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Induction of podoplanin (PDPN) expression is a critical response of macrophages to LPS stimulation or bacterial infection in sepsis, but how this key process of TLR4-stimulated PDPN upregulation is regulated and the impact of PDPN expression on macrophage function remain elusive. Here, we determined how this process is regulated in vitro and in vivo. PDPN failed to be upregulated in TLR4 stimulated macrophages deficient in adhesion and degranulation-promoting adapter protein (ADAP), which could be rescued by the reconstitution of ADAP. A distinct PDPNhi peritoneal macrophage (PM) subset, which exhibited an M2-like phenotype and enhanced phagocytic activity, was generated in WT but not in ADAP-deficient septic mice. The blockade of PDPNhi PMs mimicked the effect of ADAP deficiency, which exacerbated sepsis. Mechanistically, BTK-mediated ADAP Y571 phosphorylation worked together with mTOR to converge on STAT3 activation for the transactivation of the PDPN promoter. Moreover, agonist activation of STAT3 profoundly potentiated the PDPNhi PM subset generation and alleviated sepsis severity in mice. Together, our findings reveal a mechanism whereby ADAP resets macrophage function by controlling the TLR4-induced upregulation of PDPN as a host innate immune defense during sepsis.

Background: Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality necessitating the exploration of alternate therapeutic approaches. Tumor reactive or activated-by-cytokine killers (TRACK) are PD-L1+ highly cytolytic natural killer (NK) cells derived from umbilical cord blood NK cells and engineered to express soluble IL15 (sIL15), showing promise in preclinical studies against NSCLC.

Methods: We assessed safety, persistence, homing and cytotoxic activity in six patients with advanced, refractory, and progressing NSCLC who received a low dose of unmatched, allogeneic, off-the-shelf sIL15_TRACK NK cells. We evaluated NK cell presence and persistence with droplet digital (dd) PCR, flow cytometry, and immunofluorescent staining.

Results: sIL15_TRACK NK cells had peak measurements at one hour and became undetectable four hours after each in fusion. Cognate ligands to activating NK cell receptors were found in NSCLC. sIL15_TRACK NK cells were observed in a lung tumor biopsy seven days after the final infusion, confirming their sustainment and tumor-homing ability. They retained cytolytic function following isolation from the lung tumor. Three out of six patients achieved disease stabilization on repeat imaging, while the others progressed.

Conclusion: Unmatched, allogeneic, cryopreserved, off-the-shelf sIL15_TRACK NK cells express activating receptors, home to tumor sites that express their cognate ligands, and retain cytolytic activity after infusion, underscoring their potential as a therapeutic approach in solid tumors. At low doses, the therapy was safely administered and showed preliminary evidence of activity in three of six patients with advanced and progressive NSCLC. Additional dose escalation cohorts and co-administration with atezolizumab are planned.

Trial registration:

Clinicaltrials: gov NCT05334329FUNDING. Funding was provided by CytoImmune Therapeutics; CA266457; CA033572; CA210087.

The standard-of-care treatment of locally advanced cervical cancer includes pelvic radiation therapy with concurrent cisplatin-based chemotherapy and is associated with a 30-50% failure rate. New prognostic and therapeutic targets are needed to improve clinical outcomes. The vaginal microbiome has been linked to the pathogenesis of cervical cancer, but little is known about the vaginal microbiome in locally advanced cervical cancer as it relates to chemoradiation. In this pilot study we utilized 16S rRNA gene community profiling to characterize the vaginal microbiomes of 26 postmenopausal women with locally advanced cervical cancer receiving chemoradiation. Our analysis revealed diverse anaerobe-dominated communities whose taxonomic composition, diversity or bacterial abundance did not change with treatment. We hypothesized that characteristics of the microbiome might correlate with treatment response. Pretreatment microbial diversity and bacterial abundance were not associated with disease recurrence. We observed a greater relative abundance of Fusobacterium in patients that later had cancer recurrence, suggesting that Fusobacterium could play a role in modifying treatment response. Taken together, this hypothesis generating pilot study provides insight into the composition and dynamics of the vaginal microbiome, offering proof-of-concept for future study of the microbiome and its relationship with treatment outcomes in locally advanced cervical cancer.

The mechanisms utilized by differentiating B cells to withstand highly damaging conditions generated during severe infections, like the massive hemolysis that accompanies malaria, are poorly understood. Here we demonstrate that ROCK1 regulates B cells differentiation in hostile environments replete with PAMPs (pathogen-associated molecular patterns) and high levels of heme by controlling two key heme-regulated molecules, BACH2 and Heme-regulated eIF2a kinase (HRI). ROCK1 phosphorylates BACH2 and protects it from heme-driven degradation. As B cells differentiate, furthermore, ROCK1 restrains their proinflammatory potential and helps them handle the heightened stress imparted by the presence of PAMPs and heme by controlling HRI, a key regulator of the integrated stress response and cytosolic proteotoxicity. ROCK1 controls the interplay of HRI with HSP90 and limits the recruitment of HRI and HSP90 to unique p62/SQSTM1 complexes that also contain critical kinases like mTORC1 and TBK1, and proteins involved in RNA metabolism, oxidative damage, and proteostasis like TDP-43. Thus, ROCK1 helps B cells cope with intense pathogen-driven destruction by coordinating the activity of key controllers of B cell differentiation and stress responses. These ROCK1-dependent mechanisms may be widely employed by cells to handle severe environmental stresses, and these findings may be relevant for immune-mediated and age-related neurodegenerative disorders.

Lymphangioleiomyomatosis (LAM) is a progressive lung disease with limited treatments, largely due to an incomplete understanding of its pathogenesis. Lymphatic endothelial cells (LECs) invade LAM cell clusters, which include HMB-45-positive epithelioid cells and smooth muscle α-actin-expressing LAM-associated fibroblasts (LAMFs). Recent evidence shows that LAMFs resemble cancer-associated fibroblasts, with LAMF-LEC interactions contributing to disease progression. To explore these mechanisms, we used spatial transcriptomics on LAM lung tissues and identified a gene cluster enriched in kinase signaling pathways linked to myofibroblasts and co-expressed with LEC markers. Kinase arrays revealed elevated PDGFR and FGFR in LAMFs. Using a 3D co-culture spheroid model of primary LAMFs and LECs, we observed increased invasion in LAMF-LEC spheroids compared to non-LAM fibroblasts. Treatment with sorafenib, a multikinase inhibitor, significantly reduced invasion, outperforming Rapamycin. We also confirmed TSC2-deficient renal angiomyolipoma cells (TSC2-null AML) as key VEGF-A secretors, which was suppressed by sorafenib in both TSC2-null AML cells and LAMFs. These findings highlight VEGF-A and bFGF as potential therapeutic targets and suggest multikinase inhibition as a promising strategy for LAM.

Background: Lymphopenia and failure of lymphocytes to mount an early IFN-γ response correlate with increased mortality in COVID-19. Given the essential role of CD4 helper and CD8 cytotoxic cells in eliminating viral pathogens, this profound loss in lymphocytes may impair patients' ability to eliminate the virus. IL-7 is a pleiotropic cytokine that is obligatory for lymphocyte survival and optimal function.

Methods: We conducted a prospective, double-blind, randomized, placebo-controlled trial of CYT107, recombinant human IL-7, in 109 critically-ill lymphopenic COVID-19 patients. The primary endpoint was to assess CYT107's effect on lymphocyte recovery with secondary clinical endpoints including safety, ICU and hospital length-of-stay, incidence of secondary infections, and mortality.

Results: CYT107 was well-tolerated without precipitating a cytokine storm or worsening pulmonary function. Absolute lymphocyte counts increased in both groups without significant difference between CYT107 and placebo. COVID-19 patients receiving CYT107 but not concomitant antiviral medications, known inducers of lymphopenia, had a final lymphocyte count that was 43% greater than placebo (p=0.067). There were significantly fewer treatment-emergent adverse events in CYT107 versus placebo-treated patients (p<0.001), consistent with a beneficial drug effect. Importantly, CYT107 treated patients had 44% fewer hospital-acquired infections versus placebo-treated patients (p=0.014).

Conclusions: Given that hospital-acquired infections are responsible for a large percentage of COVID-19 deaths, this effect of CYT107 to decrease nosocomial infections could substantially reduce late morbidity and mortality in this highly lethal disease. The strong safety profile of CYT107 and its excellent tolerability provide support for trials of CYT107 in other potential pandemic respiratory viral infections.

Trial registration: NCT04379076, NCT04426201, NCT04442178, NCT04407689; NCT04927169.

Despite combination antiretroviral therapy (ART), HIV causes persistent gut barrier dysfunction, immune depletion, and dysbiosis. Further, ART interruption results in reservoir reactivation and rebound viremia. Both IL-21 and anti-α4β7 improve gut barrier functions, and we hypothesized combining them would synergize as a dual therapy to improve immunological outcomes in SIV-infected rhesus macaques (RMs). We found no significant differences in CD4+ T-cell reservoir size by intact proviral DNA assay. SIV rebounded in both dual-treated and control RMs following analytical therapy interruption (ATI), with time to rebound and initial rebound viremia comparable between groups; however, dual-treated RMs showed slightly better control of viral replication at the latest time points post-ATI. Additionally, following post-ATI, dual-treated RMs showed immunological benefits, including T-cell preservation and lower PD-1+ central memory T-cell (TCM) frequency. Notably, PD-1+ TCMs were associated with reservoir size, which predicted viral loads (VLs) post-ATI. Finally, 16S rRNA sequencing revealed better recovery from dysbiosis in treated animals, and the butyrate-producing Firmicute Roseburia predicted PD-1-expressing TCMs and VLs after ATI. PD-1+ TCMs and gut dysbiosis represent mechanisms of HIV persistence and pathogenesis, respectively. Therefore, combining IL-21 and anti-α4β7 may be an effective therapeutic strategy to improve immunological outcomes for people with HIV.

Renal osteodystrophy is commonly seen in patients with chronic kidney disease (CKD) due to disrupted mineral homeostasis. Given the impaired renal function in these patients, common anti-resorptive agents, including bisphosphonates, must be used with caution or even contraindicated. Therefore, an alternative therapy without renal burden to combat renal osteodystrophy is urgently needed. Here, we report that clinically relevant aerobic exercise significantly prevents high-turnover renal osteodystrophy in CKD mouse and patients without compromising renal function. Mechanistically, 4-week aerobic exercise in CKD mice increased expression of skeletal muscle PPARγ coactivator-1α (PGC-1α) and circulating irisin. Both exercise and irisin administration significantly activated osteoblasts, but not osteoclasts, via integrin αvβ5, thereby conferring bone quality benefits. Removal of irisin-influenced thermogenic adipose tissues or genetic ablation of uncoupling protein 1 did not alter the irisin-conferred anti-osteodystrophy effect. Importantly, in a pilot clinical study, 12-week aerobic exercise in patients with high-grade CKD significantly increased circulating irisin and prevented osteodystrophy progression, without detectable renal burden. The combination of irisin and current anti-resorptive agents effectively rescued renal osteodystrophy in mice. Our work provides mechanistic insights into the role of exercise and irisin in renal osteodystrophy, and highlights a clinically relevant, low-cost, kidney-friendly therapy for patients with this devastating disease.

Background: We aimed to characterize factors associated with the under-studied complication of cognitive decline in aging people with long-duration type 1 diabetes (T1D).

Methods: Joslin "Medalists" (n = 222; T1D ≥ 50 years) underwent cognitive testing. Medalists (n = 52) and age-matched non-diabetic controls (n = 20) underwent neuro- and retinal imaging. Brain pathology (n = 26) was examined. Relationships amongst clinical, cognitive and neuroimaging parameters were evaluated.

Results: Compared to controls, Medalists had worse psychomotor function and recall, which associated with female gender, lower visual acuity, reduced physical activity, longer diabetes duration and higher inflammatory cytokines. On neuroimaging, compared to controls, Medalists had significantly lower total and regional brain volumes, equivalent to 9 years of accelerated aging, but small vessel disease markers did not differ. Reduced brain volumes associated with female sex, reduced psychomotor function, worse visual acuity, longer diabetes duration and higher inflammation, but not with glycemic control. Worse cognitive function, lower brain volumes, and diabetic retinopathy correlated with thinning of the outer retinal nuclear layer. Worse baseline visual acuity associated with declining psychomotor function in longitudinal analysis. Brain volume mediated the association between visual acuity and psychomotor function by 57%. Brain pathologies showed decreased volumes, but predominantly mild vascular or Alzheimer's-related pathology.

Conclusion: This first comprehensive study of cognitive function, neuroimaging and pathology in aging T1D individuals demonstrated that cognitive decline was related to parenchymal rather than neurovascular abnormalities, unlike type 2 diabetes, suggestive of accelerated aging in T1D. Improving visual acuity could perhaps be an important preventive measure against cognitive decline in people with T1D.

Background: Previously, we demonstrated that changes in circulating tumor DNA (ctDNA) are promising biomarkers for early response prediction (ERP) to immune checkpoint inhibitors (ICI) in metastatic urothelial cancer (mUC). In this study, we investigated the value of whole blood immunotranscriptomics for ERP-ICI and integrated both biomarkers into a multimodal model to boost accuracy.

Methods: Blood samples of 93 patients were collected at baseline and after 2-6 weeks of ICI for ctDNA (N=88) and immunotranscriptome (N=79) analyses. ctDNA changes were dichotomized into increase or no increase, the latter including patients with undetectable ctDNA. For RNA model development, the cohort was split into a discovery (N=29), test (N=29) and validation set (N=21). Finally, RNA- and ctDNA-based predictions were integrated in a multimodal model. Clinical benefit (CB) was defined as progression-free survival beyond 6 months.

Results: Sensitivity (SN) and specificity (SP) of ctDNA increase for predicting non-CB (N-CB) was 59% and 92%, respectively. Immunotranscriptome analysis revealed upregulation of T-cell activation, proliferation and interferon signalling during treatment in the CB group, contrary to N-CB patients. Based on these differences a 10-gene RNA model was generated, reaching a SN and SP of 73% and 79% in the test and 67% and 67% in the validation set for predicting N-CB. Multimodal model integration led to superior performance with a SN and SP of 79% and 100% in the validation cohort.

Conclusion: The combination of whole blood immunotranscriptome and ctDNA in a multimodal model showed promise for ERP-ICI in mUC and accurately identified patients with N-CB.

Trial registration: 2016-3060, 2020-6778FUNDING. Eurostars grant E! 114908 - PRECISE, Paul Speth Foundation (Bullseye project).