Systems-level immunomonitoring in pediatric cancer and early life development

The immune system undergoes dynamic developmental changes throughout childhood and is simultaneously shaped by disease, nutrition, and microbial colonization. Understanding these processes in pediatric populations requires analytical approaches that capture immune complexity across multiple biological dimensions. This thesis applies systems-level immunomonitoring — integrating mass cytometry, plasma proteomics, transcriptomics, single-cell T cell receptor sequencing, and gut metagenomics — to characterize immune variation in two clinically consequential contexts: pediatric cancer and early-life immune development. In Paper I, we established the Immune Systems Against Cancer in Children (ISAC) cohort, a population-based study of 191 children (aged 0-18 years) with 34 types of solid tumors diagnosed at Karolinska University Hospital. Systems-level immunomonitoring at diagnosis — comprising 49-parameter mass cytometry on stabilized whole blood, plasma proteomics (92 proteins), and whole-blood RNA sequencing — revealed that tumor type, rather than age, is the dominant driver of immune variation in children with cancer. Children with cancer exhibited consistently elevated neutrophils and reduced NK cells and T cells compared to 295 age-matched healthy controls, with neutrophil-to-lymphocyte ratios elevated at every age interval. Tumor mutation burden correlated with CD8+ T cell infiltration in neuroblastoma and Wilms tumors. Critically, clonally expanded T cells were rare at diagnosis across all tumor types but could be induced by chemotherapy, and the transcriptional programs of expanded pediatric CD8+ T cells were strikingly similar to those in adult cancers, suggesting that limited checkpoint inhibitor efficacy in children reflects scarcity of expanded clones rather than intrinsic T cell dysfunction. Paper II focused on classical Hodgkin lymphoma (cHL), the most common malignancy in adolescents. Comparing 13 children with cHL to 11 with other lymphomas and 89 with solid tumors, we identified CCL17 and MCP-4 as diagnostic biomarkers distinguishing cHL from other pediatric cancers. Both proteins were markedly elevated in cHL and showed disease-specific regulation independent of age-related developmental trends. Longitudinal monitoring through OEPA chemotherapy revealed that greater CCL17 decline associated with adequate early treatment response, while lower baseline granzyme levels identified patients at higher risk of neutropenic fever. These findings demonstrate that plasma immune profiling could complement PET/CT-based response assessment and guide supportive care allocation in pediatric cHL. Paper III (the AMPLIO trial) evaluated a synbiotic infant formula containing seven human milk oligosaccharides and Bifidobacterium longum subsp. infantis in 240 healthy term formula-fed infants in the Philippines. A blood sub-study (n=86) using mass cytometry (>60 markers, 94 cell clusters) and plasma proteomics (358 proteins) demonstrated that synbiotic supplementation drove near-complete B. infantis colonization, reduced gut inflammation, and promoted systemic immune profiles resembling those of breastfed infants. Synbiotic-fed infants showed reduced naive CD4+ T cells and lower inflammatory protein signatures at four months, following a biphasic pattern of early immune activation and subsequent resolution consistent with immune training. Gut microbiome trajectory predicted systemic immune phenotype more strongly than feeding group assignment alone, positioning microbial colonization as a key intermediary in the gut-systemic immune axis. Synbiotic-fed infants experienced significantly fewer upper respiratory tract infections. Paper IV characterized immune development in 72 extremely preterm infants (born at 22-28 weeks) through longitudinal mass cytometry (14.3 million single cells) and serum proteomics (538 proteins) from birth to day 100. Gestational age shaped the rate but not the direction of postnatal immune maturation, with infants born at 22-24 weeks showing substantially slower normalization than those at 25-27 weeks. Mother's own milk, compared to pasteurized donor milk, significantly accelerated immune convergence toward term-born phenotypes. NK cell subclusters were the most strongly discriminating features between feeding groups: a random forest classifier achieved an AUC of 0.92, with mother's own milk promoting immunoregulatory CD56bright NK cells over cytotoxic CD56dim phenotypes. Crucially, these immune differences were largely independent of gut microbiome composition, implicating direct bioactive effects of fresh maternal milk components — including lactoferrin, secretory IgA, and epidermal growth factor — destroyed or reduced during donor milk pasteurization. Together, these four studies demonstrate the power of systems-level immunomonitoring to reveal clinically meaningful immune variation in pediatric populations. They establish that the pediatric immune system is not merely an immature version of adult immunity but a dynamically regulated system whose trajectory is shaped by tumor biology, nutritional environment, and microbial colonization. The findings identify candidate biomarkers for precision medicine in pediatric cancer, provide mechanistic insight into how early-life nutrition shapes immune development through both microbiome-dependent and microbiome-independent pathways, and generate comprehensive immune profiling resources for the research community. List of scientific papers I. Systems-level immunomonitoring in children with solid tumors to enable precision medicine. Chen, Q.* , Zhao, B.* , Tan, Z.* , Hedberg, G., Wang, J., Gonzalez, L., Mugabo, C. H., Johnsson, A., Negrini, E., Piñero Páez, L., Rodriguez, L., James, A., Bernhardsson, A. K., Reitzner, S. M., von Walden, F., O'Neill, O., Barcenilla, H., Wang, C., Davis, M. M., Carlson, L.-M., Pal, N., Blomgren, K., Herold, N., Repsilber, D., Lakshmikanth, T., Kogner, P., Ljungblad, L., Brodin, P. Cell 188, 1425-1440.e11 (2025). https://doi.org/10.1016/j.cell.2024.12.014 II. Systemic Immune Alterations in Paediatric Classical Hodgkin Lymphoma With CCL17 and MCP-4 as Diagnostic and Predictive Biomarkers. Hedberg, G., Chen, Q. Lakshmikanth, T., Herold, N., Kogner, P., Brodin, P., Ljungblad, L. EJHaem 7, e70228 (2026). https://doi.org/10.1002/jha2.70228 III. Infant formula with a novel synbiotic blend of 10 human milk oligosaccharides, bovine milk derived oligosaccharides, and B. infantis LMG11588 optimizes markers of immune development in healthy infants: a double-blind, randomized, controlled study. Chen, Q., Capeding, M. R. Z., Trinidad-Aseron, M., Tenorio-Quiñones, V. M. R., Barcenilla, H., Gonzalez, L., Johnsson, A., Hays, N. P., Darwish, N., Fumero, C., Li, H., Mondragon, A., Chung, C. K., Rasmussen, J. A., Moll, J. M., Stauch, W., Schwenk, J., Lakshmikanth, T., Sprenger, N., Noti, M., Brodin, P.* [Manuscript] IV. Immune development differs between preterm newborns fed mothers' own milk and donor milk. Tan, Z., Zhong, W., Danielsson, H., Arzoomand, A., Lakshmikanth, T., Chen, Q., Mikes, J., Wang, J., Chen, Y., James, A., Nilsson, A. K., Elfvin, A., Brusselaers, N., Portlock, T., Lundgren, P., Sävman, K., Wackernagel, D., Hansen-Pupp, I., Uhlén, M., Hellström, A., Brodin, P. iScience 28, 112918 (2025). https://doi.org/10.1016/j.isci.2025.112918 *These authors contributed equally.