Naïve T cells, unconventional NK and NKT cells, and highly responsive monocyte-derived macrophages characterize human cord blood

Immunobiology 219 (2014) 756–765

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Naïve T cells, unconventional NK and NKT cells, and highly responsive monocyte-derived macrophages characterize human cord blood María C. López a,∗ , Brent E. Palmer b , David A. Lawrence a a b

Wadsworth Center, New York State Department of Health, Empire State Plaza, PO Box 0509, Albany, NY 12201-0509, USA Department of Medicine, University of Colorado, Denver, CO 80262, USA

a r t i c l e

i n f o

Article history: Received 22 November 2013 Received in revised form 22 April 2014 Accepted 3 June 2014 Available online 11 June 2014 Keywords: Cord blood Cytokines IL-6 Monocytes Naïve T cells NK cells TLRs

a b s t r a c t This study compares the human immune systems of neonates and adults. Flow cytometric analysis was used to study the cellular phenotypes of cord blood (CB) and adult peripheral blood (APB). Luminex analysis was used to determine the levels of cytokines in cell culture supernatants. Our findings indicate that T cells in CB were mainly naïve and thus less responsive to PMA/ionomycin with the synthesis of cytokines. The percentages of CD3+ CD4+ CD25high and of CD3+ CD4+ CD25dim cells expressing chemokine receptors were different between CB and APB. TLR1, TLR6 and TLR9 expressions on NK and NKT cells also differed between CB and APB. CB monocyte-derived macrophages responded better than APB macrophages to TLR ligands with increased secretion of inflammatory cytokines, especially IL-6. The high levels of the inflammatory cytokines in cell culture supernatants of CB were mainly due to higher numbers of responsive macrophages, since dendritic cell numbers were lower in CB than APB. © 2014 Elsevier GmbH. All rights reserved.

Introduction It is well known that human neonates and young children are more susceptible to a myriad of infectious agents than adults (Levy et al., 2004; Velilla et al., 2006). Additionally, the neonatal immune system is usually shifted more toward Th2 than Th1 immunity (Zaghouani et al., 2009). Unfortunately, analysis of a neonate’s immune system is quite challenging due to the technical difficulties associated with working with a very small sample of peripheral blood as can be obtained from a newborn. Hence, the analysis of CB has become crucial in an attempt to understand the neonatal immune system. Several studies have shown that CB cells differ from APB cells in their expression of surface markers and in their functionality (Levy et al., 2004; Paloczi 1999). For example, Treg cells are more abundant in CB than in APB, and they are truly naïve Tregs since they have not encountered any foreign antigen (Chang et al., 2005; Tanaka et al., 2007). Beyond the focus on Treg analysis, lately, several studies have investigated the general differences

Abbreviations: APB, adult peripheral blood; CB, cord blood; HIFBS, heatinactivated fetal bovine serum; HSC, hematopoietic stem cells; PMA/ION, phorbol myristate acetate/ionomycin; TLR, toll-like receptor; PBMC, peripheral blood mononuclear cells. ∗ Corresponding author. Tel.: +1 5184864173. E-mail address: [email protected] (M.C. López). http://dx.doi.org/10.1016/j.imbio.2014.06.001 0171-2985/© 2014 Elsevier GmbH. All rights reserved.

between CB and APB in order to understand why neonates are less capable of fighting pathogens than adults (Black et al., 2013; Camacho-Gonzalez et al., 2013; Prendergast et al., 2012). It is noteworthy that CB NK cells can become activated in the transplanted recipient when they encounter remaining leukemic cells (Tanaka et al., 2007; Velardi et al., 2009). Our previous study had shown that the lymphocyte (CD45positive/side light scatter low) population of CB can be divided into two gates based upon the intensity of CD45 expression: CD45high and CD45dim (López et al., 2009). The proportions of T and B cells in the CD45high gate resembled those obtained from the single lymphocyte population of adult individuals; whereas, the CD45dim population of CB was enriched in B and NK cells (López et al., 2009). Furthermore, CD3+ T cells and CD3+ CD56+ NKT cells within the CD45dim gate of CB presented a bimodal pattern of staining for TLR4, perhaps associated with a differential response to pathogens between neonatal and adult blood (Black et al., 2013; CamachoGonzalez et al., 2013; López et al., 2009; Prendergast et al., 2012). The first aim of this study was to analyze the levels of memory and naïve T cells in CB and APB and to assess their ability to express chemokine receptors and TLRs and to secrete cytokines. The second aim was to further characterize NK and NKT cell expression of typical NK markers and TLRs. The third aim was to compare the ability of CB and APB cells to respond to TLR ligands with the secretion of cytokines. The fourth aim was to establish the phenotype of the cytokine expressing cells stimulated with TLR ligands.

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Materials and methods Cord blood and adult peripheral blood CB samples, not useful for banking due to low amount of CB or HSC/ml, were obtained from the University of Colorado Cord Blood Bank, ClinImmune Stem Cell Laboratory (Brian Freed PhD, director; Aurora, CO 80045); samples were collected into bags containing 35 ml citrate phosphate dextrose (CPD) anticoagulant solution (Baxter Healthcare code 4R0837MC, Deerfield, IL). Although collection bags always contain the same amount of anticoagulant, the amount of blood collected strictly depends on each individual CB collection, making it impossible to present data as absolute numbers of cells; therefore results are presented as percentages. Non-identified CB samples were shipped overnight at room temperature, and received less than 48 h after collection. It has been already shown in our previous study that transportation from Colorado to New York did not affect CB viability (López et al., 2009); for the current study, an aliquot of the CB was assessed on site at Colorado so that shipping and time differences could also be assessed for functional changes. The APBs of normal volunteers from Albany, New York, that were used for comparison with CB were also collected into CPD. APB samples were collected early in the morning and analyzed the following day later in the afternoon. The Institutional Review Board of the New York State Department of Health approved this study.

Blood sample staining Whole CB and APB samples were stained with fluorochromeconjugated antibodies for 15 min in the dark and at room temperature, using a non-wash procedure. At the end of the incubation, 1× BD FACSTM lysing solution (BD Bioscience, San Jose, CA) was added to the samples and incubation continued for a further 15 min under the same conditions. Then, samples were immediately run in a FACSCanto® (BD Bioscience) flow cytometer. The following antibody combinations were used for surface marker analysis: AlexaFluor 488-CD4/PE-CD45RO/PerCP-CD45/PE-Cy7-CD3/ APC-CD45RA; AlexaFluor 488-CD8/PE-CD45RO/PerCP-CD45/PEFITC-CD3/PE-CD4/PerCP-CD45/PE-Cy7Cy7-CD3/APC-CD45RA; CD197 (CCR7)/APC-CD184 (CXCR4)/APC-Cy7-CD25; FITC-CD3/ PE-CD4/PerCP-CD45/PE-Cy7-CD197 (CCR7)/AlexaFluor 647-CCR9/ APC-Cy7-CD25; AlexaFluor 488-CD8/PE-NKp30/PerCP-CD45/PECy7-CD56/AlexaFluor 647-CD3/APC-Cy7-CD16; AlexaFluor 488CD8/PE-NKp44/PerCP-CD45/PE-Cy7-CD56/AlexaFluor 647-CD3/ FITC-CD3/PE-CD8/PerCP-CD45/PE-Cy7-CD56/ APC-Cy7-CD16; APC-NKp46/APC-Cy7-CD16; FITC-CD3/PE-CD8/PerCP-CD45/PECy7-CD56/APC-CD11c/APC-Cy7-CD16; FITC-CD3/PE-CD8/PerCPFITC-CD3/PECD45/PE-Cy7-CD56/APC-CD62L/APC-Cy7-CD16; CD8/PerCP-CD45/PE-Cy7-CD56/APC-CD94/APC-Cy7-CD16; FITCCD3/PE-CD8/PerCP-CD45/PE-Cy7-CD56/AlexFluor 647-CD103/ APC-Cy7-CD16; AlexaFluor 488-CD8/PE-TLR1/PerCP-CD45/PECy7-CD56/AlexaFluor 647-CD3/APC-Cy7-CD16; FITC-TLR6/PECD16/PerCP-CD45/PE-Cy7-CD56/AlexaFluor 647-CD3/APC-Cy7CD16. All tubes contained anti-human CD45 antibody. Antibodies were always used following manufacturers’ recommendations. The complete list of antibodies used, indicating fluorochromes, clones, and manufacturer is shown in Supplementary Table 1. Fluorochrome-conjugated antibodies were obtained from BD Bioscience, Beckman-Coulter (Indianapolis, IN), eBioscience (San Diego, CA), and Imgenex (San Diego, CA). To identify dendritic cells the following antibodies were used: FITC-lineage cocktail 1 (antibodies anti: CD3, CD14, CD16, CD19, CD20, and CD56)/PECD123/PerCP-HLA DR/APC-CD11c; red cells were lysed with 1× BD FACSTM lysing solution; the cell suspension was washed after

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red cell lysis and cells were resuspended in 1× BD FACSTM lysing solution. Cultured cells were first stained with antibodies recognizing surface markers, red cells were lysed with 1× BD FACSTM lysing solution, fixed with BD Cytofix/CytopermTM for 20 min on ice, permeabilized with BD Perm/WashTM for further 20 min on ice, followed by the anti-cytokine antibody for 30 min. Afterwards, cells were washed twice with BD Cytofix/CytopermTM , and once with PBS/2% heat-inactivated fetal bovine serum (HIFBS), and finally resuspended in 1× BD FACSTM lysing solution, and run immediately. Cells cultured for the determination of cytoplasmic cytokines were stained with the following antibody combinations: FITC-CD3/PE-CD8/PerCP-CD45/PE-Cy7-CD56/APCIL-2/APC-Cy7-CD16; FITC-CD3/PE-CD8/PerCP-CD45/PE-Cy7-CD56/ APC-IL-4/APC-Cy7-CD16; FITC-CD3/PE-CD8/PerCP-CD45/PE-Cy7CD56/AlexaFluor-647-IFN-␥/APC-Cy7-CD16; FITC-CD3/PE-CD4/ PerCP-CD45/AlexaFluor 647-IFN-␥; FITC-CD3/PE-CD4/PerCPCD45/APC-IL-2; FITC-CD3/PE-CD4/PerCP-CD45/APC-IL-4; and PETNF-␣/PerCP-CD45. The same staining protocol was used to stain cells expressing cytoplasmic TLR9, with the following antibody combination: AlexaFluor 488-CD8/PE-TLR9/PerCP-CD45/PE-Cy7CD56/AlexaFluor 647-CD3/APC-Cy7-CD16. Flow cytometry analysis The FACSCanto® flow cytometer (BD Bioscience) at the Wadsworth Center is an instrument for clinical use, and it is used according to New York State CLEP regulations; instrument linearity is checked on a monthly basis and fluorescent 7-color setup beads (BD Bioscience) are used for daily calibration. All samples were run with the same forward scatter (FSC) and side scatter (SSC) conditions, and a fixed PMT voltage for each fluorochrome. The cells were first gated on SSC and CD45 expression to differentiate lymphocytes and monocytes. Flow cytometry data were collected with FACSDiva® software (BD Bioscience). CB and APB cell cultures CB and APB were centrifuged at 150 × g for 10 min to separate red cells, and the supernatants were additionally centrifuged at 330 × g for 15 min to collect all leukocytes. Cells were counted and cell suspensions were set up at final concentration of 1 × 106 cells/ml in RPMI1640 supplemented with 10% HIFBS. Three different types of cell cultures were set up. First, cells were cultured for 4 h at 37 ◦ C in the presence of 50 ng/ml phorbol 12-myristate 13acetate (PMA) (Sigma, Saint Louis, MO), 500 ng/ml ionomycin (ION) (Sigma), and 10 ␮l/ml of brefeldin A (Epicentre Biotechnologies Madison, WI) to measure the ability of T-cell subsets to synthesize IL-2, IL-4 and IFN-␥ using flow cytometric analysis. Second, cells were cultured for 24 h at 37 ◦ C in the presence of toll-like receptor (TLR) ligands to measure cytokine secretion in cell culture supernatants. TLR ligands (InVivoGen, San Diego, CA) used and final concentrations are summarized in Supplementary Table 2. Third, cells were cultured for 5 h in the presence of TLR2-TLR6 (FSL-1, Pam2CSK4) or TLR2-TLR1 (Pam3CSK4) ligands and 10 ␮l/ml of brefeldin A to measure the synthesis of TNF-␣ by monocytes using flow cytometric analysis. Luminex analysis Cell culture supernatants were analyzed using Fluorokine MAP human base kit A that allows the simultaneous determination of multiple cytokines (R&D Systems, Minneapolis, MN). Cytokines analyzed included: IL-1␣, IL-1␤, IL-6, IL-10, G-CSF and TNF-␣. Plates

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Fig. 1. Light scatter and CD45 vs. SSC plots showing the proportions of the leukocyte populations of CB (A) and APB (E). The lymphocytic and monocytic areas are enlarged to show the unique CD45high and CD45dim lymphocytic populations in CB (B) and the single population in APB (F). Light scatter plots for CB (C) and APB (G) are also shown as well as the relative percentages for each leukocytic population (D and H).

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were analyzed in a Luminex100 . Results are expressed as picograms of cytokine per ml of cell culture supernatant. Statistical analysis Data were analyzed with SigmaPlot 11.2 software (Systat Software Inc, San Jose, CA) using one-way analysis of variance or the Kruskal–Wallis one-way analysis of variance as determined by the software, followed by Tukey’s test, or Dunn’s method, when three groups (APB, CD45high and CD45dim ) were considered. The exact level of significance for the analysis of variance is presented for each figure; the tests were set up at p < 0.05. Mann–Whitney Rank Sum Test, also from SigmaPlot 11.2, was used when only two groups were compared. Results Our previous study demonstrated significant differences for several lymphocyte subsets between CB and APB (López et al., 2009). Furthermore, it was shown that plotting CD45 vs. SSC allowed defining two distinct lymphocytic populations in CB, which were CD45high and CD45dim (López et al., 2009). CD45high expression is the normal phenotype of adult lymphocytes and is routinely used with low SSC to gate lymphocytes from other leukocytes. This study follows on the same type of flow cytometric analysis for CB samples with differential gating of lymphocytes into CD45high and CD45dim populations. Dim expression of CD45 can also be observed in hematopoietic stem cells that are CD34+ either in CB or in adult bone marrow (Southerland et al., 2003). Moreover, mouse CD3+ CD4+ CD8+ thymic cells express lower levels of CD45 than either CD3+ CD4+ CD8− or CD3+ CD4− CD8+ thymocytes (López & Lawrence, data not shown), thence implying that the CD45dim SSClow population is more immature. The CD45high and CD45dim gated populations analyzed are shown in Fig. 1. CD45 vs. SSC plots for CB and APB are shown (Fig. 1A and E), and the monocyte and lymphocyte populations are enlarged to clearly differentiate CD45high and CD45dim populations in CB from the lymphocyte gate of APB (Fig. 1B and F). Light scatter (FSC vs. SSC) plots are also shown for CB and APB (Fig. 1C and G) as well as the percentages for each gated population of the representative samples (Fig. 1D and H). Dendritic cells were identified as lineage negative cells expressing either HLA-DR and CD123 or HLA-DR and CD11c (Fig. 2A), and their proportions were significantly lower in CB, as shown in Fig. 2B. There was a slight increase in the proportion of B cells in CB, as previously reported (López et al., 2009), but this increase was not significant when the CD45high and the CD45dim gate were joined together in only one gate for CB (Fig. 2C), because as seen in Fig. 1D, the total percentage of events in the CD45dim gate was low. The percentage of monocytes was significantly higher in CB than in APB (Fig. 2C). No significant immunophenotypic differences were observed between the fresh CB assessed in Colorado and the 48 h samples analyzed in Albany (data not shown), which is in agreement with our previous study (López et al., 2009). CB has fewer memory (CD45RO+ ) and more naïve (CD45RA+ ) T cells than APB Both CD45high and CD45dim populations of CD4+ (Fig. 3A) and CD8+ (Fig. 3B) T cells of CB had more CD45RA+ cells than CD45RO+ cells. This difference was statistically significant when the gated CD45high population of CB was compared with that of APB. Although the CD4 and CD8 percentages cannot be compared since different bloods were assayed, it is surprising that CD8+ , but not CD4+ , T cells of APB had more CD45RA+ cells than CD45RO+ cells. Furthermore, it has been suggested that lymphocytes with less expression of CD45

Fig. 2. Proportion of dendritic cells, B lymphocytes and macrophages in CB () and APB (). To study dendritic cells, lineage negative cells were gated on the FITClineage negative population vs. FSC plot, and the proportions of HLA DR+ CD123+ and HLA DR+ CD11c+ cells were determined (A). The proportions of both subsets of dendritic cells are shown for CB (n = 18) and APB (n = 5) (B). The proportion of macrophages gated on a CD45 vs. SSC plot and of B cells stained with CD19 are shown for CB (n = 12) and APB (n = 11) (C). Statistical analysis was done using Mann–Whitney Rank Sum Test, for dendritic cells p < 0.004; for monocytes p < 0.007.

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Fig. 3. Memory and naïve CD4+ (A) and CD8+ (B) T cells in CB and APB. The proportions of memory (CD45RO+ ) and naïve (CD45RA+ ) T cells in APB (), CB (CD45high : ), and CB (CD45dim : ) (CB n = 7, APB n = 7 for CD3+ CD4+ and CB n = 11, APB n = 10 for CD3+ CD8+ ) are shown. Statistical analysis was performed using one-way ANOVA at the p < 0.05 level, followed by either Dunn’s or Tukey analysis; * indicates significant difference from APB.

are more immature; however, surprisingly, the CD45dim populations of CD4+ and CD8+ T cells of the CB had a greater proportion of CD45RO+ cells. As described before (López et al., 2009), the CD45high population represents the majority (96%) of the lymphocytes in CB. CB cells have significantly less ability to secrete IL-2 and IFN after stimulation It is known that only cells that previously have been activated by antigen presenting cells (APCs) are effector memory T cells and can express rapidly (within 4 h) mRNA specific for a myriad of cytokines; thus, upon stimulation with PMA/ION in the presence of brefeldin A, only CD45RO+ cells should show positive staining using this technique. CB and APB cells were stimulated in vitro for 4 h, and only APB cells were capable of secreting significant amounts of cytokines, such as IL-2 and IFN-␥ (Fig. 4). Furthermore, the differences observed between CB and APB cells ability to synthesize cytokines is not the consequence of cells being transported overnight and thus losing their functionality, since no statistical differences were observed when cultures were performed at both sites (Supplementary Table 3). CB has CD3+ CD4+ CD25high cells and CD3+ CD4+ CD25dim T cells that differ from those of APB with regard to expression of chemokine receptors As shown (Fig. 3) for lymphocytes, in general, the majority of the T cells in CB are naïve since the newborn has not been exposed long enough to the multitude of antigens that can be encountered outside the womb. Therefore, it was important to investigate whether or not the CD3+ CD4+ CD25high referred to as Tregs in CB expressed the same pattern of chemokine receptors as those in APB, since the expression of chemokine receptors is required for the cells to migrate in response to a chemokine gradient. We defined CD3+ CD4+ T cells (Fig. 5A) expressing high or dim/negative CD25 (Fig. 5B). We examined the expression of CCR7 (CD197), CCR9, and

Fig. 4. T, NK and NKT cell synthesis of cytokines after short-term (4 h) culture in the presence of PMA/ION and brefeldin A [APB () n = 8; CB n = 6 (CD45high : ; CD45dim : )]. Statistical analysis was performed using one-way ANOVA at the p < 0.05 level, followed by either Dunn’s or Tukey analysis; * indicates significant difference from APB.

CXCR4 (CD184) on CD3+ CD4+ CD25high and on CD3+ CD4+ CD25dim cells. The proportion of CD3+ CD4+ CD25high expressing CCR7 or CCR9 and of CD3+ CD4+ CD25dim expressing CCR7 or CXCR4 or CCR7 and CXCR4 or CCR7 and CCR9 was higher in APB (Fig. 5C). On the contrary, when the proportion of CD3+ CD4+ CD25high cells expressing CCR7 and CCR9 was analyzed, the proportion of double positive cells was highest among the CB CD45dim cells (Fig. 5C). NK and NKT cells in CB exhibit different expression levels of NK markers and TLRs than those in APB Several studies suggested that one of the benefits of using hematopoietic stem cells from CB for transplantation is that the contaminating NK cells are more capable of destroying any remaining leukemic cells than cells from adult bone marrow implying that NK cells in CB are functionally different from APB NK cells (Velardi et al., 2009). NK cells are recognized as CD3− CD56+ cells; they can also express CD16. CD56+ CD16− NK cells secrete high levels of cytokines and have little cytotoxic activity; the opposite is true for CD56+ CD16+ NK cells (Moretta et al., 2008). The proportion of NK cells (CD3− CD56+ ) expressing CD11c, CD94 and NKp46 and those expressing CD11c or CD62L were higher in APB than those of the CD45high population in CB (Fig. 6A). On the contrary, the proportion of CD3− CD56+ CD16+ NK cells expressing NKp30 in CB was higher than those in APB (Fig. 6A). The proportion of CD3+ CD56+ NKT cells in CB expressing NKp30, NKp44 or CD103 was higher than those in APB (Fig. 6B). However, short term cultures of blood in the presence of PMA/ION and brefeldin A indicated that NKT cells of CB are much less able to synthesize IL-2 and IL-4, and NK cells

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Fig. 6. Expression of NKp30, NKp40, NKp44, CD11c, CD62L, CD94 and CD103 on NK and NKT cells. Flow cytometric analysis of NK (A) and NKT (B) cells in CB (n = 9–15) CD45high ( ), CD45dim (), and APB () (n = 5–10). Statistical analysis was done using Kruskal–Wallis one way analysis of variance on ranks followed by Dunn’s method, p < 0.05, *APB vs. either CB CD45high or CB CD45dim . Fig. 5. Expression of CD184 (CXCR4), CCR7 and CCR9 in CB and APB CD3+ CD4+ CD25high+ and CD3+ CD4+ CD25dim/− cells. In order to define Tregs, the lymphocyte gate (CD45 vs. SSC) as shown in Fig. 1 was further gated on the CD3+ CD4+ population (A), which was gated on the CD25+ and CD25− populations (B). Flow cytometric analysis of chemokine receptors of CD3+ CD4+ CD25high and CD3+ CD4+ CD25− in CB (n = 14) CD45high ( ), CD45dim () and APB () (n = 9–10) is shown (C). Statistical analysis was done using Kruskal–Wallis one way analysis of variance on ranks followed by Dunn’s method, p < 0.05, *APB vs. either CB CD45high or CB CD45dim ; # CB CD45high vs. CB CD45dim .

(Fig. 7A). On the contrary, the proportions of CD3− CD56+ and CD3− CD56+ CD16+ NK cells expressing TLR9 were lower in CB than those in APB (Fig. 7A). As shown for NK cells, the proportion of CD3+ CD56+ NKT cells expressing TLR1 or TLR6 was higher in CB than those in APB (Fig. 7B). Like NK cells, the proportion of CD3+ CD8+ T cells expressing TLR6 and TLR9 was greater and fewer in CB than in APB, respectively (Fig. 7C).

(CD3− CD56+ CD16− ) of CB did not synthesize IFN-␥ (Fig. 4), indicating generalized differences in the responsiveness of the cells in CB and in APB. It is well known that TLRs expressed by cells of the innate immune system (macrophages, neutrophils, dendritic cells, and NK cells) can recognize pathogens and initiate an immune response (Takeda and Akira, 2005; Marcenaro et al., 2008). T lymphocytes also directly recognize pathogens or pathogen-derived antigens through TLRs in the absence of antigen-presenting cells (Burton et al., 2010). Our previous work showed significant differences in the expression of TLR2 and TLR4 in T and NKT cells between CB and APB (López et al., 2009). In this report, we detected differential expression of TLR1, TLR6 and TLR9 in CB and APB. The proportions of CD3− CD56+ and of CD3− CD56+ CD16+ NK cells expressing TLR1 were higher in CB than those in APB (Fig. 7A). Moreover, the proportions of CD3− CD56+ , CD3− CD56+ CD16− and CD3− CD56+ CD16+ NK cells expressing TLR6 were higher in CB than those in APB

PBMCs of CB secrete higher levels of inflammatory cytokines after stimulation with TLR ligands than PBMCs of APB Neonates usually have an immature immune system, which is in agreement with the data of (Figs. 3 and 4), in that CB had more CD45RA+ cells than CD45RO+ cells and little ability to secrete IL-2 or IFN␥. CD3+ CD56+ NKT cells and CD3− CD56+ CD16− NK cells of CB, which are considered part of the innate immune system, also failed to synthesize certain cytokines when compared with APB cells (Fig. 4). Nonetheless, less is known about the innate immune system of newborns. Several studies have indicated that neonates are more susceptible to infections than older children and adults. This could be the consequence of an immature adaptive immune system or of an inadequate response from the innate immune system. To address the most likely scenario, we cultured mononuclear cells from CB or APB for 24 h in the presence of several TLR ligands (Supplementary Table 2), and measured cytokine secretion in the

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Fig. 7. Expression of TLR1, TLR6 and TLR9 on NK, NKT and CD3+ CD8+ T cells. Flow cytometric analysis of NK (A), NKT (B), and CD3+ CD8+ (C) T cells expressing TLR1, TLR6 and TLR9 in CB (n = 7). CD45high ( ) CD45dim (), and APB () (n = 8). Statistical analysis was done using Kruskal–Wallis one way analysis of variance on ranks followed by Dunn’s method, p < 0.05, *APB vs. either CB CD45high or CB CD45dim ; # CB CD45high vs. CB CD45dim .

cell culture supernatants (Fig. 8). It was assumed that the immune response to a TLR ligand was similar to the immune response against a pathogen capable of binding a specific TLR. After preliminary studies (data not shown), we concluded that 6 cytokines (IL-1␣, IL-1␤, IL-6, IL-10, G-CSF, and TNF-␣) could be detected in supernatants of CB and APB using the Luminex technology after cells were stimulated by TLR ligands for 24 h. Interestingly, CB supernatants had a higher level of cytokine secretion for nearly all cytokines analyzed in response to different TLR ligands (Fig. 8). The cytokine secreted in the highest amounts was IL-6 except when TLR7 ligand R837 was used and then IL-1␤ was the prominent cytokine (Fig. 8 I).

Fig. 8. Cytokine secretion levels after stimulating CB () and APB () cells with TLR2 (A–C), TLR3 (D), TLR4 (F, G), TLR5 (H), TLR7 (I), and TLR9 (E, J) ligands. Cytokine levels of CB and APB supernatants were quantified as described in the methodology. (CB n = 17, APB n = 10). Statistical differences between CB and APB cytokine levels were analyzed with the Mann–Whitney Rank Sum Test, the exact level of signification is included for each sample pairs: *p < 0.001; # p < 0.005; + p < 0.05.

In order to determine if monocyte-derived macrophages were the source of the secreted cytokines such as TNF-␣, which can also be secreted by T and NK cells, CB and APB cells were cultured for 5 h in the presence of TLR2 ligands (FSL-1, Pam2CSK4 and Pam3CSK4) and brefeldin A. This approach demonstrated detection of cytoplasmic TNF-␣ and IL-6 in macrophages using flow cytometric analysis (Fig. 9; Supplementary Fig. 1). TLRs are expressed by several types of leukocytes; therefore, it was important to establish that the increased secretion of several cytokines as shown (Fig. 8) was due at least, in part, to macrophages, and not due to increases in the proportions of dendritic cells (Fig. 2B) or B cells (Fig. 2C) in CB, or to direct stimulation of CD3+ CD4+ T cells (Fig. 9 D and E and

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Fig. 9. Macrophages synthesizing TNF-␣ and IL-6 after stimulation with TLR2 ligands. CB () and APB () cells were cultured with TLR2 ligands and the proportion of monocyte-derived macrophages secreting TNF-␣ was studied using flow cytometric analysis (A). (CB n = 12, APB n = 3). Side scatter vs. TNF-␣ plot gated on macrophages (B) shows negligible spontaneous synthesis of TNF-␣ by macrophages in culture without TLR ligands (control) and increased expression of TNF-␣ after FSL-1 stimulation (C). Histogram analysis for TNF-␣ gated on CD3+ CD4+ (light gray) and macrophages (dark gray) for control (D) and FSL-1 (E) treated cells shows that TNF-␣ is only synthesized by macrophages after FSL-1 stimulation. Mann–Whitney Rank Sum Test was used; *p < 0.05. Macrophages and not lymphocytes are responsible for the synthesis of IL-6 in culture for APB (F, H) and CB cells (G, I).

Supplementary Fig. 1). Furthermore, in our culture system macrophages are also the main source of IL-6 as shown in Fig. 9F–I. Although the percentage of macrophages synthesizing IL-6 in CB (Fig. 9I) was slightly smaller than the percentage observed in APB (Fig. 9H) there are more macrophages in CB than in APB (Fig. 2C).

Discussion This report demonstrates the existence of a significantly higher proportion of naïve T cells (CD45RA+ ) in CB than in APB. These results are in agreement with other studies (reviewed in Paloczi 1999), and especially those of Köhler et al. (2011), who have

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recently compared cord blood with the peripheral blood of mothers from a semi-urban population in Gabon, Central Africa. Interestingly, Köhler et al. (2011) found that 35.2% of the CD4+ cells in non-malaria-infected mothers expressed CD45RA, and we show that our control population had 35.8% of the CD4+ T cells expressing CD45RA, suggesting that the proportion of naïve CD4+ T cells is globally extremely well conserved in adults. Because the majority of cells in CB were demonstrated to be naïve, it was not possible to induce a recall response after stimulating these cells with PMA/ION since, as mentioned earlier; a rapid response requires presence of memory T cells. Thus, for the 4 h PMA/ION response of CB, elicitation of IL-2 and IFN-␥ synthesis was negligible. Taking into account that the fetus is usually protected from the outside world by the placenta, and therefore has not encountered any antigen and/or pathogen in normal circumstances, CD45RA+ T cells in the fetus are truly naïve assuming no uterine or nosocomial infections. This is an important difference to take into account when comparing CD45RA+ T cells of CB with those in peripheral blood that may have recently encountered antigen; but may not yet have become memory cells, although they are capable of secreting IL-2 and IFN␥ except at lower levels than CD45RO+ T cells, as described by other authors (Mascher et al., 1999). Our results, showing the presence of naïve cells together with studies showing higher numbers of Tregs in CB (Chang et al., 2005; Tanaka et al., 2007), partially explain why neonates are more susceptible to infection (Levy et al., 2004; Velilla et al., 2006), suggesting that vaccination should be considered whenever possible to promote earlier development of memory responses. Moreover, our study has shown that the proportion of Tregs and non-Treg (CD3+ CD4+ CD25dim ) T cells expressing chemokine receptors in CB and APB was different (Fig. 5C). The expression of specific chemokine receptors is required to facilitate the migration of leukocytes in response to chemokine gradients. Certain chemokines, such as CCL25/TECK, are released by intestinal epithelial cells, which favors the recruitment of CCR9expressing lymphocytes to the gut (Kunkel et al., 2000). It has been reported that CCR7+ Tregs inhibit the adoptive transfer of diabetes in mice due to their ability to migrate into the pancreas (Szanya et al., 2002). Cells expressing CXCR4 (CD184) migrate in response to SDF1/CXCL12 secreted by bone marrow cells (Wei et al., 2006). Therefore, our data suggest that neonatal CD4+ T cells have a differential ability to respond to chemokines when compared with adult T cells. The presence of higher proportions of T cells expressing several chemokine receptors suggest that CB cells are ready to migrate to tissues to initiate their terminal differentiation at effector sites. This finding could be a consequence of the naïve stage of CD4+ T cells in CB vs. memory cells in APB. Since the immune system of neonates is not fully mature, it is usually believed to be less capable of combating infections. It can be stated that neonates have a developing adaptive immune system that is in the process of recognizing antigens and switching from a naïve stage to a memory stage. This process requires antigenpresenting cells, in order to promote T cell maturation. At this early age, neonates nearly exclusively depend on their innate immune system, namely monocytes and neutrophils, when pathogens are encountered (Levy et al., 2004; Velilla et al., 2006). It is important to remember that NK cells are at the interface between the adaptive and the innate immune systems. It has been shown that the NK cells in CB present a different phenotype when compared with those in APB, and that their ability to secrete IFN-␥ is reduced (Luevano et al., 2012). The present study also showed phenotypical differences in CB NK cells (Fig. 6A), and confirms the inability of CB NK cells to secrete IFN-␥ (Fig. 4). Adult NK cells are known to be major producers of IFN-␥ (Andoniou et al., 2008). Our previous study showed an increase in the percentage of T cells (CD3+ CD4+ and CD3+ CD8+ ) expressing TLR2 in the CD45dim fraction of CB and differences in TLR4 expression on T, NK, and NKT cells (López et al., 2009). These

findings encouraged us to continue analyzing the expression of several TLRs on T, NK and NKT cells since pathogens are recognized by immune cells via their TLRs. Although, years ago it was considered that only cells in the innate immune system expressed TLRs; more recent work has shown that all cells of the immune system express TLRs, and that NK cells can recognize Mycobacterium tuberculosis through TLR2 (Marcenaro et al., 2008), as well as T lymphocytes can recognize Schistosoma mansoni eggs through TLR2 (Burton et al., 2010). Furthermore, it has recently been shown that it is possible to correlate higher proportions of lung CD8+ T cells expressing specific TLRs with pathogenesis in smokers suffering chronic obstructive pulmonary disease (Freeman et al., 2013). Thus, we propose that naïve T cells and NK cells in the neonate can recognize pathogens/antigens via TLRs, without the need for conventional antigen-presenting cells, such as dendritic cells. Interestingly, we found a decrease in the proportion of dendritic cells in CB (Fig. 2B). Furthermore, monocytes could be the main antigenpresenting cells available for the neonate, since we observed an increase in their percentage (Fig. 2C). Our results demonstrate that TLR1 and TLR6 are expressed by a higher proportion of NK and NKT cells in CB than in APB; conversely more NK cells expressed TLR9 in APB (Fig. 7A). Similar differences were observed for the expression of TLR6 and TLR9 on CD3+ CD8+ T cells (Fig. 7C). In order to mimic ex vivo the interaction between the immune system and pathogens, we cultured cells in the presence of several TLR ligands. Interestingly, cells from CB responded to this stimulation with higher secretion of cytokines than those from APB. It could be speculated that, this increased response may be required by the innate immune system to compensate for the lack of an adaptive immune system. Nevertheless, the observed increase in IL-1␤ and IL-6 secretion may not be the best choice to induce a protective response; they might induce an inflammatory response and divert the adaptive immune system preferentially toward Th2 type responses (Kollmann et al., 2009; Levy 2007). The production of IL-6 also could be a way to decrease the number of Tregs, which are increased in neonates, and to promote increased Th17 responses for transient inflammation to abrogate infection (Kimura and Kishimoto, 2010; Nyirenda et al., 2011). Fortunately, even though the stimulation of neonatal monocytes provoked a higher secretion of inflammatory cytokines, this process will likely decline since the life spans of most members of the innate immune system are considerably shorter than those of lymphocytes, especially memory T cells that can survive for years. Furthermore, memory responses are not elicited by the interaction between pathogens and innate immune cells, in the absence of an adaptive immune system. Therefore, even if the neonate survives the attack from the pathogen, the experience will not prepare the host for a second attack. Recent data shows that if the infection started in the uterus, the levels of IL-6 in cord blood can be used as predictors of early neonatal sepsis (Cernada et al., 2012). The ability of monocytes from neonates to respond in a differential fashion than monocytes from adults to the same stimuli could be crucial to define the overall immune response in neonates. A recent publication, using a different methodological approach compared neonatal and adult whole blood responses to either whole bacterium, which can signal through several TLRs, or cytokines, such as IL-6. This study indicated that phosphorylation of NF-B was enhanced in neonates thence facilitating an inflammatory immune response instead of a Th1 type protective immune response (Nupponen et al. 2013). In conclusion, our data demonstrated that T cells in CB are mainly naïve, secrete negligible levels of IL-2 and IFN-␥, and have a differential expression of chemokine receptors that will favor their migration to different sites when compared with T cells in APB, confirming that neonatal adaptive immune system is immature. Our data also show differences between the neonatal and adult innate immune system due to lower numbers of dendritic cells and

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different expression of TLR1, TLR6 and TLR9 on NK and NKT cells. Finally, CB monocytes seem to be the most TLR-responsive population, and therefore are likely the most responsive neonatal cells to pathogens with increased secretion of proinflammatory cytokines (IL-1␤, IL-6, and TNF␣), in an attempt to compensate for the lack of an adaptive immune system. Conflict of interest The authors declare no conflict of interest. Acknowledgments The authors greatly appreciate the help provided by all members of the University of Colorado Cord Blood Bank to ensure the timely shipping of cord blood specimens. The skilled technical work of Sharon Sen in Dr. Palmer’s lab is greatly appreciated. This study was funded by New York State Department of Health. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.imbio. 2014.06.001. References Andoniou, C.E., Coudert, J.D., Degli-Esposti, M.A., 2008. Killers and beyond: NK-cellmediated control of immune responses. Eur. J. Immunol. 38, 2938–2942. Black, S., Margarit, I., Rappuoli, R., 2013. Preventing newborn infection with maternal immunization. Sci. Transl. Med. 5, 1–4. Burton, O.T., Gibbs, S., Miller, N., Jones, F.M., Wen, L., Dunne, D.W., Cooke, A., Zaccone, P., 2010. Importance of TLR2 in the direct response of T lymphocytes to Schistosoma mansoni antigens. Eur. J. Immunol. 40, 2221–2229. Camacho-Gonzalez, A., Spearmen, P.W., Stoll, B.J., 2013. Neonatal infectious diseases: evaluation of neonatal sepsis. Pediatr. Clin. North Am. 60, 367–389. Cernada, M., Badia, N., Modesto, V., Alonso, R., Mejias, A., Golombek, A., Vento, M., 2012. Cord blood interleukin-6 as a predictor of early-onset neonatal sepsis. Acta Paediatr. 101, e203–e207. Chang, C.C., Satwani, P., Oberfield, N., Vlad, G., Simpson, L.L., Cairo, M.S., 2005. Increased induction of allogeneic-specific cord blood CD4+ CD25+ regulatory T (Treg) cells: a comparative study of naïve and antigenic-specific cord blood Treg cells. Exp. Hematol. 33, 1508–1520. Freeman, C.M., Martinez, F.J., Han, M.L.K., Washko Jr., G.R., McCubbrey, A.L., Chensue, S.W., Arenberg, D.A., Meldrum, C.A., McCloskey, L., Curtis, J.L., 2013. Lung CD8+ T cells in COPD have increased expression of bacterial TLRs. Respir. Res. 14 (13), http://dx.doi.org/10.1186/1465-9921-14-13. Kollmann, T.R., Crabtree, J., Rein-Weston, A., Blimkie, D., Thommai, F., Wang, X.Y., Lavoie, P.M., Furlong, J., Fortuno III, E.S., Hajjar, A.M., Hawkins, N.R., Self, S.G., Wilson, C.B., 2009. Neonatal innate TLR-mediated responses are distinct from those of adults. J. Immunol. 183, 7150–7160. Kimura, A., Kishimoto, T., 2010. IL-6: regulator of Treg/Th17 balance. Eur. J. Immunol. 40, 1830–1835. Köhler, C., Adegnika, A.A., van der Linden, R., Luty, A.J.F., Kremsner, P.G., 2011. Phenotypic characterization of mononuclear blood cells from pregnant Gabonese and their newborns. Trop. Med. Int. Health 16, 1061–1069.

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