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Cord blood proliferative responses to inhaled allergens: Is there a phenomenon?

2000; Elsevier BV; Volume: 106; Issue: 3 Linguagem: Inglês

10.1067/mai.2000.109427

1097-6825

Thomas A.E. Platts‐Mills, Judith A. Woodfolk,

Neonatal Respiratory Health Research

Lymphocytes develop in the human fetus during the first trimester, and the fetus is capable of making immune responses from about 20 weeks' gestation onward. These responses have been demonstrated against infectious organisms, such as cytomegalovirus and toxoplasmosis, but also against parasites, such as microfilaria, and antigens derived from food.116th ed. Infections of the neonatal infant. WB Saunders, Philadelphia2000: 538-543Google Scholar, 2Zeiger RS Development and prevention of allergic disease in childhood.in: 3rd ed. Allergy principles and practice. Volume II. The C.V. Mosby Co, St. Louis1988: 930-968Google Scholar Establishing that the fetus has made a response to an infection is normally based on measurement of IgM antibodies in the cord blood because IgM is not passed from the mother to the fetus. However, evidence for fetus infection with the parasite filaria has been obtained by measuring IgE antibodies (ie, another isotype that does not cross the placenta).3Weil GJ Hussain R Kumaraswami V et al.Prenatal allergic sensitization to helminth antigens in offspring of parasite-infected mothers.J Clin Invest. 1983; 71: 1124-1129Crossref PubMed Scopus (90) Google Scholar For other antigens (eg, food antigens), evidence has been obtained from antigen-specific proliferative responses of cord blood mononuclear cells in vitro.4Kobayashi Y Kondo N Shinoda S Agata H Fukutomi O Orii T Predictive values of cord blood IgE and cord blood lymphocyte responses to food antigens in allergic disorders during infancy.J Allergy Clin Immunol. 1994; 94: 907-916Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 5Szepfalusi Z Nentwich I Gerstmayr M et al.Prenatal allergen contact with milk proteins.Clin Exp Allergy. 1997; 27: 28-35Crossref PubMed Scopus (136) Google Scholar In each case, the presence of a positive response is taken as evidence that the fetus was exposed in utero.2Zeiger RS Development and prevention of allergic disease in childhood.in: 3rd ed. Allergy principles and practice. Volume II. The C.V. Mosby Co, St. Louis1988: 930-968Google Scholar, 3Weil GJ Hussain R Kumaraswami V et al.Prenatal allergic sensitization to helminth antigens in offspring of parasite-infected mothers.J Clin Invest. 1983; 71: 1124-1129Crossref PubMed Scopus (90) Google Scholar, 4Kobayashi Y Kondo N Shinoda S Agata H Fukutomi O Orii T Predictive values of cord blood IgE and cord blood lymphocyte responses to food antigens in allergic disorders during infancy.J Allergy Clin Immunol. 1994; 94: 907-916Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 5Szepfalusi Z Nentwich I Gerstmayr M et al.Prenatal allergen contact with milk proteins.Clin Exp Allergy. 1997; 27: 28-35Crossref PubMed Scopus (136) Google Scholar Recently, several groups have reported proliferative responses of cord blood mononuclear cells to inhalant allergens.6Jones A Miles E Warner J Colwell B Bryant T Warner J Fetal peripheral blood mononuclear cell proliferative responses to mitogenic and allergenic stimuli during gestation.Pediatr Allergy Immunol. 1996; 7: 109-116Crossref PubMed Scopus (247) Google Scholar, 7Prescott SL Macaubas C Holt BJ et al.Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T cell responses toward the TH2 cytokine profile.J Immunol. 1998; 160: 4730-4737PubMed Google Scholar, 8Van Duren-Schmidt K Pichler J Ebner C et al.Prenatal allergen contact with aeroallergens.Pediatr Res. 1997; 41: 1-4Crossref PubMed Scopus (91) Google Scholar Before considering the data, it is important to distinguish between food allergens and inhalants. The mother may consume as much as 100 g of cow's milk protein per day, and in keeping with this, there are clear reports of infants who have symptoms of milk allergy because of cow's milk protein passing into breast milk.9Delespesse G Sarfati M Lang G Sehon AH Prenatal and neonatal synthesis of IgE.Allergy. 1983; 18: 83-95Google Scholar There is also good evidence that infants can be symptomatically allergic to milk shortly after birth, and cord blood mononuclear cell proliferative responses to cow's milk with stimulation indices of greater than 5 have been reported.2Zeiger RS Development and prevention of allergic disease in childhood.in: 3rd ed. Allergy principles and practice. Volume II. The C.V. Mosby Co, St. Louis1988: 930-968Google Scholar, 4Kobayashi Y Kondo N Shinoda S Agata H Fukutomi O Orii T Predictive values of cord blood IgE and cord blood lymphocyte responses to food antigens in allergic disorders during infancy.J Allergy Clin Immunol. 1994; 94: 907-916Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 5Szepfalusi Z Nentwich I Gerstmayr M et al.Prenatal allergen contact with milk proteins.Clin Exp Allergy. 1997; 27: 28-35Crossref PubMed Scopus (136) Google Scholar By contrast, the daily exposure to pollen or dust mite allergens has been estimated to be approximately 10 ng per day.10Marsh DG Allergens and the genetics of allergy.in: The antigens. Volume III. Academic Press, New York1975: 271-360Google Scholar, 11Platts-Mills TAE Mitchell EB Tovey ER Chapman MD Wilkins SR Airborne allergen exposure, allergen avoidance and bronchial hyperreactivity.in: Third International SymposiumAsthma: physiology, immunopharmacology and treatment. Academic Press, Inc, London1984: 297-314Google Scholar There is no published evidence about infants being born allergic to inhalant allergens. Furthermore, there is no serologic evidence to support the idea that the fetus has made an immune response to allergens inhaled by the mother.2Zeiger RS Development and prevention of allergic disease in childhood.in: 3rd ed. Allergy principles and practice. Volume II. The C.V. Mosby Co, St. Louis1988: 930-968Google ScholarIn this issue of the Journal , Szepfalusi et al12Szepfalusi Z Pichler J Elsasser S et al.Transplacental priming of the human immune system with environmental allergens can occur early in gestation.J Allergy Clin Immunol. 2000; 106: 530-536Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar present a very impressive body of data testing the proliferation of mononuclear cells from preterm and term cord blood. The antigens they used were recombinant proteins derived from the pollen of Timothy grass (Phl p 1) and birch trees (Bet v 1). The study had two major objectives: (1) to identify at what stage in pregnancy transplacental priming to these antigens could be identified and (2) to investigate the relationship between cord blood responses and maternal exposure to the relevant antigens. The results provide an opportunity to consider two aspects of the literature on cord blood responses. First, how should in vitro proliferation data be evaluated to reach conclusions about development of an immune response or priming? Second, how convincing is the evidence that cord blood responses are related to exposure of the mother?When lymphocytes are isolated and cultured ex vivo, there are many factors that can influence proliferation. These include the density of cells in the culture well, antibiotics and other chemicals in the medium, and the serum added.13Kopp M Pichler J Halmerbauer G et al.Culture conditions for the detection of allergen-specific T-cell reactivity in cord blood: influence of cell number.Pediatr Allergy Immunology. 2000; 11: 4-11Crossref PubMed Scopus (14) Google Scholar, 14Rawle FC Mitchell EB Platts-Mills TAE T cell responses to the major allergen from the house dust mite Dermatophagoides pteronyssinus , antigen P1: comparison of patients with asthma, atopic dermatitis, and perennial rhinitis.J Immunol. 1984; 133: 195-201PubMed Google Scholar (Cultures can be carried out with FCS, autologous serum, human AB serum, or serum-free media.) There is no doubt that cord blood cells can divide rapidly as judged by stimulation indices obtained with agents such as PHA or IL-2 (see Table II in Szepfalusi et al12Szepfalusi Z Pichler J Elsasser S et al.Transplacental priming of the human immune system with environmental allergens can occur early in gestation.J Allergy Clin Immunol. 2000; 106: 530-536Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Clearly, any factor that induces production of cytokines such as IL-2 within the cultures would stimulate responses nonspecifically, and any antigen with mitogenic activity would act in a similar manner. When evaluating antigen-specific proliferative responses of cord blood cells, it is important to consider the nature of the immature T-cell repertoire. The precursor frequency of a given antigen-specific T cell in the mature repertoire has been estimated to be 1 per 105 cells, although this value may vary considerably.15Dunbar PR Ogg GS Chen J Rust N van der Bruggen P Cerundolo V Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood.Curr Biol. 1998; 8: 413-416Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar Thus when plating cells at relatively low cell numbers (1 × 105 per well), some wells may contain no antigen-specific cells purely by chance, even if the individual is immune. Assuming an immune response is mounted in utero, the lower prevalence of CD45RO memory T cells in human cord blood compared with adult cells (approximately 12% vs approximately 55%16D'Arena G Musto P Cascavilla N et al.Flow cytometric characterization of human umbilical cord blood lymphocytes: immunophenotypic features.Haematologica. 1998; 83: 197-203PubMed Google Scholar) makes it less likely that proliferative responses would be consistently present in all culture wells. Under these circumstances, evaluating responses on an individual basis depends on having an adequate number of replicates for both the antigen and control wells.17Haselden BM Kay B Larche M Immunoglobulin E–independent major histocompatibility complex-restricted T cell peptide epitope-induced late asthmatic reactions.J Exp Med. 1999; 189: 1885-1894Crossref PubMed Scopus (311) Google Scholar, 18Woodfolk JA, Sung SSJ, Benjamin DC, Lee JK, Platts-Mills TAE. Distinct human T cell repertoires mediate immediate and delayed type hypersensitivity to the Trichophyton antigen, Tri r 2. J Immunol. In press.Google Scholar Experiments with allergenic peptides derived from the cat allergen Fel d 1 and the dermatophyte antigen Tri r 2 have established the importance of using high numbers of replicates (ie, approximately 12) in standard proliferation assays. This is important for statistical evaluation both for individual responses and for comparing different experimental groups. It could be argued that this approach is not necessary for assays with intact antigen (containing multiple T-cell epitopes), which are capable of stimulating polyclonal cellular responses. Nevertheless, our experience with whole antigen has demonstrated large variations in counts per minute (cpm) among replicate wells, supporting the argument that increased numbers of replicates are required for assessing the significance of proliferative responses.19Woodfolk JA Human T cell repertoires associated with distinct immune responses: studies using recombinant proteins and antigenic peptides from Trichophyton rubrum [thesis]. University of Virginia, Charlottesville2000Google Scholar By using stimulation indices (SI) to evaluate responses, the negative results from some individuals are often expressed as less than 2, implying that there is no negative response.20Bjorksten B Holt BJ Baron-Hay MJ Munir AKM Holt PG Low-level exposure to house dust mites stimulates T-cell responses during early childhood independent of atopy.Clin Exp Allergy. 1996; 26: 775-779Crossref PubMed Scopus (33) Google Scholar, 21Upham JW Holt BJ Baron-Hay MJ et al.Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium.Clin Exp Allergy. 1995; 25: 634-642Crossref PubMed Scopus (118) Google Scholar With high variation between replicates, SI values of 2 will occur by chance, and their significance can only be assessed by comparing them with both the positive and negative responses seen in cultures from other individuals studied. An alternative approach to establishing the significance of a response is to repeat cultures over a period of time. Obviously, handling cord blood places severe limitations on either replicates or repeat cultures; however, repeat cultures have been reported under two circumstances. Miles et al22Miles EA Jones AC Warner JA Warner JO Lymphocyte proliferative responses to allergens and mitogens during the first year in babies born to atopic parents.J Allergy Clin Immunol. 1995; 95: 381Google Scholar in Southampton repeated cultures at 6 months of age, at which time the responses to mites were negative. In the present report from Vienna, in two cases cord blood was sampled twice in utero, and the cultures were negative on each occasion (Fig 2 in Szepfalusi et al12Szepfalusi Z Pichler J Elsasser S et al.Transplacental priming of the human immune system with environmental allergens can occur early in gestation.J Allergy Clin Immunol. 2000; 106: 530-536Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). An important feature of antigen-specific lymphocyte proliferation in vitro is that there are consistent dose responses. Although decreased responses at higher doses can occur, they are typically seen when the response to the lower dose of antigen is strongly positive. The data shown for samples obtained by chordocentesis do not show convincing dose responses to either of the antigens tested. In many studies T-cell proliferation data are presented as change in counts per minute (or change in disintegrations per minute) above background. At first glance, these data may look convincing; however, in at least one study, background counts of up to 20,000 cpm for cord blood cultures were obtained.13Kopp M Pichler J Halmerbauer G et al.Culture conditions for the detection of allergen-specific T-cell reactivity in cord blood: influence of cell number.Pediatr Allergy Immunology. 2000; 11: 4-11Crossref PubMed Scopus (14) Google Scholar Thus even when relatively high changes in counts per minute (ie, approximately 10,000) are reported, the results cannot be interpreted without including background data. Furthermore, the results for background counts reinforce the importance of incorporating sufficient replicates of the antigen and control cultures.In several studies the cord blood responses to dust mite allergens have been qualitatively greater than those reported with pollen allergens. However, many of these studies have used crude dust mite extracts (rather than purified antigens) and the serum-free medium Aim V.7Prescott SL Macaubas C Holt BJ et al.Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T cell responses toward the TH2 cytokine profile.J Immunol. 1998; 160: 4730-4737PubMed Google Scholar, 20Bjorksten B Holt BJ Baron-Hay MJ Munir AKM Holt PG Low-level exposure to house dust mites stimulates T-cell responses during early childhood independent of atopy.Clin Exp Allergy. 1996; 26: 775-779Crossref PubMed Scopus (33) Google Scholar, 21Upham JW Holt BJ Baron-Hay MJ et al.Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium.Clin Exp Allergy. 1995; 25: 634-642Crossref PubMed Scopus (118) Google Scholar These culture conditions have produced proliferative responses in a large proportion of tested individuals in several different populations. In Australia, using dust mite extract and serum-free medium, Upham et al21Upham JW Holt BJ Baron-Hay MJ et al.Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium.Clin Exp Allergy. 1995; 25: 634-642Crossref PubMed Scopus (118) Google Scholar concluded from proliferation data that there was universal sensitization of normal adults to mite antigens.21Upham JW Holt BJ Baron-Hay MJ et al.Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium.Clin Exp Allergy. 1995; 25: 634-642Crossref PubMed Scopus (118) Google Scholar Using the same culture conditions, lymphocytes from 18 of 19 two- to three-year-old children living in Sweden with very low mite exposure (ie, <0.6 μg/g of dust) responded in vitro.20Bjorksten B Holt BJ Baron-Hay MJ Munir AKM Holt PG Low-level exposure to house dust mites stimulates T-cell responses during early childhood independent of atopy.Clin Exp Allergy. 1996; 26: 775-779Crossref PubMed Scopus (33) Google Scholar Finally, another study showed that under the same experimental conditions, proliferation was equally common in cord blood from low-risk and high-risk neonates.7Prescott SL Macaubas C Holt BJ et al.Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T cell responses toward the TH2 cytokine profile.J Immunol. 1998; 160: 4730-4737PubMed Google Scholar Use of this serum-free medium in other laboratories has consistently suppressed specific responses, except those to mite antigen.23Slunt JB Taketomi EA Platts-Mills TAE Human T cell responses to Trichophyton tonsurans : inhibition using the serum free medium Aim V.Clin Exp allergy. 1997; 27: 1184-1192Crossref PubMed Scopus (12) Google Scholar The conclusion from these experiments appears to be that mite extracts can be mitogenic and that this effect is more marked in serum-free conditions.Using Bet v 1 and Phl p 1 as antigens makes it possible to ask directly whether the proliferative responses observed were influenced by exposure of the mother to the relevant pollen.22Miles EA Jones AC Warner JA Warner JO Lymphocyte proliferative responses to allergens and mitogens during the first year in babies born to atopic parents.J Allergy Clin Immunol. 1995; 95: 381Google Scholar As the authors recognize, the answer is clearly negative; that is, proliferative responses were no more likely to occur if the mother had been exposed. With dust mite allergen, evaluating exposure is more complex because each home is different, and seasonal variations are less well defined than pollen seasons. However, at least 3 groups have reported on the relationship between dust mite exposure at home and cord blood mononuclear cell proliferation in response to mite allergen.24Warner JO The prediction and prevention of childhood asthma.in: Asthma—a link between environment, immunology and the airways. Hogrefe & Huber Publishers, Seattle1999: 22-26Google Scholar, 25Smillie FI Elderfield AJ Cain G et al.Cord blood lymphoproliferative responses in neonates with defined atopic risk and maternal indoor allergen exposure.J Allergy Clin Immunol. 1999; 103 ([abstract]): S109Google Scholar, 26Chan-Yeung M Ferguson A Chan H et al.Umbilical cord blood mononuclear cell proliferative response to house dust mite does not predict the development of allergic rhinitis and asthma.J Allergy Clin Immunol. 1999; 104: 317-321Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar In each case the results have been negative; that is, the responses have not been greater or more common with samples coming from infants whose mothers had higher exposure at home. The conclusion at present has to be that neither for pollens nor for dust mites is there convincing evidence that the responses seen in cord blood are related to maternal exposure. It has recently been suggested that the weak responses in cord blood reflect the development of the T-cell repertoire in response to diverse antigen exposure.27Holt PG Macaubas C Stumbles PA Sly PD The role of allergy in the development of asthma.Nature. 1999; 402: B12-B17Crossref PubMed Scopus (427) Google Scholar If so, these responses may be biologically interesting, but they have not been shown to be relevant to understanding how infants become allergic.The very low quantities of allergen inhaled (ie, approximately 10 ng per day) pose serious problems in understanding how the infant receives sufficient antigen to make a response. It is possible that antigen transfer to the fetus could be facilitated (or blocked) by maternal IgG antibody. However, if maternal IgG antibody facilitated transfer of antigen to the infant, the mother would have a greater effect on the development of allergic responses than the father, and this is not so; those studies showing a larger effect of the mother's history on the development of asthma do not extend to the subsequent development of allergic responses.28Warner JA Jones CA Williams TJ Warner JO Maternal programming in asthma and allergy.Clin Exp Allergy. 1998; 28: 35-38Crossref PubMed Scopus (37) Google Scholar, 29Bergmann RL Edenharter G Bergmann KE et al.Predictability of early atopy by cord blood-IgE and parental history.Clin Exp Allergy. 1997; 27: 752-760Crossref PubMed Scopus (183) Google Scholar Certainly by the age of 8 to 10 years, when the atopic phenotype is well established, the father's allergic history has the same influence as the mother's allergic history. A simple calculation of the quantity of allergen that could possibly enter the mother's serum per day suggests that this is approximately 1 ng/L, which would be very difficult to measure. It is equally difficult to see how the infant could make an immune response to quantities of this kind.Objective evidence for an immune response to inhalant allergens is unusual under the age of 2 years. Tests that can be repeated consistently include skin tests and assays of IgG or IgE antibodies in serum.30Sporik RB Holgate ST Platts-Mills TAE Cogswell J Exposure to house dust mite allergen (Der p I) and the development of asthma in childhood: a prospective study.N Engl J Med. 1990; 323: 502-507Crossref PubMed Scopus (1437) Google Scholar, 31Rowntree S Cogswell JJ Platts-Mills TAE Mitchell EB Development of IgE and IgG antibodies to food and inhalant allergens in children at risk of allergic disease.Arch Dis Child. 1985; 60: 727-735Crossref PubMed Scopus (179) Google Scholar Indeed, the relationship between sensitization in early childhood and exposure to dust mite or cockroach allergen in homes makes it clear that allergen exposure plays a critical role in the development of allergic responses.32Platts-Mills TAE Vervloet D Thomas WR Aalberse RC Chapman MD Indoor allergens and asthma.J Allergy Clin Immunol. 1997; 100 (Co-chairmen) (Third International Workshop, Cuenca Spain): S1-S24Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar By contrast, the evidence about sensitization or priming of the fetus is unconvincing. The modest proliferation responses reported cannot be analyzed statistically because there are too few replicates and too much random variation in the system. It is always possible that proliferation or cytokine production in vitro reflects some form of weak priming related to the generation of immune diversity in the fetus; however, these events do not appear to be related to allergen exposure. At present, we would conclude, at least for inhalant allergens, that the proliferative responses are not consistent and not related to exposure of the mother, and they do not predict outcome for the infant in terms of either sensitization or disease. Given the very low quantities of allergen and the fact that objective evidence of sensitization is unusual under the age of 2 years, it is very unlikely that infants make a response to inhalant allergens in utero. Lymphocytes develop in the human fetus during the first trimester, and the fetus is capable of making immune responses from about 20 weeks' gestation onward. These responses have been demonstrated against infectious organisms, such as cytomegalovirus and toxoplasmosis, but also against parasites, such as microfilaria, and antigens derived from food.116th ed. Infections of the neonatal infant. WB Saunders, Philadelphia2000: 538-543Google Scholar, 2Zeiger RS Development and prevention of allergic disease in childhood.in: 3rd ed. Allergy principles and practice. Volume II. The C.V. Mosby Co, St. Louis1988: 930-968Google Scholar Establishing that the fetus has made a response to an infection is normally based on measurement of IgM antibodies in the cord blood because IgM is not passed from the mother to the fetus. However, evidence for fetus infection with the parasite filaria has been obtained by measuring IgE antibodies (ie, another isotype that does not cross the placenta).3Weil GJ Hussain R Kumaraswami V et al.Prenatal allergic sensitization to helminth antigens in offspring of parasite-infected mothers.J Clin Invest. 1983; 71: 1124-1129Crossref PubMed Scopus (90) Google Scholar For other antigens (eg, food antigens), evidence has been obtained from antigen-specific proliferative responses of cord blood mononuclear cells in vitro.4Kobayashi Y Kondo N Shinoda S Agata H Fukutomi O Orii T Predictive values of cord blood IgE and cord blood lymphocyte responses to food antigens in allergic disorders during infancy.J Allergy Clin Immunol. 1994; 94: 907-916Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 5Szepfalusi Z Nentwich I Gerstmayr M et al.Prenatal allergen contact with milk proteins.Clin Exp Allergy. 1997; 27: 28-35Crossref PubMed Scopus (136) Google Scholar In each case, the presence of a positive response is taken as evidence that the fetus was exposed in utero.2Zeiger RS Development and prevention of allergic disease in childhood.in: 3rd ed. Allergy principles and practice. Volume II. The C.V. Mosby Co, St. Louis1988: 930-968Google Scholar, 3Weil GJ Hussain R Kumaraswami V et al.Prenatal allergic sensitization to helminth antigens in offspring of parasite-infected mothers.J Clin Invest. 1983; 71: 1124-1129Crossref PubMed Scopus (90) Google Scholar, 4Kobayashi Y Kondo N Shinoda S Agata H Fukutomi O Orii T Predictive values of cord blood IgE and cord blood lymphocyte responses to food antigens in allergic disorders during infancy.J Allergy Clin Immunol. 1994; 94: 907-916Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 5Szepfalusi Z Nentwich I Gerstmayr M et al.Prenatal allergen contact with milk proteins.Clin Exp Allergy. 1997; 27: 28-35Crossref PubMed Scopus (136) Google Scholar Recently, several groups have reported proliferative responses of cord blood mononuclear cells to inhalant allergens.6Jones A Miles E Warner J Colwell B Bryant T Warner J Fetal peripheral blood mononuclear cell proliferative responses to mitogenic and allergenic stimuli during gestation.Pediatr Allergy Immunol. 1996; 7: 109-116Crossref PubMed Scopus (247) Google Scholar, 7Prescott SL Macaubas C Holt BJ et al.Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T cell responses toward the TH2 cytokine profile.J Immunol. 1998; 160: 4730-4737PubMed Google Scholar, 8Van Duren-Schmidt K Pichler J Ebner C et al.Prenatal allergen contact with aeroallergens.Pediatr Res. 1997; 41: 1-4Crossref PubMed Scopus (91) Google Scholar Before considering the data, it is important to distinguish between food allergens and inhalants. The mother may consume as much as 100 g of cow's milk protein per day, and in keeping with this, there are clear reports of infants who have symptoms of milk allergy because of cow's milk protein passing into breast milk.9Delespesse G Sarfati M Lang G Sehon AH Prenatal and neonatal synthesis of IgE.Allergy. 1983; 18: 83-95Google Scholar There is also good evidence that infants can be symptomatically allergic to milk shortly after birth, and cord blood mononuclear cell proliferative responses to cow's milk with stimulation indices of greater than 5 have been reported.2Zeiger RS Development and prevention of allergic disease in childhood.in: 3rd ed. Allergy principles and practice. Volume II. The C.V. Mosby Co, St. Louis1988: 930-968Google Scholar, 4Kobayashi Y Kondo N Shinoda S Agata H Fukutomi O Orii T Predictive values of cord blood IgE and cord blood lymphocyte responses to food antigens in allergic disorders during infancy.J Allergy Clin Immunol. 1994; 94: 907-916Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 5Szepfalusi Z Nentwich I Gerstmayr M et al.Prenatal allergen contact with milk proteins.Clin Exp Allergy. 1997; 27: 28-35Crossref PubMed Scopus (136) Google Scholar By contrast, the daily exposure to pollen or dust mite allergens has been estimated to be approximately 10 ng per day.10Marsh DG Allergens and the genetics of allergy.in: The antigens. Volume III. Academic Press, New York1975: 271-360Google Scholar, 11Platts-Mills TAE Mitchell EB Tovey ER Chapman MD Wilkins SR Airborne allergen exposure, allergen avoidance and bronchial hyperreactivity.in: Third International SymposiumAsthma: physiology, immunopharmacology and treatment. Academic Press, Inc, London1984: 297-314Google Scholar There is no published evidence about infants being born allergic to inhalant allergens. Furthermore, there is no serologic evidence to support the idea that the fetus has made an immune response to allergens inhaled by the mother.2Zeiger RS Development and prevention of allergic disease in childhood.in: 3rd ed. Allergy principles and practice. Volume II. The C.V. Mosby Co, St. Louis1988: 930-968Google Scholar In this issue of the Journal , Szepfalusi et al12Szepfalusi Z Pichler J Elsasser S et al.Transplacental priming of the human immune system with environmental allergens can occur early in gestation.J Allergy Clin Immunol. 2000; 106: 530-536Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar present a very impressive body of data testing the proliferation of mononuclear cells from preterm and term cord blood. The antigens they used were recombinant proteins derived from the pollen of Timothy grass (Phl p 1) and birch trees (Bet v 1). The study had two major objectives: (1) to identify at what stage in pregnancy transplacental priming to these antigens could be identified and (2) to investigate the relationship between cord blood responses and maternal exposure to the relevant antigens. The results provide an opportunity to consider two aspects of the literature on cord blood responses. First, how should in vitro proliferation data be evaluated to reach conclusions about development of an immune response or priming? Second, how convincing is the evidence that cord blood responses are related to exposure of the mother? When lymphocytes are isolated and cultured ex vivo, there are many factors that can influence proliferation. These include the density of cells in the culture well, antibiotics and other chemicals in the medium, and the serum added.13Kopp M Pichler J Halmerbauer G et al.Culture conditions for the detection of allergen-specific T-cell reactivity in cord blood: influence of cell number.Pediatr Allergy Immunology. 2000; 11: 4-11Crossref PubMed Scopus (14) Google Scholar, 14Rawle FC Mitchell EB Platts-Mills TAE T cell responses to the major allergen from the house dust mite Dermatophagoides pteronyssinus , antigen P1: comparison of patients with asthma, atopic dermatitis, and perennial rhinitis.J Immunol. 1984; 133: 195-201PubMed Google Scholar (Cultures can be carried out with FCS, autologous serum, human AB serum, or serum-free media.) There is no doubt that cord blood cells can divide rapidly as judged by stimulation indices obtained with agents such as PHA or IL-2 (see Table II in Szepfalusi et al12Szepfalusi Z Pichler J Elsasser S et al.Transplacental priming of the human immune system with environmental allergens can occur early in gestation.J Allergy Clin Immunol. 2000; 106: 530-536Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Clearly, any factor that induces production of cytokines such as IL-2 within the cultures would stimulate responses nonspecifically, and any antigen with mitogenic activity would act in a similar manner. When evaluating antigen-specific proliferative responses of cord blood cells, it is important to consider the nature of the immature T-cell repertoire. The precursor frequency of a given antigen-specific T cell in the mature repertoire has been estimated to be 1 per 105 cells, although this value may vary considerably.15Dunbar PR Ogg GS Chen J Rust N van der Bruggen P Cerundolo V Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood.Curr Biol. 1998; 8: 413-416Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar Thus when plating cells at relatively low cell numbers (1 × 105 per well), some wells may contain no antigen-specific cells purely by chance, even if the individual is immune. Assuming an immune response is mounted in utero, the lower prevalence of CD45RO memory T cells in human cord blood compared with adult cells (approximately 12% vs approximately 55%16D'Arena G Musto P Cascavilla N et al.Flow cytometric characterization of human umbilical cord blood lymphocytes: immunophenotypic features.Haematologica. 1998; 83: 197-203PubMed Google Scholar) makes it less likely that proliferative responses would be consistently present in all culture wells. Under these circumstances, evaluating responses on an individual basis depends on having an adequate number of replicates for both the antigen and control wells.17Haselden BM Kay B Larche M Immunoglobulin E–independent major histocompatibility complex-restricted T cell peptide epitope-induced late asthmatic reactions.J Exp Med. 1999; 189: 1885-1894Crossref PubMed Scopus (311) Google Scholar, 18Woodfolk JA, Sung SSJ, Benjamin DC, Lee JK, Platts-Mills TAE. Distinct human T cell repertoires mediate immediate and delayed type hypersensitivity to the Trichophyton antigen, Tri r 2. J Immunol. In press.Google Scholar Experiments with allergenic peptides derived from the cat allergen Fel d 1 and the dermatophyte antigen Tri r 2 have established the importance of using high numbers of replicates (ie, approximately 12) in standard proliferation assays. This is important for statistical evaluation both for individual responses and for comparing different experimental groups. It could be argued that this approach is not necessary for assays with intact antigen (containing multiple T-cell epitopes), which are capable of stimulating polyclonal cellular responses. Nevertheless, our experience with whole antigen has demonstrated large variations in counts per minute (cpm) among replicate wells, supporting the argument that increased numbers of replicates are required for assessing the significance of proliferative responses.19Woodfolk JA Human T cell repertoires associated with distinct immune responses: studies using recombinant proteins and antigenic peptides from Trichophyton rubrum [thesis]. University of Virginia, Charlottesville2000Google Scholar By using stimulation indices (SI) to evaluate responses, the negative results from some individuals are often expressed as less than 2, implying that there is no negative response.20Bjorksten B Holt BJ Baron-Hay MJ Munir AKM Holt PG Low-level exposure to house dust mites stimulates T-cell responses during early childhood independent of atopy.Clin Exp Allergy. 1996; 26: 775-779Crossref PubMed Scopus (33) Google Scholar, 21Upham JW Holt BJ Baron-Hay MJ et al.Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium.Clin Exp Allergy. 1995; 25: 634-642Crossref PubMed Scopus (118) Google Scholar With high variation between replicates, SI values of 2 will occur by chance, and their significance can only be assessed by comparing them with both the positive and negative responses seen in cultures from other individuals studied. An alternative approach to establishing the significance of a response is to repeat cultures over a period of time. Obviously, handling cord blood places severe limitations on either replicates or repeat cultures; however, repeat cultures have been reported under two circumstances. Miles et al22Miles EA Jones AC Warner JA Warner JO Lymphocyte proliferative responses to allergens and mitogens during the first year in babies born to atopic parents.J Allergy Clin Immunol. 1995; 95: 381Google Scholar in Southampton repeated cultures at 6 months of age, at which time the responses to mites were negative. In the present report from Vienna, in two cases cord blood was sampled twice in utero, and the cultures were negative on each occasion (Fig 2 in Szepfalusi et al12Szepfalusi Z Pichler J Elsasser S et al.Transplacental priming of the human immune system with environmental allergens can occur early in gestation.J Allergy Clin Immunol. 2000; 106: 530-536Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). An important feature of antigen-specific lymphocyte proliferation in vitro is that there are consistent dose responses. Although decreased responses at higher doses can occur, they are typically seen when the response to the lower dose of antigen is strongly positive. The data shown for samples obtained by chordocentesis do not show convincing dose responses to either of the antigens tested. In many studies T-cell proliferation data are presented as change in counts per minute (or change in disintegrations per minute) above background. At first glance, these data may look convincing; however, in at least one study, background counts of up to 20,000 cpm for cord blood cultures were obtained.13Kopp M Pichler J Halmerbauer G et al.Culture conditions for the detection of allergen-specific T-cell reactivity in cord blood: influence of cell number.Pediatr Allergy Immunology. 2000; 11: 4-11Crossref PubMed Scopus (14) Google Scholar Thus even when relatively high changes in counts per minute (ie, approximately 10,000) are reported, the results cannot be interpreted without including background data. Furthermore, the results for background counts reinforce the importance of incorporating sufficient replicates of the antigen and control cultures. In several studies the cord blood responses to dust mite allergens have been qualitatively greater than those reported with pollen allergens. However, many of these studies have used crude dust mite extracts (rather than purified antigens) and the serum-free medium Aim V.7Prescott SL Macaubas C Holt BJ et al.Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T cell responses toward the TH2 cytokine profile.J Immunol. 1998; 160: 4730-4737PubMed Google Scholar, 20Bjorksten B Holt BJ Baron-Hay MJ Munir AKM Holt PG Low-level exposure to house dust mites stimulates T-cell responses during early childhood independent of atopy.Clin Exp Allergy. 1996; 26: 775-779Crossref PubMed Scopus (33) Google Scholar, 21Upham JW Holt BJ Baron-Hay MJ et al.Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium.Clin Exp Allergy. 1995; 25: 634-642Crossref PubMed Scopus (118) Google Scholar These culture conditions have produced proliferative responses in a large proportion of tested individuals in several different populations. In Australia, using dust mite extract and serum-free medium, Upham et al21Upham JW Holt BJ Baron-Hay MJ et al.Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium.Clin Exp Allergy. 1995; 25: 634-642Crossref PubMed Scopus (118) Google Scholar concluded from proliferation data that there was universal sensitization of normal adults to mite antigens.21Upham JW Holt BJ Baron-Hay MJ et al.Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium.Clin Exp Allergy. 1995; 25: 634-642Crossref PubMed Scopus (118) Google Scholar Using the same culture conditions, lymphocytes from 18 of 19 two- to three-year-old children living in Sweden with very low mite exposure (ie, <0.6 μg/g of dust) responded in vitro.20Bjorksten B Holt BJ Baron-Hay MJ Munir AKM Holt PG Low-level exposure to house dust mites stimulates T-cell responses during early childhood independent of atopy.Clin Exp Allergy. 1996; 26: 775-779Crossref PubMed Scopus (33) Google Scholar Finally, another study showed that under the same experimental conditions, proliferation was equally common in cord blood from low-risk and high-risk neonates.7Prescott SL Macaubas C Holt BJ et al.Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T cell responses toward the TH2 cytokine profile.J Immunol. 1998; 160: 4730-4737PubMed Google Scholar Use of this serum-free medium in other laboratories has consistently suppressed specific responses, except those to mite antigen.23Slunt JB Taketomi EA Platts-Mills TAE Human T cell responses to Trichophyton tonsurans : inhibition using the serum free medium Aim V.Clin Exp allergy. 1997; 27: 1184-1192Crossref PubMed Scopus (12) Google Scholar The conclusion from these experiments appears to be that mite extracts can be mitogenic and that this effect is more marked in serum-free conditions. Using Bet v 1 and Phl p 1 as antigens makes it possible to ask directly whether the proliferative responses observed were influenced by exposure of the mother to the relevant pollen.22Miles EA Jones AC Warner JA Warner JO Lymphocyte proliferative responses to allergens and mitogens during the first year in babies born to atopic parents.J Allergy Clin Immunol. 1995; 95: 381Google Scholar As the authors recognize, the answer is clearly negative; that is, proliferative responses were no more likely to occur if the mother had been exposed. With dust mite allergen, evaluating exposure is more complex because each home is different, and seasonal variations are less well defined than pollen seasons. However, at least 3 groups have reported on the relationship between dust mite exposure at home and cord blood mononuclear cell proliferation in response to mite allergen.24Warner JO The prediction and prevention of childhood asthma.in: Asthma—a link between environment, immunology and the airways. Hogrefe & Huber Publishers, Seattle1999: 22-26Google Scholar, 25Smillie FI Elderfield AJ Cain G et al.Cord blood lymphoproliferative responses in neonates with defined atopic risk and maternal indoor allergen exposure.J Allergy Clin Immunol. 1999; 103 ([abstract]): S109Google Scholar, 26Chan-Yeung M Ferguson A Chan H et al.Umbilical cord blood mononuclear cell proliferative response to house dust mite does not predict the development of allergic rhinitis and asthma.J Allergy Clin Immunol. 1999; 104: 317-321Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar In each case the results have been negative; that is, the responses have not been greater or more common with samples coming from infants whose mothers had higher exposure at home. The conclusion at present has to be that neither for pollens nor for dust mites is there convincing evidence that the responses seen in cord blood are related to maternal exposure. It has recently been suggested that the weak responses in cord blood reflect the development of the T-cell repertoire in response to diverse antigen exposure.27Holt PG Macaubas C Stumbles PA Sly PD The role of allergy in the development of asthma.Nature. 1999; 402: B12-B17Crossref PubMed Scopus (427) Google Scholar If so, these responses may be biologically interesting, but they have not been shown to be relevant to understanding how infants become allergic. The very low quantities of allergen inhaled (ie, approximately 10 ng per day) pose serious problems in understanding how the infant receives sufficient antigen to make a response. It is possible that antigen transfer to the fetus could be facilitated (or blocked) by maternal IgG antibody. However, if maternal IgG antibody facilitated transfer of antigen to the infant, the mother would have a greater effect on the development of allergic responses than the father, and this is not so; those studies showing a larger effect of the mother's history on the development of asthma do not extend to the subsequent development of allergic responses.28Warner JA Jones CA Williams TJ Warner JO Maternal programming in asthma and allergy.Clin Exp Allergy. 1998; 28: 35-38Crossref PubMed Scopus (37) Google Scholar, 29Bergmann RL Edenharter G Bergmann KE et al.Predictability of early atopy by cord blood-IgE and parental history.Clin Exp Allergy. 1997; 27: 752-760Crossref PubMed Scopus (183) Google Scholar Certainly by the age of 8 to 10 years, when the atopic phenotype is well established, the father's allergic history has the same influence as the mother's allergic history. A simple calculation of the quantity of allergen that could possibly enter the mother's serum per day suggests that this is approximately 1 ng/L, which would be very difficult to measure. It is equally difficult to see how the infant could make an immune response to quantities of this kind. Objective evidence for an immune response to inhalant allergens is unusual under the age of 2 years. Tests that can be repeated consistently include skin tests and assays of IgG or IgE antibodies in serum.30Sporik RB Holgate ST Platts-Mills TAE Cogswell J Exposure to house dust mite allergen (Der p I) and the development of asthma in childhood: a prospective study.N Engl J Med. 1990; 323: 502-507Crossref PubMed Scopus (1437) Google Scholar, 31Rowntree S Cogswell JJ Platts-Mills TAE Mitchell EB Development of IgE and IgG antibodies to food and inhalant allergens in children at risk of allergic disease.Arch Dis Child. 1985; 60: 727-735Crossref PubMed Scopus (179) Google Scholar Indeed, the relationship between sensitization in early childhood and exposure to dust mite or cockroach allergen in homes makes it clear that allergen exposure plays a critical role in the development of allergic responses.32Platts-Mills TAE Vervloet D Thomas WR Aalberse RC Chapman MD Indoor allergens and asthma.J Allergy Clin Immunol. 1997; 100 (Co-chairmen) (Third International Workshop, Cuenca Spain): S1-S24Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar By contrast, the evidence about sensitization or priming of the fetus is unconvincing. The modest proliferation responses reported cannot be analyzed statistically because there are too few replicates and too much random variation in the system. It is always possible that proliferation or cytokine production in vitro reflects some form of weak priming related to the generation of immune diversity in the fetus; however, these events do not appear to be related to allergen exposure. At present, we would conclude, at least for inhalant allergens, that the proliferative responses are not consistent and not related to exposure of the mother, and they do not predict outcome for the infant in terms of either sensitization or disease. Given the very low quantities of allergen and the fact that objective evidence of sensitization is unusual under the age of 2 years, it is very unlikely that infants make a response to inhalant allergens in utero.