), and the great majority (63.2%–98.5% [median, 87.7%]) expressed CXCR4. Coexpression of CCR5 and CXCR4 was noted on 26.4%–72.8% (median, 48.7%) of breast milk CD4+ T lymphocytes (table 1). In contrast, 7.4%–20.2% (median, 9.8%) of CD4+ T cells from peripheral blood expressed CCR5, and 0.8%–20.2% (median, 9.1%) coexpressed CCR5 and CXCR4 (P = .01 and P = .007, respectively) (figure 4). Moreover, the geometric mean channel fluorescence of CCR5 was significantly higher in breast milk CD4+ T cells, compared with in peripheral blood CD4+ T cells, indicating that breast milk CD4+ T cells had higher levels of CCR5 per cell (median, 44.2 in milk vs. 10.7 in blood; P < .001). The distribution of CCR5 and CXCR4 expression on CD4+ T cells in the breast milk and blood of a representative woman is shown in figure 4.

Figure 3.
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Figure 3.

C chemokine receptor 5 (CCR5) expression by CD4+ or CD8+ T lymphocytes in breast milk and peripheral blood. Lymphocytes in peripheral blood and first-week breast milk obtained from HIV-negative women (top) and HIV-positive women (bottom) who were delivered of their infants at Grady Memorial Hospital, Atlanta, Georgia, were stained with anti-CD3, anti-CD4, and anti-CD8 antibodies, followed by staining with anti-CCR5.

Figure 4.
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Figure 4.

Representative dot plots from breast milk (BM) and peripheral blood (PB) of a healthy, uninfected woman and an HIV-infected woman, demonstrating increased C chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) expression of CD4+ (top) and CD8+ T cells (bottom) in BM. Note that CD4+CCR5+ T cells are preserved in HIV infection.

Increase in effector memory nonterminally differentiated CD8+ T cells in breast milk of HIV-uninfected women. As has been previously noted by other investigators [6–10], the CD8+ T cell population in breast milk also exhibited characteristics of a nonnaive, activated population (77.9%–99.2% [median, 88.6%] CD45RA−, 27.6%–60.2% [median, 43.6%] HLA-DR+, and 16.9%–86% [median, 65.9%] CCR5+) (table 1). Moreover, expression of the CD57 marker, a characteristic of terminal differentiation and replicative senescence among effector CD8+CD45RO+ T cells, was the same in breast milk and peripheral blood (1.2%–10% [median, 3.8%] vs. 0.6%–4% [median, 1.8%]; P = .8), indicating that CD8+ T cells in breast milk represent predominantly and preferentially an antigenexperienced but not terminally differentiated effector cell population (figure 5).

Figure 5.
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Figure 5.

CD57 expression by CD8+CD45RO+ antigen-experienced T lymphocytes in breast milk and peripheral blood. Lymphocytes from peripheral blood and first-week breast milk obtained from HIV-negative women (top) and HIV-positive women (bottom) who were delivered of their infants at Grady Memorial Hospital, Atlanta, Georgia, were stained with anti-CD3 and anti-CD8 antibodies, followed by staining with anti-CD45RO and anti-CD57. CD57 expression characterizes terminally differentiated effector lymphocytes.

Comparative analysis of CD4+ and CD8+ T cell subsets in breast milk and peripheral blood of HIV-positive and HIVnegative women: preservation of CCR5+ CD4+ T cells in breast milk. In HIV-infected women, relative preservation of CD4+ T cells was noted in breast milk, even though peripheral blood CD4+ T cells were markedly lower than in HIV-uninfected women (figure 1). Percentages of CD4+ T cells in blood were significantly lower in HIV-infected women (1.7%–50.9% [median, 36.6%]) than in uninfected women (42.3%–69.5% [median, 52%]) (P<.01), but, in breast milk, the percentage of CD4+ T cells in HIV-infected women (5.9%–41.1% [median, 14.6%]) was not significantly different from that of uninfected women (4.4%–53.5% [median, 15.3%]) (P = .06). Unexpectedly, the proportion of CCR5+CD4+ T cells was also preserved and, indeed, was slightly higher in breast milk of HIV-infected, compared with uninfected women (35.7%–88.2% [median, 77.9%] vs. 44.1%–77.7% [median, 61.2%]; P = .056) (figure 4). Moreover, percentages of activated HLA-DR—expressing CD4+ T cells were higher in the breast milk of HIV-infected women (50.9%–92.6% [median, 67.7%]) (P = .05) (figure 2) than in uninfected women; CD45RA− memory CD4+ T cells were the same in HIV-infected and HIV-uninfected women (86.6%–99.1% [median, 97.1%] vs. 71.6%–99.1% [median, 95.8%], respectively; P = .85). Combined, these data indicate that effector (CCR5+) and memory (CD45RA−CD4+) T cells are preserved in breast milk during chronic HIV infection. Percentages of CCR5+CD8+ T cells also increased in HIV infection in breast milk (P<.01), as they also did in blood (P = .02) (figure 4), consistent with the increased level of immune activation at the chronic stage of HIV infection. Ki67+CD8+ T cells were also higher in the breast milk of HIVinfected women than in that of uninfected ones (P = .02) (table 1). Among the CD8+CD45RO+ antigen-experienced cells, it was predominantly the CD57− that increased with HIV infection in breast milk, although not significantly; in blood, the CD57+ subset significantly increased (figure 5). The expression of the mucosal homing marker CD103 in breast milk T lymphocytes did not change significantly with HIV infection (table 1).

Table 1.
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Table 1.

Surface marker expression comparison between breast milk (BM) and peripheral blood (PB) cells.

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Discussion

The phenotype of CD4+ T cells in breast milk had not been previously examined. The present study demonstrates that the majority of CD4+ T cells in breast milk coexpress high levels of both CCR5 and CXCR4, the major coreceptors required for HIV attachment and entry [11]. In addition, many CD4+ T cells in breast milk have an activated phenotype. These activated CD4+CCR5+ T cells lack CD45RA expression, which suggests that they are not naive cells. This phenotype of CD4+ T cells is similar to that found in other mucosal sites such as the gastrointestinal tract and vagina [11–13] and indicates that breast milk lymphocytes express characteristics of mucosal monocytes, rather than peripheral blood mononuclear cells [12]. Taken together with the high expression level of the mucosal homing marker CD103, these data argue for a mucosal origin of breast milk lymphocytes. They further indicate that breast milk, like the intestinal tract and vagina, contains the optimal viral target cells necessary for virus transmission and establishment of HIV infection. However, in contrast to these other mucosal sites, where a profound depletion of these cells is observed very early in HIV infection (or simian immunodeficiency virus infection in macaques) [11, 14], there is no apparent loss of CD4+CCR5+ T cells in breast milk, making it unique in this respect among mucosal sites.

The preservation of CD4+CCR5+ T cells in breast milk during HIV infection is thus of particular interest, especially because these cells appear to be phenotypically ideal targets for infection. The patients we studied were clinically stable on HAART regimens, maintaining low, and in some cases undetectable, viral loads. However, these patients had not achieved CD4+ T cell reconstitution in the periphery. Indeed, CD4+ T cell percentages in blood were much lower in HIV-infected than in HIV-uninfected women. However, these percentages in breast milk were no different between HIV-infected and HIV-uninfected women. Studies of patients who are not receiving HAART who have uncontrolled viremia and of patients at various stages of HIV infection, including early infection, will be needed to further follow the CD4+ T cell dynamics during HIV infection at this mucosal site. Nevertheless, our data provide an intriguing insight into breast milk lymphocyte homeostasis, particularly because, in the gut, CD4+CCR5+ T cells are known to reconstitute poorly even after 3 years of HAART [15]. Because the mammary gland is not exposed to foreign antigens to the same extent as the gastrointestinal or vaginal mucosae, it might be hypothesized that the CCR5+CD4+ T cells in breast milk represent resting CCR5+CD4+ T cells that might be resistant to killing by HIV [14]. Future studies including sampling of both gut and breast milk in the same individuals are needed to understand whether the mammary gland is a site preferentially sheltered from HIV replication. Indeed, levels of HIV RNA in breast milk are generally 1–2 logs10 lower than those in plasma [16]. Another caution when interpreting these results is the fact that we did not analyze absolute lymphocyte counts, because of marked variability in cell yield among different individuals and because cell counts in breast milk are known to dramatically decrease during established lactation, compared with colostrum; it was thus thought that percentages might represent more stable parameters.

The activated profile of the CD8+ T cell population in breast milk has been described elsewhere [9, 10] (reviewed in [1]); moreover, HIV and other pathogen-specific responses have been detected in breast milk of infected women, arguing for a role in protection of the infant from such insults [17–19]. Indeed, HIV-specific CD8+ T cells are found in the breast milk with greater frequency than in the blood of HIV-infected women [8]. Our findings of increased activation among breast milk CD8+ T lymphocytes corroborate these results and further indicate that CD8+ T cells in breast milk—being primarily CCR5+, CD45RA−, CD57−, and CCR7−—appear to have a predominantly early intermediate phenotype according to the scheme of postthymic T cell differentiation proposed by Appay and Rowland-Jones [20] or mostly effector memory cells according to the classification proposed by Sallusto et al. [21]. In comparison, the phenotype of CD8+ T lymphocytes in blood appears, on the whole, further “advanced” along the differentiation pathway, having a higher proportion of CD57 and a lower proportion of CCR5 expression. This is even more exaggerated during HIV infection, in which blood CD8+ subset is enriched in CD57-expressing cells, indicating accumulation of terminal effectors. In contrast, in breast milk, such shifting is not observed with HIV infection, and the main CD8+ phenotype in breast milk is CD57−CD45RO+, perhaps implying enhanced functional competence of breast milk CD8+ T cells.

CD4+ and CD8+ T cells in breast milk present an overall profile of immune activation. Indeed, breast milk lymphocytes appear to have the immunophenotype of those of an inflammatory exudate; their cellular constitution is similar to inflammatory exudates of gastric or intestinal inflammation [22, 23], the middle ear effusion [24], inflamed synovial fluid [25], or lung lavage fluid during active tuberculosis [26]. Viewed from this point, the preservation of CD4+CCR5+ T cells in the breast milk of HIV-infected women might reflect the increased immune activation at this site and serve a teleological role in enhancing the infant's immune defenses. Regarding the origin of these activated cells at this early stage of lactation, we hypothesize that they are preferentially recruited in the mammary gland rather than produced by in situ proliferation in the breast. Whether the presence of these cells in breast milk might increase the risk of HIV transmission to the infant or, on the contrary, help protect from mucosal infections is uncertain. In the present study we analyzed the colostrum and, particularly in the case of HIV-infected women, the first expressed milk. It is possible that established lactation may bring on changes in levels of immune activation and the cellular composition of breast milk and, in the case of HIV infection, might lead to changing frequencies of HIV+ cells and immune depletion. Our own observations indicate that the profile of immune activation noted in the colostrum is also observed at other stages of lactation in HIV-uninfected women, ranging from 2 to 12 months (data not shown); however, additional studies are needed to determine the differences in CD4+ and CD8+ T lymphocyte subsets in breast milk at various stages of HIV disease. Because our study population was rather small, further studies will be helpful in confirming our findings.

Worldwide, transmission of HIV infection to the infant via breast feeding remains a major mode of childhood HIV acquisition (reviewed in [1]). The exact cellular targets and mechanisms involved in breast milk transmission are uncertain. As has been clearly demonstrated by the present study, the majority of CD4+ T cells in the breast milk of women are activated and express high levels of both CCR5 and CXCR4. These cells appear to be preserved in the breast milk of women at the chronic stage of HIV infection. These observations are different from what has been observed in other mucosal sites, where, even during chronic infection, CD4+CCR5+ T cells remain decreased [14, 27]. It is plausible, therefore, to speculate that these cells are protected from infection; however, what role they play in the transmission of HIV infection to the infant or protection thereof remains to be elucidated.

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Acknowledgment

We thank the Center for AIDS Research Immunology Core Laboratory, Emory University School of Medicine, for expert technical assistance.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Centers for Disease Control and Prevention.

  • The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the funding agency.

  • Received July 31, 2006.
  • Accepted November 4, 2006.
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  1. J Infect Dis. (2007) 195 (7): 965-972. doi: 10.1086/512082
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