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Naive and Vaccinated Rhesus MacaquesinImmunodeficiency Virus Vaginal Exposure

NK Cell Responses to Simian

Reeves, R. Paul Johnson and Ashley T. HaaseJ. Southern, Katherine Masek-Hammerman, R. KeithPerkey, Lucy Qu, Stephen Wietgrefe, Mary Zupancic, Peter Liang Shang, Anthony J. Smith, Lijie Duan, Katherine E.

http://www.jimmunol.org/content/193/1/277doi: 10.4049/jimmunol.14004172014;

2014; 193:277-284; Prepublished online 4 JuneJ Immunol 

Referenceshttp://www.jimmunol.org/content/193/1/277.full#ref-list-1

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The Journal of Immunology

NK Cell Responses to Simian Immunodeficiency VirusVaginal Exposure in Naive and Vaccinated Rhesus Macaques

Liang Shang,* Anthony J. Smith,*,1 Lijie Duan,* Katherine E. Perkey,* Lucy Qu,*

Stephen Wietgrefe,* Mary Zupancic,* Peter J. Southern,* Katherine Masek-Hammerman,†,2

R. Keith Reeves,† R. Paul Johnson,† and Ashley T. Haase*

NK cell responses to HIV/SIV infection have been well studied in acute and chronic infected patients/monkeys, but little is known about

NK cells during viral transmission, particularly in mucosal tissues. In this article, we report a systematic study of NK cell responses to

high-dose vaginal exposure to SIVmac251 in the rhesus macaque female reproductive tract (FRT). Small numbers of NK cells were

recruited into the FRT mucosa following vaginal inoculation. The influx of mucosal NK cells preceded local virus replication and

peaked at 1 wk and, thus, was in an appropriate time frame to control an expanding population of infected cells at the portal of entry.

However, NK cells were greatly outnumbered by recruited target cells that fuel local virus expansion and were spatially dissociated

from SIV RNA+ cells at the major site of expansion of infected founder populations in the transition zone and adjoining endocervix.

The number of NK cells in the FRT mucosa decreased rapidly in the second week, while the number of SIV RNA+ cells in the FRT

reached its peak. Mucosal NK cells produced IFN-g and MIP-1a/CCL3 but lacked several markers of activation and cytotoxicity,

and this was correlated with inoculum-induced upregulation of the inhibitory ligand HLA-E and downregulation of the activating

receptor CD122/IL-2Rb. Examination of SIVDnef-vaccinated monkeys suggested that recruitment of NK cells to the genital mucosa

was not involved in vaccine-induced protection from vaginal challenge. In summary, our results suggest that NK cells play, at most,

a limited role in defenses in the FRT against vaginal challenge. The Journal of Immunology, 2014, 193: 277–284.

Although antiretroviral therapies have converted HIV-1infection to a chronic and largely manageable diseasefor many individuals, it is clear that ending the pandemic

depends onmore effectivemeasures for prevention, particularly for theprincipal route of sexual mucosal transmission and in the population ofyoung women in the pandemic’s epicenter in sub-Saharan Africa,who are the most at risk for heterosexual acquisition of HIV-1 (1). Tothat end, we have been seeking design principles for development ofan effective vaccine to prevent HIV-1 transmission to women in theSIV-rhesus macaque nonhuman primate model. We report the resultsof an investigation into the role of NK cells in preventing transmis-sion by analyses of their responses to high-dose vaginal challengewith pathogenic SIVmac251 in naive animals, as well as animalsvaccinated with the live attenuated vaccine, SIVΔnef.We focused these studies on the immunological and virological

events of transmission in the early stages of infection at the portal ofentry for two reasons. First, it was shown that the SIV-infected

founder populations were initially quite small in the monkey fe-male reproductive tract (FRT) mucosal tissues, despite vaginal ex-posure to high doses of SIVmac251 (2, 3). Consequently, thereshould be an opportunity for pre-existing or rapidly induced innateor adaptive immune responses to operate at high E:T ratios toprevent or restrict the establishment of the initial virus-replicatingpopulation. Second, because local expansion of the infectedfounder population usually precedes the dissemination of infectioninto the circulation and the establishment of a robust self-propagatingsystemic infection in mainly lymphoid tissues (2, 3), pre-existing andrapid immune responses could efficiently prevent or attenuate sys-temic infection by constraining this local expansion.The innate immune system, particularly NK cells, could

potentially play important roles in this early window of op-portunity as a rapid responder population that could kill the infectedcells through either Ab-dependent cellular cytotoxicity or Ab-independent (NK cytotoxicity) mechanisms (4). NK cells alsoexpress b-chemokines, including MIP-1a/CCL3, MIP-1b/CCL4,and RANTES/CCL5, which could inhibit continued propagationof infection by blocking viral entry (4, 5). Furthermore, preser-vation of NK cell functions has been associated with improveddisease outcome in patients and monkeys chronically infectedwith HIV-1 and SIV, respectively (6–8). Increased NK cell activityhas been correlated with protection in HIV-1 highly exposed se-ronegative subjects (9). Studies on the activating and inhibitoryreceptors on NK cells revealed a protective correlation betweencertain KIR and HLA polymorphisms (10–13), as well as a rolefor NK cells in controlling viral infection in vivo based on HIV-1adaptation to KIR receptors (14). However, little is known aboutNK cell responses in mucosal tissues, particularly at the earlystage of SIV/HIV mucosal transmission. We address, in part, thatgap in our knowledge of the role of NK cells in protecting the FRTin early infection by analyzing and comparing NK cell responsesin the lower FRT mucosa to high-dose vaginal challenge with

*Department of Microbiology, Medical School, University of Minnesota, Minneap-olis, MN 55455; and †New England Primate Research Center, Harvard MedicalSchool, Southborough, MA 01772

1Current address: Medical Education Review Program, DeVry Medical International,Freeport, Grand Bahama, The Bahamas.

2Current address: Pfizer Worldwide Research and Development, Cambridge, MA.

Received for publication February 12, 2014. Accepted for publication April 21, 2014.

This work was supported by the International AIDS Vaccine Initiative and NationalInstitutes of Health Grants AI071306, AI090735, AI095985, and RR00168 (currentlyOD011103).

Address correspondence and reprint requests to Dr. Ashley T. Haase, Department ofMicrobiology, Medical School, University of Minnesota, MMC 196, 420 DelawareStreet S.E., Minneapolis, MN 55455. E-mail address: haase001@umn.edu.

Abbreviations used in this article: AT-2–SIV, AT-2–inactivated SIV; FRT, femalereproductive tract; ISH, in situ hybridization; TZ, transformation zone; WT-SIV, wildtype SIV.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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pathogenic SIVmac251 in naive animals, as well as in animalsvaccinated with the live attenuated vaccine, SIVΔnef.

Materials and MethodsTissues from Dnef-vaccinated and/or SIV-infected animals

Archived genital tissues from previous studies of SIV high-dose vaginalinfection (2) and SIV Dnef vaccines (15, 16) were used in this study. Briefly,fresh tissues obtained at necropsy were fixed in 4% paraformaldehyde,Streck tissue fixative, or SAFEFIX II (Fisher Scientific, Kalamazoo MI)Tissue Fixative and embedded in paraffin, as previously reported (2).

Immunohistochemistry

Single and double immunohistochemical staining, fluorescent immuno-histochemical staining, and quantitative image analysis on confocal mi-croscopy were performed as described elsewhere (17, 18). The primary Absused in this study are summarized in Table I.

In situ hybridization

SIV RNA was detected in paraformaldehyde-fixed and paraffin-embeddedtissues by in situ hybridization (ISH), as previously described (2). Briefly,5-mm sections were deparaffinized in xylene, rehydrated in PBS, and per-meabilized sequentially in HCl, digitonin, and proteinase K. The sectionswere then acetylated and hybridized to 35S-labeled SIV-specific ribop-robes. After wash and digestion with ribonucleases, the sections werecoated with nuclear-track emulsion before exposure and development. Forfluorescent ISH, digoxigenin-labeled SIV-specific riboprobes were used,followed by sequential staining with Goat Anti-Digoxigenin Abs (Roche,Indianapolis, IN) and Donkey Anti-Goat Abs conjugated with Alexa Fluor555 (Invitrogen, Eugene, OR) (17, 18).

Statistical tests

The Wilcoxon rank-sum test was used to measure the variations in NK cell,macrophage, and SIV RNA+ cell densities over the course of infection,vaccination, and challenge. The paired t test was used to compare thenumber of NK cells between vaginal and cervical tissues from the sameanimal. The Spearman rank correlation test was used to analyze the rela-tionship between mucosal NK cells and local SIV RNA+ cells. Statisticalanalyses were carried out using Prism 4 software.

ResultsNK cell responses in naive animals: location of NKG2A+CD32

NK cells

We systematically examined early NK cell responses in the vaginaland cervical mucosa of naive unvaccinated rhesus macaque FRTs

FIGURE 1. Spatial distribution of NK cells in the lower FRT mucosa of

rhesus macaques. (A) NK cells in rhesus macaques were defined as

NKG2A+CD32 using immunohistochemistry (original magnification

3200). NK cells primarily were located in the submucosal area in endo-

cervix (B), ectocervix (C), and vagina (D). (B) There were remarkably few

NK cells in the palmate folds of the endocervix. All images were taken

from stained sections of tissues collected 7 d postinfection. NKG2A

(green), CD3 (red). In (B–D), NK cells were labeled as green dots for better

visibility at the magnification shown (original magnification 3200).

FIGURE 2. NK cells are recruited into the lower FRT mucosa after

vaginal challenge. The density of NK cells in the genital mucosa was

quantified in cervical (A) and vaginal (B) tissues. (C) In the same indi-

vidual animals, there were more mucosal NK cells in the cervix than va-

gina. (D) However, even at the peak of recruitment, NK cells were

significantly outnumbered by recruited CD4 T cells and macrophages.

Data in (D) are from tissues collected 7 d postinfection. Each point or line

represents an individual animal.

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within 4 wk of high-dose vaginal inoculation of SIVmac251 (1–23105 TCID50). NK cells, defined as NKG2A+CD32 cells (Fig. 1A),as previously reported (19–23), were detected in fixed andparaffin-embedded necropsy samples using immunohistochemis-try (see Table I for Ab information). In general, NK cells primarilywere found in vaginal and cervical submucosa (Fig. 1B–D). Incontrast to gut mucosal tissues (24), we did not find intraepithelialNK cells in vagina or cervix, and we found very few NK cells at

the site where infected founder populations have been consistentlynoted (2, 3) in the palmate folds of endocervical tissues in any ofthe 32 cervical tissues examined (Fig. 1B).

NK cell responses in naive animals: rapid expansion and thencontraction following vaginal challenge

We quantified the number of NK cells in vaginal (48 animals) andcervical (32 animals) tissues in naive animals prior to and following

FIGURE 3. (A) AT-2–SIV was as potent as WT-SIV in recruiting NK cells into the FRT. Each point represents an individual animal. (B) Macrophages (CD68+)

and fibroblasts (vimentin+) were the major CXCL10/IP-10–expressing cell populations in the FRT mucosa (original magnification3200). (C) These CXCL10+ cells

were in close proximity to NKG2A+ NK cells in the submucosa. All images were from tissues collected 7 d postinfection. Original magnification 3200.

Table I. Primary Abs used in this study

Abs Clone (Catalog No.) Tissue Fixation Ag Retrieval

NKG2A Epitomics (T3308) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minGranzyme H Sigma (HPA029200) PFA 1 mM EDTA buffer (pH 8) 98˚C 20 minGranzyme B Spring Bioscience (E2580) PFA 1 mM EDTA buffer (pH 8) 98˚C 20 minIFN-g Abcam (ab25101) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minIL-2Rb (CD122) Novus Biologicals (NBP1-19140) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minHLA-E MEM-E/06 (ab3984) PFA 1 mM EDTA buffer (pH 8) 98˚C 20 minCD3 AbD Serotec (MCA 1477) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minCCL3/MIP-1a Neomarkers (RB-10489-P) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minCCL4/MIP-1b R&D (AF-271-NA) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minCCL5/RANTES R&D (AF-278-NA) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minKi67 Neomarkers (RM9106) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minHLA-DR/DQ/DP DAKO (M0073) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minCD69 Novacastra (NCL-CD69 CH11) SAFEFIX II 1 mM EDTA buffer (pH 8) 98˚C 20 minCD38 Vector (VP-C348 SPC32) SAFEFIX II 1 mM EDTA buffer (pH 8) 98˚C 20 minIP10 R&D Systems (AF-266-NA) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minCD107a LifeSpan (LS-B580) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minCD68 DAKO (KP1 M0814) PFA 10 mM citrate buffer (pH 6) 98˚C 20 minVimentin Neomarkers (RB-9063) PFA 10 mM citrate buffer (pH 6) 98˚C 20 min

PFA, paraformaldehyde.

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SIV vaginal challenge. As shown in Fig. 2A and 2B, NK cells wererapidly recruited into the cervical and vaginal mucosa after vag-

inal inoculation, with significantly higher numbers in cervix

compared with vagina (cervical median = 1017, vaginal median =

319) at the end of the first week (Fig. 2C). However, even at the

peak of expansion, NK cells were greatly outnumbered by ex-

panded populations of the CD4+ T cells that fuel local virus ex-

pansion (2, 3), as well as by macrophages (Fig. 2D).Although small foci of SIV RNA+ cells have been detected in

the cervix as early as 3–4 d (2, 25) postvaginal inoculation, it

seemed unlikely that viral replication per se would be the driving

force for the early NK cell influx into the genital mucosa, given

the magnitude of local infection. The recruitment of NK cells to

cervical tissues of animals vaginally inoculated with infectious

wild type SIV (WT-SIV) or AT-2–inactivated SIV (AT-2–SIV) is

consistent with this conclusion. As shown in Fig. 3A, the densities

of NK cells were comparable between WT-SIV and AT-2–SIV

groups through 4 d after vaginal inoculation. Moreover, the de-

crease in the numbers of mucosal NK cells in both vagina and

cervix during the second week (the peak of infection in the FRT),

when only 13.6% (cervix) and 24.8% (vagina) (percentage of

median) of the peak values of NK cells remained (Fig. 2A, 2B),

also argues against viral replication–driven NK cell recruitment.NK cells are most likely recruited by chemokine expression in

the FRT. Because CXCL10/IP-10 is well known as a potent NK cell

chemoattractant, we examined its expression profile in the FRT

mucosa of infected animals. Macrophages (CD68+) and fibroblasts

(vimentin+) were the major CXCL10-producing cell populations

in the genital mucosa (Fig. 3B). These CXCL10+ cells residedclose to the basal layer of epithelium and often were found inclose proximity to the majority of NKG2A+ NK cells in thesubmucosa (Fig. 3C). We favor local recruitment of NK cellsby these CXCL10+ cells rather than recruitment by CXCL10 inthe inoculum (26), which we would expect to elicit a generaland immediate recruitment of NK cells to the mucosal border,rather than the observed focal and delayed recruitment 3 d afterexposure.

NK cell responses in naive animals: relationship between NKcells and SIV RNA+ cells

We next investigated the potential role of NK cells recruited in thefirst week of infection in containing local viral replication by

examining the density and spatial relationships of the mucosal

NK cells and SIV RNA+ cells. We enumerated SIV RNA+ cells

detected by ISH and showed that SIV RNA+ cells were barely

detectable in the first week and then increased to peak in the

second week (Fig. 4A, 4B). Because the mucosal NK cells peaked

in the first week, when the local expansion of infected founder foci

of infected cells had just begun to expand, there was an expected

negative correlation between the densities of SIV RNA+ cells and

NK cells, which was significant in cervix but not vagina (Fig. 4C,4D). However, in montage images of the transformation zone(TZ), where SIV RNA+ cells are consistently concentrated in earlyinfection (2, 3), there was complete spatial separation of NKG2A+

CD32 NK and SIV RNA+ cell populations (Fig. 5). Indeed, in allanimals examined, the SIV RNA+ cells were always located in the

FIGURE 4. Increase in the density of SIV RNA+ cells in cervix and vagina and negative correlation with the density of mucosal NK cells at the end of the first

week following vaginal inoculation. (A and B) SIV RNA+ cells detected by ISH were enumerated in cervix and vagina. Each point represents an individual

animal. (C and D) In cervical and vaginal tissues, the densities of NK cells correlated negatively with the densities of SIV RNA+ cells at the end of the first week.

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endocervix close to the TZ where there were few, if any, NK cells(Fig. 1B). Although these images are snapshots of interactions ofcells in FRT tissues, the spatial dissociation between NK cells andSIV RNA+ cells (Fig. 5) does not support the hypothesis thatrecruited NK cells contain infection by contact-dependent mech-anisms in the endocervix and TZ where expanding founder pop-ulations of infected cells have been consistently documented (2, 3).However, this spatial dissociation does not exclude a possible rolefor NK cells in eliminating infected cells at sites close to NK cellsbefore the SIV RNA reaches a detectable level.

NK cell responses in naive animals: functional and activationmarkers of NK cells in FRT tissues

We examined the chemokine and cytokine expression profiles ofcervical NK cells at 7 d postvaginal inoculation for evidence thatmight indirectly support the hypothesis that NK cells play a pro-tective role in containing local infection following vaginal exposureto SIV. The majority of cervical NK cells produced IFN-g (74.3–98.1%, median 80.3%, n = 7) (Fig. 6A), as well as the HIV-1inhibitory b-chemokine MIP-1a (CCL3) (Fig. 6B), but not MIP-

1b (Fig. 6E) or RANTES (Fig. 6F) (data not shown). Only ap-proximately one in four cervical NK cells expressed granzyme B(10.3–29.2%, median 25.4%, n = 7) (Fig. 6C), and less than half ofthe NK cells were positive for the more abundant NK cytotoxiceffector (27), granzyme H (14.6–42.6%, median 38.5%, n = 7)(Fig. 6D). Moreover, the NK cells were negative for the degran-ulation marker, CD107a (Fig. 6G), and also negative for the pheno-typic markers of activation and replication Ki67, CD69, CD38, andHLA-DR (Fig. 6H–K).

FIGURE 5. Spatial dissociation between cervical NK cells and SIV

RNA+ cells. Montage images of the density and spatial distribution of

mucosal NK cells and SIV RNA+ cells in entire cervical tissue sections. (A

and B) NK cells and SIV RNA+ cells are too small to be visible in montage

images of entire tissue section at the resolution shown. Therefore, the NK

cells and SIV RNA+ cells were labeled as green and red dots, respectively.

All images were from tissues collected 7 d postinfection. Original mag-

nification 3200.

FIGURE 6. Cervical NK cells express IFN-g (A), MIP-1a (CCL3) (B),

granzyme B (C), and granzyme H (D). However, the majority are CCL42

(E), CCL52 (F), CD107a2 (G), Ki672 (H), CD692 (I), CD382 (J), and

HLA-DR2 (K). Original magnification 3200.

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In seeking an explanation for the latter phenotype, we hadnoticed in unpublished microarray analyses, in which we com-

pared transcriptional profiles of cervical tissues from uninfected

and vaginally infected animals, that expression of the HLA-E

(Mamu-E) inhibitory ligand of NKG2A receptors was upregulat-

ed in cervical tissues within 4 d after virus infection (Fig. 7A)

(A.J. Smith, S.W. Wietgrefe, L. Shang, C.S. Reilly, P.J. Southern,

K.E. Perkey, L. Duan, H. Kohler, S. Muller, J. Robinson, J.V. Carlis,

Q. Li, R.P. Johnson, and A.T. Haase, unpublished observations).

By immunohistochemical staining, HLA-E+ cells were detectable

as soon as 24 h postchallenge (Fig. 7B). These HLA-E+ cells

included Langerhans cells in the genital epithelium and cells in the

submucosa close to epithelium, where NK cells usually reside

(Fig. 7B). Consistent with the inhibitory function of HLA-E, the

resident NK cell population was CD122 (IL-2Rb) negative (Fig. 7C)

and, therefore, would be unable to respond to IL-2 and IL-15. Thus,

collectively, the cervical NK cells recruited by SIV vaginal inocula-

tion lacked many markers of activation and cytotoxic function that

might be expected for an effector population.

FRT NK cell density does not correlate with protection bySIVmac239Dnef vaccination

SIVmac239Dnef vaccination was shown to protect against high-dose vaginal challenge (15, 16, 28) (e.g., vaccinated animals either

had sterilizing immunity or lower peak and set point viral loads

compared with unvaccinated animals at 20 wk postvaccination;

R.K. Reeves, manuscript in preparation). We evaluated potential NK

cell contributions to this protection by asking whether vaccinated

animals had denser vaginal and cervical NK cell populations

compared with naive animals prior to and after vagina challenge.

To the contrary, we found that the cervical and vaginal NK cell

populations prior to challenge were not statistically different be-

tween naive and vaccinated animals (Fig. 8A, 8B), and, surpris-

ingly, high-dose vaginal challenge of vaccinated animals with

pathogenic SIVmac251 did not induce the influx of NK cells or

macrophages (Fig. 8C) into the genital mucosa that we had docu-

mented (Fig. 1) in unvaccinated animals.

DiscussionIn this article, we report characterization of the NK cell response toSIV infection in the female genital mucosa of rhesus macaques

after a high-dose vaginal exposure. In this animal model, the first 3–4 d postinfection define a time frame of vulnerability for the virusin establishing and expanding infected founder populations and,thus, a window of opportunity for the host immune system to

inhibit viral replication and prevent mucosal transmission. Wefound that NK cells were recruited into the genital mucosa in thiswindow of opportunity by mechanisms that can be attributed, atleast in part, to cells in the FRT expressing CXCL10/IP-10, the

most potent NK cell chemoattractant (29), in close proximity toNK cells. The peak values of recruited cervical NK cells at 7 dpostvaginal exposure correlated negatively with the number ofSIV RNA+ cells in cervix. Moreover, most of the NK cells in the

FRT mucosa were positive for IFN-g and MIP-1a/CCL3, a b-chemokine that was shown to block viral entry in culture (4, 5).Collectively, these results initially provided evidence to support

the hypothesis that the NK cells recruited into the genital mucosacould contribute to containing SIV infection in the early windowof opportunity at the portal of entry.However, we document aspects of the quantity, location, and

quality of observed mucosal NK cell responses that are inconsistentwith NK cells as effective gatekeepers to prevent viral transmission.First, only a very small number of NK cells was recruited into thegenital mucosa relative to the orders of magnitude greater re-

cruitment of CD4 T cells that fuel the local expansion of viruses andinfected cells prior to systemic dissemination and infection (2, 25).The numbers of NK cells also declined at the peak of virus rep-

lication in the lower FRT tissues, similar to the decreased numbersof NK cells in the gut mucosa of SIV-infected animals 2–4 wkfollowing SIV vaginal transmission (23, 30). Thus, the effectivein vivo ratio of NK effector cells/infected targets was even more

unfavorable for a protective role.Second, the spatial dissociation between recruited NK cells and

expanding populations of SIV RNA+ cells in the endocervix doesnot support a model in which mucosal NK cells kill their targets

FIGURE 7. (A) Microarray analysis comparison of gene expression in uninfected versus infected cervical tissue reveals upregulation of HLA-E ex-

pression in cervical tissues after vaginal challenge. (B) The HLA-E–expressing cells in cervical–vaginal submucosa as early as 24 h postinfection include

Langerhans cells in epithelium (right panel) (DAB staining, original magnificaiton 3100). (C) NKG2A+CD32 NK cells in cervical mucosa were CD122

(IL-2Rb) negative (original magnification 3200).

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by cell–cell contact mechanisms, such as Ab-dependent cellularcytotoxicity and death receptor–mediated cytotoxicity (31). Third,the recruited NK cells lacked many phenotypic markers associatedwith a functioning effector population, such as evidence of de-granulation (CD107a), activation (CD38, CD69, and HLA-DR),and other cytokines/chemokines previously associated with anactivated effector population of NK cells, although CD69 andKi67 expression on NK cells in blood and gut has been reported(23, 32). In the FRT, we show increased expression of the inhib-itory HLA-E ligand and downregulation of activating receptorCD122 following vaginal exposure to SIV, highlighting the rapidlyaltered balance between activating and inhibitory signals during

mucosal transmission, as was shown in other acute and chronicinfections (33–35).SIVmac239Dnef vaccination provides robust protection against

subsequent WT-SIV challenge by parenteral and mucosal routes(16, 28). Although safety issues have precluded advancing this andother live attenuated viruses as candidates for an effective HIV-1vaccine, understanding the correlates of protection could providecritical insights to guide HIV-1 vaccine development. We havebeen focusing particularly on identifying correlates associatedwith the Dnef vaccine–mediated maturation of protection, definedby little protection if animals are challenged vaginally at 5 wkpostvaccination but substantial protection at 15–20 wk or even 40wk postvaccination. In this study, we assessed the role of NK cellsin the FRT mucosa in mediating the vaccine-induced protectionin animals that have been vaccinated for 20 wk (versus naiveanimals). Surprisingly, NK cell numbers in the FRT mucosa ofanimals vaccinated 20 wk prior to challenge were comparable tothose in naive animals. Even more remarkably, we did not observeany influx of NK cells or macrophages into the FRT mucosa in thevaccinated animals following high-dose vaginal challenge. Weshow elsewhere that the formation of immune complexes fol-lowing vaginal challenge, as well as the interaction of theseimmune complexes with FcR for IgG, FcgRIIb, induces an in-hibitory program that blocks CD4 T cell recruitment (A.J. Smith,S.W. Wietgrefe, L. Shang, C.S. Reilly, P.J. Southern, K.E. Perkey,L. Duan, H. Kohler, S. Muller, J. Robinson, J.V. Carlis, Q. Li,R.P. Johnson, and A.T. Haase, manuscript in preparation). This in-hibitory program may similarly be involved in blocking NK cell,as well as macrophage, recruitment. Thus, our studies collectivelyprovide only limited evidence of correlations between NK cells inthe FRT mucosa of naive animals and containment of infection at theportal of entry and no evidence for a role for NK cells in protectionagainst vaginal challenge in SIVmac239Dnef-vaccinated animals.

AcknowledgmentsWe thank Dr. Ronald Desrosiers for providing SIVmac239Δnef; JacquelineGillis, Fay Eng Wong, and Elizabeth Curran for assistance with tissue

processing; and Dr. Angela Carville and other members of the New Eng-

land Primate Research Center Division of Veterinary Resources for expert

animal care.

DisclosuresThe authors have no financial conflicts of interest.

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FIGURE 8. NK cell population densities in the FRT mucosa do not

correlate with SIVmac239Dnef vaccine protection before or after high-

dose vaginal challenge. NK cells in the cervical (A) and vaginal (B) mu-

cosa were quantified by immunohistochemistry in tissues from naive,

SIVmac251 vaginally infected, SIVmac239Dnef-vaccinated, and vacci-

nated plus vaginally challenged rhesus macaques. There was no correlation

between NK cell density prior to and after vaginal challenge with pro-

tection, and NK cell densities did not increase following challenge in the

vaccinated animals. (C) Macrophage recruitment also was inhibited in the

vaccinated animals. *Not significant.

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