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SummaryCerebrovascular damage coexists with Alzheimer’s disease (AD) pathology and increases AD risk. However, it is unclear whether endothelial progenitor cells reduce AD risk via cerebrovascular repair. By using the Framingham Heart Study (FHS) offspring cohort, which includes data on different progenitor cells, the incidence of AD dementia, peripheral and cerebrovascular pathologies, and genetic data (n = 1,566), we found that elevated numbers of circulating endothelial progenitor cells with CD34+CD133+ co-expressions had a dose-dependent association with decreased AD risk (HR = 0.67, 95% CI: 0.46-0.96, p = 0.03) after adjusting for age, sex, years of education, andAPOEε4. With stratification, this relationship was only significant among those individuals who had vascular pathologies, especially hypertension (HTN) and cerebral microbleeds (CMB), but not among those individuals who had neither peripheral nor central vascular pathologies. We applied a genome-wide association study (GWAS) and found that the number of CD34+CD133+ cells impacted AD risk depending on the homozygous genotypes of two genes:KIRREL3rs580382 CC carriers (HR = 0.31, 95% CI: 0.17-0.57, pKIRREL3rs4144611 TT carriers (HR = 0.29, 95% CI: 0.15-0.57, pEXOC6Brs61619102 CC carriers (HR = 0.49, 95% CI: 0.31-0.75, pKIRREL3TT/CT or GG/GT carriers andEXOC6BGG/GC carriers. Our findings suggest that circulating CD34+CD133+ endothelial progenitor cells can be therapeutic in reducing AD risk in the presence of cerebrovascular pathology, especially inKIRREL3andEXOC6Bgenotype carriers.

Hematopoietic stem cells (HSCs) reside at the top of the hematopoietic hierarchy and can give rise to all the mature blood cell types in our body, while at the same time maintaining a pool of HSCs through self-renewing divisions. This potential is reflected in their functional definition as cells that are capable of long-term multi-lineage engraftment upon transplantation. While all HSCs meet these criteria, subtle differences exist between developmentally different populations of these cells. Here we present a comprehensive overview of traditional and more recently described markers for phenotyping HSCs and their downstream progeny. To address the need to assess the growing number of surface molecules expressed in various HSC-enriched fractions at different developmental stages, we have developed an extensive multi-parameter spectral flow cytometry panel to phenotype hematopoietic stem and multipotent progenitor cells (HSC/MPPs) throughout development. In this study we then employ this panel to comprehensively profile the HSC compartment in the human fetal liver (FL), which is endowed with superior engraftment potential compared to postnatal sources. Spectral cytometry lends an improved resolution of marker expression to our comprehensive approach, allowing to extract combinatorial expression signatures of several relevant HSC/MPP markers to precisely characterize the HSC/MPP fraction in a variety of tissues.

Lung cancer (LC) is the leading cause of cancer death worldwide. Lung squamous cell carcinoma (LSCC) is the second most prevalent subtype of LC, accounting for approximately 22% of cases. It is strongly associated with smoking, with approximately 90% of cases attributable to cigarette smoke exposure. Despite the strong association with smoking, not all smokers will develop lung cancer. From this, two key questions arise: which patients will develop the lesions preceding LSCC, and among those, which will progress to frank carcinoma? Stratification of at-risk patients for diversion into intervention and prevention strategies, such as increased monitoring and chemical interception, remains an urgent need, and a greater understanding of the factors contributing to progression of LSCC premalignancy is critical in addressing this. Previous evidence suggests a role for the immune system in the development of preinvasive disease, and we hypothesize that immune differences between at-risk smokers may also play a role in which patients progress on to frank LSCC and which do not. Using the N-Nitrosotris-(2-chloroethyl) urea (NTCU) carcinogen-induced murine model of LSCC, we sought to explore the immune contexture of LSCC premalignancy in both NTCU-sensitive and NTCU-resistant mouse strains. We immunophenotyped mouse lungs via flow cytometry and imaging mass cytometry over the course of disease initiation and progression to carcinoma. We found that although the frequency of γδ T cells in control animals were the same between sensitive and resistant mice, in sensitive mice the frequency of γδ T cells and the expression of activation/exhaustion markers increased with histological progression of PMLs, along with a decrease in the frequency of cytotoxic CD8+ T cells. Given that pro-tumor γδ T cells have been established to mediate airway inflammation via recruitment of myeloid cells (e.g., macrophages, neutrophils, and myeloid derived suppressor cells) and to influence maturation/exhaustion of T cells, these findings suggest γδ T cells potentially contribute to the early establishment of an inflammatory and immunosuppressive microenvironment in developing LSCC premalignancy. By better understanding how immune alterations within the precancerous microenvironment contribute to lesion progression, immunomodulatory intervention agents can be developed and deployed in at-risk patients to ameliorate the development of frank lung squamous cell carcinoma and ultimately reduce LC mortality. Citation Format: Táchira Pichardo, Anna Belkina, Jennifer Snyder-Cappione, Jennifer Beane, Sarah Mazzilli. γδ T cells contribute to an inflammatory and immunosuppressive microenvironment in lung squamous cell carcinoma tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P045.

While childhood-onset asthma is extensively modeled in labs and includes TH2 cell driven-, steroid-responsive, eosinophilic etiology, little is understood about late-onset asthma which presents as severe, steroid-resistant, and destructive neutrophilic disease with poor outcomes. Lack of appropriate disease models widens this knowledge gap leading to fewer therapies and poorer quality of life for neutrophilic asthma patients. Here, we discover that while conventional models of allergic asthma (relying on naïve sensitized mice acutely challenged with aerosolized ovalbumin) induces eosinophilic asthma, transient and recurrent aeroallergenic exposure over extended durations (as would occur in humans progressing into adulthood) reprograms the lung myeloid and lymphoid landscape to instigate neutrophilic asthma. Mice with such lung history harbor diverse clusters of lung-resident CD4+ TRM cells including a novel RORγt-negative-IL17A+ TH17 subset; the latter detectable in asthmatic adult human lungs. On allergen reencounter, these RORγt-negative TH17 cells, rapidly secrete IL-17A which signals into lung epithelial and stromal cells to express CXCL5 and induce neutrophilic asthma including peribronchial neutrophilia, vascular leakage and lung damage. We find that lung epithelial antigen presentation is crucial to regulate disease severity by skewing CD4+ TRM phenotypes in asthmatic lungs. Specifically, epithelial MHC-II supports TH1 TRM cells and IFNγ secretion; the latter being a potent suppressor of IL-17A-induced CXCL5 and airway neutrophilia. Thus, using a relevant model of the disease, we identify an ‘epithelium-lymphocyte-neutrophil’ circuitry as a critical regulator of late-onset neutrophilic asthma. Supported by NIH grants including HL147461 to F.T.K., HL142199 to K.A.B., HL136725 to M.R.J., GM120060 and HL111449 to L.J.Q., AI115053, HL135756, and HL137081 to J.P.M. and T32 HL007035 for support of trainees.

There is a critical need to identify strategies for lung cancer prevention, as it remains the leading cause of cancer-related death in the US. A critical barrier to developing new approaches to prevent lung cancer is the lack of understanding of key molecular and cellular changes that lead to cancer initiation and progression. Our group has initiated a Pre-Cancer Genome Atlas (PCGA) to define the early molecular alterations that drive premalignant progression of lung cancer. This work has identified transcriptomic alterations in immune pathways associated with progression of bronchial dysplasia (BD) that werefurther validated by immunofluorescent staining showing shifts in immune populations in adjacent bronchial biopsy sections. A challenge to further explore interactions between epithelial and immune cells driving lesion progression is the lack of in vivo models of premalignant transitions ofBD that progress into lung squamous cell carcinoma (LUSC). However, in the NTCU mouse model of LUSC, mice treated with carcinogen develop lesions histologically comparable to human BD. The NTCU-induced murine model serves to act as a robust model for LUSC premalignancy, however, the molecular and cellular alterations driving disease in this model have not been well characterized. This study seeks to investigate the molecular pathogenesis of NTCU-induced BD progression to carcinoma to that of human BD progression to determine the pre-clinical utility of the model for intervention studies. RNA was isolated from normal, dysplastic and tumor tissue from NTCU-treated mice and sequenced (n=22). Gene expression alterations associated with histological grade were identified using regression models. Concordance between the murine and human data was investigated using GSEA (Gene Set Enrichment Analysis). We identified 503 genes differentially expressed with increasing histological severity in the mouse dysplasia (FDR Citation Format: Sarah A. Mazzilli, Kahkeshan Hijazi, Riley Pihl, Anna Belkina, Xiaohui Zhang, Gang Liu, Marc Lenburg, Christopher Stevenson, Avrum Spira, Jennifer Beane. The molecular characterization of a murine lung premalignancy model for lung cancer interception [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 660.

T cell inflammation plays critical roles in the development of obesity-associated type 2 diabetes (T2D). B cells support T cell-mediated inflammation in T2D, but the mechanisms underlying lymphocyte cross-talk and the relative importance of T cell inflammation in human T2D remain untested. Using peripheral blood mononuclear cells (PBMC) and/or purified cells from T2D and non-diabetic (ND) subjects, we show that B cell contact upregulates a pro-inflammatory CD4+ Th17 T cell program in T2D. Neutralization of either B cell CD80/86 or the downstream production of T cell IL-17A/F significantly decreased production of classical diabetogenic cytokines (TNFα, IL-6) in samples from T2D subjects. Although monocytes, B cells and T cells produced TNFα in response to treatment of PBMCs with αCD3/CD28, monocyte TNFα production was independent of IL-17A/F, while IL-17F was critical for maximal TNFα production by both T and B cells. IL-17A/F neutralization also reduced IL-17A and IL-17F production by CD4+ T cells, indicating Th17 function is auto-regulated. We conclude that human B cell co-stimulation broadly supports Th17 cells, which in turn stimulate lymphocytes (but not monocytes) to produce the classical diabetogenic cytokine TNFα.

Chronic inflammation likely underlies the predisposition of type 2 diabetes (T2D) patients to periodontitis (PD), and the exacerbation of each disease when both are present. Several lines of evidence indicate that pro-inflammatory B cells promote T2D and PD individually, but the possibility that B cells are a key link between T2D to PD is untested. We compared outcomes from oral P. gingivalis challenge of lean WT or B cell-null mice to outcomes from mice that were obese and insulin resistance prior to oral challenge. Obese WT mice responded to oral P. gingivalis challenge with significant periodontal bone loss, while obese B cell-null mice were completely protected from PD. By contrast, lean WT and B cell-null mice suffer similar periodontal bone loss in response to oral pathogen. B cells from obese/insulin resistant hosts also support oral osteoclastogenesis, and both oral and systemic production of inflammatory cytokines, including the PD-associated cytokines TNF-α and MIP-2. B cells furthermore impact adipose tissue inflammation in obese P. gingivalis-infected hosts. Taken together, these data show that fundamentally different mechanisms regulate PD in lean and obese hosts, with B cells promoting PD only if the hosts are “primed” by obesity. These results justify more intense analysis of obesity-associated changes in B cells that predispose PD in human T2D, and may indicate the use of B cell depletion to alleviate PD and T2D simultaneously.

New treatment strategies are greatly needed for both acute and chronic inflammatory diseases. A novel triazolediazepine-fused ring compound JQ1 was recently reported to disrupt interactions between acetylated chromatin and dual bromodomains of BET proteins Brd4 and Brd2. Brd2 knockdown mice demonstrate reduced inflammatory responses, and various Brd2 shRNA-expressing cells cannot fully respond to inflammatory stimuli. Our HYPOTHESIS was that JQ1 disrupts BRD interactions to ablate inflammatory responses in a similar manner. RESULTS. We treated RAW264.7 cells and BMDMs with JQ1, stimulated them with LPS, and assayed mRNA of cytokine genes and cytokine levels in culture medium (TNFa, IL-6, IL-10, MCP-1). We stimulated mouse B cells with E. coli LPS and Pam3CSK4 to see the effect of JQ1 on TLR-mediated response. Finally, we subjected C57BL/6 mice to a lethal dose of LPS and treated them with 50 mg/kg of JQ1. We recorded survival and measured cytokine response in the serum 20,40,60,90,120, and 240 min after LPS injection. We found that JQ1 treatment dramatically reduces inflammatory responses in vitro and in vivo, downregulating expression and production of key NFkB-dependent cytokines. In B cells, JQ1 reduced proliferative effects of TLR2 and TLR4 stimulation. 90% of JQ1-treated mice survived endotoxic shock while 0% survived in the non-treated group. JQ1 ablated serum inflammatory cytokines within 1 hr. Thus JQ1 is a potent, novel, epigenetically-acting anti-inflammatory.

Lymphocytes play key roles in the chronic inflammation critical for T2D pathogenesis. T2D patients have an elevated ratio of pro- to anti-inflammatory T cells, and B cells that fail to produce anti-inflammatory IL-10. New data show that B cells from the diet-induced obesity (DIO) mouse model of T2D secrete a pro-inflammatory balance of cytokines, including relatively low IL-10 production, which mirrors our findings in T2D patients. DIO B cells do not secrete auto-antibodies, as evidenced by anti-nuclear antigen staining. Complementary metabolic studies show that B cell-null μMT mice resist the development of glucose intolerance (but not obesity) in response to DIO. Taken together, these data support the conclusion that a pro-inflammatory B cell cytokine balance, rather than antibodies, promotes T2D. To further define roles for B cells in T2D, we immunophenotyped DIO μMT splenocytes. Regulatory T cells (Tregs) expand in DIO μMT, while Tregs fail to expand in WT mice. These data are consistent with new human studies showing B cells from T2D (but not healthy) subjects support pro-inflammatory T cells, and indicate clinical relevance of published studies showing decreased Th1 function in DIO μMT vs. WT mice. We conclude B cells are critical regulators of inflammation in T2D due to 1. Secretion of a pro-inflammatory cytokine balance; and 2. An ability to promote pro-inflammatory T cell function. Thus B cell depletion may represent a valuable tool in the T2D clinic.