OIT-BRAVE Questionnaire: Development and clinical implementation of a screening instrument for patient-reported difficulties during oral immunotherapy.

The OIT-BRAVE questionnaire was developed to serve as a clinical screening tool to identify patients who may be experiencing adverse effects with oral immunotherapy.

Immune cell profiles associated with human exposure to perfluorinated compounds (PFAS) suggest changes in natural killer, T helper, and T cytotoxic cell subpopulations.

Per- and polyfluoroalkyl substances (PFAS) constitutes a group of highly persistent man-made substances. Recent evidence indicates that PFAS negatively impact the immune system. However, it remains unclear how different PFAS are associated with alterations in circulating leukocyte subpopulations. More detailed knowledge of such potential associations can provide better understanding into mechanisms of PFAS immunotoxicity in humans. In this exploratory study, associations of serum levels of common PFAS (perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS)) and immune cell profiles of peripheral blood mononuclear cells, both with and without immunostimulation, were investigated. High-dimensional single cell analysis by mass cytometry was done on blood leukocytes from fifty participants in the Norwegian human biomonitoring EuroMix study. Different PFAS were associated with changes in various subpopulations of natural killer (NK), T helper (Th), and cytotoxic T (Tc) cells. Broadly, PFAS concentrations were related to increased frequencies of NK cells and activated subpopulations of NK cells. Additionally, increased levels of activated T helper memory cell subpopulations point to Th2/Th17 and Treg-like skewed profiles. Finally, PFAS concentrations were associated with decreased frequencies of T cytotoxic cell subpopulations with CXCR3+ effector memory (EM) phenotypes. Several of these observations point to biologically plausible mechanisms that may contribute to explaining the epidemiological reports of immunosuppression by PFAS. Our results suggest that PFAS exposures even at relatively low levels are associated with changes in immune cell subpopulations, a finding which should be explored more thoroughly in a larger cohort. Additionally, causal relationships should be confirmed in experimental studies. Overall, this study demonstrates the strength of profiling by mass cytometry in revealing detailed changes in immune cells at a single cell level.

Progressive accumulation of hyperinflammatory NKG2Dlow NK cells in early childhood severe atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic comorbidities remain ill-defined. Here, analysis of circulating NK cells in a longitudinal early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D, which was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aeroallergens, a risk factor for development of asthma. Individual-level longitudinal analysis in a subset of children revealed coincident reduction of NKG2D on NK cells with acquired or persistent sensitization, and this was associated with impaired skin barrier function assessed by transepidermal water loss. Low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine tumor necrosis factor-α. These observations provide important insights into a potential mechanism underlying the development of allergic comorbidity in early life in children with AD, which involves altered NK cell functional responses, and define an endotype of severe AD.

Risk subgroups and intervention effects among infants at high risk for peanut allergy: A model for clinical decision making.

BACKGROUND: The Learning Early About Peanut Allergy (LEAP) trial showed that early dietary introduction of peanut reduced the risk of developing peanut allergy by age 60 months in infants at high risk for peanut allergy. In this secondary analysis of LEAP data, we aimed to determine risk subgroups within these infants and estimate their respective intervention effects of early peanut introduction. METHODS: LEAP raw data were retrieved from ITNTrialShare.org. Conditional random forest was applied to participants in the peanut avoidance arm to select statistically important features for the classification and regression tree (CART) analysis to group infants based on their risk of peanut allergy at 60 months of age. Intervention effects were estimated for each derived risk subgroup using data from both arms. Our main model was generated based on baseline data when the participants were 4-11 months old. Specific IgE measurements were truncated to account for the limit of detection commonly used by laboratories in clinical practice. RESULTS: The model found infants with higher predicted probability of peanut allergy at 60 months of age had a similar relative risk reduction, but a greater absolute risk reduction in peanut allergy with early introduction of peanut, than those with lower probability. The intervention effects were significant across all risk subgroups. Participants with baseline peanut sIgE ≥0.22 kU/L (n = 78) had an absolute risk reduction of 40.4% (95% CI 27.3, 51.9) whereas participants with baseline peanut sIgE<0.22 kU/L and baseline Ara h 2 sIgE <0.10 kU/L (n = 226) had an absolute risk reduction of 6.5% (95% CI 2.6, 11.0). These findings were consistent in sensitivity analyses using alternative models. CONCLUSION: In this study, risk subgroups were determined among infants from the LEAP trial based on the probability of developing peanut allergy and the intervention effects of early peanut introduction were estimated. This may be relevant for further risk assessment and personalized clinical decision-making.

Antigen-specific decidual CD8+ T cells include distinct effector memory and tissue-resident memory cells.

Maternal decidual CD8+ T cells must integrate the antithetical demands of providing immunity to infection while maintaining immune tolerance for fetal and placental antigens. Human decidual CD8+ T cells were shown to be highly differentiated memory T cells with mixed signatures of dysfunction, activation, and effector function. However, no information is present on how specificity for microbial or fetal antigens relates to their function or dysfunction. In addition, a key question, whether decidual CD8+ T cells include unique tissue-resident memory T cells (Trm) or also effector memory T cell (Tem) types shared with peripheral blood populations, is unknown. Here, high-dimensional flow cytometry of decidual and blood CD8+ T cells identified 2 Tem populations shared in blood and decidua and 9 functionally distinct Trm clusters uniquely found in decidua. Interestingly, fetus- and virus-specific decidual CD8+ Trm cells had similar features of inhibition and cytotoxicity, with no significant differences in their expression of activation, inhibitory, and cytotoxic molecules, suggesting that not all fetus-specific CD8+ T cell responses are suppressed at the maternal-fetal interface. Understanding how decidual CD8+ T cell specificity relates to their function and tissue residency is crucial in advancing understanding of their contribution to placental inflammation and control of congenital infections.

Progressive accumulation of hyperinflammatory NKG2D low NK cells in early childhood severe atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic co-morbidities remain ill-defined. Herein, analysis of circulating NK cells in a longitudinal early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D, which was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aero allergens, a risk factor for development of asthma. Individual-level longitudinal analysis in a subset of children revealed co-incident reduction of NKG2D on NK cells with acquired or persistent sensitization, and this was associated with impaired skin barrier function assessed by transepidermal water loss. Low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine TNF-α. These observations provide important insights into a potential mechanism underlying the development of allergic co-morbidity in early life in children with AD which involves altered NK-cell functional responses, and define an endotype of severe AD.

Proinflammatory polarization of monocytes by particulate air pollutants is mediated by induction of trained immunity in pediatric asthma.

BACKGROUND: The impact of exposure to air pollutants, such as fine particulate matter (PM), on the immune system and its consequences on pediatric asthma, are not well understood. We investigated whether ambient levels of fine PM with aerodynamic diameter ≤2.5 microns (PM2.5 ) are associated with alterations in circulating monocytes in children with or without asthma. METHODS: Monocyte phenotyping was performed by cytometry time-of-flight (CyTOF). Cytokines were measured using cytometric bead array and Luminex assay. ChIP-Seq was utilized to address histone modifications in monocytes. RESULTS: Increased exposure to ambient PM2.5 was linked to specific monocyte subtypes, particularly in children with asthma. Mechanistically, we hypothesized that innate trained immunity is evoked by a primary exposure to fine PM and accounts for an enhanced inflammatory response after secondary stimulation in vitro. We determined that the trained immunity was induced in circulating monocytes by fine particulate pollutants, and it was characterized by the upregulation of proinflammatory mediators, such as TNF, IL-6, and IL-8, upon stimulation with house dust mite or lipopolysaccharide. This phenotype was epigenetically controlled by enhanced H3K27ac marks in circulating monocytes. CONCLUSION: The specific alterations of monocytes after ambient pollution exposure suggest a possible prognostic immune signature for pediatric asthma, and pollution-induced trained immunity may provide a potential therapeutic target for asthmatic children living in areas with increased air pollution.

B cell repertoire in children with skin barrier dysfunction supports altered IgE maturation associated with allergic food sensitization.

The skin is a major immune organ and skin barrier dysfunction is a major risk factor for the development of the inappropriate immune response seen in allergic disease. Skin barrier disruption alters the landscape of antigens experienced by the immune system and the downstream impacts on the antibody repertoire remain poorly characterized, particularly for the IgE isotype responsible for allergic specificity and in early life, when allergic disease is developing. In this study, we sequenced antibody gene repertoires from a large and well-characterized cohort of children with atopic dermatitis and found that food sensitization was associated with lower mutation frequencies in the IgE compartment. This trend was abrogated in children living with pets during the first year of life. These results elucidate potential molecular mechanisms underlying the protective effects of pet ownership and non-antiseptic environs reported for allergic disease, and the hygiene hypothesis more broadly. We also observed increased IgE diversity and increased isotype-switching to the IgE isotype, suggesting that B cell development, particularly isotype-switching, is heavily altered in the those with food allergen sensitizations relative to those without food allergen sensitizations. Unlike for food antigens, aeroallergen sensitization exhibited no effect on IgE mutation or diversity. Consistent patterns of antibody rearrangement were associated with food allergen sensitization in subjects with atopic dermatitis. Thus, we propose the Immune Repertoire in Atopic Disease (IRAD) score, to quantify this repertoire shift and to aid clinically in patient diagnosis and risk stratification.

Food Allergy Characteristics Associated With Coexisting Eosinophilic Esophagitis in FARE Registry Participants.

BACKGROUND: Eosinophilic esophagitis (EoE) can coexist in individuals with food allergy. OBJECTIVE: To evaluate the characteristics of food-allergic patients with and without coexisting EoE using a large food allergy patient registry. METHODS: Data were derived from 2 Food Allergy Research & Education, Inc, Patient Registry surveys. A series of multivariable regression models were used to evaluate associations between demographic, comorbidity, and food allergy characteristics and the likelihood of reporting EoE. RESULTS: Five percent (n = 309) of registry participants (n = 6074; ages <1 year->80 years, mean, 20.20 ± 15.37 years) reported having EoE. The odds of having EoE were significantly greater in male participants (adjusted odds ratio [aOR], 1.3; 95% CI, 1.04-1.72) and those with comorbid asthma (aOR, 2.0; 95% CI, 1.55-2.49), allergic rhinitis (aOR, 1.8; 95% CI, 1.37-2.22), oral allergy syndrome (aOR, 2.8; 95% CI, 2.09-3.70), food protein-induced enterocolitis syndrome (aOR, 2.5; 95% CI, 1.34-4.84), and hyper-IgE syndrome (aOR, 7.6; 95% CI, 2.93-19.92), though not atopic dermatitis (aOR, 1.3; 95% CI, 0.99-1.59), when adjusting for demographics (sex, age, race, ethnicity, and geographic location). Those with a greater number of food allergies (aOR, 1.3; 95% CI, 1.23-1.32), more frequent food-related allergic reactions (aOR, 1.2; 95% CI, 1.11-1.24), previous anaphylaxis (aOR, 1.5; 95% CI, 1.15-1.83), and health care utilization for food-related allergic reactions (aOR, 1.3; 95% CI, 1.01-1.67)-specifically intensive care unit admission (aOR, 1.2; 95% CI, 1.07-1.33)-were more likely to have EoE after controlling for demographics. However, no significant difference in ever using epinephrine for food-related allergic reactions was detected. CONCLUSIONS: These self-reported data showed that coexisting EoE is associated with an increased number of food allergies, food-related allergic reactions per year, and measures of reaction severity, calling attention to the likely increased health care needs of food-allergic patients with EoE.

Pro-Inflammatory Alterations of Circulating Monocytes in Latent Tuberculosis Infection.

Background: Latent tuberculosis infection (LTBI) has been associated with increased cardiovascular risk. We investigated the activation and pro-inflammatory profile of monocytes in individuals with LTBI and their association with coronary artery disease (CAD). Methods: Individuals 40-70 years old in Lima, Peru, underwent QuantiFERON-TB testing to define LTBI, completed a coronary computed tomography angiography to evaluate CAD, and provided blood for monocyte profiling using flow cytometry. Cells were stimulated with lipopolysaccharide to assess interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α responses. Results: The clinical characteristics of the LTBI (n = 28) and non-LTBI (n = 41) groups were similar. All monocyte subsets from LTBI individuals exhibited higher mean fluorescence intensity (MFI) of CX3CR1 and CD36 compared with non-LTBI individuals. LTBI individuals had an increased proportion of nonclassical monocytes expressing IL-6 (44.9 vs 26.9; P = .014), TNF-α (62.3 vs 35.1; P = .014), and TNF-α+IL-6+ (43.2 vs 36.6; P = .042). Among LTBI individuals, CAD was associated with lower CX3CR1 MFI on classical monocytes and lower CD36 MFI across all monocyte subsets. In multivariable analyses, lower CD36 MFI on total monocytes (b = -0.17; P = .002) and all subsets remained independently associated with CAD in LTBI. Conclusions: Individuals with LTBI have distinct monocyte alterations suggestive of an exacerbated inflammatory response and tissue migration. Whether these alterations contribute to cardiovascular disease pathogenesis warrants further investigation.

Mass cytometry analysis of blood from peanut-sensitized tolerant and clinically allergic infants.

IgE-mediated food allergies in infants are a significant health concern, with peanut allergy being of particular interest due to its prevalence and severity. Among individuals who produce peanut-specific IgE some experience no adverse reaction on peanut consumption. This asymptomatic phenotype is known as sensitized tolerance. To elucidate the immune environment of peanut sensitized tolerant and clinically allergic one-year-olds, high-dimensional mass cytometry was conducted as part of the HealthNuts study. The resulting data includes peripheral blood mononuclear cells from 36 participants encompassing non-allergic, peanut sensitized with tolerance, and clinically peanut allergic infants. The raw mass cytometry data is described here and freely available for reuse through the Immunology Database and Analysis Portal (ImmPort). Additional allergy information and serum vitamin D levels of the participants were measured and are also included in the data upload. These high-dimensional mass cytometry data, when combined with clinical information, offer a broad immune profile of peanut allergic and sensitized tolerant infants.

CD8+ T cell differentiation status correlates with the feasibility of sustained unresponsiveness following oral immunotherapy.

While food allergy oral immunotherapy (OIT) can provide safe and effective desensitization (DS), the immune mechanisms underlying development of sustained unresponsiveness (SU) following a period of avoidance are largely unknown. Here, we compare high dimensional phenotypes of innate and adaptive immune cell subsets of participants in a previously reported, phase 2 randomized, controlled, peanut OIT trial who achieved SU vs. DS (no vs. with allergic reactions upon food challenge after a withdrawal period; n = 21 vs. 30 respectively among total 120 intent-to-treat participants). Lower frequencies of naïve CD8+ T cells and terminally differentiated CD57+CD8+ T cell subsets at baseline (pre-OIT) are associated with SU. Frequency of naïve CD8+ T cells shows a significant positive correlation with peanut-specific and Ara h 2-specific IgE levels at baseline. Higher frequencies of IL-4+ and IFNγ+ CD4+ T cells post-OIT are negatively correlated with SU. Our findings provide evidence that an immune signature consisting of certain CD8+ T cell subset frequencies is potentially predictive of SU following OIT.

CyAnno: a semi-automated approach for cell type annotation of mass cytometry datasets.

MOTIVATION: For immune system monitoring in large-scale studies at the single-cell resolution using CyTOF, (semi-)automated computational methods are applied for annotating live cells of mixed cell types. Here, we show that the live cell pool can be highly enriched with undefined heterogeneous cells, i.e. 'ungated' cells, and that current semi-automated approaches ignore their modeling resulting in misclassified annotations. RESULT: We introduce 'CyAnno', a novel semi-automated approach for deconvoluting the unlabeled cytometry dataset based on a machine learning framework utilizing manually gated training data that allows the integrative modeling of 'gated' cell types and the 'ungated' cells. By applying this framework on several CyTOF datasets, we demonstrated that including the 'ungated' cells can lead to a significant increase in the precision of the 'gated' cell types prediction. CyAnno can be used to identify even a single cell type, including rare cells, with higher efficacy than current state-of-the-art semi-automated approaches. AVAILABILITY AND IMPLEMENTATION: The CyAnno is available as a python script with a user-manual and sample dataset at https://github.com/abbioinfo/CyAnno. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Novel application of a discrete time-to-event model for randomized oral immunotherapy clinical trials with repeat food challenges.

The evaluation of double-blind, placebo-controlled food challenges (DBPCFC) generally focuses on a participant passing a challenge at a predetermined dose, and does not consider the dose of reaction for those who fail or are censored due to study discontinuation. Further, a number of food allergy trials have incorporated multiple DBPCFCs throughout the duration of the study in order to evaluate changes in reaction over time including sustained unresponsiveness from treatment. Outcomes arising from these trials are commonly modeled using Chi-squared or Fisher's exact tests at each time point. We propose applying time-to-event methodology to food allergy trials in order to exploit the inherent granularity of challenge outcomes that additionally accommodates repeated DBPCFCs. Specifically, we consider dose-to-failure for each study challenge and extend the cumulative tolerated dose across challenges to result in a dose-time axis. A discrete time-to-event framework is applied to the dose-time outcome to assess the efficacy of treatment across the entire study period. We illustrate ideas with data from the Peanut Oral Immunotherapy Study: Safety, Efficacy and Discovery (POISED) trial, conducted at Stanford University, which evaluated the efficacy of oral immunotherapy on desensitization and sustained unresponsiveness in peanut allergic children and adults. We demonstrate the advantages of time-to-event approaches for assessing the efficacy of treatment over time and incorporating information for those who failed or were lost to follow up. Further, we introduce a dose-time outcome that is interpretable to clinicians and allows for examination of such outcomes over time.

Peanut oral immunotherapy in a pediatric allergy clinic: Patient factors associated with clinical outcomes.

BACKGROUND: Additional information is needed to inform optimal patient selection, expected outcomes, and treatment end points for clinical peanut oral immunotherapy (OIT). OBJECTIVE: To provide insight into the optimal patient selection, expected outcomes, and treatment end points for clinical peanut oral immunotherapy by analyzing a real-world peanut OIT cohort. METHODS: Records were reviewed for 174 children undergoing peanut OIT at a pediatric allergy clinic. Patient age, peanut skin prick test results, and peanut-specific immunoglobulin E (sIgE) results, with inclusion of additional foods in OIT, were analyzed for correlations with OIT outcomes. RESULTS: To date, 144 patients have achieved maintenance dosing, 50 of whom transitioned to ad lib twice-weekly peanut ingestion. A total of 30 discontinued OIT. In addition, 47 patients who underwent multifood OIT had no significant difference in reactions (FDR-adjusted P = .48) or time-to-reach maintenance (FDR-adjusted P = .48) compared with those on peanut OIT alone. Age at initiation inversely correlated with achievement of maintenance: 92% of patients 0.5 to less than 5 years, 81% of those 5 to less than 11 years, and 70% of those 11 to less than 18 years reached and continued maintenance (P = .01). Baseline peanut-sIgE level positively correlated with number of reactions during updosing (P < .001) and maintenance (P = .005), though it was not significantly different in patients achieving successful maintenance vs those who discontinued OIT (P = .09). Furthermore, 66% of patients experienced greater than or equal to 1 adverse reaction during OIT. Of those on ad lib peanut ingestion, 2 reported mild reactions after lapses in peanut consumption. CONCLUSION: Clinical peanut OIT has similar outcomes to research protocols. OIT can be successful in older children and those with high peanut-sIgE levels, though these factors affect outcomes. Clinical and laboratory criteria can guide successful transition to intermittent ad lib peanut consumption.

Immune changes beyond Th2 pathways during rapid multifood immunotherapy enabled with omalizumab.

BACKGROUND: Multifood oral immunotherapy (mOIT) with adjunctive anti-IgE (omalizumab, XOLAIR® ) treatment affords safe, effective, and rapid desensitization to multiple foods, although the specific immune mechanisms mediating this desensitization remain to be fully elucidated. METHODS: Participants in our phase 2 mOIT trial (NCT02643862) received omalizumab from baseline to week 16 and mOIT from week 8 to week 36. We compared the immune profile of PBMCs and plasma taken at baseline, week 8, and week 36 using high-dimensional mass cytometry, component-resolved diagnostics, the indirect basophil activation test, and Luminex. RESULTS: We found (i) decreased frequency of IL-4+ peanut-reactive CD4+ T cells and a marked downregulation of GPR15 expression and CXCR3 frequency among γδ and CD8+ T-cell subsets at week 8 during the initial, omalizumab-alone induction phase; (ii) significant upregulation of the skin-homing receptor CCR4 in peanut-reactive CD4+ T and Th2 effector memory (EM) cells and of cutaneous lymphocyte-associated antigen (CLA) in peanut-reactive CD8+ T and CD8+ EM cells; (iii) downregulation of CD86 expression among antigen-presenting cell subsets; and (iv) reduction in pro-inflammatory cytokines, notably IL-17, at week 36 post-OIT. We also observed significant attenuation of the Th2 phenotype post-OIT, defined by downregulation of IL-4 peanut-reactive T cells and OX40 in Th2EM cells, increased allergen component-specific IgG4/IgE ratio, and decreased allergen-driven activation of indirectly sensitized basophils. CONCLUSIONS: This exploratory study provides novel comprehensive insight into the immune underpinnings of desensitization through omalizumab-facilitated mOIT. Moreover, this study provides encouraging results to support the complex immune changes that can be induced by OIT.

Altered immune cell profiles and impaired CD4 T-cell activation in single and multi-food allergic adolescents.

BACKGROUND: Approximately 5% of adolescents have a food allergy, with peanut and tree nut allergies the most common. Having two or more food allergies in adolescence also doubles the risk of any adverse food reaction, and is associated with increased dietary and social burden. Investigations of immune function in persistently food allergic children are rare. OBJECTIVE: In the present study, we aimed to investigate the immune mechanisms that underlie food allergy in adolescence. METHODS: We used high-dimensional flow cytometry, unsupervised computational analysis and functional studies to comprehensively phenotype a range of non-antigen-specific immune parameters in a group of well-characterized adolescents with clinically defined single peanut allergy, multi-food allergy and aged-matched non-food allergic controls. RESULTS: We show that food allergic adolescents have higher circulating proportions of dendritic cells (p = .0084, FDR-adjusted p = .087, median in no FA: 0.63% live cells, in FA: 0.93%), and higher frequency of activated, memory-like Tregs relative to non-food allergic adolescents (p = .011, FDR-adjusted p = .087, median in no FA: 0.49% live cells, in FA: 0.65%). Cytokine profiling revealed that CD3/CD28 stimulated naïve CD4 T cells from food allergic adolescents produced less IL-6 (p = .0020, FDR-adjusted p = .018, median log2 fold change [stimulated/unstimulated] in no FA: 3.03, in FA: 1.92) and TNFα (p = .0044, FDR-adjusted p = .020, median in no FA: 9.16, in FA: 8.64) and may secrete less IFNγ (p = .035, FDR-adjusted p = .11, median in no FA: 6.29, in FA: 5.67) than naïve CD4 T cells from non-food allergic controls. No differences between clinical groups were observed for LPS-stimulated monocyte secretion of cytokines. CONCLUSIONS: These results have important implications for understanding the evolution of the immune response in food allergy throughout childhood, revealing that dendritic cell and T-cell signatures previously identified in early life may persist through to adolescence.

Aging and CMV discordance are associated with increased immune diversity between monozygotic twins.

BACKGROUND: Broadly, much of variance in immune system phenotype has been linked to the influence of non-heritable factors rather than genetics. In particular, two non-heritable factors: aging and human cytolomegavirus (CMV) infection, have been known to account for significant inter-individual immune variance. However, many specific relationships between them and immune composition remain unclear, especially between individuals over narrower age ranges. Further exploration of these relationships may be useful for informing personalized intervention development. RESULTS: To address this need, we evaluated 41 different cell type frequencies by mass cytometry and identified their relationships with aging and CMV seropositivity. Analyses were done using 60 healthy individuals, including 23 monozygotic twin pairs, categorized into young (12-31 years) and middle-aged (42-59 years). Aging and CMV discordance were associated with increased immune diversity between monozygotic twins overall, and particularly strongly in various T cell populations. Notably, we identified 17 and 11 cell subset frequencies as relatively influenced and uninfluenced by non-heritable factors, respectively, with results that largely matched those from studies on older-aged cohorts. Next, CD4+ T cell frequency was shown to diverge with age in twins, but with lower slope than in demographically similar non-twins, suggesting that much inter-individual variance in this cell type can be attributed to interactions between genetic and environmental factors. Several cell frequencies previously associated with memory inflation, such as CD27- CD8+ T cells and CD161+ CD4+ T cells, were positively correlated with CMV seropositivity, supporting findings that CMV infection may incur rapid aging of the immune system. CONCLUSIONS: Our study confirms previous findings that aging, even within a relatively small age range and by mid-adulthood, and CMV seropositivity, both contribute significantly to inter-individual immune diversity. Notably, we identify several key immune cell subsets that vary considerably with aging, as well as others associated with memory inflation which correlate with CMV seropositivity.