Potent Simian Immunodeficiency Virus-Specific Cellular
Immune Responses in the Breast Milk of Simian
Immunodeficiency Virus-Infected, Lactating Rhesus Monkeys1
Sallie R. Permar,2*†Helen H. Kang,* Angela Carville,‡Keith G. Mansfield,‡
Rebecca S. Gelman,§Srinivas S. Rao,¶James B. Whitney,* and Norman L. Letvin*
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 breast milk was found to be similar to that in natural lactation breast milk. Hormone-induced lactating
monkeys were inoculated i.v. 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 1–2 logs lower than plasma viral load through peak and set point of viremia. Surprisingly, whereas 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. The Journal of Immunology, 2008, 181:
infant morbidity and mortality in the developing world (1, 2).
Moreover, poor access to clean water in the developing world lim-
its the safety of infant replacement feeding. However, HIV is ver-
tically transmitted via breast milk, and mother-to-child transmis-
sion 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 (4–7), advanced maternal HIV disease
(8–10), 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
he benefits of breastfeeding, including optimal nutrition
and protection against gastrointestinal and respiratory in-
fections, are well established and significantly improve
plasma viral load, are associated with a high risk of HIV trans-
mission to infants (9, 13–15). Mucosally transmitted virus is ex-
posed to distinct immune responses specific to the mucosal com-
partment (16–19). Virus in genital tract, semen, and breast milk
appears genetically divergent from that in the peripheral blood
(20–23), indicating that local immune responses shape the evolu-
tion of compartmentalized virus.
Because late mother-to-child transmission of HIV occurs in a
small minority of breastfeeding infants born to HIV-infected moth-
ers, the majority of infants remain protected from transmission
despite ongoing low-dose exposure to the virus, raising the possi-
bility 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 Abs in breast milk have been associated
with transmission in some (24), but not all studies (25). HIV-spe-
cific 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 character-
istics of breast milk cellular immunity compared with systemic
cellular immunity have not been defined.
The SIV/rhesus monkey model is ideal to study viral-specific
mucosal cellular immunity, because 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
*Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, MA 02115;†Division of Infectious Disease, Children’s Hos-
pital Boston, Harvard Medical School, Boston, MA 02115;‡New England Regional
Primate Research Center, Harvard Medical School, Southboro, MA 01772;§Dana
Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; and¶Vaccine
Research Center, National Institutes of Health, Bethesda, MD 20892
Received for publication February 22, 2008. Accepted for publication June 20, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the Center for HIV/AIDS Vaccine Immunology (to
S.R.P. and N.L.L.; R01AI067854), the Fred Lovejoy Research and Education Fund
(to S.R.P.), the Children’s Hospital House Officer Research Award (to S.R.P.), the
Pediatric Infectious Disease Society/St. Jude Children’s Hospital Basic Science Re-
search Award (to S.R.P.), and the Harvard Center for AIDS Research (to R.S.G.;
2Address correspondence and reprint requests to Dr. Sallie R. Permar, 330 Brookline
Avenue, Research East, Beth Israel Deaconess Medical Center, Boston, MA 02115.
E-mail address: email@example.com
The Journal of Immunology
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 immu-
nologic 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 i.m. route for 2
mo to induce mammary gland maturation. The dopamine antagonist, hal-
operidol, was administered orally after 2 mo of hormone treatment to raise
serum prolactin levels. All four monkeys began lactating within 6 wk 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 i.m. route immediately before 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: 2 mg/kg estradiol cypionate i.m. bimonthly, 5 mg/kg medroxy-
progesterone acetate weekly, 0.15 mg/kg haloperidol by mouth twice daily.
Mammary gland biopsies were performed on each animal more than 3
mo after the initiation of lactation. Tissue samples were sectioned and
stained with H&E. Breast milk was also manually collected once from each
of 19 naturally lactating female breeder rhesus monkeys that were between
2 and 36 wk postpartum. Breast milk samples with visible blood contam-
ination or less than 100 CD3?cells were not included in the cellular anal-
yses (n ? 5).
Four female Mamu-A*01?rhesus monkeys were selected for the study
after PCR-based MHC typing. Following induction of lactation by hor-
mone treatment, the monkeys were i.v. inoculated with SIVmac251. Blood
and breast milk samples were collected three times per week during the
first 4 wk 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 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, 500 g
of methylene blue chloride (EMD Chemicals), 56 ml of 95% ethyl alcohol
(Pharmaco), and 40 ml of xylene (Fisher Scientific) were mixed and left
standing for 24 h at 4°C, and then 4 ml of glacial acetic acid (Fisher
Scientific) was added through a filter. A total of 10 ?l of fresh whole breast
milk was spread on a 1-cm circle on a glass cell count slide (Bellco Glass)
and allowed to air dry. The slide was fixed with methanol for 5 s and
stained for 2 min. After drying, the slide was washed gently in tap water,
air dried, and viewed under magnification of ?100. Three counts of 100
cells/sample were performed by two independent counters and then
Phenotyping, tetramer staining, and intracellular cytokine
staining of breast milk and PBMC
Breast milk samples were separated into fat, supernatant, and cellular frac-
tions by centrifugation at 60 ? g for 20 min. 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. A total of 1 ? 106
PBMC isolated from EDTA-anticoagulated blood and the breast milk cell
pellet were stained with anti-CD4 Amcyan (L200; BD Biosciences), anti-
CD8?Alexa700 (RPA-T8; BD Biosciences), anti-CD28 PerCP-Cy5.5
(28.2; Beckman Coulter), anti-CD95 FITC (DX2; BD Biosciences), and
anti-CD3 allophycocyanin-Cy7 (SP34.2; BD Biosciences). Naive and
memory lymphocyte subsets were defined by anti-CD95 and anti-CD28
staining (naive, CD28?, CD95?; effector memory (EM),3CD28?, CD95?;
central memory (CM), 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 Abs: anti-TNF-? Pacific Blue (Mab11; eBioscience), anti-IFN-?
PE-Cy7 (B27; BD Biosciences), and anti-IL-2 allophycocyanin (MQ1-
17H12; BD Biosciences). PBMC and breast milk cells from SIV-infected
animals were also stained with soluble Mamu-A*01 PE- or allophycocya-
nin-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
IgM and IgG levels were measured in duplicate using monkey-specific
ELISA kits (Alpha Diagnostics) and standards per protocol. Breast milk
supernatant was diluted between 1/10 and 1/2,000 for the assays. The IgA
and secretory IgA (sIgA) levels were measured by coating a 96-well poly-
styrene plate with anti-IgA Ab (Rockland), 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 1/1,000 (for IgA) or 1/100
and 1/102,400 (for sIgA) and incubated for 2 h. Ab was detected after 1-h
incubation with an HRP-conjugated anti-IgA Ab (Alpha Diagnotics) or an
HRP-conjugated anti-sIgA (Nordic) and addition of ABTS-2 peroxidase
substrate system (Kirkegaard & Perry Laboratories). 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
2-fold dilutions with a cutoff 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 h, and
the viral pellet was stored in RNA later (Ambion). RNA from plasma and
the breast milk viral pellet was isolated using the QIAamp viral RNA kit
(Qiagen), according to protocol, with the final product resuspended in 60
?l of RNase-free water. A total of 25 ?l of the RNA suspension was used
in a reverse-transcriptase reaction containing SuperScript III RT enzyme
(Invitrogen) and the Gag-specific primer 5?-GCAATATCTGATCCT
GACGGCTC-3?, according to manufacturer’s protocol. A quantity
amounting to 10 ?l of the resulting cDNA was used in a real-time PCR
using the Taqman EZ RT-PCR kit (Applied Biosystems), as well as a
Gag-specific labeled probe (5?-FAM-CTTCCTCAGTGTGTTTCACTT
CATCTGGTGCATTC-3? and 5?-CAGTAGGTGTCTCTGCACTATCT
GTTTTG-3?). Reactions were performed in duplicate on the 7700 ABI
PRISM Sequence Detector (Applied Biosystems) at 95°C for 10 min, then
50 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 30 s. 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 viral 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 first undetectable standard). RNA
copies per milliliter were determined by dividing the copy number by the
volume of breast milk or plasma.
Ig 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 natural lactation (NL)
and hormone-induced lactation (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 produc-
tion 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 monkeys reveals skewing
of breast milk lymphocytes toward a memory phenotype
T lymphocyte subsets in breast milk were compared with those in
peripheral blood using paired samples from 14 healthy, naturally
lactating rhesus monkeys. Although the percentage of CD4?and
CD8?T lymphocytes was similar between blood and breast milk
3Abbreviations used in this paper: EM, effector memory; CM, central memory; HIL,
hormone-induced lactation; NL, natural lactation; sIgA, secretory IgA.
3644SIV-SPECIFIC CELLULAR IMMUNE RESPONSE IN BREAST MILK
(Table I), breast milk had a lower median percentage of naive
CD4?(7.1 vs 45.8%; Fig. 1A) and CD8?(2.3 vs 26.2%; Fig. 1B)
T lymphocytes than the blood and significantly lower naive lym-
phocyte ratios (percentage of naive/percentage of naive ? percent-
age of memory; Table I) in both CD4?(p ? 0.001) and CD8?
(p ? 0.0004) subsets. Accordingly, the median percentages of CM
and EM lymphocytes were higher in the breast milk than the blood
in both CD4?(79.8 vs 52.2% CM; 10.8 vs 2.3% EM; Fig. 1A) and
CD8?(70.9 vs 42.9% EM; 23.6 vs 12.4% CM; Fig. 1B) T lym-
phocyte subsets. Breast milk also demonstrated a significantly
higher CD4?EM ratio (percentage of EM/(percentage of EM ?
percentage of CM)) (Table I; p ? 0.001) than peripheral blood,
indicating a higher percentage of CD4?effector cells in the mem-
ory lymphocyte subset in the breast milk when compared with
peripheral blood. In contrast, the CD8?EM ratio was similar in
breast milk and peripheral blood (Table I).
Ig classes were also compared between peripheral blood and
breast milk in 11 healthy, naturally lactating rhesus monkeys be-
tween 8 and 12 wk postpartum. As expected, breast milk had a
significantly lower median concentration of IgM (14 ?g/ml vs
1,971 ?g/ml; p ? 0.001), IgG (49 ?g/ml vs 25,539 ?g/ml; p ?
0.001), and IgA (196 ?g/ml vs 14,767 ?g/ml; p ? 0.001) Ab
compared with peripheral blood (Fig. 2). As anticipated, IgA was
the major Ig class in breast milk.
Cellular and humoral immunologic constituents in breast milk
during HIL and NL are similar
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 mon-
keys underwent HIL. All four animals began lactating after 2 mo
of estrogen and medroxyprogesterone injections and 4–6 wk of
dopamine antagonist therapy. Histologic evidence of mammary
gland development was achieved within 12 wk of hormone treat-
ment in all animals (data not shown). The distribution of the HIL
breast milk cells (n ? 4) ?2 mo after initiation of lactation was
more similar to the reported cellular content of early milk (32–36)
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/macro-
phage content of the HIL breast milk was similar, whereas early
human milk is expected to have a substantial monocyte/macro-
phage predominance (36).
Comparisons of lymphocyte subsets in breast milk of hor-
mone-induced, lactating monkeys 12 wk after initiation of lac-
tation (n ? 4) and naturally lactating monkeys between 2 and
36 wk postpartum (n ? 14) were performed to support the use
of the hormone-induced, lactating female monkeys for studies
milk lymphocytes are skewed more
than PBLs toward a memory pheno-
milk lymphocyte phenotypes is similar
in hormone-induced and naturally lac-
tating monkeys. CD4?(A) and CD8?
(B) T lymphocytes with a CM (CD95?,
CD28?), EM (CD95?, CD28?), and
naive (CD95?, CD28?) phenotype
were quantitated in breast milk (open
symbols) and the peripheral blood
(closed symbols). CD4?(A) and CD8?
(B) T lymphocytes with CM, EM, and
naive phenotype in breast milk of rhe-
sus monkeys during HIL (open sym-
bols) and NL (closed symbols). The
solid line indicates the median value.
Rhesus monkey breast
Table I. The frequencies of naive and memory CD4?and CD8?T lymphocytesa
2-Sided p Valueb
CD4 naive ratioc
CD4 effector memory ratiod
CD8 naive ratioe
CD8 effector memory ratiof
aAlthough breast milk and peripheral blood have similar percentages of CD4?and CD8?T lymphocytes, breast milk has
a significantly lower percentage of naive CD4?and CD8?T lymphocytes and a higher percentage of effector memory CD4?
bValue of p was determined using the exact Wilcoxon sign rank test for paired values, and the bolded p values remained
significant after adjusting for multiple comparisons.
cCD4 naive ratio is defined as percentage of CD4 naive (CD4?CD95?CD28?)/percentage of naive ? percentage of memory
dCD4 effector memory ratio is defined as percentage of CD4 effector memory (CD4?CD95?CD28?)/percentage of effector
memory ? percentage of central memory (CD95?, CD28?).
eCD8 naive ratio is defined as percentage of CD8 naive (CD8?CD95?CD28?)/percentage of naive ? percentage of memory
fCD8 effector memory ratio is defined as percentage of CD8 effector memory (CD8?CD95?CD28?)/percentage of effector
memory ? percentage of central memory (CD95?, CD28?).
3645The Journal of Immunology
of adaptive immunity. A similar skewing toward a memory phe-
notype was seen in HIL and NL breast milk in both the CD4?
(medians of 84.7 vs 79.8% CM and 10.5 vs 10.8% EM; Fig. 1A)
and CD8?(medians of 36.6 vs 23.6% CM and 57.3 vs 70.9%
EM; Fig. 1B) lymphocyte populations. Accordingly, the HIL
and NL breast milk EM ratios in CD4?(median of 0.119 vs
0.109; p ? 0.65) and CD8?(median of 0.747 vs 0.597; p ?
0.19) were comparable. Although the median naive ratio of
CD4?T lymphocytes trended toward significantly different in
HIL and NL breast milk (0.004 vs 0.72; p ? 0.034), none of the
comparisons of lymphocyte subsets and naive or EM lympho-
cyte ratios in HIL and NL breast milk remained significant after
adjustment for multiple comparisons.
In comparing the Ab classes between hormone-induced, lactat-
ing monkeys 12 wk after initiation of lactation and naturally lac-
tating monkeys between 8 and 12 wk postpartum, the median con-
centration of IgM was found to be similar in HIL and NL breast
milk (6.8 vs 14.1 ?g/ml; Fig. 2A). In contrast, the median IgG
(257.4 and 48.6 ?g/ml; p ? 0.001; Fig. 2B) and IgA concentration
(807.5 vs 195.9 ?g/ml; p ? 0.026; Fig. 2C) were consistently
higher in the HIL breast milk. As expected, the median titer of
sIgA was ?1 log higher in HIL breast milk than in NL breast milk
(HIL breast milk median titer, 9,600; range, 3,200–102,400 vs 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 sIgA rather than IgA
translocated from serum. Although the higher IgA concentra-
tion in HIL breast milk did not remain significant after adjust-
ment for multiple comparisons, the IgG concentration remained
significantly higher in HIL breast milk compared with NL
breast milk. This finding may be explained by the relative im-
maturity of HIL milk compared with NL milk and a higher Ab
content that might be expected in early milk compared with
mature milk (37, 38).
Functional Gag-specific CD8?T lymphocyte cellular immunity
in breast milk of chronically SIV-infected rhesus monkeys
To confirm that Ag-specific cellular immunity could be monitored
in breast milk of rhesus monkeys, breast milk lymphocytes from
two chronically SIV-infected, lactating Mamu A*01?rhesus mon-
keys were stained with tetrameric complexes specific for the im-
munodominant 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-? and IFN-? secretion after Gag p11C stimulation was also
demonstrated in breast milk lymphocytes of chronically SIV-in-
fected 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 Ag-specific cellular immunity in breast milk
during acute and chronic SIV infection.
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) Ig class concentration in breast milk (open
symbols) and the peripheral blood (closed symbols). IgM (A), IgG (B), and
IgA (C) concentrations in HIL and NL breast milk. The solid line indicates
the median value.
Rhesus monkey Ig concentrations are lower in breast milk
breast milk chronically SIV-infected, lactating rhesus
monkeys generate a functional immune response con-
sisting of minimal IL-2, but significant TNF-? and
IFN-? intracellular cytokine production. Cytokine pro-
duction in peripheral blood (A) and breast milk (C)
CD8?T lymphocytes after no stimulation or Gag p11C
peptide stimulation (B and D) in a chronically SIV-in-
fected, lactating rhesus monkey.
SIV-specific CD8?T lymphocytes in
3646SIV-SPECIFIC CELLULAR IMMUNE RESPONSE IN BREAST MILK
SIV viral load is 1–2 logs lower in breast milk than plasma at
both peak and set point of viremia
Although plasma viral load peaked as expected on day 10 after
SIV inoculation (Fig. 4A), breast milk viral load peaked slightly
later, between days 14 and 21 (Fig. 4B). The peak of viral load in
the breast milk (median, 8.3 ? 105; range, 3.8 ? 105–1.0 ? 107)
was 1–2 logs lower than the peak of viral load in the plasma (me-
dian, 8.2 ? 107; range, 1.8 ? 107–1.9 ? 108). This differential was
maintained after viral set point (defined as day 50 after inocula-
tion) with breast milk viral load (median, 2.4 ? 104; range, 4.0 ?
103–1.0 ? 105) remaining 1–2 logs lower than plasma viral load
(median, 1.8 ? 106; range, 4.7 ? 104–2.7 ? 106). The breast milk
viral load trended toward significantly lower than plasma viral load
at both peak and set point in this study of only four animals (both
p ? 0.12).
SIV-specific CD8?T lymphocyte response is significantly higher
in breast milk than blood during acute SIV infection
Although 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. 5, A–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 ?2–3 times higher than in blood in all animals at the peak of
the response (median, 14.4%; range, 11.1–17.3%) (Fig. 5, A–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 ?2–3 times higher in breast milk (median,
37.2%; range, 27.6–44.3%) than in blood (median, 15.4%; range,
sponses 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 RNA levels in
plasma (A) and breast milk supernatant (B) during acute
SIV infection were quantitated by SIV Gag RT-PCR.
SIV-specific CD8?T lymphocyte re-
T lymphocytes in breast milk (E)
and blood (f) that bind Tat TL8
(A–D) and Gag p11C (E–H) tetram-
ers during acute SIV infection. Per-
centages of Tat TL8 (I–L) and Gag
p11C (M–P) tetramer?CD8?T
lymphocytes in breast milk (E) are
displayed temporally with breast
milk viral load (Œ).
Percentage of CD8?
3647The Journal of Immunology
14–23.1%) at the peak of the response in three of four animals
(Fig. 5, E–H). This Gag-specific CD8?T lymphocyte response in
breast milk remained higher than in blood after viral set point in all
four monkeys. In this pilot study of four animals, there is a trend
toward significantly higher Gag- and Tat-specific CD8?T lym-
phocyte response in breast milk compared with 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 ap-
proached 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 Ag-specific cellular immune response ranged from
2 to 289 cells/?l breast milk (median ? 27 cells/?l), whereas the
absolute number of Tat TL8 CD8?T lymphocytes in peripheral
blood varied between 39 and 450 cells/?l blood (median ? 156
cells/?l). Likewise, the absolute number of Gag p11C-specific
CD8?T lymphocytes ranged from 3 to 316 cells/?l breast milk
(median ? 27 cells/?l), and the absolute number of Gag p11C-
specific CD8?T lymphocytes in peripheral blood fell between 49
and 430 cells/?l blood (median ? 197 cells/?l) at the peak of the
Gag p11C-specific cellular immune response.
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 lym-
phocyte response in breast milk occurred near the time of the ini-
tial decline in breast milk viral load after the peak of viral repli-
cation (Fig. 5, I–L). Although 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-spe-
cific CD8?T lymphocytes in breast milk than in plasma (Fig. 5,
M–P). The percentage of CD8?T lymphocytes in breast milk that
produced TNF-? and IFN-? after stimulation with Gag p11C on
day 115 after SIV (Fig. 3) inoculation trended toward 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 pri-
mate 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 breed-
ing cycles and care of infant monkeys, and ease of sample collec-
tion. Lymphocyte subsets were similar in HIL and NL breast milk,
indicating that Ag-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 Ig (37, 38), macrophage/monocyte, and neutro-
phil content (32–35) than mature milk, because the artificial lac-
tation protocol most 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, sug-
gesting that the HIL breast milk may be on the continuum between
early milk and mature milk. Because 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 Ag-specific
mucosal immunity, because lymphocytes and Ab 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. Although blood contami-
nation of manually collected breast milk is a concern, our data are
consistent with the detection of breast milk-specific humoral and
cellular immune responses, because the defined phenotype of the
lymphocytes and the Ab content of the rhesus monkey breast milk
were distinct from the blood and consistent with previous descrip-
tions of human breast milk immune responses (26, 35, 37, 38, 43).
Although 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 have not been
evaluated. This study has documented a robust SIV-specific cel-
lular immunity in breast milk that parallels the kinetics of the
CD8?T lymphocyte response in the blood, but is 2–3 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
Ag burden, because the viral load in the breast milk remained at
least 1 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 re-
ported in the gastrointestinal and genital tracts during acute infec-
tion 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 (16–18). 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 pe-
ripheral blood (47, 48). Although control of viral replication in
mucosal compartments is most likely affected by both anatomic
and immunologic factors, the breast milk SIV-specific CD8?T
lymphocytes most 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 lympho-
cyte 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
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. Because 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
3648SIV-SPECIFIC CELLULAR IMMUNE RESPONSE IN BREAST MILK
macrophage/dendritic cells and activated CD4?T lymphocytes.
These cell types are in high concentration in early milk (34) and
most likely play an important role in the transmission of HIV via
breastfeeding. Additionally, the infant is exposed to a high fre-
quency of HIV-specific maternal CD8?T lymphocytes that may
be absorbed by the infant gastrointestinal tract, because animal
studies have demonstrated the absorption of maternal lymphocytes
into the gastrointestinal mucosa (50, 51), as well as the blood-
stream (52), of suckling neonates. Furthermore, maternal lympho-
cytes transmitted in breast milk may play an active role in the
developing neonatal immune system, because evidence of acqui-
sition of maternal mitogen and Ag-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 interven-
tion to enhance this breast milk immune response may be a viable
strategy for prevention of mother-to-child transmission via
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.
The authors have no financial conflict of interest.
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