Lothert, Petra K.

ABSTRACT Objective Fetal surgery (FS) for spina bifida (SB) repair improves infant outcomes; however, spontaneous preterm birth (sPTB) is common. We aimed to evaluate maternal and fetal immune responses following FS for SB repair. Method Pregnant participants undergoing fetoscopic SB repair at 24–26 weeks of gestational age (GA) were prospectively recruited and compared to GA matched low‐risk controls. Blood samples were collected at baseline, 2‐week post‐surgery (POD14), and delivery. Cytokine analysis, flow cytometry, and T cell receptor (TCR) sequencing were performed. Results sPTB occurred in 5/6 of the FS group (0/6 of controls). Maternal blood in the FS group demonstrated increased alpha‐fetoprotein (AFP) and anti‐inflammatory IL‐1RA at baseline and POD14, increased pro‐inflammatory IL‐8 only post‐operatively, and elevated growth factors throughout. T cell phenotypes following FS revealed reduced TH17 frequency but increased activation receptors on cytotoxic T cells. At delivery, the FS cohort had reduced Th2 and Th17 helper T cells and CD4+ memory T cells, but CD8 T cells still showed increased activation. No TCR expansion was detected post‐surgery, suggesting antigen‐independent activation. Cord blood in FS repaired neonates had increased AFP, IL‐8, IL‐1RA. Conclusion Systemic immune changes following FS, particularly T cell maturation and activation, were observed. Further characterization of these mechanisms may help predict and prevent surgery‐induced PTB, improving outcomes.

Abstract Introduction: Deletion in the short arm of chromosome 17 (del17p) is a strong, negative prognostic biomarker found in about eight percent of patients with untreated chronic lymphocytic leukemia (CLL). CLL patients with del17p exhibit resistance to therapy, accelerated disease progression, and global immunodeficiency. It is unclear the extent to which global immunodeficiency impacts disease course and therapy outcomes. The goal of this study was to document the blood immune repertoire of patients with untreated CLL who have del17p and associate with clinical features, time to first treatment (TFT), and overall survival (OS). Methods: Biobanked blood samples from treatment-naïve del17p CLL patients (n = 58) and age-matched controls (n = 5) were obtained. B cells and T cells were analyzed with comprehensive, cell type-specific flow cytometry panels and visualized with unbiased dimensionality reduction. Lab values were log-transformed and compared using t-tests and one-way ANOVAs. TFT was calculated from date of sample collection to date of first CLL-directed therapy or last known untreated date; OS was calculated from date of sample collection to death or last known alive status. Cox proportional hazards regression analyses (results reported as hazard ratio [HR] and 95% confidence interval [CI]) were employed to identify associations of lab values with OS and TFT. Results: Patients with del17p CLL had increased proportions of T regulatory cells (Tregs) among total CD4+ T cells compared to controls (CLL 11.1%, control 5.7%, p = 0.05). A higher proportion of CD8+ T cells was associated with longer TFT (HR 0.57, 95%CI 0.36-0.89); in contrast, higher proportions of TIGIT+ (HR 1.83, 95%CI 1.03-3.28) and TIM3+ conventional (non-Treg) CD4+ T cells were associated with shorter TFT (HR 1.70, 95%CI 1.02-2.84). A higher proportion of PD1+ CD8+ T cells was associated with shorter OS (HR 1.79, 95%CI 1.05-3.06), in addition to PD1+ conventional CD4+ T cell proportion (HR 2.21, 95%CI 1.06-4.60). CLL B cells expressed PD-1 and inducible T cell costimulator (ICOS) at significantly lower median fluorescence intensities (MFIs) than control B cells (p < 0.0001 for both). The CXCR5+ MFI (HR 1.0002, 95%CI 1.0001-1.003) and CD23+ MFI (HR 1.88, 95%CI 1.01-3.50) of CLL B cells were associated with shorter TFT. Discussion: In this cohort of treatment-naïve del17p CLL patients, we determined that patients with higher proportions of exhausted CD4+ and CD8+ T cell phenotypes had significantly shorter OS. Cytotoxic T cell proportion positively associated with TFT, whereas proportions of CD4+ T cell exhaustion phenotypes negatively associated with TFT. Higher CXCR5+ and CD23+ MFIs of CLL B cells also associated with shorter TFT. The preliminary findings from our study suggest that the blood immune repertoire has strong associations with clinically relevant outcomes such as time to first treatment and overall survival in patients with untreated del17p CLL. Citation Format: Ethan J. Katcher, Kim A. Gwin, Sameer A. Parikh, Kari G. Rabe, Petra K. Lothert, Preeti Trisal, Esteban Braggio, Susan L. Slager, Nicholas Strong, Daniel L. Van Dyke, Zhiquan Wang, C. Chris Huang, Anita K. Gandhi, Kay L. Medina, Neil E. Kay. Correlating the blood immune repertoire and clinical features of del17p CLL patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB345.

Abstract The intestinal epithelial barrier is vital in preventing translocating bacteria and subsequent systemic infections. This protection is provided by host factors, by the intestinal microbiota, and, during early life, by breast milk. While the beneficial functions of breast milk are numerous to the nursing infant, it has been observed that proteins such as antibodies and growth factors are secreted in higher concentrations early during lactation and decrease as the milk matures. One such growth factor, epidermal growth factor (EGF), regulates the development of colonic goblet cell-associated antigen passages (GAPs). Decrease of EGF through disruption of synchronous breastfeeding or deletion of the epidermal growth factor receptor from goblet cells alters the kinetics of GAP formation, which can serve as a passage for pathogenic bacteria. In a murine model of early GAP formation utilizing an inhibitor of EGF receptor, we observed a variety of gut-resident pathogens were able to utilize GAPs to disseminate systemically and cause disease, including Escherichia coli, Enterococcus faecalis, Enterobacter cloacae, and Pseudomonas aeruginosa. In human studies, we found a strong correlation between concentrations of EGF in the diet and stool of very low birth weight infants. Notably, formula-fed infants contained significantly less EGF in both diet and stool. Future work will assess EGF in donor milk, which we find has highly variable EGF concentration. This work has strong implications for how diet influences risk of developing infections from gut-resident pathogens and how maternal EGF present in breastmilk is synchronized to the newborn infant to provide appropriate protection in early life. Supported by grants from NIH (AI144721, DK122187)

The current paradigms of known peripheral immune abnormalities in B-Chronic Lymphocytic Leukemia (CLL) are not able to consistently explain patient complications and it is difficult to correct a given CLL patient's immune status. Here, we expand on our initial report demonstrating bone marrow (BM) hematopoietic dysfunction in untreated CLL patients (Manso et al., Leukemia volume 33, pages 638-652, 2019). CLL patient BM had significantly reduced frequencies and short-term functional capacity of hematopoietic stem and progenitor cells (HSPCs). Additionally, the remaining progenitors exhibited increased protein levels of the key hematopoietic transcriptional regulators GATA-2 and PU.1. We further evaluated the frequency and function of myeloid stem cells from controls and untreated CLL patients by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays. Over the 5 week duration of the assay, we observed delayed and partial recovery of myelopoiesis from CLL-derived HSPCs (Figure 1A). These data suggest that removal of HSPCs from the CLL leukemic microenvironment partially recovers their ability to sustain myelopoiesis. A known inflammatory mediator and hematopoiesis-modulating cytokine that is constitutively produced by CLL cells, TNF-α, induced increased expression of GATA-2 and PU.1 in specific HSPC subsets and reduced formation of short-term colony forming units in vitro. Addition of TNF-α to LTC-IC assays resulted in a striking ablation of myelopoiesis in a dose-dependent manner, partially reproducing the ex vivo results (Figure 1B). To further assess the direct impact of CLL cells on HSPC biology, isolated HSPCs from controls were exposed in vitro to leukemic CLL cells. The co-culture induced overexpression of GATA-2 and PU.1 in distinct HSPC populations, recapitulating our ex vivo findings (Figure 1C-D). When cell-cell contact was inhibited by use of Transwell inserts, an intermediate increase in GATA-2 and PU.1 was observed, highlighting the contributions of both soluble mediators and cell-cell contact to HSPC alterations. In both direct and Transwell co-culture conditions, overexpression of GATA-2 and PU.1 was reversed when TNF-α was neutralized (Figure 1E-F). Taken together, these findings indicate a significant role for CLL-derived TNF-α in HSPC modulation and expand our previous observations of BM dysfunction in untreated CLL patients. This data offers new molecular insight into the contribution of the leukemic microenvironment to altered hematopoiesis, contributing to immunodeficiency in CLL, and identifies TNF-α as a potential therapeutic target for correction of hematopoiesis in CLL disease. Disclosures Ding: Merck: Research Funding; DTRM Biopharma: Research Funding. Parikh:AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Novak:Celgene Coorperation: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board.