Our study aimed to profile the human innate immune response to wide range of well-defined TLR ligands over the first 2 years of life. Using a well-established, robust, high-throughput profiling platform, we were able to follow a large number of children from birth to two years of age. We detected a pattern that suggests the existence of age-specific responses rather than a global, linear progression from a neonatal to an adult pattern. Specifically, we found an increase in the production of IFN-α2 that reached adult response levels after stimulation with TLR7/8 or TLR9 ligands by 1-year of age. This was also confirmed at the single-cell level, in that pDC acquired adult-like cytokine response patterns, including degree of polyfunctionality, by 1 year of age. This stood in contrast to the infants' capacity to produce IL-12p70, which in response to TLR3 and TLR7/8 increased from birth to 1 and again to 2 years of age, but even then never reached adult levels. And while the ability to produce TNF-α or IL-1β reached adult levels after TLR2/1 and TLR4 stimulation, it in fact decreased following stimulation with TLR7/8 to below adult levels by 1 year of age. IL-6, IL-12p40, IL-23 and IL-10 following TLR2/1, TLR4, and TLR7/8 (and TLR9 for IL-10) stimulation decreased by 1 year of age from the neonatal high level, and continued to decrease to either reach or even drop below adult levels of response. These findings suggest potentially important time periods during which vaccination may result in biased immune responses, or particular windows of vulnerability to specific pathogens. Deciphering the mechanisms underlying our observations is likely to yield important insights, and constitutes the necessary next step investigating innate immune ontogeny.
Most studies examining early life innate immune function have focused on cord blood 
. Only few previous studies have analyzed postnatal innate immune development 
. Our study design offered several unique advantages over these previous studies. For example, ours was the only study that examined the ontogeny of innate immune responses in a truly longitudinal fashion–i.e. following the same subjects over time–this likely reduces the variability due to genetic heterogeneity 
. Furthermore, we were able to enroll, retain and analyze high numbers of our subjects at each time point for each assay, allowing well-powered statistical analysis. Our study also was the first to analyze the postnatal ontogeny of the innate immune system using ICS in all 4 major APC subsets at the same time in the same sample, and the first to interrogate a wide-range of specific TLR responses using the currently most comprehensive innate profiling platform by coupling flow cytometry with a multiplexed bead array approach. Lastly, our cohort was followed over the same time period that the elevated risk for infectious diseases related morbidity and mortality has repeatedly been observed—the first 2 years of life 
. The same time period also covers the span that most childhood vaccines are given 
Our analysis of the cellular composition of the human APC compartment in MC revealed several significant changes over the first 2 years of life, namely the juxtaposed increase of B cells and decrease of monocytes from birth to 1- and 2-years of age 
. We also observed a steady increase in cDC no increase in pDC over the first 2 years of life. Nguyen et al.
and Belderbos et al.
had looked at WB 
, while we analyzed the innate cell content in MC. WB contains neutrophils, the predominant white blood cell in peripheral blood; neutrophil numbers are known to drop from a high in early life 
leading to a potential relative increase in monocytes, cDC and pDC, precisely as described by Nguyen et al.
and Belderbos et al. 
. The changes in the composition of APC in MC over the first few years of life we describe here may impact the cytokine quantification in bulk culture supernatant as shown in .
Nguyen et al.
followed children from birth up to only 1-year of age by analyzing the impact of TLR4 and TLR9 stimulation on APC surface maturation and cytokine secretion in WB cultures 
. With respect to the 2 TLRs examined by Nguyen et al.
, our findings are in full agreement with theirs, but importantly, our studies extend them to 2 years of age and include several additional qualitatively and quantitatively important insights. For example, similar to us, Nguyen et al.
describe that the TNF-α response to LPS reaches adult levels by 1-year of age, and that the LPS induced IL-6 production drops from a high at birth to lower levels at 1-year of age. In our study, we extended this observation to show that this drop in IL-6 production in response to LPS continued up until at least 2-years of age, in fact to a level below that of the adult. Similarly to our results, Nguyen et al.
also found no striking change in the very high early-life LPS induced IL-10 production by 1-year of age. Again, our findings extended this through our discovery of a pronounced drop in LPS induced IL-10 production to occur between 1- and 2-years of age to a level significantly below that of adults. Similar to our findings, Nguyen et al.
detect a significant drop in CpG-A induced IL-6 and IL-10 production between birth and 1-year of age; again, we observed this drop to continue further between years 1 and 2 of life, and to also occur after TLR7/8 stimulation. Both our results and those of Nguyen et al.
indicate that the early-life IL-12p70 response to LPS reaches adult-levels by 1-year of age. However, we found that even then the amount of IL-12p70 produced in response to LPS was still markedly lower as compared to, e.g., pI:C or 3M-003. Finally, for the high IL-12p70-inducing TLRs (TLR3 and TLR7/8), while production of IL-12p70 increased after birth, it remained at a significantly lower level than adults even up to 2-years of age.
The fact that our data and that of Nguyen et al.
support each other up to at least 1 year post birth, is not only reassuring, but also produces a significant insight that neither study alone had addressed: a comparison of MC to WB responses. For cord blood vs. adult peripheral blood we had previously completed the direct MC vs. WB comparison, and found there to be striking and significant qualitative and quantitative differences in TLR responses between neonatal vs. adult MC and WB 
. The fact that our current observations using MC and those of Nguyen et al.
who used WB 
uncovered the same trends over the first year of life suggests that the impact of soluble factors, such as adenosine 
or other cells contained in WB on the TLR response, while important around birth 
, may be less important by 1-year of age. Another study investigating the innate responses to TLR stimulation from newborn up to 1-mo old infants confirmed, in a direct comparison of MC to WB, that the impact of soluble factors or other cells contained in WB (as compared to MC) on TLR responses disappears by a month after birth 
. However, in vivo
exposure to e.g. different concentrations of adenosine may still influence our in vitro
results, as may age-dependent differences in the sensitivity of infant mononuclear cells to adenosine 
Belderbos et al.
, similar to our and Nguyen's observations, also describes a rapid postnatal increase in IFN-α2 production in response to TLR7/8 stimulation, and an LPS-induced increase in IL-12p70 production by 1 month but only to levels still below those of the adult 
. However, they analyzed the cytokine response in WB culture supernatant after stimulation with LPS following IFN-γ priming, and used concentrations of pI:C and CpG-A much higher than ours. The latter aspect, the need for higher pI:C and CpG-A concentrations when stimulating WB vs. MC is consistent with our previously made observation of a lack of response of APC in WB at concentrations that maximally stimulate APC in MC 
. Using this much higher concentration of pI:C in WB cultures, Belderbos et al.
describe a rapid increase to adult levels of IL-12p70 production by 1-mo of age 
. While we also detected an increase in pI:C-induced IL-12p70 production postnatally, we did not see this level reach those of adults even by 2- years of age. This difference may relate to the higher concentration of pI:C, or may indicate changes between 1-mo and 1-2-years of life. The observation by Belderbos et al.
regarding TLR9 induced IL-10 production is in line with our and Nguyen's observation, in that it appears to rapidly drop off from a neonatal high 
. Similar to Belderbos et al.
, we did not detect a drop in IL-10 production in response to LPS stimulation from the high cord levels by 1 year of age; we did however observe a striking drop of IL-10 produced in response to LPS to below adult levels between 1- and 2-years of age.
Two other studies investigating the postnatal ontogeny of innate immune responses in humans come from the same group 
. Similar to us, this group used MC; but their MC were cryopreserved and thawed prior to TLR stimulation, while ours were stimulated fresh within 4 hours of collecting the blood. Furthermore, this group stimulated with LPS only after IFN-γ priming, while our stimulations occurred without prior priming. Despite these differences in experimental setup, Upham et al.
describe an IL-12p70 production pattern consistent with our findings, in that even by 12 years of age, IL-12p70 appears to still not be produced at levels comparable to adults, while early life elevated IL-10 production reaches the low adult level by at least 5 years of age 
. Yerkovich et al.
observe a level of IL-6 and IL-10 production at birth that is similar to the adult, but drops off by 1- and even further by 2-years of age 
, which agrees with our findings. Neither Yerkovich et al.
nor Upham et al.
used any of our other TLR stimuli, limiting the extent to which these studies can be compared to ours 
Yerkovich et al.
and Upham et al.
are also the only other studies that attempt to identify the cellular source of the cytokines produced in postnatal samples after TLR stimulation. Upham et al.
approached this through depletion, and determined that most of the IL-12p70 after LPS/IFN-γ priming is made by dendritic cells 
. Yerkovich et al.
employed ICS, but limited staining to only 4 parameters, namely CD3, CD14, TNF-α and IL-6. Similar to our ICS results, they determined that monocytes in cord blood produce high levels of both, TNF-α and IL-6 
. We are not aware of any other study that conducted the extensive polychromatic single-cell TLR induced cytokine analysis via intracellular cytometry that we describe here; our approach to combine this type of ICS with the bulk culture supernatant analysis is thus the most comprehensive innate immune profiling study in early life to date. Interestingly, our findings comparing ICS and multiplex bead-array were largely congruent. For example, neonatal monocytes in MC samples contained higher percentages of cells making IL-12/23p40 than adult monocytes, but by 1 year of age, this had started to drop for both, ICS and for secreted IL-12/23p40 measured via multiplex bead-array. The same was true for IL-6 production. Similarly, monocytes and cDCs from 1- and 2- year olds contained higher percentages of cells making TNF-α after TLR2/1 or TLR4 stimulation as compared to the neonatal sample of the same subjects; this was also the case analyzing the culture supernatant for TNF-α. And lastly, the amount of IFN-α2 detected in the culture supernatant after TLR7/8 or TLR9 stimulation increased dramatically from birth to reach or exceed adult levels by 1-year of age; as IFN-α2 is mostly produced by pDC, this increase in IFN-α2 production was also detected by ICS in pDC. We thus were able to confirm, at the single-cell level our observations made in the bulk-culture supernatants.
Our in vitro
findings with innate cells from newborns and infants found in blood and after exposure to adult serum may not accurately reflect the response to microbes or vaccine adjuvants found in tissues of an infant. However, several observations suggest that our findings described in this study are at least consistent with clinical presentations of the human newborn and infant. For example, infection with HSV results in the most severe morbidity and mortality if it occurs before 1-month of life 
; as it is IFN-α2 that is most relevant to protection from HSV infection, the rapid increase in IFN-α2 production early in life may explain this clinical observation 
. The delay in IFN-γ and IL-12p70 production beyond 2 years of life is consistent with the known heightened susceptibility beyond 2 years of age to infection with microbes such as TB, where protection is known to depend on robust IFN-γ and IL-12p70 production 
. The change of the age-specific predominant innate immune response we observed may also impact vaccine immune response preferences. For example, BCG immunization at birth has been shown to support a stronger Th17 adaptive T cell response as compared to BCG given at 4 months of age 
, while a delay in BCG vaccination for several months post-birth results in a somewhat stronger Th1 type T cell response 
; this observation is entirely consistent with the preferential production of Th17 supporting innate cytokines around birth we detected.
In summary, we present here the results of the most comprehensive longitudinal innate immune profiling study covering the first 2 years of human life. Our findings contradict the notion that the ontogeny of the innate immune response to TLR stimulation linearly progresses from birth to adulthood. Instead, it appears that age-specific regulatory mechanisms are in place governing the TLR response by the major human APC subsets. With the precise knowledge of the most pertinent ontogenic changes in TLR responses in hand, we are now well positioned to initiate a targeted interrogation of the underlying molecular mechanisms governing early life innate immune ontogeny. This, in turn, will form the rational basis on which to attempt age-specific and age-appropriate interventions aimed at improving the immune-mediated protection of this highly vulnerable population.