The present prospective study, approved by the Ethics Review Board of the Ottawa Hospital, is derived from an ongoing birth-cohort study of the influence of indoor environmental factors on respiratory illness during the first 2 years of life. The study began in 1997 and is being conducted in the province of Prince Edward Island, Canada, which has a population of approximately 150,000. All physicians who practice obstetrics in the province participated in the study. Women in the third trimester of pregnancy received letters from their physicians' offices describing the study and requesting participation. Those who expressed interest to their doctor were contacted by telephone by a member of the research team to obtain informed consent. Excluded from the study are babies born more than 4 weeks prematurely, those with neonatal respiratory difficulties requiring prolonged hospitalization at birth, and those whose families expect to change residence within 2 years of the child's birth. Because of resource constraints, we recruited approximately 40 consecutive newborns each year. Baseline socio-demographic information and family histories were obtained.

The participating parents maintained a daily symptom diary on large multipurpose calendars on which they recorded the presence or absence of symptoms of respiratory illness in the index child. For this study, four symptoms defining a respiratory-illness event were tracked daily: stuffy nose, coughing, wheezing, and shortness of breath. "Wheezing" referred to audible high-pitched musical sounds during breathing, and shortness of breath was defined by the parents' perception of rapid or laboured breathing. Each study family was phoned every 2 weeks to document information from the diary. If the parents had omitted to record symptoms on a daily basis, they provided information for that 2-week period based on recall.

We used the method of Samet to define a respiratory illness event [2]. A respiratory illness event began when 2 consecutive days with at least one of the above four symptoms occurred and ended when there were 2 consecutive symptom-free days. This was done to identify discrete acute illness events as opposed to persistent ongoing symptoms such as a chronically runny nose. There were no doctors' visits, physical examinations, cultures, or blood tests to confirm an infectious event, nor did we collect information on nonrespiratory illness events. The primary outcome of interest was the number of respiratory illness events per year averaged over the 2 years of follow-up.

Lymphocyte phenotyping was performed in participating children at the age of 2 years. Peripheral blood samples were collected in a 5 mL Vacutainer containing ethylenediaminetetraacetic acid. One hundred microlitres of whole blood were incubated with 5 μL of fluorescein isothiocyanate (FITC), phycoerythrin (PE) or phycoerythrincyanin 5.1 (PC5) anti-CD3 (UCHT1), CD4 (13B8.2), CD8 (B9.11), HLA-DR (Immu-35), CD38 (T16), CD25 (B1.49.9), CD45RA(ALB11), CD45RO (UCHL1), CD62L (DREG56) (all from Beckman Coulter, Fullerton, CA, USA), anti- CD69 (L78), and CD29 (MAR4) (Pharmingen, Mississauga, ON) mABs for 25 minutes. The antibody combinations were as follows: CD3-FITC/CD4-PE/CD8-PC5, CD4-PE/HLA-DR-PC5/CD38-FITC, CD8-PE/HLA-DR-PC5/CD38-FITC, CD4-PE/CD25-PC5/CD69-FITC, CD8-PE/CD25-PC5/CD69-FITC, CD4-PC5/CD45RA-P/CD62L-FITC, CD8-PC5/CD45RA-P/CD62L-FITC, CD4-PC5/CD45RO-FITC/CD29-PE, and CD8-PC5/CD45RO-FITC/CD29-PE. Autofluorescence and colour compensation tubes were also included.

The samples were then lysed with the Immuno-Prep reagent system on a Coulter Multi-Q-Prep workstation. Ten thousand lymphocytes were acquired on a Beckman Coulter EPICS ALTRA flow cytometer, and the data was analyzed with EXPO2 analysis software. Lymphocyte subsets were distinguished from nonlymphoid cells by light scattering profiles [3], and the expression of the various cell surface molecules was determined on CD4⁺ and CD8⁺ T cells.

The illness rate per year was correlated with clinical data, and the percentage of lymphocytes expressing one or more cell surface antigens was measured at age 2 years with Pearson's correlation coefficient. Results were then adjusted for any characteristics that were either associated with the incidence of illness or the lymphocyte subpopulation distributions. Multivariate analysis was not performed, and because this was an exploratory study, p values were not corrected for the multiple comparisons.

With a greater appreciation of the significance of various lymphocyte subpopulations and the availability of monoclonal antibodies to identify distinct subsets, characterizing the distribution of subsets and how they are affected by age has to be the subject of a limited number of studies. The most thorough study was recently published by Shearer and colleagues, who evaluated lymphocyte subsets in a cross-sectional study of 807 healthy children from birth to 18 years of age [6]. Their findings, which are consistent with ours and those of others [9, 10], were that compared to adolescents and adults [6, 11], children have a relatively larger proportion of CD4⁺ T cells expressing CD45RA or both CD45RA and CD62L, indicating that the majority of circulating T cells are antigen naïve.1 Interestingly, the children we studied also had a greater proportion of both CD4⁺ and CD8⁺ T cells that expressed CD38 (typically considered a marker of immune activation) as compared to adults [11], which is also consistent with the observations of others [6, 12]. Whether this is an age-related phenomenon and whether there are specific environmental factors that explain this finding remain to be established.

Within our population of healthy children, a higher rate of upper respiratory illness events was associated with a higher percentage of activated T cells as indicated by coexpression of CD38 or HLA-CD45RO. We concede that despite being statistically significant, the r values are low, suggesting that the event rates contributed only a small part to the lymphocyte profiles. Our definition of a respiratory event is based on parents' observations of very young children, and we presume that the events are infectious; however, we did no studies to confirm this. Further, because we did not collect information on nonrespiratory illness events, we were unable to assess how this might have affected the distribution of lymphocytes at age 2 years and the magnitude of the correlation coefficients. Although the alterations in the lymphocyte distributions may be the cause of the increase in illness rates, the increase in the activation markers (CD38, RO) reflect antigen exposure and therefore makes it more likely that the respiratory events influenced the distribution of T cells.

Our major observation was an increased percentage of activated CD8 cells expressing CD38 or CD45RO in those children who had a greater frequency of respiratory events. In an earlier study, we demonstrated that residential fungal exposure altered the biologic host response as measured by an increase in the percentage of total lymphocytes expressing CD45RO and CD29 [1]. We have no evidence that the alteration in the distribution of T-cell subsets in children is associated with an alteration in lymphocyte function.

However, the possible consequence of increased microbial challenge early in life has been summarized in the "hygiene" hypothesis [13, 14], which proposes that lack of microbial challenge early in life promotes the later development of atopic diseases, such as hay fever and asthma, and diminishes the later expression of autoimmune diseases such as rheumatoid arthritis. How this occurs is not known. One proposed mechanism is an alteration in the balance between mutually antagonistic subsets of CD4-positive helper T cells in favour of the T helper 2 (Th2) response mediating allergic responses and against the Th1 response mediating autoimmune disease. Nevertheless, the results of three large population-based studies argue against this interpretation by finding concomitant increases in both Th1-mediated autoimmune diseases and Th2-mediated allergic diseases [15–17]. Bach proposed that a loss of regulatory function may explain the mechanism of the hygiene hypothesis [18]. Humans and animals with autoimmune and atopic diseases are noted for deficient T-suppressor cell function [19]. A property of activated CD8 lymphocytes is regulation (suppression) of immune responses [13, 18, 20]. In mice, T-regulatory cells express CD8 and HLA-DR (MHC II)19 and can suppress CD4-mediated autoimmunity [21–23]. According to this interpretation of the hygiene hypothesis, microbial challenge will enhance suppressor function and diminish the later development of all categories of immune-mediated (hypersensitivity) diseases. However, whether human CD8⁺ T cells expressing CD38 or CD45RO have a regulatory (suppressor) function remains to be established.

We have described characteristics of peripheral lymphocytes in a group of 2-year-old children. In this study and in an earlier study [1], we believe we have demonstrated that antigen exposure results in a change in immunophenotype from a naive to activated memory status and that the exact changes vary depending on the stimulus, such as fungal exposure (as in the earlier study) [1] and viruses (as in this study). However, much further research is recommended, to confirm the directionality of the associations, the effects in repertoire (distribution of lymphocytes) by single and repeated exposures, and the functions of the lymphocyte subpopulations in question. Much more information is needed prior to embarking on population health planning directed at influencing the distribution of circulating lymphocytes at a young age in order to influence susceptibility to diseases at a later age.