Breastfeeding modulates neonatal innate immune responses: a prospective birth cohort study.

BACKGROUND: Neonatal Toll-like receptor (TLR) responses are biased toward Th2-polarizing responses at birth and rapidly mature toward more balanced responses during the first month of life. Postnatal TLR maturation may be guided by environmental exposure. AIMS: To determine the environmental determinants of neonatal TLR function. MATERIALS AND METHODS: A prospective birth cohort study was performed in 291 healthy term neonates. Mode of delivery, breastfeeding, birth month, siblings, pets and parental smoking were analyzed in relation to neonatal innate immune parameters at the age of 1 month. Whole blood concentrations of innate immune cells were measured by flow cytometry. In vitro TLR-mediated cytokine production was determined by ELISA. RESULTS: Breastfeeding was the major determinant of neonatal innate immunity, associated with 5 (31%) of neonatal innate immune parameters, of which the association with TLR7-mediated IL-10 production was most significant (76 pg/ml in breastfed neonates vs. 293 pg/ml in formula-fed neonates, p = 0.001). Of innate immune variables, TLR3-mediated IL-12p70 production was highly associated with environmental exposures (pets, breastfeeding and mode of delivery), whereas TLR9-mediated cytokine responses were not associated with any environmental factor. CONCLUSION: Neonatal innate immune responses are differentially modulated by environmental exposure in the first month of life. The protective effect of breastfeeding against subsequent infections and atopy might be explained by its innate immune modulatory effects in the first month of life.

Low neonatal Toll-like receptor 4-mediated interleukin-10 production is associated with subsequent atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) and respiratory syncytial virus lower respiratory tract infection (RSV LRTI) are common diseases during early life. Impaired Th1-cell polarizing Toll-like receptor (TLR) responses play an important role in the pathogenesis of both diseases. Neonatal TLR-mediated production of Th1-type cytokines is decreased at birth, but rapidly increases during the first month of life. OBJECTIVE: To determine whether decreased TLR-mediated production of Th1-polarizing cytokines, at the age of 1 month is associated with subsequent AD or RSV LRTI. METHODS: A prospective healthy birth cohort study was performed. Whole blood concentrations of innate immune cells and TLR-mediated cytokine responses were measured at the age of 1 month in 291 neonates. AD was determined by a physician questionnaire at the age of 1 year and RSV LRTI was defined as parent-reported respiratory symptoms and presence of RSV RNA in a nose-throat specimen. RESULTS: Of participating neonates, 45 (15%) developed AD and 41 (14%) developed RSV LRTI. Risks of AD and RSV LRTI were not associated (χ(2) , P = 1.00). AD was associated with decreased concentrations of basophils (7.6 vs. 14.0 × 10(6) /mL, P = 0.002) and plasmacytoid dendritic cells (17.0 vs. 20.5 × 10(6) /mL, P = 0.04), increased concentrations of NK-cells (79.7 vs. 45.1 × 10(6) /mL, P = 0.03), and twofold lower TLR4-mediated IL-10 production (P = 0.001). In contrast, RSV LRTI was associated neither with neonatal concentrations of innate immune cells, nor with TLR-mediated TNF-α, IL-12p70, IL-10 or IFN-α production. CONCLUSIONS AND CLINICAL RELEVANCE: Atopic dermatitis, but not RSV LRTI, is associated with distinct pre-symptomatic differences in the innate immune system. We hypothesize that decreased neonatal IL-10-mediated immune regulation during early life might play a causal role in the initiation of AD.

Skewed pattern of Toll-like receptor 4-mediated cytokine production in human neonatal blood: low LPS-induced IL-12p70 and high IL-10 persist throughout the first month of life.

Newborns are highly susceptible to infectious diseases, which may be due to impaired immune responses. This study aims to characterize the ontogeny of neonatal TLR-based innate immunity during the first month of life. Cellularity and Toll-like receptor (TLR) agonist-induced cytokine production were compared between cord blood obtained from healthy neonates born after uncomplicated gestation and delivery (n=18), neonatal venous blood obtained at the age of one month (n=96), and adult venous blood (n=17). Cord blood TLR agonist-induced production of the Th1-polarizing cytokines IL-12p70 and IFN-alpha was generally impaired, but for TLR3, 7 and 9 agonists, rapidly increased to adult levels during the first month of life. In contrast, TLR4 demonstrated a slower maturation, with low LPS-induced IL-12p70 production and high IL-10 production up until the age of one month. Polarization in neonatal cytokine responses to LPS could contribute to neonatal susceptibility to severe bacterial infection.

Identification of strong interleukin-10 inducing lactic acid bacteria which down-regulate T helper type 2 cytokines.

BACKGROUND: Decreased exposure to microbial stimuli has been proposed to be involved in the increased prevalence of atopic disease. Such a relationship was indicated by enhanced presence of typical probiotic bacteria in the intestinal flora correlating with reduced prevalence of atopic disease. Recent clinical trials suggested that probiotic bacteria may decrease and prevent allergic symptoms, but which (different) species or strains may contribute is poorly understood. OBJECTIVE: We sought to select probiotic bacteria by their ability to modulate in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs), to make a rational choice from available strains. METHODS: PBMCs, purified monocytes, and lymphocytes from healthy donors were co-cultured with 13 different strains of probiotic bacteria. The effect of lactic acid bacteria (LAB) on different cell populations and effects on cytokine production induced by the polyclonal T cell stimulator phytohaemagglutinin (PHA) was evaluated by measuring T helper type 1, T helper type 2 (Th2), and regulatory cell cytokines in culture supernatants by multiplex assay. RESULTS: PBMCs cultured with different strains produced large amounts of IL-10 and low levels of IL-12p70, IL-5, and IL-13. In PHA-stimulated PBMC cultures, the tested strains decreased the production of Th2 cytokines. Neutralizing IL-10 production resulted in partial to full restoration of Th2 cytokine production and concurred with an increase in pro-inflammatory cytokines such as IL-12p70 and TNF-alpha. Within the PBMCs, the CD14(+) cell fraction was the main source of IL-10 production upon interaction with LAB. CONCLUSION: Our results indicate that certain strains of lactobacilli and bifidobacteria modulate the production of cytokines by monocytes and lymphocytes, and may divert the immune system in a regulatory or tolerant mode. These specific strains may be favorable to use in prevention or treatment of atopic disease.

Isolation of an immunodominant viral peptide that is endogenously bound to the stress protein GP96/GRP94.

Heat shock protein gp96 primes class I restricted cytotoxic T cells against antigens present in the cells from which it was isolated. Moreover, gp96 derived from certain tumors functions as an effective vaccine, causing complete tumor regressions in in vivo tumor challenge protocols. Because tumor-derived gp96 did not differ from gp96 isolated from normal tissues, a role for gp96 as a peptide carrier has been proposed. To test this hypothesis, we analyzed whether such an association of antigenic peptides with gp96 occurs in a well-defined viral model system. Here we present the full characterization of an antigenic peptide that endogenously associates with the stress protein gp96 in cells infected with vesicular stomatitis virus (VSV). This peptide is identical to the immunodominant peptide of VSV, which is also naturally presented by H-2Kb major histocompatibility complex class I molecules. This peptide associates with gp96 in VSV-infected cells regardless of the major histocompatibility com- plex haplotype of the cell. Our observations provide a biochemical basis for the vaccine function of gp96.

The role of self peptides in the allogeneic cross-reactivity of CTLs.

This study presents data relevant to understanding the molecular and structural basis for the cross-reactivity of many CTLs on multiple MHC targets. Five anti-H-2Kb alloreactive CTL clones derived from B6.C-H-2bm1 (bm1), B6.C-H-2bm8 (bm8), and B6.C-H-2bm11 (bm11) mice and a Sendai virus-specific H-2Kb-restricted CTL clone were studied. Self peptides extracted from Kb molecules were fractionated by HPLC and tested for their ability to be recognized on RMA/s (H-2b) cells by those clones. For each alloreactive clone, a single dominant peptide peak was found to sensitize target cells. In addition to recognizing peptides presented by the Kb molecule, the five alloreactive clones and the one Sendai virus-specific clone all showed cross-reactivities on a panel of Kbm mutant cells in a peptide-dependent manner. Two CTL clones, one alloreactive and one virus specific, cross-recognized Kbm targets by each responding to a unique self peptide in the context of the mutant MHC molecules. Our data underscore the prevalent idea that TCR-alpha beta have an inherent structural capability to react with several peptide/MHC structural patterns other than the original peptide/MHC pattern that might have been used to select that TCR. The high incidence of cross-reactivity seems to reflect a feature of the mechanism of positive selection in the thymus and the need for T cells in the repertoire to have an expanded capability for responding to a wide variety of foreign Ags.

Diversity of T cell receptors specific for the VSV antigenic peptide (N52-59) bound by the H-2Kb class I molecule.

As an approach to determine the structural basis of interactions between T cell receptors (TCRs) and MHC class I/peptide complexes, the fine specificities of a panel of vesicular stomatitis virus (VSV)-specific CTL clones recognizing the antigenic peptide (nucleoprotein 52-59) and the class I (Kb) molecule were correlated with the TCR primary structure. Each TCR showed a distinct interaction pattern with N52-59 and the Kb molecule. The large majority of the TCRs expressed by the panel of CTL clones used V beta 13 gene segments that had randomly recombined with D beta and J beta gene segments. The alpha chains were from randomly assorted V alpha and J alpha gene segments. Thus, the panel was found to be a highly heterogeneous set of TCRs, each member of which appeared to have an unique surface interface area, the recognition site, that interacted with a complementary surface formed by the single peptide bound in the class I antigenic groove.

Differential binding of a minor histocompatibility antigen peptide to H-2 class I molecules correlates with immune responsiveness.

Minor histocompatibility (H) Ag are recognized in the context of MHC class I (K/D) molecules and can constitute a strong barrier to tissue transplantation. The products encoded by the MHC (H-2 in mice) have been shown recently to be Ag-presenting molecules that bind foreign and self peptides in their peptide binding sites. Different class I molecules preferentially bind different arrays of endogenous peptides for presentation to CTL. Previous studies showed that the Kb-restricted CTL response to one minor histocompatibility Ag, H-4, varied in different Kb mutants. One possible basis for this variation might be that the response is regulated by the level of binding of an H-4 peptide by class I molecules. To analyze this possibility, we initiated studies to identify the H-4 peptide that is included in the array of self peptides. The complete mixture of peptides eluted from Kb molecules of H-4+ tumor cells was able to sensitize H-4- targets for lysis by H-4-specific CTL. The presence of a specific H-4 peptide was confirmed when the radiolabeled peptide mix eluted from Kb molecules was separated by HPLC. Only one peak in the profile of H-4+ tumor peptides was capable of sensitizing H-4- targets for lysis by H-4-specific CTL; this active peak was absent in H-4 mutant cells. Peptide mixtures eluted from a panel of Kb mutant molecules extracted from H-4+ lymphocytes varied in their capacities to sensitize targets for H-4-specific lysis and the relative sensitization correlated with the demonstrated capacity of mutant mice to generate H-4-specific CTL. Therefore, the highly specific genetic control of the T cell response to the H-4 minor histocompatibility Ag appears to be due to the differential binding of an H-4 peptide by class I molecules.

Presentation of antigenic peptides by MHC class I molecules.

The basis for the immune response against intracellular pathogens is the recognition by cytotoxic T lymphocytes of antigenic peptides derived from cytosolic proteins, which are presented on the cell surface by major histocompatibility complex (MHC) class I molecules. The understanding of MHC class I-restricted peptide presentation has recently improved dramatically with the elucidation of the structural basis for the specificity of peptide binding to MHC class I molecules and the identification of proteins encoded in the class II region of the MHC that are putatively involved in the production of peptides and their transport into the endoplasmic reticulum, where they assemble with class I molecules.

Synthetic peptide libraries in the determination of T cell epitopes and peptide binding specificity of class I molecules.

Major histocompatibility complex (MHC) class I molecules combine with short peptides of defined length and sequence. Here we describe an approach that may be used in the analysis of peptide preference of different allelic MHC class I molecules, and in the determination of T cell epitopes. We produced synthetic "peptide libraries" of limited complexity by standard peptide chemistry. Using these peptide mixtures we show that H-2 Kb molecules can accommodate both 8- and 9-residue peptides, whereas Db molecules are unable to combine with peptides shorter than 9 amino acids present in these libraries. When these peptide mixtures are used to provide "fingerprints" of Db molecules and mutants thereof, both loss and gain of the ability to combine with certain peptides is observed. For the Kbm1 mutant a strong influence of amino acid substitutions in class I molecules on the peptides selected is observed. In these synthetic peptide mixtures, the presence of a specific T cell epitope, known to be represented once, can be detected. This approach may be extended to the identification of new T cell epitopes from larger peptide libraries.

Vesicular stomatitis virus antigenic octapeptide N52-59 is anchored into the groove of the H-2Kb molecule by the side chains of three amino acids and the main-chain atoms of the amino terminus.

This study describes an analysis of the interaction of individual amino acid residues of the vesicular stomatitis virus (VSV) nucleocapsid antigenic octapeptide (N52-59; Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu) with the H-2Kb molecule and T-cell receptors (TCRs). Tyr-3, Tyr-5, and Leu-8 were the positions in the peptide found to be H-2Kb contact residues by analyzing single alanine-substituted peptides in a competition assay with a Kb-restricted antigenic nonapeptide of Sendai virus. Arg-1, Gly-2, Val-4, Gln-6, and Gly-7 of the peptide were identified as putative TCR contact residues by testing the peptide analogs for their capacity to sensitize targets for VSV-specific cytolytic T-lymphocyte clones. The octamer N52-59 was the optimal length of the peptide required for binding to Kb. This peptide length requirement and the finding of an irregular interspersing of major histocompatibility complex and TCR contact residues are most consistent with the conclusion that the peptide is in an extended conformation in the antigen binding groove. Furthermore, data on binding of truncated peptides show that, although the Arg-1 side chain has been assigned as a TCR contact residue, the main-chain atoms of the N-terminal amino group are most likely involved in interacting with the major histocompatibility complex molecule. A panel of H-2Kb point mutants was constructed to explore the effect of altered amino acid residues on the binding of N52-59. Mutants with amino acid substitutions along the floor of the groove all bound the VSV peptide but modulated its interaction with Kb, apparently causing subtle changes in the spatial arrangement of some specific TCR contact residues in the peptide.

The structure of the antigen-binding groove of major histocompatibility complex class I molecules determines specific selection of self-peptides.

We have examined the effect of diversity in the antigen-binding groove of the Kb, Db, Kbm1, and Kbm8 major histocompatibility complex (MHC) class I molecules on the set of self-peptides they present on the cell surface, by using a procedure we recently developed in our laboratory to isolate endogenously processed peptides bound to MHC class I molecules. We found that such naturally processed peptides are 7-10 amino acids long. A major motif of tyrosine and phenylalanine residues at positions three and five was found for peptides binding to Kb. The availability of Kb mutant molecules Kbm1 and Kbm8, each with localized clustered changes in the antigen-binding cleft, allowed us to probe the effect of such small alterations on peptide selection. We found that such changes in different regions in the antigen-binding groove exert an absolute effect by changing subsets of self-peptides bound to these MHC molecules. In the Kbm1 mutant, the binding of the characteristic major set of Kb-associated peptides with tyrosine at position three or both positions three and five is abrogated, although this MHC molecule still binds peptides with tyrosine at position seven; the latter peptides also bind to Kb. Kbm8 shares the major Tyr-3, Tyr-5 peptide set that binds to Kb but does not bind the peptides with tyrosine at position seven. Thus differences in binding selectivity in Kbm1 and Kbm8 appear to be the major determinant for the observed alterations in in vivo immune responses.

Isolation of an endogenously processed immunodominant viral peptide from the class I H-2Kb molecule.

Using an approach for isolating and characterizing peptide fractions that are intracellularly associated with major histocompatibility complex class I molecules, the major peptide recognized by cytotoxic T cells specific for the vesicular stomatitis virus has been isolated from the H-2Kb molecule of infected cells. This endogenously processed octapeptide is allele-specific as it does not bind to H-2Db molecules, and contains the core sequence of the epitope of the nucleocapsid protein of the vesicular stomatitis virus identified by testing with exogenous synthetic peptides.

Specificity of anti-H-2 class I antibodies induced by syngeneic immunization with Sendai virus-treated cells is regulated by the mouse MHC and viral antigens. No evidence for MHC-restricted virus-specific antibodies.

In a previous study, we searched for Sendai virus (SV)-specific antibodies that were restricted in their binding by self-major histocompatability complex (MHC) antigens. In C57BL/6 (B6; H-2b) mice, most of the sera obtained after i.p. injections with syngeneic SV-coated (SV+) spleen cells contained auto- and alloreactive lymphocytotoxic antibodies directed against H-2 class I molecules, but no viral-specific, MHC-restricted antibodies. Here we report that syngeneic immunization with SV+ cells regularly induced H-2-specific antibodies in various mouse strains. From a total of 12 strains tested, only the B10.S (H-2s) strain appeared to be a low responder. The immune responses are of two types: (i) mice of some strains produce autoreactive antibodies and a broad variety of alloreactive antibodies; and (ii) mice of some strains produce only narrow or widely alloreactive antibodies. Because most of the strains differ only in the H-2 region, the patterns observed are regulated by the MHC. To locate the genes involved in the induction of H-2-specific antibodies more precisely, two B6 mutant strains, bm1 (Kb mutant) and bm13 (Db mutant), were immunized with syngeneic SV+ cells. The results suggest that the H-2Db region plays an important role in the induction and specificity of the lymphocytotoxic H-2 class I-specific antibodies present in sera of H-2b mice after syngeneic immunization with SV+ cells. The role of SV in the induction of H-2-specific antibodies was studied in B6 mice after injections of syngeneic cells coated with liposomes bearing the F and HN proteins of SV. The results suggest that SV surface glycoproteins as well as internal proteins are directly involved in regulating the specificity of anti-H-2 antibodies present in sera after syngeneic immunization with SV+ cells. This study does not support the concept that antigen-specific, MHC-restricted antibodies are a part of the B-cell repertoire.