We previously showed associations between season-of-birth, thymic size and functional changes during early infancy, with those born during the harvest/low infection season having larger thymi and enhanced T cell production. To test the hypothesis that these early life events are amplified in adults to reflect the season of birth effects on the reported adult mortality, we first assumed that as infants, the young adults studied here, were exposed to some of the same seasonal pressures which existed previously, although current improvements in the socioeconomic conditions of today are consistent with milder environmental pressures. The improved socioeconomic conditions coupled with the repeated exposure of environmental stressors across the seasons has the potential to obscure previously reported season of birth differences. Unlike the earlier findings in the babies,  we did not observe season-of-birth associations with thymic output in the young adults. In addition to the overall improvements in the socioeconomic conditions enjoyed today, it is also possible that the cumulative impact of chronic nutritional deprivation and repeated infections over the years leading to adolescence obscured season-of-birth differences on thymic output which may have pre-existed at infancy. The hungry/high infection season is associated with greater mortality rates;  therefore it is also possible that the worst affected individuals died before reaching adolescence which may be a source of bias. It support of our data, the lower TREC concentrations in the young adults compared to the reports in the babies,  are consistent with reduced thymic output in advancing age.
Despite the lack of season of birth effects on thymic output, lower CD4+ as well as CD3+ counts were associated with hungry/high infection season births. Considering the central role of CD4+ cells in the immune system, [35–38] this implies worse immunity for adults born in the hungry/high infection season. When season of collection was controlled for, (i.e. sampling done in a different season to the subject's birth) this did not significantly alter the findings, although sample sizes were substantially reduced. Nonetheless, 67.9% (38/56) for the CD4+ T cell subset were collected during the hungry/high infection season months of July to December, therefore it is possible that the raised CD4+ and CD3+ counts for those born in the harvest/low infection season (but had some of their samples collected in the hungry/high infection season) were partly influenced by more peripheral T cell proliferation. Antigenic load which drives T cell proliferation is higher in the hungry/high infection season when infectious burden is heavier; and malaria which peaks here during the hungry/high infection season, is likely to impose further immune pressure accompanied by changes in T cell counts as reported in human studies[40, 41]. Further studies specifically testing the season of collection are needed to verify this.
The lack of season of birth effects on thymic output may reflect its higher tolerance threshold, than the CD4+ and CD3+ subsets, which may be more sensitive to pressures from environmental elements. In support of this, dietary zinc was associated with and differential rises of up to 24% and 64% CD4+ and CD3+ lymphocyte counts respectively.
Similarly to the CD4+ and CD3+ counts, the season of birth effects on specific TCRVβ types suggested differential sensitivities to elements of the environment. The reduced usage of TCRVβ12 for those born in the hungry/high infection season (most of whose samples were also collected in the hungry/high infection season) may be related to the prevailing infections. HBV infection is endemic in this community with 10-15% of adult male deaths due to hepatocellular carcinoma which is associated with HBV infection and it has been shown that TCRVβ12 is specific for hepatitis B virus (HBV) core antigen. Those born in the hungry/high infection season may be less able to control HBV (particularly during the hungry/high infection season), possibly leading to increased liver damage. The season of birth differences observed for the TCRVβ24 which is deleted in all but 8 of the 52 subjects all of whom only lowly expressed TCRVβ24, suggested that TCRVβ24 may not be of significant value for immune protection in either season. It is possible that a 'chance' Type 1 statistical error arising from multiple testing of repeated variables may account for the season of birth difference seen, and that a bigger sample size may produce significant differences in other TCRVβ types.
Repertoire skewing is consistent with accelerated proliferation and the potential to drive telomere erosion, therefore the shorter mean telomere length for those born in the hungry/high infection season suggested that their CD8+ T cells were under proliferative pressure and at a higher risk of replicative senescence. Telomere shortening is accelerated in arterial tissue exposed to oxidative stress factors including reactive oxygen species (ROS). The endemicity of infections in this community may be expected to generate ROS to contribute to telomere shortening especially for those born in the hungry/high infection season.
To optimize the interpretation of our TREC findings at the population level, results from other study populations were used for comparison. The TREC assay which is now widely used as a marker of thymic output lacks a 'gold' standard; thus limiting the number of studies with which to compare our data. However, our results suggested that average TREC concentrations in the subjects studied may be substantially lower than those of adults elsewhere, [47, 48] implying diminished thymic output and immune capacity in this population. Persistent infectious burden rather than low thymic output may also be responsible for the lower TRECs; since elevated cell proliferation from antigenic exposure is known to dilute TREC concentrations. Our findings, at the population level, that the T cell subsets are comparable to those of healthy individuals from the sub region, [50–53] imply that poor T cell immunity may be common here. The lack of observable differences arising from the further analyses by birth weight category (higher/lower than the population median) is consistent with the overall findings but may have been confounded by the resultant reductions in numbers.
Our analyses of the T cell repertoire was meant to give an in depth evaluation of T cell immune status beyond thymic output and T cell numbers, and the extensive CD8+ TCRVβ repertoire distortions in the population indicated more severe immune challenges than was evident from the thymic output and T cell counts. Only 2 (TCRVβ12 and 15) out of 24 TCRVβ types showed an average spectra-type peak number ≥5 across all donors; compared to reports of >8 peaks in healthy individuals[54, 55]. We speculate that repertoire skewing in this population was driven by environmental stressors including the repeated persistent antigenic exposure annually and across the seasons due to the endemicity of infections [56–58] including CMV, which is associated with virus specific CD8+ T cell types and other risk factors [59, 60]. We argue that the chronic nature of the assault on the immune system of both groups may be the reason for the general distortion of the TCRVß repertoire. Significantly, the timing of exposure to environmental stressors may be more critical, the closer to the time of birth it occurs, as the thymus experiences its greatest and only growth phase in the first year of life, a period of maximum vulnerability; with the potential to generate ever-lasting impact on the thymus and the T cells it generates. Consequently the thymi of those born in the hungry/high infection season may never be adequately compensated to cope with later life demands. Conversely, thymi of those born in the harvest/low infection in a more enabling environment for development, may be endowed with a more resilient initial thymic capacity. The immune insufficiency implied by the apparent oligoclonal repertoire distortions is consistent both with the lower thymic output compared to others;[47, 48] and supported by the association of a polyclonal repertoire with a lack of antigen exposure, favourable immunity being associated with good thymic output and a broad repertoire.
Chronic HBV infection is also endemic in this community, and the publicly expressed TCRVβ12 being specific to the HLA-A2 restricted hepatitis B virus (HBV) core antigen supports a role for HBV in the marked global repertoire skewing seen. The near extinction of TCRVβ24 in the population, which has also been reported in other settings, where TCRVβ24 became notably expanded when stimulated by specific antigen, suggested that the near zero expression in our study was probably not due to lack of capacity for the TCRVβ24 clone to expand. This implies that TCRVβ24 offers little, if any, advantages in this community. As clonal expansion and cell division are accompanied by telomere erosion,[64, 65] the shorter telomere of the CD8+ compared to the CD4+ subset, supports reports that the CD8+ subset undergoes faster clonal expansion. Indeed shorter mean telomere length has previously been reported for the CD8+ compared to the CD4+ subset in healthy humans and with increasing T cell turnover. A naïve T cell is estimated to go through at least 14 cell divisions during an immune response,[68, 69] therefore the repeated infections might be expected to drive telomere shortening although human studies with which to compare our data were lacking.