Maternal Leukocytes and Infant Immune Programming during Breastfeeding

TREIMM 1652 No. of Pages 15

Trends in Immunology

Review

Maternal Leukocytes and Infant Immune Programming during Breastfeeding Amale Laouar1,* The fetal immune system develops in a rather sterile environment relative to the outside world and, therefore, lacks antigenic education. Soon after birth, the newborn is exposed to the hostile environment of pathogens. Recently, animaland limited human-based studies have indicated that help from the mother, upon transfer of leukocytes and their products via breast milk feeding, greatly assists the newborn’s immune system. Here, I discuss the newest advances on how milk leukocytes impact early life immunity, with an emphasis on the development of the infant T cell repertoire and early immune responses in the periphery and gut-associated lymphoid tissue. A deeper understanding of these novel mechanistic insights may inform potential translational approaches to improving immunity in infants.

Highlights Neonates are born with many immune components that are not fully developed. Through ingestion of breast milk, the transfer of maternal immune cells and their products, which begins in utero, continues after birth to aid the development of the neonatal immune system. A broader range of leukocyte types than previously recognized was recently identified in human breast milk. Neonatal conditions of the digestive tract enable the survival of milk cells and facilitate their trafficking to the infant’s tissues.

Early Life Immunity The capacity to mount an effective immune response depends not only on the functional roles of each of the neonatal cellular immune components, but also on their ability to adapt to a microbeladen external environment (e.g., differentiating what is to be tolerated from microbes and what is to be eliminated through host immune responses). Mammalian innate immunity is fast (seconds to minutes), but its duration of action is short (3–5 days) [1,2]. By contrast, adaptive immunity takes longer to activate (4–7 days), but its response is more specific and sustained (weeks to years) [1]. Faced with an immature adaptive immune system that has not had the time to build up the necessary memory and repertoires to permit such discrimination and specific immunity, the neonatal defense relies more on innate immune cells for protection. However, neonatal innate immune cells are not capable of efficient microbial killing, rendering newborn infants more vulnerable to infectious agents [2–4]. Other important events also occur during the neonatal phase. For instance, the gut is presented with two new simultaneous tasks that are both critical for survival: (i) digestion and absorption of nutrients; and (ii) discrimination and defense upon each microbial encounter. The intestinal immune system is not fully functional at birth. Studies of mice and pre- and full-term human infants have shown that postnatal microbial colonization contributes to the development of the immune system; indeed, deficiency in gut microbiota colonization, by germ-free husbandry, significantly delays this development [5]. For example, only a few B cells with antibody-producing capacity are present in the neonatal gut and blood of humans and mice [2,6,7]; it takes up to 30 days postpartum, a time-frame paralleling the expansion of the human gut microbial colonization, for the infant to produce sufficient antibody titers to ensure protection [6]. Available help from the mother, via the transfer of immunity by feeding on breast milk, assists the infant immune system during this susceptible time [8–14]. Through the ingestion of breast milk, the transfer of maternal immune cells and their products, which formerly starts in utero [15,16], continues after birth to aid the development of the neonatal immune system. Historically, breast milk was thought to pass immunity to the newborn only by virtue of its high secretory Trends in Immunology, Month 2020, Vol. xx, No. xx

Infants acquire immune protection from milk lymphocytes that originate from the gut and upper respiratory mucosa of the mother. These maternal cells have important roles in the protection and education of the developing immune system. Transferred maternal immunity via milk leukocytes affects short and long-term infant immune functions, shedding light on the potential ability of these maternal cells to modulate immune-related pediatric diseases.

1

Surgery Department and the Child Health Institute of New Jersey, Robert Wood Johnson Medical School-Rutgers University, 89 French Street, New Brunswick, NJ 08901, USA

*Correspondence: [email protected] (A. Laouar).

https://doi.org/10.1016/j.it.2020.01.005 © 2020 Elsevier Ltd. All rights reserved.

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immunoglobulin (Ig) content [17]. However, abundant numbers of leukocytes of the maternal immune system in human breast milk were revealed during the late 1960s [18,19]. The relationship of these maternal cells with the neonate was first demonstrated during the early 1980s [20], and subsequent murine studies often focused on the immune phenotype of milk cells [8–11]. Compared with systemic counterparts, human [21–24] and murine [9,10] milk leukocytes exhibit an activated and/or differentiated phenotype and express distinct migratory molecules, such as the homing molecules to the gut (α4β7) and upper respiratory tract (α4β1) [24]. However, only a small number of murine studies have assessed the transfer of breast milk leukocytes to infant tissues during normal breastfeeding and investigated their potential functions in early life immunity. Murine studies investigating the physiological transfer of milk leukocytes to foster-nursed pups by syngeneic or congeneic lactating dams revealed that milk lymphocytes were able to survive the challenging conditions of the digestive tract, infiltrate the gut mucosa, and pass to the blood circulation into different early life tissues [8–11,15]. Some of the latest advances suggest that the transfer of immunocompetent lymphocytes, via nursing, contributes to the maintenance of humoral immunity [10,24,25], the modulation of the immune response in the periphery and gut-associated lymphoid tissue (GALT; see Glossary) [9,26], as well as, potentially, the development of the infant T cell repertoire [8,11]. Here, I provide a comprehensive analysis of milk leukocyte trafficking to infant immune sites and discuss the newest advances on how this maternal immune transfer might impact short and long-term immune functions. Acquiring better knowledge of these lactational programming aspects is critical to advancing our understanding of immune development during infancy and the putative mechanisms of interference during early immune responses.

Major Types of Maternal Leukocytes in Breast Milk Fresh breast milk contains numerous types of hematopoietic-derived immune cells of maternal origin [27–30]. Compiled data from human and murine studies indicate that myeloid cells are predominant in breast milk (N80% of total leukocytes), followed by lymphocytes (b20% of total leukocytes), the latter being mainly T cells (~80%) [9,27,31]. Of note, the composition of milk immune cells is dynamic, changing with the maturation stage of the breast milk [27,32,33], the health status of the breastfeeding dyad [31,34,35], and other maternal conditions [32] (Box 1). Recently identified in human breast milk are natural killer (NK) cells, NK-T cells, innate lymphoid cells (ILCs), immature granulocytes, regulatory T cells (Tregs), and myeloid precursors [27–32,36]. Given that flow cytometry has limited sensitivity for the detection of cells that appear at extremely low numbers, other immune cell subsets, particularly those expressing little or no CD45, have not been readily identifiable, but might include immature or rare leukocyte types [37]. Of note, human milk myeloid and lymphoid cells appear to be more activated and/or differentiated than their blood counterparts [21,38]. For instance, by contrast to the two major T cell subsets found in the periphery, milk T lymphocytes almost exclusively comprise an extralymphoid cell population in which most CD4+ and CD8+ T cells are effector memory cells [21,23,39,40]. Similarly, human milk B cells exhibit a different phenotype from their systemic counterparts [24]: fewer naive B lymphocytes with higher percentages of class-switched IgD-memory B cells and plasma cells (PCs) have been found in breast milk relative to the blood compartment. Such a phenotype suggests that most milk B lymphocytes have undergone terminal PC differentiation [24].

Trafficking of Breast Milk Leukocytes and Significance to the Suckling Infant Previously, breast milk leukocytes were thought to have little biological importance for the infant because it was assumed that these cells could not survive in the acidic environment of the stomach. This idea has been challenged by numerous animal studies analyzing the accumulation of milk leukocytes in different infant tissues of mice [8–11,15], lambs and rats [46], baboons [47], 2

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Glossary Agammaglobulinemia: a group of inherited immune deficiencies characterized by low titers of antibodies in the blood due to a gene defect that blocks the growth of mature B lymphocytes. Class-switched IgD-memory B cells: cells that have undergone antibody class-switching in germinal centers in lymph nodes, or other secondary lymphoid tissues. Colostrum: form of milk produced by the mammary glands during late pregnancy and the few days after giving birth. Dam: female animal used to suckle the pups of another female. Enteromammary pathway: form of mother–neonate communication that is established during breastfeeding to enable the translocation of internal bacteria from mother to neonate and vice versa. Gut-associated lymphoid tissue (GALT): immune site where gut antigen sampling occurs and immune responses are initiated. Its overall role is to manage immune responses to the gut microbiota while maintaining a potent adaptive immune response to protect the host from enteric pathogens. Hypogammaglobulinemia: disease characterized by depressed serum titers of antibodies due to congenital or acquired causes. Given that antibodies are important immune components that help the body fight pathogens, newborns with hypogammaglobulinemia often get infections that may lead to potentially serious complications. Maternal educational immunity: potential new form of transferred immunity from mother to her newborn during physiological breastfeeding. This process may affect the development of the infant T cell repertoire in response to maternal milk leukocyte instruction. Microchimerism: presence of a small number of genetically distinct cells in the host individual. In humans (and perhaps in all placental mammals), the most common form is fetomaternal microchimerism, whereby cells from a fetus pass through the placenta and establish cell lineages within the mother. Microchimerism also occurs during breast milk feeding and in most pairs of twins in cattle. Mucosa-associated lymphoid tissue (MALT): system of small concentrations of lymphoid tissue found in various

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Box 1. Factors Affecting Breast Milk Leukocyte Content Several studies [27,41,42], with the exception of [43] in humans, indicate that preterm breast milk contains higher concentrations of maternal leukocytes compared with term milk. This milk leukocyte surge might be a compensatory immune mechanism to provide more protection to premature neonates [44]. Another key factor that influences milk leukocyte content is the health status of the breastfeeding dyad. For instance, a recent human study showed that breast milk samples from asthmatic mothers differed from those from nonasthmatics in that they contained a higher proportion of polymorphonuclear cells and their lymphocytes exhibited higher expression of the IgE receptor CD23 [34]. In addition, several studies [31,32,35,45], with the exception of [27] in humans, showed that, during periods of infection of either the mother or the infant or both, a rapid increase in milk leukocytes occurred relative to healthy mother–child dyads, but returned to baseline concentrations upon recovery. Such infections included infant conditions such as measles, influenza virus and GI infections, as well as maternal conditions, such as breast mastitis [32]. Whether changes to milk leukocyte prevalence and/or activation status due to these health conditions result in altered immune effects of breastfeeding is unclear and requires further study. The stage of lactation has perhaps the major impact on milk leukocyte content [27,32,33]. The first stage is characterized by the presence of colostrum, which, in most mammalian species, occurs during pregnancy and lasts for a few days after birth. Human transitional milk fully replaces colostrum within 3–5 days and lasts for 2 weeks postpartum. This milk has increasing amounts of lactose and fat, resulting in a higher caloric density of the milk to meet the infant’s growth demands. Mature milk replaces the transitional milk at the end of the second week, continues until it is fully mature at 4–6 weeks postpartum, and remains constant through the remainder of the lactation period [44]. In general, the quantity of immune components in breast milk diminishes drastically in parallel with the developing immune system and digestive tract [27,44]. In this regard, human colostrum has always been found to contain a considerable number of viable leukocytes [range 32 175–784 080 [31]; 8470–1 510 000 [27] per ml milk), while transitional and mature milk forms are characterized by low leukocyte content (less than one tenth of the colostral leukocyte content) when both the mother and/or the infant are healthy [27,31]. However, with infection of either the mother or the infant, the transfer of milk cells increases rapidly to reach astronomical numbers (billions) of viable leukocytes per day [31].

and pigs [48]. Of note, all these studies were conducted at the beginning of the breastfeeding period, before the functional development of the gastrointestinal (GI) tract of the infant or pup is completed. Thus, I propose that there are three major challenges that milk leukocytes must overcome during their transfer from the aerodigestive tract to their site distribution in early life tissues: milk cells must: (i) survive the conditions of the upper digestive tract; (ii) cross the intestinal barrier and enter the blood circulation; and, finally, (iii) avoid rejection by the host immune system once they reach their site distribution in infant tissues. However, in line with this, neonatal organisms have opportunely found ways to circumvent these challenges (Box 2). (See Box 3.) Demonstration of milk leukocyte trafficking to infant tissues in humans is hampered by an inability to obtain internal organ samples in a noninvasive manner for research purposes. Thus, wellconducted studies in animal models are particularly relevant and essential to study the immunology of lactation. To date, not many animal studies have investigated the role of the physiological transfer of maternal leukocytes via breast milk and its functional significance for early life immunity [8–11,15]. Traditionally, studies investigating this lactational aspect used nonphysiological inoculation protocols using nasogastric tubes or direct injection of concentrated and/or radiolabeled breast milk leukocytes into the digestive tract of starved young infant animals, such as pigs, lambs, rats, and baboons [46–48,66]. However, these methods exemplify neither the number nor the status of leukocytes physiologically found in breast milk. In addition, these studies, although interesting, have been unable to show any benefit of foster nursing on survival or immune competence in animals, because of the short time frames examined after the ingestion of milk cells. To track the physiological transfer of milk leukocytes to suckling infants and examine the functional consequence of this transfer once recipients are adults, recent studies utilized foster-nursing mouse models in which syngeneic CD45.1/ CD45.2 B6 dyads and/or transgenic (tg) dams expressing GFP were often used [8,9,15,26]. These studies demonstrated the physiological transfer of active milk leukocytes into the blood circulation of the young, and movement to, and engraftment in, different infant tissues (Figure 1). Of note, compiled data from these in vivo studies showed the transfer of very small frequencies of milk leukocyte types to early life tissues [8–11]. Indeed,

submucosal membrane sites of the body, such as the GI tract, nasopharynx, lung, eye, and skin. The role of MALT is to initiate immune responses to specific antigens encountered along all mucosal surfaces. Nasopharynx-associated lymphoid tissue (NALT): mucosal inductive site for the upper respiratory tract. Its role is to protect the body from airborne pathogens and other aerosol agents. Parietal cells: specialized cells of the gastric glands that contain an extensive secretory network from which hydrochloric acid (HCl) is secreted by active transport into the stomach. Pre-B cell stage: immature B cells that are sensitive to antigen binding. Successfully selected cells (if they do not bind self-antigen in the bone marrow) exit the marrow and become mature naive B cells. Regulatory T cells (Tregs): subset of T lymphocytes with a role in regulating or suppressing other cells in the immune system. Tregs modulate the immune system, maintain tolerance to selfantigens, and contribute to preventing autoimmune diseases. Th1: subset of helper T cells (Th cells, CD4+) that primarily produce interferon (IFN)-γ and interleukin (IL)-2; tend to generate responses against intracellular pathogens, such as bacteria and viruses. Th2: subset of helper T cells (Th cells, CD4+) that produce IL-4, IL-5, IL-6, IL-10, and IL-13, and tend to produce immune responses against helminths and other extracellular parasites. Th-antigen-presenting cells (Th-APCs): APCs that can transfer the functional antigen-MHC I and II complexes and co-stimulatory molecules, such as CD80, onto CD4+T helper cells; hence termed ‘Th-APCs’. CD4+ T helper cells that acquired the functional antigen–MHC I and II complexes and the costimulatory molecules (e.g., CD80) via APCs; hence termed Th-APCs Type IV hypersensitivity or delayedtype hypersensitivity (DTH) response: a clear example is the sensitization by subcutaneous injection of a suitable antigen, followed by challenge of the same antigen upon injection at a distant site. Given that there are only a few primed cells likely to be in any region of tissue at any given time, it takes some time for the inflammatory response to develop, hence, the term ‘delayed type hypersensitivity (DTH) response’.

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Box 2. Challenges Facing Milk Leukocytes during Their Transfer to Infant Tissues First Challenge: Surviving the Conditions of the Upper Digestive Tract Studies showed that infant saliva reacts with breast milk to produce a combination of biochemical metabolites that triggers fundamental changes in the composition of the milk [49–53]. This biochemical process may have a key role in promoting the survival of milk leukocytes by protecting these cells from acid injury while limiting the growth of pathogens [49–53]. In addition, during infancy, digestive enzymes and gastric acidity are weaker [54,55]. One of the reasons for this includes a diminished activity of parietal cells at birth [56]. Therefore, this decreased acidity together with the high buffering capacity of breast milk is a likely combination for the production of a medium of a sufficiently high pH, thereby producing a window of opportunity for milk cell survival. Second Challenge: Infiltrating the Intestinal Barrier The gut mucosa forms a physical, biochemical, and immune barrier that blocks or regulates the transfer of the gut luminal contents to the interstitial submucosal tissue. During the neonatal period, the intestinal mucosa exhibits increased permissiveness to macromolecules and cells from the gut lumen to blood [57]. Of note, it is possible that gut permeability temporarily increases beyond the neonatal period in response to the slow introduction of food, which is normally intended to complement breastfeeding beginning at ~6 months after birth [58]. It is currently unknown whether the second phase of increased gut ‘leakiness’ is another window of opportunity for maternal immunity transfer to older infants. Third Challenge: Avoiding Rejection by the Neonatal Immune System Harboring foreign cells from another individual is called microchimerism, a process that occurs during pregnancy through the placenta, and postnatally, by blood transfusion and organ transplantation [16,59]. In rodents and non-human primates, nesting of breast milk cells into pup tissues has been demonstrated, either as a long-lived cell population [16,59] or as a transient homing in neonatal organs [8]. Therefore, this phenomenon was defined as breast feeding-induced maternal microchimerism. Such a process is thought to induce tolerance to non-inherited maternal antigens (NIMAs). In response to NIMA stimulation, neonatal CD4+ T cells undergo mainly regulatory T cell differentiation that suppresses antimaternal effector T cells [16,60]. This outcome may explain why breastfed offspring become tolerant to a maternal, but not a paternal, antigen-expressing allograft [61]. Of note, this groundbreaking work goes back to that by Peter Medawar and Ray Owen [62,63], who explored the immunological basis of tissue transplantation between different strains of animals and how foreign antigens could be perceived as self if provided in utero (reviewed in [64,65]).

microchimerism is defined as the presence of low concentrations of cells that are genetically distinct from those of the host individual [67]. Examples of low breast feeding-induced maternal microchimerism include the small-scale presence of murine milk-derived IgG-secreting cells in the spleen (b1% [10]), cytotoxic T cells in Peyer’s patches (PP; b0.6% [9]), and CD4+MHCII+ cells in the thymus (b0.01% [8]). Therefore, using reliable analytical methodologies is vital for the accurate assessment of small or negligible microchimerism levels. For example, when using a fluorescence-based methodology, it is essential that selective panels of leukocyte markers are utilized in conjunction with the pan-leukocyte marker CD45 and a cell viability marker to more accurately assess the phenotypic characteristics of these populations. Although myeloid cells are prominently present in breast milk [9,27,31], to our knowledge, no evidence exists that these cells (e.g., dendritic cells and macrophages) cross into the murine pup tissues [8,9,26]. Of the variety of maternal leukocytes present in breast milk, only lymphocytes have been convincingly detected in early life organs of murine pups [8–10]. Some of the latest research focuses on the trafficking of milk B and T lymphocytes as well as the possible significance of their transmission to the infant.

Maternal B Lymphocyte Trafficking and Significance to the Infant The mammary gland (MG) is an effector site of the mammalian mucosal immune system [7]. Memory B cells and plasmablasts that colonize the gland late during pregnancy originate from the mucosaassociated lymphoid tissue (MALT), where they have been exposed to maternal antigens [7,68]. The major part of MALT is constituted by GALT, which is formed by aggregated (e.g., PPs) and isolated B cell follicles, and the nasopharynx-associated lymphoid tissue (NALT), which is mainly formed by the adenoids and the palatine tonsils. Both human and murine breast milk are 4

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Box 3. Topics for Future Research Although evidence concerning health effects of breast milk leukocytes continues to expand in terms of depth of understanding and quality of research, there are still numerous knowledge gaps in the field, as discussed herein. It is fundamentally important to understand the effects of maternally derived T lymphocytes on the development of the neonatal T cell repertoire during normal breastfeeding. Mature T cells and Tregs are generated in the thymus (a specialized primary immune organ that is largest and more active during infancy). A correlation between thymic development and breast milk feeding was suggested more than 20 years ago [106]. Moreover, evidence suggests that breast milk feeding increases T cell counts and influences the T cell compartment in human [107] and murine [108] infants. However, it is not clear whether this influence is mediated directly through the thymus [92] or indirectly through milk-derived T lymphocytes that home to the thymus. Hybrid resistance is the mechanism by which neonatal NK cells target hematopoietic-derived parental cells [109]. The model that describes this event is called the ‘missing self’ [109]. According to this model, one of the major functions of NK cells is to recognize and eliminate cells that fail to express correct self-MHC molecules. Normally breastfed infants receive ∼94 000–351 million leukocytes from breast milk on a daily basis [32]. During periods of infection of either the mother or the infant, the number of leukocytes ingested daily by breastfed infants can reach billions [32]. However, little is known about the mechanisms that prevent the infant immune system from destroying maternal milk cells, and, conversely, the mother’s leukocytes from attacking infant tissues. Understanding these immune aspects is central to the overall notion of immunity transfer between mother and infant. Understanding early life immune mechanisms associated with breastfeeding and gut mucosal protection is also an exciting area for future research. For example, further work is needed to determine the effect of milk CTLs on neonatal gut immunity. Such work would allow testing specific hypotheses that milk CTLs are beneficial, by actively contributing, in an additive or synergistic fashion, to the reduction of risk of intestinal infections; or, that maternal milk CTLs are deleterious, by contributing to an inflammatory response that can cause intestinal tissue damage of the young host. These hypotheses of ‘cooperation and conflict’ may also not be mutually exclusive depending on the conditions under which milk CTLs may have positive or negative effects on neonatal immunity and health.

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Figure 1. Physiological Transfer of Milk Lymphocytes to Foster-Nursed Murine Pups. Wild-type (WT) or mutant neonates are transferred to be continually breastfed by syngeneic or congeneic GFPtg dams until weaning. Syngeneic dyads with CD45.1 versus CD45.2 alleles are also used to discriminate between donor (dam) and recipient (infant) leukocytes.

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enriched in activated memory B cells that bear a particular profile of mucosal adhesion molecules α4β7+ α4β1+ and CD62L– [9,24,69]. Given that α4β7 and α4β1 integrins have an important role in lymphocyte trafficking to GALT and NALT, respectively [7,70–73], a functional link between the breast milk B cell compartment and the aerodigestive mucosa was suggested [71–73]. These findings are consistent with previous observations showing the presence of the vascular counterreceptor VCAM-1 (α4β1 ligand) and MAdCAM-1 (α4β7 ligand) in human and murine mammary glands [69,74]. A hypothesis was tested, proposing that, during lactation, Ig-secreting cells leave the intestinal and respiratory surfaces towards the MG [69]. Specifically, the recruitment of α4β1+ and α4β7+ Ig-secreting cells was compared with the expression of their vascular counterreceptors (VCAM-1 and MAdCAM-1] in the MG. Findings showed the expression of VCAM-1 in vessels within the MG. During mouse pregnancy and lactation, expression of MAdCAM-1 in the MG increased and α4β1 + and α4β7+ Ig-secreting cell recruitment during lactation correlated with this increase. Thus, these findings suggested that the recruitment of Ig-secreting cells to MG was mediated by VCAM-1/α4β1 + and MAdCAM-1/α4β7+ [69]. Role of Milk B Lymphocytes in Supplementing Low Neonatal IgA Production Human and murine IgA-secreting B lymphocytes predominantly colonize the MG [24,25], whereas IgG-secreting B cells are more frequent in the breast milk compartment [24,75]. It appears that a negative selection process in the mammary tissue may direct IgA-secreting B cells to remain in the MG, whereas IgG-secreting B lymphocytes attracted to this site, extravasate the mammary epithelium and enter milk secretions [24,25]. In this regard, CCL28, a mucosae-associated epithelial chemokine regulating the chemotaxis of cells expressing the chemokine receptors CCR10 and CCR3, was proposed as a key regulator of milk B cell motility [25,70]. Specifically, during lactation, CCL28 is upregulated and binds the CCR10 receptor expressed on IgA-secreting B cells in the mammary tissue [24]. As a result, most IgA-secreting B lymphocytes are retained in the gland, while other B cells, which exhibit weak or no attraction to CCL28, are able to traffic from subepithelial mammary tissue into milk [25] (Figure 2A). Direct evidence for CCL28/CCR10 involvement in this process has been shown in murine studies using in vivo treatment of dams with anti-CCL28 antibodies [25] and a CCR10-deficient mouse model [76]. Specifically, in vivo treatment with anti-CCL28 antibodies or CCR10 deficiency in the murine mother blocked IgA antibodysecreting B cell accumulation in the mouse MG, inhibiting IgA antibody secretion into milk and the subsequent appearance of the antibody in the GI tract of nursing neonates [25,76]. These results provide direct evidence of the involvement of CCL28/CCR10 [25,76] in conjunction with VCAM-1/α4β1 and MAdCAM-1/α4β7 [69] in IgA-secreting B lymphocyte homing and accumulation in vivo in the MG. A noteworthy observation is that, while most maternal IgA+ memory B cells are sequestered in the mammary tissue [25,70], large amounts of secretory (s) IgA enters the milk secretions from the breast [7,77]. These antibodies have a remarkable stability in external secretions (e.g., intestinal luminal content) and are thought to promote gut barrier function by limiting bacterial interaction with the intestinal epithelium and preventing microbial translocation to underlying tissues [77]. These Igs have also been reported to support the development of long-term intestinal homeostasis by regulating the mouse gut microbiota and epithelial cell gene expression [77]. Of note, infants may also acquire maternal protection from another process termed the ‘enteromammary pathway’ [32]. This mammalian process appears to be associated with the retrograde ductal flow of milk during breastfeeding, when a pathogen in the infant’s oral cavity may be transferred via the nipple to locally stimulate an immune response in the breast [31,78]. Once the mother’s immune system recognizes the pathogenic challenge through contact with the infant (e.g., the saliva), the mother produces specific Igs that are then delivered to the suckling infant via breast milk [32,79] (Figure 2B). 6

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Figure 2. Intertwining of the Maternal and Infant Immune System. (A) Milk lymphocytes originate from the gut-associated lymphoid tissue (GALT) and nasopharynxassociated lymphoid tissue (NALT) of the mother. Maternally derived T lymphocytes as well as IgG- (but not IgA)-secreting B lymphocytes enter the milk compartment. (B) During breastfeeding, the retrograde ductal flow may allow infant-derived pathogens to be transferred by the saliva via the nipple to stimulate the production of an antibody against the challenge, and then delivers that antibody to the infant through breastfeeding.

Role of Milk IgG-Producing Cells in Promoting Immune Homeostasis Maternal transmission of passive immunity occurs in most mammalian species and involves a specific transport of IgG from mother to offspring. The route and time point for IgG transport varies among different species, but in general, transport takes place either prenatally via the placenta, or postnatally via milk [17,80]. A study suggested that not only is IgG transmitted to progeny, but also that functional IgG-secreting cells can also be transferred to the neonate via milk [10]. A B cell-deficient (μ–/–) mouse strain was used to model B cell immunodeficiency found in humans [81]. Findings revealed the presence of splenic IgG-secreting cells in μ–/– pups born to μ–/– dams and transferred to a wild-type (WT) μ+/+ lactating foster dam at birth. This outcome suggested that maternal IgG-secreting cells were able to enter milk secretion Trends in Immunology, Month 2020, Vol. xx, No. xx

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and home to neonatal tissues (e.g., spleen), residing there until adulthood [10]. Of note, these milk cells appeared to be predominantly long-lived PCs [10]. In a seminal publication, researchers examined the issue of PC longevity and estimated the life span of these cells to be more than 1 year in mice [82]. Of note, one PC can secrete 10 000 Ig molecules per second [83]; therefore, even small numbers of transferred PCs might contribute to the production of a large amount of antibodies before their discharge from the infant’s body [83]. This, in turn, could provide the suckling infant with a sustainable and adequate amount of serum IgG for defense against early life infections [10]. However, in μ–/– mice, the B cell compartment is not occupied by endogenously produced B cells. Thus, the accumulation of transferred milk B cells might be expected to be greater in B cell-deficient (μ–/–) than in B cell-sufficient (WT) mice. This does not necessarily mean that maternal PC transmission cannot occur in WT mice, but suggests that the frequency of PC transmission will be lower in WT than in μ–/– mice [10]. However, because PCs are potent [83] and long-lived IgG producers [82], the transfer of a small number of these cells might be sufficient to supplement the pup with adequate and sustainable serum IgG for protection. Of note, the phenotype of μ–/– mice used in this study is similar to that of humans with a certain type of agammaglobulinemia. In both cases, there is no detectable progression of B cell differentiation beyond the pre-B cell stage due to a mutation in the IgM heavy chain gene, leading to profound hypogammaglobulinemia and increased susceptibility to early-life infections [81,84,85]. Based on these observations and study outcomes [10], it is tempting to speculate that the survival of pups and young infants with hypogammaglobulinemia might be facilitated by milk transmission of long-lived IgG-secreting PCs, although this possibility warrants further and robust testing. The importance of maternal IgG has traditionally been ascribed to the provision of immunity against a variety of pathogenic microbial species, thereby protecting neonates from potentially fatal infections [86]. Recent reports have now provided compelling evidence that healthy mice generate a broad IgG response to symbiotic microbial species [87,88]. In one report, B cellsufficient (μ+/+) mouse pups were foster-nursed by a B cell-deficient (μ–/–) lactating dam at birth to determine whether anticommensal IgG antibodies were acquired maternally via milk. Outcomes showed that breast milk was a primary source of anticommensal IgG and that these antibodies promoted the suppression of mucosal T helper (Th) cell responses to newly encountered symbiotic microbes, thus helping the establishment of host–microbial symbiosis during early life [87]. Furthermore, new discoveries of neonatal immune mechanisms indicate that maternal IgG is also a potent inducer of neonatal tolerance [14]. Specifically, a murine study investigated the effects of maternal allergen sensitization in the offspring and their susceptibility to developing food allergies [14]. Outcomes showed that breastfeeding by ovalbumin (OVA)-sensitized mothers was sufficient to induce oral tolerance in pups [14]. This protection was shown to be mediated by neonatal crystallizable fragment receptor (FcRn)-dependent transfer of maternal IgG and OVA immune complexes (IgG-IC) via breast milk and the induction of OVA-specific Tregs in offspring [14]. Overall, these recent advances identify milk-derived IgGs as key factors that contribute to establishing host– microbial symbiosis and protecting newborn infants against infections, and possibly, also against allergies (Figure 3, Key Figure), although this will require further investigation.

Transfer of Maternal T Cells via Breast Milk Feeding and Significance to the Infant Potential Role of Milk CD4+ T Helper Cells in Modulating T Cell-Mediated Hypersensitivity The use of nontransgenic neonates foster-nursed by syngeneic GFPtg dams enabled the detection of milk GFP+ CD4+ T lymphocytes in the thymus and spleen of suckling pups [26]. The functional significance of this transfer was subsequently examined in a mouse model of T cell-mediated immune response not involving antibodies [type IV hypersensitivity or delayed-type hypersensitivity (DTH)] [26]. Specifically, male and female B6 neonates were 8

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Key Figure

Potential Effects of Breast Milk Leukocytes on Early Life Immunity

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Figure 3. Summary of breakthroughs on the transfer of cellular immunity from mother to infant during normal breastfeeding and its functional significance in the programming of early life immunity. The text in bold highlights developments during the past 5 years. All animal findings described here were obtained from studies using inbred mouse strains that were kept in a sterile environment, a condition that does not represent how humans live. Moreover, substantial differences in immunophenotype and function have been reported between murine strains, and even greater immune discrepancies exist between murine and human species due to evolutionary differences between the two species, as well as differing cellular composition of tissues. Given these caveats, we can only speculate based on results from murine studies. Despite these shortcomings, animal experimentation remains an invaluable tool in studying pre- and postnatal immunology due to ethical concerns for humans, especially newborns.

foster-nursed by the same B6 GFPtg dams that were either sensitized or not to Candida albicans, an opportunistic microbe that causes most fungal infections in humans [89]. When the foster nursed pups reached the age of 8 weeks, they were sensitized with an intradermal injection of C. albicans into both flanks and then challenged 7 days later with C. albicans protein antigen in the footpad that was, a day later, examined for swelling as a readout of DTH response [26]. Outcomes of maternal T cell transfer via breastfeeding on the DTH response in suckling pups were surprising: nursing by a sensitized dam amplified a subsequent DTH response to C. albicans in

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females and yet suppressed it in males [26]. This gender-specific immunomodulation appears to occur in Th1- (C57BL/6 [26]) but not in Th2- (BALB/c [11]) biased animals and does not appear to be a simple function of the major male and female sex hormones, since both testosterone and 17β-estradiol are suppressive of DTH [26,90,91]. It is not yet known whether this phenomenon might occur in humans. Along these lines, a human study showed the presence of tuberculinreactive T cells in the blood of breastfed infants of tuberculin-positive mothers, but essentially none were found in the breastfed infants from tuberculin-negative mothers [13]. However, only eight out of 13 infants gained tuberculin reactivity from the positive mothers, a finding that might be explained by the other five being male infants [26]. If this speculation is proven to be true, robust clinical evaluation weighing the pros and cons of nursing male versus female children by mothers with genetically linked hypersensitivity diseases (e.g., eczema) or those in regions of the world with endemic DTH-eliciting diseases (e.g., tuberculosis) might be warranted, although this might represent a contentious and debatable matter [26]. Potential Role of Milk Th-APCs in Influencing the T Cell Repertoire Development Presence of milk T lymphocytes in the murine pup thymus suggests that these cells are able to influence the development of the T cell repertoire [26]. This hypothesis was first stipulated [15], and then later tested in two successive studies [8,11]. In the initial study [8], a new form of transferred maternal immunity by suckling an immunized dam was uncovered (Figure 3). Specifically, fosternursing of murine pups by tuberculin- immunized dams led to the development of cytotoxic (CD8+) T cells in nonimmunized foster pups that were specific for the antigen (tuberculin) against which the foster dam was immunized [8]. Outcome-based interpretations suggested that, to create this immunity, maternal Th-antigen-presenting cells (Th-APCs) (CD4+ T helper cells expressing MHC class II produced in the dam before pregnancy in response to tuberculin) travel into breast milk, are taken up by the pup, and accumulate in the thymus, where they might have an important role in educating the CD8+ T cell compartment [8]. The authors proposed a working model in which transferred milk Th-APCs to the thymus release the antigen, which is subsequently taken up by host CD8+ T cells, and loaded onto the correct pup MHC [8]. By young adulthood, all immune cells responding to the antigen against which the foster dam has been vaccinated are the product of pup thymic immune cells [8]. Given that the transferred immunity lasted well into adulthood in this study, the authors proposed that this immunity process had an effect on the development of the CD8+ T cell repertoire in response to maternal Th-APCs instruction; hence the term ‘maternal educational immunity’ was coined [8]. This is a conceptually interesting lactational process but will require further and robust validation in other animal strains and species (Box 3). If proven to be a generalized phenomenon, this maternal–infant immunity form might be central to understanding early life immune mechanisms of protection and tolerance. For example, such a process might be important for the neonatal immune adaptation to persistent intracellular pathogens that might exist in the shared maternal environment [92]. Relevant to the induction of tolerance, a murine study showed that tolerance to airborne antigens could be efficiently transferred from mother to neonate via nursing [93]. Specifically, mouse pups born from naïve mothers were foster-nursed by nonsensitized or OVA-sensitized mothers that were exposed or not, to OVA aerosols during lactation. Subsequently, 6–8-week-old fostered mice were sensitized, challenged with OVA aerosols, and analyzed for features of allergic airway disease. Outcomes revealed that exposition of sensitized mothers to OVA aerosols during lactation resulted in a profound and reproducible inhibition of allergic airway disease in the breastfed pups [93]. Nevertheless, whether maternally derived Th-APCs localized to the neonatal thymus via nursing have a role in this process (tolerance to airborne allergens) remains unknown and demands further study (Box 3). In the first demonstration of ‘maternal educational immunity’ [8], the authors used Th1-biased/MHCmatched dyads (both the dams and pups mounted robust Th1 responses to Mycobacterium 10

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tuberculosis). In a follow-up study, the authors asked two important questions: (i) did this phenomenon occur if dam and foster pups were MHC mismatched (a setting emulating the immunological interface between the human mother and her breastfed infant)?; and (ii) could maternal milk T cell transfer from a robust Th1-biased (C57BL-6/J) dam be used to improve immunity against M. tuberculosis in vulnerable Th2-biased (BALB/cJ) pups [11]? Outcomes of this study suggested that transfer of this maternal–infant immunity form also occurs in MHC-mismatched dyads and, furthermore, that long-term immunity to M. tuberculosis is more effective when acquired through breast milk of a robust Th1-biased dam than through direct immunization of some vulnerable Th2-biased pups [11]. Specifically, when compared with direct immunization of Th2-biased BALB/cJ pups, lactational transfer from a Th1-biased C57BL-6/J dam was a more efficient immunization method, because it produced a higher frequency of antigen-specific CD8+ T cells and elevated amounts of proinflammatory cytokines [e.g., interferon (IFN)γ] in foster-nursed pups [11]. If further investigated, these findings might contribute to explain, at least in part, why human breastfed infants, when compared with those fed with formula, generally have longer vaccine protection [94]; this type of phenomenon has been more evident for vaccine responses strictly dependent on T cell functionality, such as Bacillus Calmette-Guérin (BCG) responses [95]. In this regard, a study assessed the effect of breastfeeding on the immune response to BCG vaccination in human infants [95]. Their results showed that breastfeeding significantly enhanced the T cell-mediated immune response to BCG vaccine given at birth [95]. Thus, acquiring an understanding of early life immune mechanisms responsible for this new form of maternal–infant immunity represents an exciting area of research. Indeed, further work is needed to confirm the occurrence of this new lactational process in other mammalian species. If proven to be reproducible in other animal settings, it might invigorate translational work evaluating whether prepregnancy immunization in the human population might improve immunity to mycobacterial infection, a disease that kills more than 20 000 persons each day, according to the WHO Global Tuberculosis Report 2019i. Furthermore, it would be interesting to test whether wet nursing by women with robust Th1 responses might significantly enhance immunity in some vulnerable newborn infants relative to lactating women exhibiting a higher Th2 bias. Potential Role of Milk Cytotoxic CD8+ T Cells in Supporting Neonatal Gut Immunity in Defense Tasks and Homeostasis As the gestational period transitions to the early life period, the neonatal gut may be considered as a temporary, but important developmental extension to the role of the placenta during intrauterine life. At the same time, breast milk has the role of maternal blood involved in mediating and delivering maternal soluble factors and immunologically active milk cells [58]. The neonatal gut has many deficient cellular layers; for example, the lamina propria has ineffective phagocytes and lymphocytes compared with its adult counterparts: namely: (i) neonatal phagocytes are not as capable of intracellular killing of pathogens; and (ii) lymphocytes have reduced protective inflammatory functions [2,44,96]. Thus, the provision of maternal immune components via breast milk feeding are key to supplementing the inefficient neonatal gut immune defenses and promoting intestinal immune homeostasis. Homeostasis between protective inflammation and modulation of inflammation is essential to protecting the neonate against infection while limiting the intestinal damage due to inflammation. However, due to certain placental mediators (e.g., progesterone and prostaglandins), Th2-type responses are promoted during fetal stages, which then extend through the perinatal phase [97,98]. For instance, a human study showed that significantly higher concentrations of Th2 cytokines were produced during the first trimester in a healthy pregnant women group than in a group with unexplained recurrent spontaneous abortion (RSA); by contrast, significantly higher concentrations of Th1 cytokines were produced by the abortion group compared with women in the first trimester with a normal pregnancy, indicating a distinct Th2 bias in normal pregnancy and a Th1 bias in unexplained abortion [97]. Since Th1 cytokines can compromise human pregnancy and Th2 cytokines are produced at the maternal–fetal interface, it is possible that this

Clinician’s Corner In addition to providing invaluable nutritional components to infants, breast milk has an abundance of various living cells. Breastfeeding and the use of human breast milk reduce the incidence and severity of many acute and chronic illnesses, thereby reducing infant morbidity and mortality. Previously, clinicians and scientists assumed that milk leukocytes must have little biological importance for the developing immune system because it was thought that these cells could not survive in the environment of the neonatal digestive tract. Compelling evidence from numerous animal- and limited human-based studies clearly show that, through ingestion of breast milk, the transfer of maternal leukocytes continues during infancy to aid the developing immune system. In the future, it may be possible to explore the potential of maternal leukocytes in breast milk for clinical applications. For example, the survival of human infant patients with hypogammaglobulinemia (an immune disorder caused by a mutation in the IgM heavy chain gene that blocks the production of Ig-secreting B lymphocytes) might be facilitated by breast milk transmission of long-lived IgGsecreting plasma cells.

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predetermined Th2 propensity inhibits Th1 responses, improving fetal survival but impairing protective inflammatory functions of fetal and early life leukocytes, leading to tolerance [2,98]. Part of the reason for such a suppressed immune state might be to ensure that, during the critical developmental period of acquisition of adaptive immunity, the neonate is devoid of self-reactivity and, furthermore, that reactivity to symbiotic microbes does not occur [96–98]. However, this distinct feature of adaptive immunity from fetal to early life may increase the susceptibility of newborn infants to infections. A recent murine study [9] showed that help from the mother, via transfer of CD8+ cytotoxic T cells (CTLs) by feeding on breast milk, could mitigate this effect. This study utilized MHC-matched GFPtg dams that continually foster-nursed WT pups immediately postpartum [9]. Given that, in humans, the MHC in mothers is not compatible with that of their offspring due to the duality of genetic chromosomal determination from mother and father; the study was extended to examine the transfer of breast milk leukocytes into the circulation of MHC-mismatched pups (Figure 1). Outcomes showed that milk CTLs expressing the gut homing molecules CCR9 and α4β7, were taken up by intestinal PPs, survived, and were functional [9] (Box 3). Of note, it is not surprising that most milk CTLs harbor homing markers representing their GALT origin or that their site-specific distribution during normal breastfeeding is the infant GALT [9]. Functionally, transferred maternal CTLs exhibit a superior capacity to produce potent cytolytic and proinflammatory mediators [e.g., tumor necrosis factor (TNF)α, IFNγ, interleukin (IL)-18, and granzyme B] compared with those generated by breastfed pups [9]. This immunological competence is likely to contribute to the support of the inefficient gut CTLs in the growing infant because these cells are confronted by an onslaught of microbes colonizing its gastrointestinal tract (Figure 3). This murine study also established that, while most migrating milk leukocytes were CTLs, a yet unidentified CD45+ CD19– CD11b– GR1– cell subset was present and accounted for ~10% of total milk immune cells that localized to host PPs [9]. Recently, a group was able to consistently identify ILCs (these cells often reside near the lining of the aerodigestive tract [99]) in human breast milk [2,28]. Although being truly innate immune cells, ILCs have striking functional similarities with T cells and have an important role in containing infections and maintaining tissue homeostasis [100]. Furthermore, since the neonatal gut must cultivate its own microbial flora, ILCs are critical to the coevolution of the intestinal microbiome [101,102]. For example, in the healthy murine intestine, RORγt+ ILC3s are major producers of IL-22, a cytokine primarily associated with the maintenance of gut barrier function and induction of innate antimicrobial at mucosal surfaces [103]. The absence of murine IL-22-producing ILCs causes peripheral dissemination of gut commensal bacteria and systemic inflammation, which is prevented by in vivo administration of IL-22 [103]. Although the study examining breast milk leukocyte trafficking to the GI tract [9] did not directly address the possibility of maternal ILC transfer to the gut, it is reasonable to speculate that, under normal conditions of breastfeeding, milk ILCs might take up temporary residence in the gut; here, they could help protect the newborn infant from enteric infections and gut inflammation, while simultaneously regulating the establishment of the gut microbiome. These questions remain to be addressed and certainly merit further attention.

Concluding Remarks Breast milk is a dynamic living fluid, capable of modifying its composition to meet the changing immune needs of the infant. From animal- and limited human-based observations, compelling evidence suggests that leukocyte transfer from mother to infant occurs during normal breastfeeding [8–11,58,104]. These maternal immune cells are especially transmitted during the early lactation phase [7–9], a period during which the highest concentration of maternal leukocytes is present in the early milk form (colostrum), and at a time when there is low acidity and high permeability of the newborn’s GI tract [7,48,56]. Therefore, it is not surprising that low 12

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Outstanding Questions Is there an association between the transfer of milk leukocytes and pathological states of autoimmune nature? Whether production of autoreactive cells in an infant is a consequence of suckling a mother with a T cell-mediated autoimmune disease (e.g., rheumatoid arthritis and celiac disease) is unclear. Further animal and human studies should be conducted to test this possibility. Are there pros and cons of nursing male versus female children? Nursing by a sensitized murine dam amplified a subsequent DTH response in females and yet suppressed one in males. Whether this gender-specific immune modulation occurs in humans remains to be determined. What is the functional significance of milk ILCs? It is not known whether maternal ILCs transferred via breast milk take up temporary residence in an infant’s aerodigestive tract; here, they potentially aid in protecting from infections and inflammation, while regulating the establishment of the microbiome. This demands further investigation. What are the mechanisms that prevent the infant’s immune system from destroying maternal milk cells and, conversely, the mother’s leukocytes from attacking infant tissues? The functional effects of neonatal and milk leukocytes on each other and on other tissues are largely unknown and will need to be fully explored, especially in humans. Is there an association between the maturational stage of the aerodigestive tract and immunocompetence of milk leukocytes? There is a shortage of information on the functional competence of milk leukocytes, in terms of the structure and function of the GI mucosa and oral physiology. Unmasking these conjunctional aspects will be an important and interesting challenge.

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microchimerism is observed during physiological breastfeeding [8–10]. From an immunological point of view, this may be beneficial to the infant. For example, milk CTLs are potent producers of cytolytic mediators and inflammatory cytokines [9]. While the transfer of low concentrations of milk-derived CTLs may favorably supplement the immature adaptive immune system of the neonatal gut, higher microchimerism might detrimentally lead to exacerbated inflammatory responses that can cause early intestinal tissue damage. In fact, a higher presence of maternally derived cells in humans has been associated with higher rates of inflammatory diseases (e.g., maternal cells are found in a higher proportion in the blood of patients with juvenile idiopathic inflammatory myopathies compared with healthy controls [105]). In addition, considering that one PC can secrete 10 000 Ig molecules per second [83] and has a life span of more than 1 year in mice [82], the transfer of a small number of milk Ig-secreting cells might be sufficient to supplement the pup with sustainable and adequate amounts of serum Igs for defense. Overall, studies in this subfield of lactational immunology suggest that the process of breast milk-induced microchimerism results in the transfer of maternal immune information that might influence early immune responses, with short-term implications on infectious disease and allergy outcomes; this transfer might even have long-term effects on the developing T cell repertoire (Figure 3). The many functions of milk leukocytes that take up residence in early life organs constitute a fertile ground for future scientific explorations (see Outstanding Questions). In this regard, identifying and untangling the maternal–infant immune mechanisms that contribute to the infant health outcomes associated with breastfeeding will be important to advance our understanding of immune development during early infancy and may enable us to design strategies for clinical applications. For instance, breastfeeding might be used as a means to empower infant immunity through maternal sensitization or vaccination. In this regard, early tolerance induction of neonates, via breastfeeding by allergen-sensitized mothers, might be an attractive approach for primary prevention of food allergy and asthma. Moreover, establishing a program of maternal vaccination and better understanding of how maternal vaccines are transmitted or augmented by the practice of breastfeeding to the breastfed infant might be a promising approach to lessen the risk of occurrence of various infectious diseases, and prevent morbidity among newborns during the postnatal phase, until their vaccination establishes active immunity. Acknowledgements The author apologizes to all investigators whose relevant work was not included and/or discussed in-depth in this review owing to space constraints. This work was supported by the Robert Wood Johnson Foundation SG (grant # 581534 to A.L.) and the Robert Wood Johnson Foundation (grant # 67038 to the Child Health Institute of New Jersey.

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