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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC 2009 September 1.
Published in final edited form as:
PMCID: PMC2593904

Potent SIV-specific Cellular Immune Responses in the Breast Milk of SIV-infected, Lactating Rhesus Monkeys1


Breast milk transmission of HIV is a leading cause of infant HIV/AIDS in the developing world. Remarkably, only a small minority of breastfeeding infants born to HIV-infected mothers contract HIV via breast milk exposure, raising the possibility that immune factors in the breast milk confer protection to the infants who remain uninfected. To model HIV-specific immunity in breast milk, lactation was pharmacologically induced in Mamu-A*01+ female rhesus monkeys. The composition of lymphocyte subsets in hormone-induced lactation (HIL) breast milk was found to be similar to that in natural lactation (NL) breast milk. Hormone-induced lactating monkeys were inoculated intravenously with SIVmac251 and CD8+ T lymphocytes specific for two immunodominant SIV epitopes, Gag p11C and Tat TL8, and SIV viral load were monitored in peripheral blood and breast milk during acute infection. The breast milk viral load was one to two logs lower than plasma viral load through peak and set-point of viremia. Surprisingly, while the kinetics of the SIV-specific cellular immunity in breast milk mirrored that of the blood, the peak magnitude of the SIV-specific CD8+ T lymphocyte response in breast milk was more than twice as high as the cellular immune response in the blood. Furthermore, the appearance of the SIV-specific CD8+ T lymphocyte response in breast milk was associated with a reduction in breast milk viral load, and this response remained higher than that in the blood after viral set point. This robust viral-specific cellular immune response in breast milk may contribute to control of breast milk virus replication.

Keywords: AIDS, T cells, Cytotoxic, Mucosa


The benefits of breastfeeding, including optimal nutrition and protection against gastrointestinal and respiratory infections, are well-established and significantly improve infant morbidity and mortality in the developing world (1, 2). Moreover, poor access to clean water in the developing world limits the safety of infant replacement feeding. However, HIV is vertically transmitted via breast milk and mother to child transmission via breast milk remains a significant mode of HIV transmission in the developing world. Nearly 800,000 new infant HIV infections occur each year, and it is estimated that one-third to one-half of these infections are attributable to breastfeeding (3).

Risk factors for transmission of HIV via breast milk include duration of breastfeeding (47), advanced maternal HIV disease (810), and breast abnormalities, such as breast abscess, mastitis and cracked nipples (6, 11, 12). Moreover, the level of breast milk viral RNA and number of infected breast milk cells, in addition to plasma viral load, are associated with a high risk of HIV transmission to infants (9, 1315). Mucosally transmitted virus is exposed to distinct immune responses specific to the mucosal compartment (1619). Virus in genital tract, semen and breast milk appears genetically divergent from that in the peripheral blood (2023), indicating that local immune responses shape the evolution of compartmentalized virus.

As late mother to child transmission of HIV occurs in a small minority of breastfeeding infants born to HIV-infected mothers, the majority of infants remain protected from transmission despite ongoing low dose exposure to the virus, raising the possibility that HIV-specific cellular or humoral immunity in the breast milk may protect infants from HIV transmission. However, limited information exists regarding maternal breast milk compartment-specific immunity and risk of breast milk transmission. Low titer HIV-specific IgA and IgM antibodies in breast milk have been associated with transmission in some (24), but not all studies (25). HIV-specific CD8+ T cells have been demonstrated in the breast milk of HIV-infected women (26). Yet, little is known about the kinetics, magnitude or function of virus-specific cellular immunity in breast milk during acute and chronic infection. Moreover, the characteristics of breast milk cellular immunity compared to systemic cellular immunity have not been defined.

The simian immunodeficiency virus (SIV)/rhesus monkey model is ideal to study viral-specific mucosal cellular immunity, as immunodominant epitopes and viral evolution are well-defined in this model. SIV inoculation of lactating rhesus monkeys allows for investigation of mucosal virus-specific immunity and viral replication in a compartment with a direct impact on risk of infection for the developing infant. In the present study, we describe a pharmacologic induction of lactation model in rhesus monkeys. We then define the kinetics and magnitude of viral-specific cellular immunity and viral replication during acute SIV infection in these monkeys. The understanding of cellular immune function in the breast milk and its effect on local viral replication that will come from studies in this nonhuman primate model should provide a framework for developing immunologic interventions to prevent breast milk transmission of HIV.

Materials and Methods

Animals, hormone treatment and virus

Four nonpregnant, female rhesus monkeys were administered increasing doses of depot medroxyprogesterone and estrodiol by the intramuscular route for two months to induce mammary gland maturation. The dopamine antagonist, haloperidol, was administered orally after two months of hormone treatment in order to raise serum prolactin levels. All four monkeys began lactating within six weeks of haloperidol treatment and breast milk was collected by manual massage under ketamine sedation two to three times weekly. Ten units of oxytocin were administered by the intramuscular route immediately prior to sampling. Dose, route and frequency of estradiol, medroxyprogesterone, and haloperidol were optimized to achieve serum levels of estradiol, progesterone and prolactin similar to those reported during pregnancy and the postpartum period in rhesus monkeys (27, 28). The optimal dosing schedule was as follows: estradiol cypionate 2mg/kg IM bimonthly, medroxyprogesterone acetate 5mg/kg weekly, haloperidol 0.15mg/kg PO twice daily.

Mammary gland biopsies were performed on each animal more than 3 months after the initiation of lactation. Tissue samples were sectioned and stained with hematoxlin and eosin. Breast milk was also manually collected once from each of 19 naturally-lactating female breeder rhesus monkeys that were between 2 and 36 weeks postpartum. Breast milk samples with visible blood contamination or less than 100 CD3+ cells were not included in the cellular analyses (n = 5).

Four female Mamu-A*01+ rhesus monkeys were selected for the study after PCR-based MHC typing. Following induction of lactation by hormone treatment, the monkeys were intravenously inoculated with SIVmac251. Blood and breast milk samples were collected 3 times per week during the first 4 weeks after inoculation and then twice weekly until day 126 after infection. The animals were maintained in accordance with the guidelines of the Committee on Animals for the Harvard Medical School and the “Guide for the Care and Use of Laboratory Animals” (National Research Council, National Academic Press, Washington, D.C., 1996).

Breast milk leukocyte differentials

LW/NL-X stain was prepared as previously reported (29). Briefly, 500g methylene blue chloride (EMD, Darmstadt, Germany), 56 ml 95% ethyl alcohol (Pharmaco, Brookfield, CT), and 40ml xylene (Fisher, Fair Lawn, NJ) was mixed and left standing for 24 hours at 4°C, then 4ml glacial acetic acid (Fisher) was added through a filter. 10μl of fresh whole breast milk was spread on a 1cm circle on a glass cell count slide (Bellco, Vineland, NJ) and allowed to air dry. The slide was fixed with methanol for 5 seconds and stained for 2 minutes. After drying, the slide was washed gently in tap water, air dried and viewed under magnification of 100X. Three counts of 100 cells per sample were performed by two independent counters and then averaged.

Phenotyping, tetramer staining and intracellular cytokine staining of breast milk and peripheral blood mononuclear cells (PBMC)

Breast milk samples were separated into fat, supernatant and cellular fractions by centrifugation at 60 g for 20 minutes. The fat layer was removed and the supernatant was collected and stored. The cell pellet was washed once and then stained for flow cytometric analysis. 1 × 106 PBMC isolated from EDTA-anticoagulated blood and the breast milk cell pellet were stained with anti-CD4-Amcyan (L200, Becton Dickinson), anti-CD8- Alexa700 (RPA-T8, Becton Dickinson), anti-CD28-PerCP-Cy5.5 (28.2, Beckman Coulter), anti-CD95-FITC (DX2, Becton Dickinson), and anti-CD3-APC-Cy7(SP34.2, Becton Dickinson). Naive and memory lymphocyte subsets were defined by anti-CD95 and anti-CD28 staining (naive: CD28+, CD95; effector memory: CD28, CD95+; central memory: CD28+, CD95+). An amine dye (Molecular Probes) was used to distinguish live from dead cells. Intracellular cytokine staining was performed after exposure of PBMC and breast milk cells to Gag p11C peptide (CTPYDINQM) as previously described (30) with the following additional antibodies: anti-TNF-α-Pacific Blue (Mab11 eBiosciences), anti-IFN-γ-PE-Cy7 (B27, Becton Dickinson), and anti-IL-2-APC (MQ1-17H12 Becton Dickinson). PBMC and breast milk cells from SIV-infected animals were also stained with soluble Mamu-A*01-PE or APC-labeled tetrameric complexes containing Tat TL8 (TTPESANL) or Gag p11C as previously described (31). Data were collected on the LSRII instrument (BD Biosciences) with FACSdiva software and analyzed with FlowJo software.

Antibody assays

IgM and IgG levels were measured in duplicate using monkey-specific ELISA kits (Alpha Diagnositics, San Antonio, Tx) and standards per protocol. Breast milk supernatant was diluted between 1:10 and 1:2000 for the assays. The IgA and secretory IgA levels were measured by coating a 96-well polystyrene plate with anti-IgA antibody (Rockland, Gilbertsville, PA), blocked with PBS-0.25% non-fat dry milk and 0.05% Tween-20. Plasma was diluted 1:20,000 and breast milk supernatant was diluted between 1:50 and 1:1000 (for IgA) or 1:100 and 1:102,400 (for sIgA) and incubated for two hours. Antibody was detected after one hour incubation with an HRP-conjugated anti-IgA antibody (Alpha Diagnotics) or an HRP-conjugated anti-sIgA (Nordic) and addition of ABTS-2 peroxidase substrate system (KPL, Gaithersburg, MD). A standard curve for the IgA assay was created using standards from a monkey-specific IgA ELISA kit (Alpha Diagnostics) and sIgA titer was determined by serial two-fold dilutions with a cut off of twice OD reading of the PBS negative control. Both assays were read at 410 nm.

Plasma and breast milk SIV quantitative RT-PCR

Total breast milk supernatant was centrifuged at 16,100 g for 1.5 hours and the viral pellet was stored in RNA later (Ambion, Foster City, CA). RNA from plasma and the breast milk viral pellet was isolated using the QIAamp viral RNA kit (Qiagen, Valencia, CA), according to protocol, with the final product resuspended in 60 μl of RNase-free water. 25 μl of the RNA suspension was used in an RT reaction containing SuperScript III RT enzyme (Invitrogen, Carlsbad, CA) and the Gag-specific primer 5’-GCAATATCTGATCCTGACGGCTC-3’, according to manufacturer’s protocol. 10 μl of the resulting cDNA was used in a real time PCR reaction using the Taqman EZ RT-PCR kit (Applied Biosystems, Foster City, CA), as well as a Gag-specific labeled probe (5’-FAM-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-TAMRA) and flanking primers (5’-GTCTGCGTCATCTGGTGCATTC-3’ and 5’-CAGTAGGTGTCTCTGCACTATCTGTTTTG-3’). Reactions were performed in duplicate on the 7700 ABI PRISM Sequence Detector (Applied Biosystems) at 95°C for 10 minutes, then 50 cycles of 95°C for 30 seconds, 60°C for 1 minute, and 72°C for 30 seconds. An RNA standard was transcribed from a plasmid containing the SIV Gag gene using the Megascript T7 kit (Ambion), quantitated by OD and serially diluted to generate a standard curve. The sensitivity of the assay was 600 copies. Preliminary experiments demonstrated that the quantitation of breast milk load correlated well to known amount of viral RNA added to breast milk supernatant from SIV-uninfected monkeys (data not shown). For breast milk samples obtained after SIV inoculation that demonstrated a copy number below the level of detection, a value of 300 copies was assigned (mean of the last detectable and the first undetectable standard). RNA copies per milliliter was determined by dividing the copy number by the volume of breast milk or plasma.

Statistical analyses

Immunoglobulin classes, and lymphocyte subsets or subset ratios in paired blood and breast milk of naturally lactating monkeys were compared using the exact Wilcoxon sign rank. The same comparisons between NL and HIL monkey breast milk were performed using the exact Wilcoxon rank sum test. Two-sided P value interpretation of significance was adjusted for multiple comparisons using Holm’s method. Wilcoxon sign rank test was used for comparisons of SIV viral load and SIV-specific CD8+ T lymphocytes in blood and breast milk and Spearman’s correlation was used to compare intracellular cytokine production and breast milk viral load. Analyses were performed using Stata and StatXact software and graphs were made using Prism software.


Comparison of lymphocyte subsets in breast milk and peripheral blood of rhesus monkey reveals skewing of breast milk lymphocytes towards a memory phenotype

T lymphocyte subsets in breast milk were compared to those in peripheral blood using paired samples from 14 healthy, naturally-lactating rhesus monkeys. While the percentage of CD4+ and CD8+ T lymphocytes was similar between blood and breast milk (Table I), breast milk had a lower median percentage of naive CD4+ (7.1% versus 45.8%, Fig 1A) and CD8+ (2.3% versus 26.2%, Fig 1B) T lymphocytes than the blood and significantly lower naive lymphocyte ratios (% naive/% naive + % memory; Table I) in both CD4+ (p = 0.001) and CD8+ (p = 0.0004) subsets. Accordingly, the median percentages of central memory (CM) and effector memory (EM) lymphocytes were higher in the breast milk than the blood in both CD4+ (79.8% versus 52.2% CM; 10.8% versus 2.3% EM; Fig. 1A) and CD8+ (70.9% versus 42.9% EM; 23.6% versus 12.4% CM; Fig. 1B) T lymphocyte subsets. Breast milk also demonstrated a significantly higher CD4+ EM ratio (% EM/(% EM + % CM))(Table I; p = 0.001) than peripheral blood, indicating a higher percentage of CD4+ effector cells in the memory lymphocyte subset in the breast milk when compared to peripheral blood. In contrast, the CD8+ EM ratio was similar in breast milk and peripheral blood (Table 1).

Figure 1
Rhesus monkey breast milk lymphocytes are skewed more than peripheral blood lymphocytes toward a memory phenotype, however the distribution of breast milk lymphocyte phenotypes are similar in hormone-induced and naturally lactating monkeys. CD4+ (A) and ...
Table I
The Frequencies of naïve and memory CD4+ and CD8+ T lymphocytes

Immunoglobulin classes were also compared between peripheral blood and breast milk in 11 healthy, naturally-lactating rhesus monkeys between 8 and 12 weeks postpartum. As expected, breast milk had a significantly lower median concentration of IgM (14 μg/ml versus 1,971 μg/ml; p = 0.001), IgG (49 μg/ml versus 25,539 μg/ml; p = 0.001) and IgA (196 μg/ml versus 14,767 μg/ml; p = 0.001) antibody compared to peripheral blood (Fig 2A–C). As anticipated, IgA was the major immunoglobulin class in breast milk.

Figure 2
Rhesus monkey immunoglobulin concentrations are lower in breast milk than serum and levels of total IgG and IgA are higher in hormone-induced rhesus monkey breast milk than in natural breast milk of rhesus monkeys. IgM (A), IgG (B) and IgA (C) immunoglobulin ...

Cellular and humoral immunologic constituents in breast milk during hormone-induced lactation (HIL) and natural lactation (NL) are similar

In order to develop a nonhuman primate model of HIV/SIV immunity in breast milk that circumvents reliance on breeder monkeys and monkey breeding cycles, four nonpregnant, female rhesus monkeys underwent hormone induction of lactation (HIL). All 4 animals began lactating after 2 months of estrogen and medroxyprogesterone injections and 4 to 6 weeks of dopamine antagonist therapy. Histologic evidence of mammary gland development was achieved within 12 weeks of hormone treatment in all animals (data not shown). The distribution of the HIL breast milk cells (n = 4) approximately 2 months after initiation of lactation was more similar to the reported cellular content of early milk (3236) than that of mature milk, with elevated macrophage/monocyte (median 44%, range 42–78%) and neutrophil (median 39%, range: 11–40%) content. However, the neutrophil and monocyte/macrophage content of the HIL breast milk was similar, whereas early human milk is expected to have a substantial monocyte/macrophage predominance (36).

Comparisons of lymphocyte subsets in breast milk of hormone-induced, lactating monkeys 12 weeks after initiation of lactation (n = 4) and naturally-lactating monkeys between 2 and 36 weeks postpartum (n = 14) were performed to support the use of the hormone-induced, lactating female monkeys for studies of adaptive immunity. A similar skewing towards a memory phenotype was seen in HIL and NL breast milk in both the CD4+ (medians of 84.7% versus 79.8% CM and 10.5% versus 10.8% EM; Fig 1A) and CD8+ (medians of 36.6% versus 23.6% CM and 57.3% versus 70.9% EM; Fig 1B) lymphocyte populations. Accordingly, the HIL and NL breast milk effector memory ratios in CD4+ (median of 0.119 versus 0.109; p = 0.65) and CD8+ (median of 0.747 versus 0.597; p = 0.19) were comparable. While the median naive ratio of CD4+ T lymphocytes trended towards significantly different in HIL and NL breast milk (0.004 versus 0.72; p = 0.034), none of the comparisons of lymphocyte subsets and naive or effector memory lymphocyte ratios in HIL and NL breast milk remained significant after adjustment for multiple comparisons.

In comparing the antibody classes between hormone-induced, lactating monkeys 12 weeks after initiation of lactation and naturally-lactating monkeys between 8 and 12 weeks postpartum, the median concentration of IgM was found to be similar in HIL and NL breast milk (6.8 μg/ml versus 14.1 μg/ml; Fig 2A). In contrast, the median IgG (257.4 μg/ml and 48.6 μg/ml; p = 0.001; Fig 2B) and IgA concentration (807.5 μg/ml versus 195.9 μg/ml; p = 0.026; Fig 2C) were consistently higher in the HIL breast milk. As expected, the median titer of secretory IgA was approximately one log higher in HIL breast milk than in NL breast milk (HIL breast milk median titer: 9600, range: 3200 – 102400 versus NL breast milk median titer: 400, range: 200 – 1600; p = 0.0075, data not shown), suggesting that the increased IgA content in HIL breast milk is due to mucosally-derived secretory IgA rather than IgA translocated from serum. While the higher IgA concentration in HIL breast milk did not remain significant after adjustment for multiple comparisons, the IgG concentration remained significantly higher in HIL breast milk compared to NL breast milk. This finding may be explained by the relative immaturity of HIL milk compared to NL milk and a higher antibody content that might be expected in early milk compared to mature milk (37, 38).

Functional Gag-specific CD8+ T lymphocyte cellular immunity in breast milk of chronically SIV-infected rhesus monkeys

In order to confirm that antigen-specific cellular immunity could be monitored in breast milk of rhesus monkeys, breast milk lymphocytes from 2 chronically SIV-infected, lactating Mamu A*01+ rhesus monkeys were stained with tetrameric complexes specific for the immunodominant SIV epitopes Gag p11C and Tat TL8. CD8+ T lymphocytes specific for both immunodominant epitopes were demonstrated in the breast milk (data not shown). Furthermore, a functional CD8+ T lymphocyte immune response consisting of TNF-α and IFN-γ secretion after Gag p11C stimulation was also demonstrated in breast milk lymphocytes of chronically SIV-infected monkeys using standard intracellular cytokine staining techniques (30) (Fig 3). These data further validate that hormone-induction of lactation in rhesus monkeys provides an excellent model for studying antigen-specific cellular immunity in breast milk during acute and chronic SIV infection.

Figure 3
SIV-specific CD8+ T lymphocytes in breast milk chronically SIV-infected, lactating rhesus monkeys generate a functional immune response consisting of minimal IL-2, but significant TNF-α and IFN-γ intracellular cytokine production. Cytokine ...

SIV viral load is one to two logs lower in breast milk than plasma at both peak and set point of viremia

While plasma viral load peaked as expected on day 10 after SIV inoculation (Fig. 4A), breast milk viral load peaked slightly later, between day 14 and day 21 (Fig. 4B). The peak of viral load in the breast milk (median: 8.3×105; range: 3.8×105-1.0×107) was one to two logs lower than the peak of viral load in the plasma (median: 8.2×107; range: 1.8×107-1.9×108). This differential was maintained after viral set-point (defined as day 50 after inoculation) with breast milk viral load (median: 2.4×104; range: 4.0×103-1.0×105) remaining one to two logs lower than plasma viral load (median: 1.8×106; range: 4.7×104-2.7×106). The breast milk viral load trended towards significantly lower than plasma viral load at both peak and set point in this study of only 4 animals (both p = 0.12).

Figure 4
SIV-specific CD8+ T lymphocyte responses are higher in breast milk than peripheral blood during acute infection, despite viral RNA levels remaining 1–2 logs lower in breast milk than plasma during early infection at both peak and set-point. SIV ...

SIV-specific CD8+ T lymphocyte response is significantly higher in breast milk than blood during acute SIV infection

While the kinetics of the CD8+ T lymphocyte response specific for the Mamu A*01+ immunodominant epitopes Tat TL8 and Gag p11C in breast milk paralleled the blood, the magnitude of the SIV-specific cellular response was considerably higher in breast milk (Fig. 5A–H). The percentage of CD8+ T lymphocytes in breast milk specific for Tat TL8 (median: 30.4%; range: 28.3%–37.5%), a response that is important for early control of viremia, was approximately two to three times higher than in blood in all animals at the peak of the response (median: 14.4%; range: 11.1%–17.3%) (Fig. 5A–D). Additionally, the percentage of CD8+ T lymphocytes specific for Gag p11C, a response that is important for long term control of SIV viremia, was also approximately two to three times higher in breast milk (median: 37.2%; range: 27.6%–44.3%) than in blood (median: 15.4%; range: 14%–23.1%) at the peak of the response in three out of four animals (Fig. 5E–H). This Gag-specific CD8+ T lymphocyte response in breast milk remained higher than the in blood after viral set point in all four monkeys. In this pilot study of four animals, there is a trend towards significantly higher Gag and Tat-specific CD8+ T lymphocyte response in breast milk compared to blood at the peak of the response (p = 0.12). Despite considerably lower lymphocyte numbers in breast milk than in blood (39, 40), the absolute number of SIV-specific CD8+ T lymphocytes in breast milk during acute SIV infection approached a similar magnitude as the absolute number in peripheral blood at the peak of the cellular immune response. In breast milk, the absolute number of Tat TL8-specific CD8+ T lymphocytes at the peak of the antigen-specific cellular immune response ranged from 2–289 cells/μl of breast milk (median = 27 cells/μl), whereas the absolute number of Tat TL8 CD8+ T lymphocytes in peripheral blood varied between 39–450 cells/μl of blood (median = 156 cells/μl). Likewise, the absolute number of Gag p11C-specific CD8+ T lymphocytes ranged from 3–316 cells/μl of breast milk (median = 27 cells/μl) and the absolute number of Gag p11C-specific CD8+ T lymphocytes in peripheral blood fell between 49–430 cells/μl of blood (median = 197 cells/μl) at the peak of the Gag p11C-specific cellular immune response.

Figure 5
Percent CD8+ T lymphocytes in breast milk (open circles) and blood (closed squares) that bind Tat TL8 (A–D) and Gag p11C (E–H) tetramers during acute SIV-infection. Percent Tat TL8 (I–L) and Gag p11C (M–P) tetramer+ CD8 ...

Emergence of CD8+ T lymphocyte response in breast milk is associated with decline in breast milk viral load

Importantly, the appearance of the Tat TL8-specific CD8+ T lymphocyte response in breast milk occurred near the time of the initial decline in breast milk viral load after the peak of viral replication (Fig. 5I–L). While the initial decline in breast milk viral load could be explained by a simultaneous decline in plasma viral load, sustained lower breast milk viral load after viral set point was concurrent with persistent higher percentages of Gag p11C-specific CD8+ T lymphocytes in breast milk than in plasma (Fig 5M–P). The percentage of CD8+ T lymphocytes in breast milk that produced TNFα and IFNγ after stimulation with Gag p11C on day 115 after SIV (Fig. 3) inoculation trended towards an inverse association with breast milk viral load on day 112 in all monkeys that continued lactating (p = 0.33, n = 3, data not shown), further indicating a functional role for CD8+ T lymphocytes in control of breast milk viral replication.


In these experiments, we have introduced a novel nonhuman primate model for studying breast milk immunity during HIV/SIV infection. The advantages of pharmacologic-induction of lactation in rhesus monkeys include independence from both primate breeding cycles and care of infant monkeys, and ease of sample collection. Lymphocyte subsets were similar in HIL and NL breast milk, indicating that antigen-specific cellular immune response in each type of breast milk should be comparable. Not surprisingly, the HIL breast milk displayed characteristics similar to colostrum or early milk with higher immunoglobulin (37, 38), macrophage/monocyte, and neutrophil content (3235) than mature milk, as the artificial lactation protocol likely induces breast milk that is not fully mature. However, human colostrum has a macrophage/monocyte predominance that was not observed in the HIL breast milk, suggesting that the HIL breast milk may be on the continuum between early milk and mature milk. Since breast milk viral load (41) and rates of HIV transmission are higher in early lactation (42), the apparent immaturity of the HIL breast milk is advantageous for modeling the virologic and immunologic factors contributing to this period of high risk for the breastfeeding infant.

The lactation model is a valuable tool to investigate antigen-specific mucosal immunity, as lymphocytes and antibody can be collected in the fluid phase at multiple time points and immune responses can be evaluated simultaneously in the mucosal compartment and the blood. In the lactation-induction SIV/rhesus monkey model, we were able to quantitate epitope-specific T lymphocytes, as well as characterize functional, ex vivo-stimulated cellular responses without the high background activation that often limits the ability to monitor cellular immune responses. While blood contamination of manually-collected breast milk is a concern, our data are consistent with the detection of breast milk-specific humoral and cellular immune responses, as the defined phenotype of the lymphocytes and the antibody content of the rhesus monkey breast milk were distinct from the blood and consistent with previous descriptions of human breast milk immune responses (26, 35, 37, 38, 43).

While HIV-specific CD8+ T lymphocytes have previously been demonstrated in breast milk of HIV-infected women (44), the kinetics and function of this population of cells has not been evaluated. This study has documented a robust SIV-specific cellular immunity in breast milk that parallels the kinetics of the CD8+ T lymphocyte response in the blood, but is two to three times higher at the time of both peak and set point of viremia. The robust SIV-specific cellular response in the breast milk was not related to high antigen burden, as the viral load in the breast milk remained at least one log lower than that in the blood throughout acute infection.

The high frequency of SIV-specific T lymphocytes in breast milk measured during acute and chronic SIV infection was not predicted. The SIV-specific CD8+ T lymphocyte response reported in the gastrointestinal and genital tracts during acute infection is less than or equal to the CD8+ T lymphocyte response in the blood (18, 19, 45, 46), despite intense viral replication in those mucosal compartments. The robust mucosal CD8+ T lymphocyte response in breast milk provides further evidence that the mucosal compartments are immunologically distinct (1618). Moreover, the high levels of cellular immunity in breast milk during acute infection may contribute to prevention of viral transmission from mothers to infants during breastfeeding.

It is well-established that the SIV-specific CD8+ T lymphocyte response is essential for the control of viral replication in the peripheral blood (47, 48). While control of viral replication in mucosal compartments are likely affected by both anatomic and immunologic factors, the breast milk SIV-specific CD8+ T lymphocytes likely play a key role in the containment of breast milk viral load because of several supporting observations. First, the breast milk viral load remained appreciably less than the viral load in plasma throughout acute and chronic SIV infection in association with a particularly high SIV-specific CD8+ T lymphocyte response in the breast milk. Second, the initial containment of breast milk viral load occurred near the time of the emergence of the Tat TL8-specific CD8+ T lymphocyte response in the breast milk, although this association may be explained by the concurrent reduction in plasma viral load. Finally, maintenance of low breast milk viral load after viral set point was coincident with a sustained high level Gag p11C-specific CD8+ T lymphocyte response in breast milk.

The potency of the breast milk SIV-specific cellular immune response during acute SIV infection may contribute to containing mother to child transmission of the virus. As high breast milk HIV RNA viral load is associated with infant breast milk transmission (9, 15, 49), cellular immune containment of viral shedding in breast milk could contribute to a reduction in risk of transmission via breastfeeding. HIV-specific cytotoxic CD8+ T lymphocytes in breast milk are expected to reduce breast milk viral load by eliminating local cellular reservoirs of virus, including macrophage/dendritic cells and activated CD4+ T lymphocytes. These cell types are in high concentration in early milk (34) and likely play an important role in the transmission of HIV via breastfeeding. Additionally, the infant is exposed to a high frequency of HIV-specific maternal CD8+ T lymphocytes that may be absorbed by the infant gastrointestinal tract, as animal studies have demonstrated the absorption of maternal lymphocytes into the gastrointestinal mucosa (50, 51), as well as the bloodstream (52), of suckling neonates. Furthermore, maternal lymphocytes transmitted in breast milk may play an active role in the developing neonatal immune system, as evidence of acquisition of maternal mitogen and antigen-specific cellular responses via breastfeeding exists in both experimental animal (53) and human (34, 54, 55) studies. Further investigation of the function of the high frequency viral-specific CD8+ T lymphocytes in breast milk is certainly warranted. Through these studies, maternal intervention to enhance this breast milk immune response may be a viable strategy for prevention of mother to child transmission via breastfeeding.


We thank Adam Buzby, Kevin Carlson, Saran Bao and Michelle Lifton for their technical assistance. We also thank Corrine Welt, Dan Barouch and Barton Haynes for their generous assistance and advice.


1This work was supported by the Center for HIV/AIDS Vaccine Immunology (S.R.P. and N.L.L.; R01AI067854), the Fred Lovejoy Research and Education Fund (S.R.P.), the Children’s Hospital House Officer Research Award (S.R.P.), the Pediatric Infectious Disease Society/St. Jude Children’s Hospital Basic Science Research Award (S.R.P.), and the Harvard Center for AIDS Research (R.S.G; P30AI060354).


The authors have no financial conflict of interest.

3Abbrevations used in this manuscript include: natural lactation (NL); hormone-induced lactation (HIL); central memory (CM); effector memory (EM)


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