Proteomics-Based Mapping of Bronchopulmonary Dysplasia-Associated Changes in Noninvasively Accessible Oral Secretions.

OBJECTIVE: To determine if oral secretions (OS) can be used as a noninvasively collected body fluid, in lieu of tracheal aspirates (TA), to track respiratory status and predict bronchopulmonary dysplasia (BPD) development in infants born <32 weeks. STUDY DESIGN: This was a retrospective, single center cohort study that included data and convenience samples from week-of-life (WoL) 3 from 2 independent preterm infant cohorts. Using previously banked samples, we applied our sample-sparing, high-throughput proteomics technology to compare OS and TA proteomes in infants born <32 weeks admitted to the Neonatal Intensive Care Unit (NICU) (Cohort 1; n = 23 infants). In a separate similar cohort, we mapped the BPD-associated changes in the OS proteome (Cohort 2; n = 17 infants including 8 with BPD). RESULTS: In samples collected during the first month of life, we identified 607 proteins unique to OS, 327 proteins unique to TA, and 687 overlapping proteins belonging to pathways involved in immune effector processes, neutrophil degranulation, leukocyte mediated immunity, and metabolic processes. Furthermore, we identified 37 OS proteins that showed significantly differential abundance between BPD cases and controls: 13 were associated with metabolic and immune dysregulation, 10 of which (eg, SERPINC1, CSTA, BPI) have been linked to BPD or other prematurity-related lung disease based on blood or TA investigations, but not OS. CONCLUSIONS: OS are a noninvasive, easily accessible alternative to TA and amenable to high-throughput proteomic analysis in preterm newborns. OS samples hold promise to yield actionable biomarkers of BPD development, particularly for prospective categorization and timely tailored treatment of at-risk infants with novel therapies.

Effective early antiretroviral therapy in perinatal-HIV infection reduces subsequent plasma inflammatory profile.

BACKGROUND: The long-term immunologic effects of antiretroviral therapy (ART) in children with perinatally-acquired HIV (PHIV) have not been fully elucidated. Here, we investigated how the timing of ART initiation affects the long-term immune profile of children living with PHIV by measuring immunomodulatory plasma cytokines, chemokines, and adenosine deaminases (ADAs). METHODS: 40 PHIV participants initiated ART during infancy. 39 participant samples were available; 30 initiated ART ≤6 months (early-ART treatment); 9 initiated ART >6 months and <2 years (late-ART treatment). We compared plasma cytokine and chemokine concentrations and ADA enzymatic activities between early-ART and late-ART treatment 12.5 years later and measured correlation with clinical covariates. RESULTS: Plasma concentrations of 10 cytokines and chemokines (IFNγ, IL-12p70, IL-13, IL-17A, IL-IRA, IL-5, IL-6, and IL-9 as well as CCL7, CXCL10), ADA1, and ADA total were significantly higher in late-ART compared to early-ART treatment. Furthermore, ADA1 was significantly positively correlated with IFNγ, IL-17A, and IL-12p70. Meanwhile, total ADA was positively correlated with IFNγ, IL-13, IL-17A, IL-1RA, IL-6, and IL-12p70 as well as CCL7. CONCLUSIONS: Elevation of several pro-inflammatory plasma analytes in late-ART despite 12.5 years of virologic suppression compared to early-ART treatment suggests that early treatment dampens the long-term plasma inflammatory profile in PHIV participants. IMPACT: This study examines differences in the plasma cytokine, chemokine, and ADA profiles 12.5 years after treatment between early (≤6months) and late (>6 months and <2 years) antiretroviral therapy (ART) treatment initiation in a cohort of European and UK study participants living with PHIV. Several cytokines and chemokines (e.g., IFNγ, IL-12p70, IL-6, and CXCL10) as well as ADA-1 are elevated in late-ART treatment in comparison to early-ART treatment. Our results suggest that effective ART treatment initiated within 6 months of life in PHIV participants dampens a long-term inflammatory plasma profile as compared to late-ART treatment.

Implications of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Pandemic on the Epidemiology of Pediatric Respiratory Syncytial Virus Infection.

Respiratory viral infections account for a large percentage of global disease and death. Respiratory syncytial virus is a seasonal virus affecting immunologically vulnerable populations, such as preterm newborns and young infants; however, its epidemiology has changed drastically during the coronavirus disease 2019 pandemic. In this perspective, we discuss the implications of coronavirus disease 2019 on respiratory syncytial virus seasonality patterns and mitigation efforts, as well as the urgent need for vaccination as a preventive tool.

T cells expressing the transcription factor PLZF regulate the development of memory-like CD8+ T cells.

Several gene-deficiency models promote the development of innate CD8(+) T cells that have diverse T cell antigen receptors (TCRs) but have a memory phenotype and rapidly produce cytokines. We demonstrate here that similar cells developed in mice deficient in the transcription factor KLF2. However, this was not due to intrinsic deficiency in KLF2 but instead was due to interleukin 4 (IL-4) produced by an expanded population of T cells expressing the transcription factor PLZF. The development of innate CD8(+) T cells in mice deficient in the tyrosine kinase Itk and coactivator CBP was also attributable to this IL-4-dependent mechanism. Finally, we show that the same mechanism drove the differentiation of innate CD8(+) T cells in BALB/c mice. Our findings identify a previously unknown mechanism of regulation of CD8(+) T cells via the production of IL-4 by PLZF(+) T cells.

Normalizing the environment recapitulates adult human immune traits in laboratory mice.

Our current understanding of immunology was largely defined in laboratory mice, partly because they are inbred and genetically homogeneous, can be genetically manipulated, allow kinetic tissue analyses to be carried out from the onset of disease, and permit the use of tractable disease models. Comparably reductionist experiments are neither technically nor ethically possible in humans. However, there is growing concern that laboratory mice do not reflect relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside. Laboratory mice live in abnormally hygienic specific pathogen free (SPF) barrier facilities. Here we show that standard laboratory mouse husbandry has profound effects on the immune system and that environmental changes produce mice with immune systems closer to those of adult humans. Laboratory mice--like newborn, but not adult, humans--lack effector-differentiated and mucosally distributed memory T cells. These cell populations were present in free-living barn populations of feral mice and pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting that the environment is involved in the induction of these cells. Altering the living conditions of mice profoundly affected the cellular composition of the innate and adaptive immune systems, resulted in global changes in blood cell gene expression to patterns that more closely reflected the immune signatures of adult humans rather than neonates, altered resistance to infection, and influenced T-cell differentiation in response to a de novo viral infection. These data highlight the effects of environment on the basal immune state and response to infection and suggest that restoring physiological microbial exposure in laboratory mice could provide a relevant tool for modelling immunological events in free-living organisms, including humans.

Ontogeny of plasma cytokine and chemokine concentrations across the first week of human life.

INTRODUCTION/BACKGROUND & AIMS: Early life is marked by distinct and rapidly evolving immunity and increased susceptibility to infection. The vulnerability of the newborn reflects development of a complex immune system in the face of rapidly changing demands during the transition to extra-uterine life. Cytokines and chemokines contribute to this dynamic immune signaling network and can be altered by many factors, such as infection. Newborns undergo dynamic changes important to health and disease, yet there is limited information regarding human neonatal plasma cytokine and chemokine concentrations over the first week of life. The few available studies are limited by small sample size, cross-sectional study design, or focus on perturbed host states like severe infection or prematurity. To characterize immune ontogeny among healthy full-term newborns, we assessed plasma cytokine and chemokine concentrations across the first week of life in a robust longitudinal cohort of healthy, full-term African newborns. METHODS: We analyzed a subgroup of a cohort of healthy newborns at the Medical Research Council Unit in The Gambia (West Africa; N = 608). Peripheral blood plasma was collected from all study participants at birth (day of life (DOL) 0) and at one follow-up time point at DOL 1, 3, or 7. Plasma cytokine and chemokine concentrations were measured by bead-based cytokine multiplex assay. Unsupervised clustering was used to identify patterns in plasma cytokine and chemokine ontogeny during early life. RESULTS: We observed an increase across the first week of life in plasma Th1 cytokines such as IFNγ and CXCL10 and a decrease in Th2 and anti-inflammatory cytokines such as IL-6 and IL-10, and chemokines such as CXCL8. In contrast, other cytokines and chemokines (e.g. IL-4 and CCL5, respectively) remained unchanged during the first week of life. This robust ontogenetic pattern did not appear to be affected by gestational age or sex. CONCLUSIONS: Ontogeny is a strong driver of newborn plasma-based levels of cytokines and chemokines throughout the first week of life with a rising IFNγ axis suggesting post-natal upregulation of host defense pathways. Our study will prove useful to the design and interpretation of future studies aimed at understanding the neonatal immune system during health and disease.

Decrease in Numbers of Naive and Resting B Cells in HIV-Infected Kenyan Adults Leads to a Proportional Increase in Total and Plasmodium falciparum-Specific Atypical Memory B Cells.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with B cell activation and exhaustion, and hypergammaglobulinemia. How these changes influence B cell responses to coinfections such as malaria is poorly understood. To address this, we compared B cell phenotypes and Abs specific for the Plasmodium falciparum vaccine candidate apical membrane Ag-1 (AMA1) in HIV-infected and uninfected adults living in Kenya. Surprisingly, HIV-1 infection was not associated with a difference in serum AMA1-specific Ab levels. HIV-infected individuals had a higher proportion of total atypical and total activated memory B cells (MBCs). Using an AMA1 tetramer to detect AMA1-specific B cells, HIV-infected individuals were also shown to have a higher proportion of AMA1-specific atypical MBCs. However, this proportional increase resulted in large part from a loss in the number of naive and resting MBCs rather than an increase in the number of atypical and activated cells. The loss of resting MBCs and naive B cells was mirrored in a population of cells specific for an Ag to which these individuals were unlikely to have been chronically exposed. Together, the data show that changes in P. falciparum Ag-specific B cell subsets in HIV-infected individuals mirror those in the overall B cell population, and suggest that the increased proportion of atypical MBC phenotypes found in HIV-1-infected individuals results from the loss of naive and resting MBCs.

The proapoptotic function of Noxa in human leukemia cells is regulated by the kinase Cdk5 and by glucose.

The BH3-only protein, Noxa, is induced in response to apoptotic stimuli, such as DNA damage, hypoxia, and proteasome inhibition in most human cells. Noxa is constitutively expressed in proliferating cells of hematopoietic lineage and required for apoptosis in response to glucose stress. We show that Noxa is phosphorylated on a serine residue (S(13)) in the presence of glucose. Phosphorylation promotes its cytosolic sequestration and suppresses its apoptotic function. We identify Cdk5 as the Noxa kinase and show that Cdk5 knockdown or expression of a Noxa S(13) to A mutant increases sensitivity to glucose starvation, confirming that the phosphorylation is protective. Both glucose deprivation and Cdk5 inhibition promote apoptosis by dephosphorylating Noxa. Paradoxically, Noxa stimulates glucose consumption and may enhance glucose turnover via the pentose phosphate pathway rather than through glycolysis. We propose that Noxa plays both growth-promoting and proapoptotic roles in hematopoietic cancers with phospho-S(13) as the glucose-sensitive toggle switch controlling these opposing functions.

Behavioral, virologic, and immunologic factors associated with acquisition and severity of primary Epstein-Barr virus infection in university students.

BACKGROUND: University students were studied prospectively to determine the incidence of and risk factors for acquisition of primary Epstein-Barr virus (EBV) infection and the virologic and immune correlates of disease severity. METHODS: EBV antibody-negative freshmen participated in monthly surveillance until graduation. If antibodies developed, proximate samples were assayed for viral load by polymerase chain reaction. Lymphocyte and natural killer (NK) cell numbers and activation were measured by flow cytometry, and plasma cytokine levels were measured by a multiplex assay. RESULTS: Of 546 students screened, 202 (37%) were antibody negative; 143 antibody-negative students were enrolled. During a median of 3 years of observation, 66 subjects experienced primary infection. Of these, 77% had infectious mononucleosis, 12% had atypical symptoms, and 11% were asymptomatic. Subjects reporting deep kissing with or without coitus had the same higher risk of infection than those reporting no kissing (P < .01). Viremia was transient, but median oral shedding was 175 days. Increases were observed in numbers of NK cells and CD8(+) T-cells but not in numbers of CD4(+) T-cells during acute infection. Severity of illness correlated positively with both blood EBV load (P = .015) and CD8(+) lymphocytosis (P = .0003). CONCLUSIONS: Kissing was a significant risk for primary EBV infection. A total of 89% of infections were symptomatic, and blood viral load and CD8(+) lymphocytosis correlated with disease severity.

Plasma adenosine deaminase-1 and -2 activities are lower at birth in Papua New Guinea than in The Gambia but converge over the first weeks of life.

Introduction: Dynamic cellular and molecular adaptations in early life significantly impact health and disease. Upon birth, newborns are immediately challenged by their environment, placing urgent demands on the infant immune system. Adenosine deaminases (ADAs) are enzymatic immune modulators present in two isoforms - ADA-1 and ADA-2. Infants exhibit low ADA activity, resulting in high plasma adenosine concentrations and a consequent anti-inflammatory/anti-Th1 bias. While longitudinal studies of plasma ADA have been conducted in infants in The Gambia (GAM), little is known regarding ADA trajectories in other parts of the world. Methods: Herein, we characterized plasma ADA activity in an infant cohort in Papua New Guinea (PNG; n=83) and compared to ontogeny of ADA activity in a larger cohort in GAM (n=646). Heparinized peripheral blood samples were collected at day of life (DOL) 0, DOL7, DOL30, and DOL128. Plasma ADA-1, ADA-2, and total ADA activities were measured by chromogenic assay. Results: Compared to GAM infants, PNG infants had significantly lower ADA-1 (0.9-fold), ADA-2 (0.42-fold), and total ADA (0.84-fold) activities at birth which converged by DOL30. Discussion: Overall, discovery of a distinct baseline and a consistent pattern of increasing plasma ADA activity in early life in two genetically and geographically distinct populations validates and extends previous findings on the robustness of early life immune ontogeny.

Breastfeeding and Neonatal Age Influence Neutrophil-Driven Ontogeny of Blood Cell Populations in the First Week of Human Life.

The first few days of life are characterized by rapid external and internal changes that require substantial immune system adaptations. Despite growing evidence of the impact of this period on lifelong immune health, this period remains largely uncharted. To identify factors that may impact the trajectory of immune development, we conducted stringently standardized, high-throughput phenotyping of peripheral white blood cell (WBC) populations from 796 newborns across two distinct cohorts (The Gambia, West Africa; Papua New Guinea, Melanesia) in the framework of a Human Immunology Project Consortium (HIPC) study. Samples were collected twice from each newborn during the first week of life, first at Day of Life 0 (at birth) and then subsequently at Day of Life 1, 3, or 7 depending on the randomization group the newborn belongs to. The subsequent analysis was conducted at an unprecedented level of detail using flow cytometry and an unbiased automated gating algorithm. The results showed that WBC composition in peripheral blood changes along patterns highly conserved across populations and environments. Changes across days of life were most pronounced in the innate myeloid compartment. Breastfeeding, and at a smaller scale neonatal vaccination, were associated with changes in peripheral blood neutrophil and monocyte cell counts. Our results suggest a common trajectory of immune development in newborns and possible association with timing of breastfeeding initiation, which may contribute to immune-mediated protection from infection in early life. These data begin to outline a specific window of opportunity for interventions that could deliberately direct WBC composition, and with that, immune trajectory and thus ontogeny in early life. This trial is registered with NCT03246230.

DNA Methylation signatures underpinning blood neutrophil to lymphocyte ratio during first week of human life.

Understanding of newborn immune ontogeny in the first week of life will enable age-appropriate strategies for safeguarding vulnerable newborns against infectious diseases. Here we conducted an observational study exploring the immunological profile of infants longitudinally throughout their first week of life. Our Expanded Program on Immunization - Human Immunology Project Consortium (EPIC-HIPC) studies the epigenetic regulation of systemic immunity using small volumes of peripheral blood samples collected from West African neonates on days of life (DOL) 0, 1, 3, and 7. Genome-wide DNA methylation and single nucleotide polymorphism markers are examined alongside matched transcriptomic and flow cytometric data. Integrative analysis reveals that a core network of transcription factors mediates dynamic shifts in neutrophil-to-lymphocyte ratios (NLR), which are underpinned by cell-type specific methylation patterns in the two cell types. Genetic variants are associated with lower NLRs at birth, and healthy newborns with lower NLRs at birth are more likely to subsequently develop sepsis. These findings provide valuable insights into the early-life determinants of immune system development.

Predictive gene expression signature diagnoses neonatal sepsis before clinical presentation.

BACKGROUND: Neonatal sepsis is a deadly disease with non-specific clinical signs, delaying diagnosis and treatment. There remains a need for early biomarkers to facilitate timely intervention. Our objective was to identify neonatal sepsis gene expression biomarkers that could predict sepsis at birth, prior to clinical presentation. METHODS: Among 720 initially healthy full-term neonates in two hospitals (The Gambia, West Africa), we identified 21 newborns who were later hospitalized for sepsis in the first 28 days of life, split into early-onset sepsis (EOS, onset ≤7 days of life) and late-onset sepsis (LOS, onset 8-28 days of life), 12 neonates later hospitalized for localized infection without evidence of systemic involvement, and 33 matched control neonates who remained healthy. RNA-seq was performed on peripheral blood collected at birth when all neonates were healthy and also within the first week of life to identify differentially expressed genes (DEGs). Machine learning methods (sPLS-DA, LASSO) identified genes expressed at birth that predicted onset of neonatal sepsis at a later time. FINDINGS: Neonates who later developed EOS already had ∼1000 DEGs at birth when compared to control neonates or those who later developed a localized infection or LOS. Based on these DEGs, a 4-gene signature (HSPH1, BORA, NCAPG2, PRIM1) for predicting EOS at birth was developed (training AUC = 0.94, sensitivity = 0.93, specificity = 0.92) and validated in an external cohort (validation AUC = 0.72, sensitivity = 0.83, and specificity = 0.83). Additionally, during the first week of life, EOS disrupted expression of >1800 genes including those influencing immune and metabolic transitions observed in healthy controls. INTERPRETATION: Despite appearing healthy at birth, neonates who later developed EOS already had distinct whole blood gene expression changes at birth, which enabled the development of a 4-gene predictive signature for EOS. This could facilitate early recognition and treatment of neonatal sepsis, potentially mitigating its long-term sequelae. FUNDING: CIHR and NIH/NIAID.

Kruppel-like factor 2 is required for trafficking but not quiescence in postactivated T cells.

The transcription factor Kruppel-like factor 2 (KLF2) was proposed to regulate genes involved in cell cycle entry and T cell trafficking; however, the physiological role of its expression in postactivated T cells is not well defined. Previous studies suggested that the cytokines IL-2 and IL-15 differentially regulate KLF2 re-expression in postactivation T cells and that these cytokines also influence effector versus memory T cell differentiation. Using conditional and inducible KLF2-knockout model systems, we tested the specific role of KLF2 expression in activated CD8(+) T cells cultured with these cytokines. KLF2 was required for effective transcription of sphingosine-1-phosphate receptor-1 (S1P(1)) and CD62L in postactivation T cells. However, although different cytokines dramatically altered the expression of cell-cycle-related genes, endogenous KLF2 had a minimal impact. Correspondingly, KLF2-deficient T cells showed dysregulated trafficking but not altered proliferative characteristics following in vivo responses to Ag. Thus, our data help to define KLF2-dependent and -independent aspects of activated CD8(+) T cell differentiation and argue against a physiological role in cell cycle regulation.

Progress and problems in understanding and managing primary Epstein-Barr virus infections.

Epstein-Barr virus (EBV) is a gammaherpesvirus that infects a large fraction of the human population. Primary infection is often asymptomatic but results in lifelong infection, which is kept in check by the host immune system. In some cases, primary infection can result in infectious mononucleosis. Furthermore, when host-virus balance is not achieved, the virus can drive potentially lethal lymphoproliferation and lymphomagenesis. In this review, we describe the biology of EBV and the host immune response. We review the diagnosis of EBV infection and discuss the characteristics and pathogenesis of infectious mononucleosis. These topics are approached in the context of developing therapeutic and preventative strategies.

Molecular Determinants of the Early Life Immune Response to COVID-19 Infection and Immunization.

Clinical manifestations from primary COVID infection in children are generally less severe as compared to adults, and severe pediatric cases occur predominantly in children with underlying medical conditions. However, despite the lower incidence of disease severity, the burden of COVID-19 in children is not negligible. Throughout the course of the pandemic, the case incidence in children has substantially increased, with estimated cumulative rates of SARS-CoV-2 infection and COVID-19 symptomatic illness in children comparable to those in adults. Vaccination is a key approach to enhance immunogenicity and protection against SARS-CoV-2. Although the immune system of children is functionally distinct from that of other age groups, vaccine development specific for the pediatric population has mostly been limited to dose-titration of formulations that were developed primarily for adults. In this review, we summarize the literature pertaining to age-specific differences in COVID-19 pathogenesis and clinical manifestation. In addition, we review molecular distinctions in how the early life immune system responds to infection and vaccination. Finally, we discuss recent advances in development of pediatric COVID-19 vaccines and provide future directions for basic and translational research in this area.

Krüppel-like factor 2 regulates trafficking and homeostasis of gammadelta T cells.

gammadelta T cells are generated in the thymus and traffic to secondary lymphoid organs and epithelial surfaces, where they regulate immune responses. alphabeta T cells require sphingosine 1-phosphate receptor type 1 (S1P(1)) and CD62L for thymic emigration and circulation through secondary lymphoid organs. Both of these genes are regulated by the transcription factor Krüppel-like factor 2 (KLF2) in conventional alphabeta T cells. It is unclear if gammadelta T cells use similar mechanisms. In this study, we show that thymic gammadelta T cells express S1P(1) and that it is regulated by KLF2. Furthermore, KLF2 and S1P(1)-deficient gammadelta T cells accumulate in the thymus and fail to populate the secondary lymphoid organs or gut, in contrast to the expectation from published work. Interestingly, KLF2 but not S1P(1) deficiency led to the expansion of a usually rare population of CD4(+) promyelocytic leukemia zinc finger(+) "gammadelta NKT" cells. Thus, KLF2 is critically important for the homeostasis and trafficking of gammadelta T cells.

Urine Proteomics for Noninvasive Monitoring of Biomarkers in Bronchopulmonary Dysplasia.

INTRODUCTION: Current techniques to diagnose and/or monitor critically ill neonates with bronchopulmonary dysplasia (BPD) require invasive sampling of body fluids, which is suboptimal in these frail neonates. We tested our hypothesis that it is feasible to use noninvasively collected urine samples for proteomics from extremely low gestational age newborns (ELGANs) at risk for BPD to confirm previously identified proteins and biomarkers associated with BPD. METHODS: We developed a robust high-throughput urine proteomics methodology that requires only 50 µL of urine. We utilized the methodology with a proof-of-concept study validating proteins previously identified in invasively collected sample types such as blood and/or tracheal aspirates on urine collected within 72 h of birth from ELGANs (gestational age [26 ± 1.2] weeks) who were admitted to a single Neonatal Intensive Care Unit (NICU), half of whom eventually developed BPD (n = 21), while the other half served as controls (n = 21). RESULTS: Our high-throughput urine proteomics approach clearly identified several BPD-associated changes in the urine proteome recapitulating expected blood proteome changes, and several urinary proteins predicted BPD risk. Interestingly, 16 of the identified urinary proteins are known targets of drugs approved by the Food and Drug Administration. CONCLUSION: In addition to validating numerous proteins, previously found in invasively collected blood, tracheal aspirate, and bronchoalveolar lavage, that have been implicated in BPD pathophysiology, urine proteomics also suggested novel potential therapeutic targets. Ease of access to urine could allow for sequential proteomic evaluations for longitudinal monitoring of disease progression and impact of therapeutic intervention in future studies.

Plasma Adenosine Deaminase (ADA)-1 and -2 Demonstrate Robust Ontogeny Across the First Four Months of Human Life.

Background: Human adenosine deaminases (ADAs) modulate the immune response: ADA1 via metabolizing adenosine, a purine metabolite that inhibits pro-inflammatory and Th1 cytokine production, and the multi-functional ADA2, by enhancing T-cell proliferation and monocyte differentiation. Newborns are relatively deficient in ADA1 resulting in elevated plasma adenosine concentrations and a Th2/anti-inflammatory bias compared to adults. Despite the growing recognition of the role of ADAs in immune regulation, little is known about the ontogeny of ADA concentrations. Methods: In a subgroup of the EPIC002-study, clinical data and plasma samples were collected from 540 Gambian infants at four time-points: day of birth; first week of life; one month of age; and four months of age. Concentrations of total extracellular ADA, ADA1, and ADA2 were measured by chromogenic assay and evaluated in relation to clinical data. Plasma cytokines/chemokine were measured across the first week of life and correlated to ADA concentrations. Results: ADA2 demonstrated a steady rise across the first months of life, while ADA1 concentration significantly decreased 0.79-fold across the first week then increased 1.4-fold by four months of life. Males demonstrated significantly higher concentrations of ADA2 (1.1-fold) than females at four months; newborns with early-term (37 to <39 weeks) and late-term (≥41 weeks) gestational age demonstrated significantly higher ADA1 at birth (1.1-fold), and those born to mothers with advanced maternal age (≥35 years) had lower plasma concentrations of ADA2 at one month (0.93-fold). Plasma ADA1 concentrations were positively correlated with plasma CXCL8 during the first week of life, while ADA2 concentrations correlated positively with TNFα, IFNγ and CXCL10, and negatively with IL-6 and CXCL8. Conclusions: The ratio of plasma ADA2/ADA1 concentration increased during the first week of life, after which both ADA1 and ADA2 increased across the first four months of life suggesting a gradual development of Th1/Th2 balanced immunity. Furthermore, ADA1 and ADA2 were positively correlated with cytokines/chemokines during the first week of life. Overall, ADA isoforms demonstrate robust ontogeny in newborns and infants but further mechanistic studies are needed to clarify their roles in early life immune development and the correlations with sex, gestational age, and maternal age that were observed.