Immune-related microRNAs are abundant in breast milk exosomes

Breast milk is a complex liquid rich in immunological components that affect the development of the infant's immune system. Exosomes are membranous vesicles of endocytic origin that are found in various body fluids and that can mediate intercellular communication. MicroRNAs (miRNAs), a well-defined group of non-coding small RNAs, are packaged inside exosomes in human breast milk. Here, we identified 602 unique miRNAs originating from 452 miRNA precursors (pre-miRNAs) in human breast milk exosomes using deep sequencing technology. We found that, out of 87 well-characterized immune-related pre-miRNAs, 59 (67.82%) are presented and enriched in breast milk exosomes (P < 10(-16), χ(2) test). In addition, compared with exogenous synthetic miRNAs, these endogenous immune-related miRNAs are more resistant to relatively harsh conditions. It is, therefore, tempting to speculate that these exosomal miRNAs are transferred from the mother's milk to the infant via the digestive tract, and that they play a critical role in the development of the infant immune system.     

Lactation-Related MicroRNA Expression Profiles of Porcine Breast Milk Exosomes

Breast milk is the primary source of nutrition for newborns, and is rich in immunological components. MicroRNAs (miRNAs) are present in various body fluids and are selectively packaged inside the exosomes, a type of membrane vesicles, secreted by most cell types. These exosomal miRNAs could be actively delivered into recipient cells, and could regulate target gene expression and recipient cell function. Here, we analyzed the lactation-related miRNA expression profiles in porcine milk exosomes across the entire lactation period (newborn to 28 days after birth) by a deep sequencing. We found that immune-related miRNAs are present and enriched in breast milk exosomes (p<10(-16), χ(2) test) and are generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs are present in higher numbers in the colostrums than in mature milk. It was higher in the serum of colostrum-only fed piglets compared with the mature milk-only fed piglets. These immune-related miRNA-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations are a prelude to in-depth investigations of the essential roles of breast milk in the development of the infant's immune system.     

Exosomes containing glycoprotein 350 released by EBV-transformed B cells selectively target B cells through CD21 and block EBV infection in vitro

Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes by inward budding of the endosomal limiting membrane. They have immune stimulatory-, inhibitory-, or tolerance-inducing effects, depending on their cellular origin, which is why they are investigated for use in vaccine and immune therapeutic strategies. In this study, we explored whether exosomes of different origins and functions can selectively target different immune cells in human peripheral blood. Flow cytometry, confocal laser scanning microscopy, and multispectral imaging flow cytometry (ImageStream) revealed that exosomes derived from human monocyte-derived dendritic cells and breast milk preferably associated with monocytes. In contrast, exosomes from an EBV-transformed B cell line (LCL1) preferentially targeted B cells. This was not observed for an EBV(-) B cell line (BJAB). Electron microscopy, size-distribution analysis (NanoSight), and a cord blood transformation assay excluded the presence of virions in our LCL1 exosome preparations. The interaction between LCL1-derived exosomes and peripheral blood B cells could be blocked efficiently by anti-CD21 or anti-gp350, indicating an interaction between CD21 on B cells and the EBV glycoprotein gp350 on exosomes. The targeting of LCL1-derived exosomes through gp350-CD21 interaction strongly inhibited EBV infection in B cells isolated from umbilical cord blood, suggesting a protective role for exosomes in regulating EBV infection. Our finding also suggests that exosome-based vaccines can be engineered for specific B cell targeting by inducing gp350 expression.     

Immune surveillance properties of human NK cell-derived exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56(+) and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.     

Exosomes derived from M. Bovis BCG infected macrophages activate antigen-specific CD4+ and CD8+ T cells in vitro and in vivo

Activation of both CD4(+) and CD8(+) T cells is required for an effective immune response to an M. tuberculosis infection. However, infected macrophages are poor antigen presenting cells and may be spatially separated from recruited T cells, thus limiting antigen presentation within a granuloma. Our previous studies showed that infected macrophages release from cells small membrane-bound vesicles called exosomes which contain mycobacterial lipid components and showed that these exosomes could stimulate a pro-inflammatory response in na?ve macrophages. In the present study we demonstrate that exosomes stimulate both CD4(+) and CD8(+) splenic T cells isolated from mycobacteria-sensitized mice. Although the exosomes contain MHC I and II as well as costimulatory molecules, maximum stimulation of T cells required prior incubation of exosomes with antigen presenting cells. Exosomes isolated from M. bovis and M. tuberculosis infected macrophages also stimulated activation and maturation of mouse bone marrow-derived dendritic cells. Interestingly, intranasal administration of mice with exosomes isolated from M. bovis BCG infected macrophages induce the generation of memory CD4(+) and CD8(+) T cells. The isolated T cells also produced IFN-gamma upon restimulation with BCG antigens. The release of exosomes from infected macrophages may overcome some of the defects in antigen presentation associated with mycobacterial infections and we suggest that exosomes may be a promising M. tuberculosis vaccine candidate.     

Chemokine-containing exosomes are released from heat-stressed tumor cells via lipid raft-dependent pathway and act as efficient tumor vaccine

Exosomes derived from dendritic cells or tumor cells are a population of nanometer-sized membrane vesicles that can induce specific antitumor immunity. During investigation of the effects of hyperthermia on antitumor immune response, we found that exosomes derived from heat-stressed tumor cells (HS-TEX) could chemoattract and activate dendritic cells (DC) and T cells more potently than that by conventional tumor-derived exosomes. We show that HS-TEX contain chemokines, such as CCL2, CCL3, CCL4, CCL5, and CCL20, and the chemokine-containing HS-TEX are functionally competent in chemoattracting CD11c(+) DC and CD4(+)/CD8(+) T cells both in vitro and in vivo. Moreover, the production of chemokine-containing HS-TEX could be inhibited by ATP inhibitor, calcium chelator, and cholesterol scavenger, indicating that the mobilization of chemokines into exosomes was ATP- and calcium-dependent and via a lipid raft-dependent pathway. We consistently found that the intracellular chemokines could be enriched in lipid rafts after heat stress. Accordingly, intratumoral injection of HS-TEX could induce specific antitumor immune response more efficiently than that by tumor-derived exosomes, thus inhibiting tumor growth and prolonging survival of tumor-bearing mice more significantly. Therefore, our results demonstrate that exosomes derived from HS-TEX represent a kind of efficient tumor vaccine and can chemoattract and activate DC and T cells, inducing more potent antitumor immune response. Release of chemokines through exosomes via lipid raft-dependent pathway may be a new method of chemokine exocytosis.     

Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans

In addition to sperm cells, seminal fluid contains various small membranous vesicles. These include prostasomes, membrane vesicles secreted by prostate epithelial cells. Prostasomes have been proposed to perform a variety of functions, including modulation of (immune) cell activity within the female reproductive tract and stimulation of sperm motility and capacitation. How prostasomes mediate such diverse functions, however, remains unclear. In many studies, vesicles from the seminal plasma have been categorized collectively as a single population of prostasomes; in fact, they more likely represent a heterogeneous mixture of vesicles produced by different reproductive glands and secretory mechanisms. We here characterized membranous vesicles from seminal fluid obtained from vasectomized men, thereby excluding material from the testes or epididymides. Two distinct populations of vesicles with characteristic sizes (56 ± 13 nm vs. 105 ± 25 nm) but similar equilibrium buoyant density (～1.15 g/ml) could be separated by using the distinct rates with which they floated into sucrose gradients. Both types of vesicle resembled exosomes in terms of their buoyant density, size, and the presence of the ubiquitous exosome marker CD9. The protein GLIPR2 was found to be specifically enriched in the lumen of the smaller vesicles, while annexin A1 was uniquely associated with the surface of the larger vesicles. Prostate stem-cell antigen (PSCA), a prostate-specific protein, was present on both populations, thereby confirming their origin. PSCA was, however, absent from membrane vesicles in the seminal fluid of some donors, indicating heterogeneity of prostasome characteristics between individuals.     

BCR-bound antigen is targeted to exosomes in human follicular lymphoma B-cells

BACKGROUND INFORMATION: Exosomes are small membrane vesicles secreted by several cell types during exocytic fusion of multivesicular bodies with the plasma membrane. Exosomes from tumour cells can transfer antigens from cell to cell, a property favouring antigen-specific immune responses in vitro and in vivo, and are thus an interesting putative therapeutic tool in human cancers. Exosomes have been well studied in EBV (Epstein-Barr virus)-transformed human B-cell lines; however, biological stimuli regulating exosome secretion quantitatively and/or qualitatively still remain poorly defined. RESULTS: We analysed the effect of the BCR stimulation on exosome release in the human follicular lymphoma B-cell line DOHH2. We found that BCR (B-cell receptor) triggering of DOHH2 cells induced the polarization of CD63(+) MHC class II compartments. Moreover, BCR stimulation increased the release of exosome-associated proteins in the extracellular space. Finally, we found that the BCR was expressed at the surface of exosomes, and could target a bound anti-human IgG to these vesicles. CONCLUSIONS: BCR can modulate the protein content of exosomes upon stimulation, and can target its bound antigen to these vesicles.     

MicroRNAs are exported from malignant cells in customized particles

MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body.     

MHC class II+ exosomes in plasma suppress inflammation in an antigen-specific and Fas ligand/Fas-dependent manner

Exosomes are 50- to 100-nm vesicles that are formed within the late endocytic compartment and released from a variety of cell types. Previously, we demonstrated that exosomes derived from dendritic cells transduced with adenoviral vectors expressing IL-10, IL-4, or Fas ligand (FasL) produce anti-inflammatory exosomes able to reduce inflammation in a murine paw delayed-type hypersensitivity model, suppress the onset on murine collagen-induced arthritis, and reduce the severity of established collagen-induce arthritis. In this study, we examined the ability of endogenous, blood-borne exosomes to regulate the immune response. Exosomes isolated from plasma of mice immunized to keyhole limpet hemocyanin, but not from naive or OVA-immunized mice, were able to suppress the keyhole limpet hemocyanin-specific delayed-type hypersensitivity inflammatory response. The anti-inflammatory effect was mediated by MHC class II(+) plasma exosomes that were also FasL(+) and CD11b(+), but CD11c(-). Moreover, the anti-inflammatory effect of the MHC class II(+) plasma-derived exosomes was, in part, dependent upon the presence of FasL in the exosomes and Fas in the recipient mouse. These results suggest that exosomes in the plasma, produced by MHC class II(+) and CD11b(+) cells, have the ability to suppress the immune response in an Ag-specific manner in part through a Fas/FasL-dependent manner.     

Nanovesicles from Malassezia sympodialis and host exosomes induce cytokine responses--novel mechanisms for host-microbe interactions in atopic eczema

Background

Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated.

Objective

To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis.

Methods

Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively.

Results

We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-α responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-α responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-α but not IL-4 in undepleted PBMC.

Conclusions

Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE.     

Large-scale expansion of CD3<Superscript>+</Superscript>CD56<Superscript>+</Superscript> lymphocytes capable of lysing autologous tumor cells with cytokine-rich supernatants

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMC) by the timed addition of cytokine-rich supernatants collected from allogeneic PBMC cultures stimulated with anti-CD3 monoclonal antibody (mAb) (allogeneic CD3 supernatants; ACD3S). These cytotoxic effectors belonged primarily to CD56(+) natural killer (NK) cells, and the cell subset with the greatest cytotoxic activity was an otherwise rare population of CD3(+)CD56(+) cells (NKT cells) that expand dramatically under these conditions. CD3(+)CD56(+) cytotoxic effectors were generated from the PBMC of 16 patients with several types of cancer. The CD3(+)CD56(+) cell subset expanded significantly and efficiently lysed NK- as well as lymphokine-activated killer (LAK)-sensitive targets. More importantly, ACD3S-activated CD3(+)CD56(+) cells were capable of efficiently lysing autologous tumor cells including metastatic colorectal, ovarian, breast, lung and pancreatic tumor cells as well as melanoma cells. ACD3S-expanded CD3(+)CD56(+) cells exhibited increased levels of cytoplasmic interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and perforin. CD3(+)CD56(+) cell-mediated cytotoxicity was not restricted by major histocompatibility complex (MHC) gene products, since it was not blocked by anti-MHC class I mAb but was highly inhibited in the presence of CD2- and CD18-specific mAb. These data suggest that CD3(+)CD56(+) cells expanded under the presence of ACD3S may be utilized in clinical protocols for cancer immunotherapy.     

T cell exosomes induce cholesterol accumulation in human monocytes via phosphatidylserine receptor

Activated T lymphocytes release vesicles, termed exosomes, enriched in cholesterol and exposing phosphatidylserine (PS) at their outer membrane leaflet. Although CD4(+) activated T lymphocytes infiltrate an atherosclerotic plaque, the effects of T cell exosomes on the atheroma-associated cells are not known. We report here that exosomes isolated from the supernatants of activated human CD4(+) T cells enhance cholesterol accumulation in cultured human monocytes and THP-1 cells. Lipid droplets found in the cytosol of exosome-treated monocytes contained both cholesterol ester and free cholesterol. Anti-phosphatidylserine receptor antibodies recognized surface protein on the monocyte plasma membrane and prevented exosome-induced cholesterol accumulation, indicating that exosome internalization is mediated via endogenous phosphatidylserine receptor. The production of proinflammatory cytokine TNF-alpha enhanced in parallel with monocyte cholesterol accumulation. Our data strongly indicate that exosomes released by activated T cells may represent a powerful, previously unknown, atherogenic factor.     

Distinct kinetics of memory B-cell and plasma-cell responses in peripheral blood following a blood-stage Plasmodium chabaudi infection in mice

B cell and plasma cell responses take place in lymphoid organs, but because of the inaccessibility of these organs, analyses of human responses are largely performed using peripheral blood mononuclear cells (PBMC). To determine whether PBMC are a useful source of memory B cells and plasma cells in malaria, and whether they reflect Plasmodium-specific B cell responses in spleen or bone marrow, we have investigated these components of the humoral response in PBMC using a model of Plasmodium chabaudi blood-stage infections in C57BL/6 mice. We detected memory B cells, defined as isotype-switched IgD(-) IgM(-) CD19(+) B cells, and low numbers of Plasmodium chabaudi Merozoite Surface Protein-1 (MSP1)-specific memory B cells, in PBMC at all time points sampled for up to 90 days following primary or secondary infection. By contrast, we only detected CD138(+) plasma cells and MSP1-specific antibody-secreting cells within a narrow time frame following primary (days 10 to 25) or secondary (day 10) infection. CD138(+) plasma cells in PBMC at these times expressed CD19, B220 and MHC class II, suggesting that they were not dislodged bone-marrow long-lived plasma cells, but newly differentiated migratory plasmablasts migrating to the bone marrow; thus reflective of an ongoing or developing immune response. Our data indicates that PBMC can be a useful source for malaria-specific memory B cells and plasma cells, but extrapolation of the results to human malaria infections suggests that timing of sampling, particularly for plasma cells, may be critical. Studies should therefore include multiple sampling points, and at times of infection/immunisation when the B-cell phenotypes of interest are likely to be found in peripheral blood.     

Proteomic profiling of exosomes: current perspectives

Exosomes are 40-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro. Recent studies have shown that exosomes are also found in vivo in body fluids such as blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, and breast milk. While the biological function of exosomes is still unclear, they can mediate communication between cells, facilitating processes such as antigen presentation and in trans signaling to neighboring cells. Exosome-like vesicles identified in Drosophila (referred to as argosomes) may be potential vehicles for the spread of morphogens in epithelia. The advent of current MS-based proteomic technologies has contributed significantly to our understanding of the molecular composition of exosomes. In addition to a common set of membrane and cytosolic proteins, it is becoming increasingly apparent that exosomes harbor distinct subsets of proteins that may be linked to cell-type associated functions. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo such as prions and retroviruses such as HIV suggest their participation in pathological situations. Interestingly, the recent observation that exosomes contain both mRNA and microRNA, which can be transferred to another cell, and be functional in that new environment, is an exciting new development in the unraveling exosome saga. The present review aims to summarize the physical properties that define exosomes as specific cell-type secreted membrane vesicles.     

Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ-producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100(-/-) L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100(-/-) leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100(-/-) exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.     

HIV envelope suppresses CD4+ T cell activation independent of T regulatory cells

We previously demonstrated that HIV envelope glycoprotein (Env), delivered in the form of a vaccine and expressed by dendritic cells or 293T cells, could suppress Ag-stimulated CD4(+) T cell proliferation. The mechanism remains to be identified but is dependent on CD4 and independent of coreceptor binding. Recently, CD4(+) regulatory T (Treg) cells were found to inhibit protective anti-HIV CD4(+) and CD8(+) T cell responses. However, the role of Tregs in HIV remains highly controversial. HIV Env is a potent immune inhibitory molecule that interacts with host CD4(+) cells, including Treg cells. Using an in vitro model, we investigated whether Treg cells are involved in Env-induced suppression of CD4(+) T cell proliferation, and whether Env directly affects the functional activity of Treg cells. Our data shows that exposure of human CD4(+) T cells to Env neither induced a higher frequency nor a more activated phenotype of Treg cells. Depletion of CD25(+) Treg cells from PBMC did not overcome the Env-induced suppression of CD4(+) T cell proliferation, demonstrating that CD25(+)FoxP3(+) Treg cells are not involved in Env-induced suppression of CD4(+) T cell proliferation. In addition, we extend our observation that similar to Env expressed on cells, Env present on virions also suppresses CD4(+) T cell proliferation.     

Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles

Dendritic cells constitutively secrete a population of small (50-90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1alpha) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.     

Breast milk-derived antigen-specific CD8+ T cells: an extralymphoid effector memory cell population in humans

Although mouse studies have demonstrated the presence of an effector memory population in nonlymphoid tissues, the phenotype of human CD8(+) T cells present in such compartments has not been characterized. Because of the relatively large number of CD8(+) T cells present in breast milk, we were able to characterize the phenotype of this cell population in HIV-infected and uninfected lactating women. CMV, influenza virus, EBV, and HIV-specific CD8(+) T cells as measured by the IFN-gamma ELISPOT and MHC class I tetramer staining were all present at greater frequencies in breast milk as compared with blood. Furthermore, a greater percentage of the breast milk CD8(+) T cells expressed the intestinal homing receptor, CD103, and the mucosal homing receptor CCR9. Breast milk T cells were predominantly CD45RO(+)HLADR(+) and expressed low levels of CD45RA, CD62L, and CCR7 consistent with an effector memory population. Conversely, T cells derived from blood were mainly characterized as central memory cells (CCR7(+)CD62L(+)). These results demonstrate a population of extralymphoid CD8(+) T cells with an effector memory phenotype in humans, which could contribute to enhanced local virologic control and the relative lack of HIV transmission via this route.