Changes In CD4 Research Articles

Increases in the susceptibility of human endometrial CD4+ T cells to HIV-1 infection post-menopause are not dependent on greater viral receptor expression frequency

Epidemiological evidence suggests that post-menopausal women are more susceptible to HIV infection following sexual intercourse than are younger cohorts for reasons that remain unclear. Here, we evaluated how menopause-associated changes in CD4+ T cell numbers and subsets as well as HIV coreceptor expression, particularly CCR5, in the endometrium (EM), endocervix (CX), and ectocervix (ECX) may alter HIV infection susceptibility. Using a tissue-specific mixed cell infection model, we demonstrate that while no changes in CD14+ macrophage infection susceptibility were observed, CD4+ T cell HIV-1 infection frequency increases following menopause in the EM, but not CX nor ECX. Unexpectedly, the CD4+ T cell expression of two known correlates of HIV infection susceptibly, CCR5 and integrin-α4β7, increased following menopause across all three tissues despite only being associated with increased infection frequency in EM derived CD4+ T cells. After controlling for changes in the expression of either receptor, both CCR5 and α4β7 expressing CD4+ T cells isolated from the EM of post-menopausal women remained more susceptible to HIV-1 infection than those isolated from pre-menopausal women. Shifts in T helper subset composition, including increases in Th1 frequency and decreases in Th17 and Treg frequency were also observed in the EM only following menopause, but did not correlate with increased infection frequency. Treatment of EM derived CD4+ T cells with 17β-estradiol (E2) prior to viral infection, reduced infection frequency independent of changes in either CCR5 or α4β7 expression frequency. Our results demonstrate that the susceptibility of EM derived CD4+ T cells to HIV-1 infection increases post menopause but is unlikely to be driven by increased expression frequency of either CCR5 or integrin-α4β7. These findings contribute to our understanding of how advanced age alters HIV infection risk which will become increasingly important as the human population continues to age.

Analysis of frequency changes in CD8+ regulatory T cell subsets in peripheral blood of individuals with type 1 diabetes

BackgroundThis study aims to examine the alterations and clinical significance of CD8+ regulatory T cell subsets in the peripheral blood of individuals with type 1 diabetes mellitus (T1DM).MethodsFrom January 2020 to December 2023, a study was conducted involving 40 individuals with T1DM, who visited the Department of Endocrinology at the First Affiliated Hospital of Nanjing Medical University (T1DM group). For comparison, 40 healthy individuals who underwent routine physical examinations at the same hospital during this period were selected as the control group. Peripheral blood mononuclear cells were isolated, and CD8+ T cells were labeled with CD3, CD25 and FoxP3 to analyze their subset frequencies using flow cytometry. The study examined differences in subset frequencies between the two groups and explored correlations between subset frequency, disease duration, and age of onset.ResultsThe frequencies of CD8+ CD25+, CD8+ FoxP3+, CD8+ CD25+ CD3+ and CD8+ FoxP3+ CD3+ subsets in peripheral blood mononuclear cells did not significantly differ between the healthy control group and the T1DM group (P > 0.05). In the T1DM group, the expression level of CD25 on CD8+ T cells showed no correlation with the age of onset or disease duration, and FoxP3 levels were also unrelated to the age of onset, with no statistical differences (P > 0.05). However, within the T1DM group, FoxP3 levels progressively decreased with longer disease duration, demonstrating a statistically significant negative correlation (Pearson r = -0.331, P < 0.05). In the T1DM group, the level of CD3+ CD8+ T cells expressed CD25, and there was no correlation between Foxp 3 level and age of onset, not statistically significant (P > 0.05), but the level of Foxp 3 in the T1DM group decreased with the duration of the disease, (Pearson r= − 0.363, P < 0.05).ConclusionThe levels of CD8+FoxP3+ regulatory T cells in peripheral blood mononuclear cells of patients with T1DM show a significant correlation with disease duration, suggesting that these cells may play a critical role in the progression of T1DM.

Quantification of the median fluorescence intensity of CD3 and CD4 in mycosis fungoides/Sezary syndrome versus non-neoplastic control cases in peripheral blood.

Peripheral blood involvement by MF/SS has significant implications for prognosis and treatment. Flow cytometry is commonly used to assess MF/SS by analyzing the ratio of CD26- and/or CD7-CD4 + T cells and assessment of immunophenotypic abnormalities. However, distinguishing normal from abnormal cells is not always easy. In this study, we aimed to establish quantitative thresholds to better distinguish normal CD4 + T cells from neoplastic CD4 + T cells. A retrospective analysis of flow cytometry data was performed on 30 MF/SS patients with a detectable abnormal T cell population (positive), 63 patients with suspected or confirmed cutaneous involvement without a detectable abnormal T cell population (negative), and 60 healthy controls (control). CD3 and CD4 median fluorescence intensity (MFI) was normalized to internal control subsets. Among the positive cases, 50% had CD3 expression outside ± 2 SD from the mean of the negative and control group in the CD4 + CD26- subset. The corresponding specificity of this threshold was 94%. The ± 2 SD threshold showed a sensitivity of 57% and a specificity of 94% for the CD3 intensity among the CD7-negative subset. For CD4 intensity, the ± 2 SD threshold had a sensitivity of 33.3% and specificity of 95% for the CD26-negative subset and a sensitivity of 37% and specificity of 95% for the CD7-negative subset. In our study, although changes in CD3 and CD4 intensity greater than ± 2 SD were specific for MF/SS, more subtle differences in the intensity of CD3 and CD4 should not be used as the sole abnormality to make a diagnosis of circulating MF/SS.

Tissue-Specific DNA Methylation Changes in CD8+ T Cells During Chronic Simian Immunodeficiency Virus Infection of Infant Rhesus Macaques.

Robust CD8+ T cell responses are critical for the control of HIV infection in both adults and children. Our understanding of the mechanisms driving these responses is based largely on studies of cells circulating in peripheral blood in adults, but the regulation of CD8+ T cell responses in tissue sites is poorly understood, particularly in pediatric infections. DNA methylation is an epigenetic modification that regulates gene transcription. Hypermethylated gene promoters are associated with transcriptional silencing and, conversely, hypomethylated promoters indicate gene activation. In this study, we evaluated DNA methylation signatures of CD8+ T cells isolated from several different anatomic compartments during pediatric AIDS-virus infection by utilizing the SIVmac239/251 infected infant rhesus macaque model. We performed a stepwise methylation analysis starting with total cellular DNA, to immunomodulatory cytokine promoters, to specific CpG sites within the cytokine promoters in CD8+ T cells isolated from peripheral blood, lymph nodes, and intestinal tissue during the chronic phase of infection. Tissue-specific methylation patterns were determined for transcriptionally active promoters of key immunomodulatory cytokines: interferon gamma (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNFα). In this study, we observed tissue-specific differences in CD8+ T cell modulation by DNA methylation in SIV-infected infant macaques, highlighting the importance of evaluating cells from both blood and tissues to obtain a complete picture of CD8+ T cell regulation during pediatric HIV infection.

Trajectory of beta cell function and insulin clearance in stage 2 type 1 diabetes: natural history and response to teplizumab.

We aimed to analyse TrialNet Anti-CD3 Prevention (TN10) data using oral minimal model (OMM)-derived indices to characterise the natural history of stage 2 type 1 diabetes in placebo-treated individuals, to describe early metabolic responses to teplizumab and to explore the predictive capacity of OMM measures for disease-free survival rate. OMM-estimated insulin secretion, sensitivity and clearance and the disposition index were evaluated at baseline and at 3, 6 and 12 months post randomisation in placebo- and teplizumab-treated groups, and, within each group, in slow- and rapid-progressors (time to stage 3 disease >2 or 2 years). OMM metrics were also compared with the standard AUC C-peptide. Percentage changes in CD8+ T memory cell and programmed death-1 (PD-1) expression were evaluated in each group. Baseline metabolic characteristics were similar between 28 placebo- and 39 teplizumab-treated participants. Over 12 months, insulin secretion declined in placebo-treated and rose in teplizumab-treated participants. Within groups, placebo slow-progressors (n=14) maintained insulin secretion and sensitivity, while both declined in placebo rapid-progressors (n=14). Teplizumab slow-progressors (n=28) maintained elevated insulin secretion, while teplizumab rapid-progressors (n=11) experienced mild metabolic decline. Compared with rapid-progressor groups, insulin clearancesignificantlydecreased between baseline and 3, 6 and 12 months in the slow-progressor groups in both treatment arms. In aggregate, both higher baseline insulin secretion (p=0.027) and reduced 12 month insulin clearance (p=0.045) predicted slower progression. A >25% loss of insulin secretion at 3 months had specificity of 0.95 (95% CI 0.86, 1.00) to identify rapid-progressors and correctly classified the 2 year risk for progression in 92% of participants, with a sensitivity of 0.19 (95% CI 0.08, 0.30). OMM-estimated insulin secretion outperformed AUC C-peptide to differentiate groups by treatment or to predict progression. Metabolic changes were paralleled by relative frequency of change in PD-1+ CD8+ T effector memory cells. OMM measures characterise the metabolic heterogeneity in stage 2 diabetes, identifying differences between rapid- and slow-progressors, and heterogeneous impacts of immunotherapy, suggesting the need to account for these differences when designing and interpreting clinical trials.

TMIC-20. UNLOCKING THE POTENTIAL OF HYPOXIA REDUCTION IN RESHAPING THE GLIOBLASTOMA TUMOR MICROENVIRONMENT FOR ENHANCED THERAPEUTIC SYNERGY

Abstract Glioblastoma (GBM) tumors respond poorly to nearly all cancer therapies due to their highly immunosuppressive tumor microenvironment (TME). Hypoxia exacerbates this immunosuppression by upregulating the hypoxia-inducible factor (HIF) pathway in glioma-associated myeloid cells (GAMs) which suppressively polarizes them to maintain this immune “cold” state. This hypoxia-driven programming inhibits CD8+ T cell infiltration and effector function by inducing metabolic dysfunction, thereby increasing resistance to chemotherapy and T cell immune checkpoint blockade (ICB). Targeting hypoxia thus emerges as a promising strategy to overcome such resistance. Our study suggests that GBM hypoxia ablation can convert the immunosuppressive tumor microenvironment into a more pro-inflammatory one. Tumor hypoxia ablation using the hypoxia-activated prodrug Evofosfamide significantly enhances survival in both GL261 (p&amp;lt;0.0001) and the checkpoint blockade-resistant CT2A murine GBM models (p&amp;lt;0.01). Evofosfamide treatment boosts tumor infiltrating Ki67+ proliferative CD8+ T cells (p&amp;lt;0.001) and heightens cytotoxicity, indicated by increased Granzyme B expression (p&amp;lt;0.01). It also reduces the co-expression of exhaustion markers LAG-3 and TIM-3 (p&amp;lt;0.001), signaling decreased T cell exhaustion. Antigen-specific CD8+ T cells from Evofosfamide-treated primary GBM tumors exhibit enhanced ex vivo killing capacity when co-cultured with GL261-OVA cells (p&amp;lt;0.001). Furthermore, Evofosfamide enhances microglia proliferation and repolarizes them towards a pro-inflammatory state, marked by upregulation of M1 markers such as CD80 (p&amp;lt;0.001) and downregulation of M2 markers like Arginase (p&amp;lt;0.05). Hypoxia reduction also increases chemotherapy sensitivity, synergistically improving survival when combined with the blood-brain barrier-penetrating, immunogenic chemotherapy Berubicin (CT2A; vehicle MS=26d, Evo MS=36d, Berubicin MS=55d, Combo MS=70.5d). Future research will investigate the mechanisms underlying this shift towards a pro-inflammatory state, focusing on metabolic changes in CD8+ T cells and the impact of altered HIF pathway signaling on GAM polarization following hypoxia ablation. Understanding the therapeutic potential of reversing hypoxia-mediated immunosuppression could lead to novel combination therapies capable of overcoming resistance to both chemotherapy and immunotherapy in GBM.

Dynamic Peripheral T-Cell Analysis Identifies On-Treatment Prognostic Biomarkers of Atezolizumab plus Bevacizumab in Hepatocellular Carcinoma

Introduction: Variability in response to atezolizumab plus bevacizumab (AB) treatment of hepatocellular carcinoma (HCC) underscores the critical need for the development of effective biomarkers. We sought to identify peripheral blood biomarkers reflecting response to AB treatment. Methods: We analyzed dynamic changes in peripheral blood mononuclear cells from a prospective, multicenter cohort of 65 patients with HCC, using flow cytometry to evaluate the T-cell population before and 3 weeks after the first AB treatment. Results: We found a unique response of the CD8+ T cells in terms of both frequency and phenotype, in contrast to CD4+ T cells and regulatory T cells. Notably, CD8+ T cells showed significant changes in expression of Ki-67 and T-cell immunoreceptors with Ig and ITIM domains (TIGIT). These distinct responses were observed particularly in the programmed cell death receptor-1 (PD-1)+ subpopulation of CD8+ T cells. Interestingly, the baseline differentiation status of PD-1+CD8+ T cells, particularly the central memory T-cell subset, correlated positively with greater proliferation (higher Ki-67 expression) of PD-1+CD8+ T cells after treatment. Moreover, effector memory cells expressing CD45RA correlated negatively with the increase in TIGIT+/PD-1+CD8+ T cells. The increase in TIGIT+/CD8+ T cells was associated with the development of immune-related adverse events, whereas increase in Ki-67+/PD-1+CD8+ T cells was associated with the better objective response rate. Importantly, dynamic shifts of Ki-67+/PD-1+CD8+ T cells and TIGIT+/CD8+ T cells significantly predicted progression-free survival and overall survival, as confirmed by multivariate analysis. Conclusion: These findings highlight the potential of dynamic changes in CD8+ T cells as an on-treatment prognostic biomarker. Our study underscores the value of peripheral blood profiling as a noninvasive and practical method for predicting the clinical outcomes of AB treatment in patients with HCC.

Sodium chloride in the tumor microenvironment enhances T cell metabolic fitness and cytotoxicity

The efficacy of antitumor immunity is associated with the metabolic state of cytotoxic T cells, which is sensitive to the tumor microenvironment. Whether ionic signals affect adaptive antitumor immune responses is unclear. In the present study, we show that there is an enrichment of sodium in solid tumors from patients with breast cancer. Sodium chloride (NaCl) enhances the activation state and effector functions of human CD8+ T cells, which is associated with enhanced metabolic fitness. These NaCl-induced effects translate into increased tumor cell killing in vitro and in vivo. Mechanistically, NaCl-induced changes in CD8+ T cells are linked to sodium-induced upregulation of Na+/K+-ATPase activity, followed by membrane hyperpolarization, which magnifies the electromotive force for T cell receptor (TCR)-induced calcium influx and downstream TCR signaling. We therefore propose that NaCl is a positive regulator of acute antitumor immunity that might be modulated for ex vivo conditioning of therapeutic T cells, such as CAR T cells.

A phase Ib/II study of pacritinib, an interleukin 1 receptor associated kinase 1 (IRAK1) inhibitor, in patients (pts) with solid tumors harboring the 1q21.3 copy number amplification (CNA).

43 Background: The role of IRAK1 pathway in inflammatory and immune response is established in autoimmune diseases but less well-understood in cancer. Chromosome 1q21.3 CNA detected in plasma cell-free DNA was observed in pts with advanced solid tumors, and associated with IRAK1 upregulation. In preclinical animal models, IRAK1 upregulation promoted tumorigenesis, while its inhibition led to decreased tumor growth. Pacritinib is a JAK2/FLT3 inhibitor that is FDA approved for treatment in myelofibrosis, but also have known activity against IRAK1. We hypothesize that pacritinib may be effective in disease control of pts with plasma 1q21.3 CNA. Methods: A phase Ib/II clinical trial was designed to investigate the efficacy of pacritinib in pts with treatment (rx) refractory solid tumors harboring 1q21.3 CNA. Serial pt tumor and blood samples were collected and analyzed to elucidate the effect of IRAK1 inhibition on tumor microenvironment and systemic immune modulation. Results: 330 patients were screened, 1q21.3 CNA was detected in 30.1% of pts. Highest incidence of 1q21.3 CNA positivity was detected in breast cancer (39.8%), colorectal cancer (39.7%) and lung cancer (36.1%). 12 pts were commenced on rx over 3 dose levels (DL) (DL1: n=6, DL2: n=3, DL3: n=3). At DL1 (200mg BID), no DLTs were observed, but 2 pts had G3 transaminitis attributed to drug/disease, and 1 pt had intolerable G2 rash; decision was made for dose reduction. At DL2 (200mg OM, 100mg ON), no DLTs were observed. Reescalation to 200mg BID (DL3) was carried out with no DLT observed. Pacritinib at 200mg BD was declared the recommended phase II dose in pts with solid tumors. In the dose expansion cohort, 8 pts (4 colorectal cancer, 3 hepatobiliary [HPB] cancer, 1 lung cancer) have been enrolled thus far. No objective responses were observed. Within pts with HPB tumors, 1 pt with pancreatic cancer had a progression-free survival (PFS) of 25.0 weeks, and 1 pt with cholangiocarcinoma had a PFS of 15.9 weeks. Preliminary analysis of serial tumor biopsy samples suggest an expansion of CD8+ T cell population with pacritinib rx. Serial peripheral blood mononuclear cells analysis showed no significant trend in change in CD8+ T cell population, but a predominance of PD-1+ T cells with rx, and also an increase in CD4+ T cell population. In the myeloid cell population, there appears to be decrease in dendritic cell population, but no significant change in trend of macrophage was observed. Conclusions: Pacritinib at 200mg BID is safe and tolerable in pts with solid tumors. IRAK1 inhibition appears to modulate immune cell populations both in the tumor microenvironment and systemic circulation. Dose expansion is underway, with potential signal of disease control in HPB tumors. Clinical trial information: NCT04520269 .

FLI1 in PBMCs contributes to elevated inflammation in combat-related posttraumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric condition with significant public health implications that arise following exposure to traumatic events. Recent studies highlight the involvement of immune dysregulation in PTSD, characterized by elevated inflammatory markers. However, the precise mechanisms underlying this immune imbalance remain unclear. Previous research has implicated friend leukemia virus integration 1 (FLI1), an erythroblast transformation-specific (ETS) transcription factor, in inflammatory responses in sepsis and Alzheimer's disease. Elevated FLI1 levels in peripheral blood mononuclear cells (PBMCs) have been linked to lupus severity. Yet, FLI1's role in PTSD-related inflammation remains unexplored. In our study, PBMCs were collected from Veterans with and without PTSD. We found significantly increased FLI1 expression in PBMCs from PTSD-afflicted Veterans, particularly in CD4+ T cells, with no notable changes in CD8+ T cells. Stimulation with LPS led to heightened FLI1 expression and elevated levels of inflammatory cytokines IL-6 and IFNγ in PTSD PBMCs compared to controls. Knockdown of FLI1 using Gapmers in PTSD PBMCs resulted in a marked reduction in inflammatory cytokine levels, restoring them to control group levels. Additionally, co-culturing PBMCs from both control and PTSD Veterans with the human brain microglia cell line HMC3 revealed increased inflammatory mediator levels in HMC3. Remarkably, HMC3 cells co-cultured with PTSD PBMCs treated with FLI1 Gapmers exhibited significantly lower inflammatory mediator levels compared to control Gapmer-treated PTSD PBMCs. These findings suggest that suppressing FLI1 may rebalance immune activity in PBMCs and mitigate microglial activation in the brain. Such insights could provide novel therapeutic strategies for PTSD.

Heat shock protein 90α reduces CD8+ T cell exhaustion in acute lung injury induced by lipopolysaccharide

CD8+ T-cell exhaustion is a promising prognostic indicator of sepsis-induced acute respiratory distress syndrome (ARDS). Patients with sepsis-related ARDS had reduced levels of HSP90AA1. However, whether the changes in CD8+ T cells were related to HSP90α, encoded by the HSP90AA1 gene, was unclear. This study aimed to examine the regulatory mechanism of HSP90α and its impact on CD8+ T-cell exhaustion in lipopolysaccharide (LPS)-induced acute lung injury (ALI). In this study, by conducting a mouse model of ALI, we found that one week after LPS-induced ALI, CD8+ T cells showed exhaustion characteristics. At this time, proliferation and cytokine release in CD8+ T cells were reduced. The inhibitory costimulatory factors PD-1 and Tim-3, on the other hand, were enhanced. Meanwhile, the expression of HSP90α and STAT1 decreased significantly. The in vitro studies showed that HSP90α stimulation or inhibition affected the CD8+ T-cell exhaustion phenotype. Interference with STAT1 reduced the expression of HSP90α and impaired its regulation of CD8+ T cells. The Co-Immunoprecipitation results indicated that HSP90α can directly or indirectly bind to TOX to regulate TOX expression and downstream signal transduction. In summary, by inhibiting TOX-mediated exhaustion signaling pathways, HSP90α inhibited CD8+ T-cell exhaustion in ALI. The participation of STAT1 in the regulation of HSP90α was required.

A randomized phase 2 study of combination atezolizumab and varlilumab (CDX-1127) with or without addition of cobimetinib in previously treated unresectable biliary tract cancer.

4017 Background: Combined MEK inhibition (MEKi) and PD-L1 blockade significantly improved progression-free survival (PFS) versus PD-L1 monotherapy in patients with advanced biliary tract cancer (BTC) following first-line chemotherapy (NCT03201458). Further study has shown MEKi enhances tumor immunogenicity but impairs host T cell activation/priming. The addition of immune agonists, e.g. a CD27 agonist, can rescue T cell function in the setting of systemic MEKi in vivo and may be an effective strategy to optimize MEKi’s immunomodulatory potential when used in combination with checkpoint blockade. Methods: We conducted a randomized ph 2 trial (NCT04941287) evaluating Atezolizumab in combination with the CD27 immune agonist (CDX-1127 [Varlilumab]) with or without the addition of a MEKi (Cobimetinib) in patients with unresectable, previously treated, BTC. Adults with pathologically-confirmed BTC, s/p at least 1 prior line (no more than 2) of systemic therapy, measurable disease, and ECOG PS ≤1 were enrolled. Patients who had received previous PD-[L]1 were eligible. The study was planned for 64 evaluable subjects randomized in a 1:1 ratio, stratified by BTC site, to either AV (Atezolizumab [840mg IV days 1,15])+ Varlilumab [3mg/kg IV days 1, 15]) or CAV (Cobimetinib [60mg oral daily days 1-21, off 22-28] + Atezolizumab + Varlilumab). Overall response rate (ORR) and PFS were co-primary endpoints. The primary correlative outcome was treatment-related changes in CD8+ infiltrating T cells. Results: A preplanned interim analysis based on objective response rates did not meet the threshold for continuance in either treatment arm, so the trial was closed early for futility. In total, 57 patients were enrolled (n = 29 [CAV], n = 28 [AV], 67% had intrahepatic cholangiocarcinoma, and 33% had received previous PD-[L]1. Both CAV and AV regimens were well-tolerated without new safety signals. Adding CDX-1127 did not appear to meaningful increase immunotoxicity beyond established PD-L1 toxicity profiles. ORR was 0% (CAV) and 4% (AV). With a median follow up time of 6.2 months, median PFS was 2.2 (CAV) and 1.8 (AV) months (HR 0.71 [0.40,1.25]). In PD[L]1-experienced patients, median PFS was 3.6 (CAV) and 1.7 (AV) months (HR 0.44 [0.14,1.33]). Median OS was 10.2 (CAV) and 6.1 (AV) months (HR 0.76 [0.37,1.55]). In PD[L]1-experienced patients, median OS was 6.4 (CAV) and 4.4 (AV) months for CAV and AV (HR 0.68 [0.19,2.42]). Primary correlative endpoint and updated survival will be reported at time of meeting. Conclusions: The combination of Atezolizumab and Varlilumab with or without Cobimetinib was safe but did not improve clinical outcomes in advanced stage BTC patients treated in later lines. Though not statistically significant, treatment with CAV demonstrated numerically favorable PFS/OS compared AV among immunotherapy-experienced patients. Clinical trial information: NCT03201458 .

Immune cell populations in the tumour environment following calcium electropora-tion for cutaneous metastasis: a histopathological study.

Calcium electroporation (CaEP) involves injecting calcium into tumour tissues and using electrical pulses to create membrane pores that induce cell death. This study assesses resultant immune responses and histopathological changes in patients with cutaneous metastases. The aimed cohort comprised 24 patients with metastases exceeding 5 mm. Tumours were treated once with CaEP (day 0) or twice (day 28). Biopsies were performed on days 0 and 2, with additional samples on days 7, 28, 30, 35, 60, and 90 if multiple tumours were treated. The primary endpoint was the change in tumour-infiltrating lymphocytes (TILs) two days post-treatment, with secondary endpoints evaluating local and systemic immune responses via histopathological analysis of immune markers, necrosis, and inflammation. Seventeen patients, with metastases primarily from breast cancer (14 patients), but also lung cancer (1), melanoma (1), and urothelial cancer (1), completed the study. Of the 49 lesions treated, no significant changes in TIL count or PD-L1 expression were observed. However, there was substantial necrosis and a decrease in FOXP3-expression (p = 0.0025) noted, with a slight increase in CD4+ cells but no changes in CD3, CD8, or CD20 expressions. Notably, four patients showed reduced tumour invasiveness, including one case of an abscopal response. This exploratory study indicates that CaEP can be an effective anti-tumour therapy potentially enhancing immunity. Significant necrosis and decreased regulatory lymphocytes were observed, although TIL count remained unchanged. Several patients exhibited clinical signs of immune response following treatment.

Adipose tissue-selective ablation of ADAM10 results in divergent metabolic phenotypes following long-term dietary manipulation

ABSTRACT A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10), is involved in several metabolic and inflammatory pathways. We speculated that ADAM10 plays a modulatory role in adipose tissue inflammation and metabolism. To this end, we studied adipose tissue-specific ADAM10 knock-out mice (aKO). While young, regular chow diet-fed aKO mice showed increased insulin sensitivity, following prolonged (33 weeks) high-fat diet (HFD) exposure, aKO mice developed obesity and insulin resistance. Compared to controls, aKO mice showed less inflammatory adipokine profile despite the significant increase in adiposity. In brown adipose tissue, aKO mice on HFD had changes in CD8+ T cell populations indicating a lesser inflammatory pattern. Following HFD, both aKO and control littermates demonstrated decreased adipose tissue pro-inflammatory macrophages, and increased anti-inflammatory accumulation, without differences between the genotypes. Collectively, our observations indicate that selective deletion of ADAM10 in adipocytes results in a mitigated inflammatory response, leading to increased insulin sensitivity in young mice fed with regular diet. This state of insulin sensitivity, following prolonged HFD, facilitates energy storage resulting in increased fat accumulation which ultimately leads to the development of a phenotype of obesity and insulin resistance. In conclusion, the data indicate that ADAM10 has a modulatory effect of inflammation and whole-body energy metabolism.

HIV vaccine candidate efficacy mediated by epigenetic changes in CD14+ cells, cAMP-dependent efferocytosis and immunoinhibitory DC-10

HIV vaccine candidate efficacy mediated by epigenetic changes in CD14+ cells, cAMP-dependent efferocytosis and immunoinhibitory DC-10

Spatial Profiling of Ovarian Carcinoma and Tumor Microenvironment Evolution under Neoadjuvant Chemotherapy.

This study investigates changes in CD8+ cells, CD8+/Foxp3 ratio, HLA I expression, and immune coregulator density at diagnosis and upon neoadjuvant chemotherapy (NACT), correlating changes with clinical outcomes. Multiplexed immune profiling and cell clustering analysis were performed on paired matched ovarian cancer samples to characterize the immune tumor microenvironment (iTME) at diagnosis and under NACT in patients enrolled in the CHIVA trial (NCT01583322). Several immune cell (IC) subsets and immune coregulators were quantified pre/post-NACT. At diagnosis, patients with higher CD8+ T cells and HLA I+-enriched tumors were associated with a better outcome. The CD8+/Foxp3+ ratio increased significantly post-NACT in favor of increased immune surveillance, and the influx of CD8+ T cells predicted better outcomes. Clustering analysis stratified pre-NACT tumors into four subsets: high Binf, enriched in B clusters; high Tinf and low Tinf, according to their CD8+ density; and desert clusters. At baseline, these clusters were not correlated with patient outcomes. Under NACT, tumors were segregated into three clusters: high BinfTinf, low Tinf, and desert. The high BinfTinf, more diverse in IC composition encompassing T, B, and NK cells, correlated with improved survival. PDL1 was rarely expressed, whereas TIM3, LAG3, and IDO1 were more prevalent. Several iTMEs exist during tumor evolution, and the NACT impact on iTME is heterogeneous. Clustering analysis of patients unravels several IC subsets within ovarian cancer and can guide future personalized approaches. Targeting different checkpoints such as TIM3, LAG3, and IDO1, more prevalent than PDL1, could more effectively harness antitumor immunity in this anti-PDL1-resistant malignancy.

Abstract 719: A tumor-activated PD1/IL2 bispecific molecule, designed to overcome IL-2 receptor-mediated clearance, improve tolerability and stimulate antigen-experienced CD8+ T cells in the tumor microenvironment of murine models

Abstract PD-1/PD-L1 therapies have shown significant activity across a range of tumor types, however, 70-90% of patients do not derive durable benefit. In preclinical settings, delivery of PD-1 blockade and IL-2 agonism in cis via a bispecific molecule, comprising a PD-1 antibody fused to IL-2, has demonstrated superior activity compared to delivery of individual components or their combination. Targeting of IL-2 to PD-1+ cells in cis has been shown to drive a unique differentiation path endowing CD8+ T cells with enhanced functionality. However, we show that the IL-2 component in an unmasked PD1/IL2 bispecific molecule dominates its pharmacology, driving marked peripheral activation of immune cells, rapid IL-2 receptor-mediated clearance and systemic toxicity. To address these challenges and enable tumor-specific activity, we designed a PD1/IL2 bispecific molecule that incorporates a single domain antibody-based mask to bind to the IL-2 component and maintain it in a quiescent state until activated by prevalent and dysregulated proteases in the tumor microenvironment (TME). In preclinical models, the affinity-optimized mask prevented peripheral IL-2 receptor binding, associated toxicity and receptor-mediated clearance. Improved pharmacokinetics of our tumor-activated PD1/IL2 enabled PD1 antibody-like exposures and is projected to achieve complete blockade of PD1 signaling. Tumor-activated PD1/IL2 significantly inhibited tumor growth compared to anti-PD-1 alone or vehicle and demonstrated improved tolerability compared to an unmasked PD1/IL2 bispecific in mouse models. In addition, pharmacodynamic activity of tumor-activated PD1/IL2 was restricted to the TME. Consistent with the masked design, we observed no significant change in CD8+ T cells in the periphery and significant increases in antigen specific CD8+ T cells, memory CD8+ T cells and TCF1+ stem-like CD8+ T cells in the TME. Ex vivo protease cleavage assays performed in human samples demonstrated efficient activation of tumor-activated PD1/IL2 by multiple tumor types and minimal activation in human plasma. In non-human primates, tumor-activated PD1/IL2 achieved tolerable exposures that were comparable to those of existing PD-1 blocking immunotherapies. Taken together, these preclinical data suggest that tumor-activated PD1/IL2 has the potential to improve upon existing PD-1/PD-L1 immunotherapies by inhibiting PD-1/PD-L1 axis and simultaneously directing immunostimulatory IL-2 to antigen-experienced effector cell populations in the TME. Citation Format: Ertan Eryilmaz, Wilson Guzman, Stephanie Hsiao, Parker Johnson, Lisa Quinn, Jimit Lakhani, Brendan Whalen, Rosa Quiroz, Kurt Jenkins, Bernard Lanter, Oleg Yerov, Will Scott, Justin Greene, Zhen Liu, Jason Hedges, Megan McLaughlin, Sallyann Vu, Chelsea Turcotte, Jacob Taylor, Caitlin O'Toole, Magali Pederzoli-Ribeil, Haley Duprey, Natalia Malkova, Damiano Fantini, David Crowe, Kyle Smith, Joseph Card, Janice Lee, Kerri Smith, Benjamin Nicholson, Jennifer O'Neil, Carl U. Bialucha. A tumor-activated PD1/IL2 bispecific molecule, designed to overcome IL-2 receptor-mediated clearance, improve tolerability and stimulate antigen-experienced CD8+ T cells in the tumor microenvironment of murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 719.

Differential effects of itacitinib, fedratinib, and ruxolitinib in mouse models of hemophagocytic lymphohistiocytosis

AbstractHemophagocytic lymphohistiocytosis (HLH) comprises a severe hyperinflammatory phenotype driven by the overproduction of cytokines, many of which signal via the JAK/STAT pathway. Indeed, the JAK1/2 inhibitor ruxolitinib has demonstrated efficacy in preclinical studies and early-phase clinical trials in HLH. Nevertheless, concerns remain for ruxolitinib-induced cytopenias, which are postulated to result from the blockade of JAK2-dependent hematopoietic growth factors. To explore the therapeutic effects of selective JAK inhibition in mouse models of HLH, we carried out studies incorporating the JAK1 inhibitor itacitinib, JAK2 inhibitor fedratinib, and JAK1/2 inhibitor ruxolitinib. All 3 drugs were well-tolerated and at the doses tested, they suppressed interferon-gamma (IFN-γ)–induced STAT1 phosphorylation in vitro and in vivo. Itacitinib, but not fedratinib, significantly improved survival and clinical scores in CpG–induced secondary HLH. Conversely, in primary HLH, in which perforin-deficient (Prf1−/−) mice are infected with lymphocytic choriomeningitis virus (LCMV), itacitinib, and fedratinib performed suboptimally. Ruxolitinib demonstrated excellent clinical efficacy in both HLH models. RNA-sequencing of splenocytes from LCMV-infected Prf1−/− mice revealed that itacitinib targeted inflammatory and metabolic pathway genes in CD8 T cells, whereas fedratinib targeted genes regulating cell proliferation and metabolism. In monocytes, neither drug conferred major transcriptional impacts. Consistent with its superior clinical effects, ruxolitinib exerted the greatest transcriptional changes in CD8 T cells and monocytes, targeting more genes across several biologic pathways, most notably JAK-dependent proinflammatory signaling. We conclude that JAK1 inhibition is sufficient to curtail CpG-induced disease, but combined inhibition of JAK1 and JAK2 is needed to best control LCMV-induced immunopathology.

Analysis of T-lymphocyte subsets and risk factors in children with tuberculosis

BackgroundTuberculosis (TB) is not only related to infection but also involves immune factors. This study explores the changes in T-lymphocyte subsets in children with TB who are human immunodeficiency virus (HIV)-negative and examines their relationship using chest computed tomography (CT) scans. Additionally, the study identifies risk factors for severe TB (STB) in children and establishes relevant risk prediction models. MethodsWe recruited 235 participants between 2018 and 2022, comprising 176 paediatric patients with TB who were HIV-negative and 59 age-matched children with bacterial community-acquired pneumonia (CAP). We quantitatively analysed and compared T-lymphocyte subsets between the two groups and among different types of TB infection. Both univariate and multivariate analyses of clinical and laboratory characteristics were conducted to identify independent risk factors for STB in children and to establish a risk prediction model. ResultsThe absolute counts of CD3, CD4 and CD8 T-cells in children with TB infection decreased significantly compared with bacterial CAP. The percentage of CD8 T-cells increased, whereas the percentage of CD4 T-cells did not change significantly. The absolute count of CD3, CD4 and CD8 T-cells in extrapulmonary TB (EPTB) was significantly higher than in extra-respiratory TB, with unchanged subset percentages. According to chest CT lesion classification, CD4 T-cell counts decreased significantly in S3 compared with S1 or S2, with no significant change in CD3 and CD8 T-cell counts and percentages. No significant differences were observed in lymphocyte subset counts and percentages between S1 and S2. Univariate analyses indicated that factors such as age, symptom duration, white blood cell count, platelet count, neutrophil-to-lymphocyte ratio (NLR), erythrocyte sedimentation rate, prealbumin level, albumin level, globulin level, albumin/globulin (A/G) ratio, high-sensitivity C-reactive protein (Hs-CRP) level and CD4 and CD8 T-cell counts are associated with STB. Multivariate logistic regression analysis revealed that age, Hs-CRP level, NLR, symptom duration and A/G ratio are independent risk factors for STB in children. Increased age, Hs-CRP levels and NLR, along with decreased A/G, correlate with increased susceptibility to STB. A nomogram model, based on these independent risk factors, demonstrated an area under the receiver operating characteristics curve of 0.867 (95% CI: 0.813–0.921). Internal verification confirmed the model's accuracy, with the calibration curve approaching the ideal and the Hosmer–Lemeshow goodness-of-fit test showing consistent results (χ2 = 12.212, p = 0.142). ConclusionIn paediatric patients with TB, the absolute counts of all lymphocyte subsets were considerably reduced compared with those in patients with bacterial CAP. Clinicians should consider the possibility of EPTB infection in addition to respiratory infections in children with TB who have higher CD3, CD4 and CD8 T-cell counts than the ERTB group. Furthermore, CD4 T-cell counts correlated closely with the severity of chest CT lesions. Age, symptom duration, A/G ratio, Hs-CRP level and NLR were established as independent risk factors for STB. The nomogram model, based on these factors, offers effective discrimination and calibration in predicting STB in children.

Platinum-based neoadjuvant chemotherapy upregulates STING/IFN pathway expression and promotes TILs infiltration in NSCLC.

To evaluate the effects of platinum-based neoadjuvant chemotherapy (NACT) on the STING/IFN pathway and tumor-infiltrating lymphocytes (TILs) in non-small cell lung cancer (NSCLC), as well as clinicopathological factors affecting patient survival. A total of 68 patients aged 34-77 years with NSCLC who received neoadjuvant chemotherapy and surgical treatment from March 2012 to February 2019 were reviewed, and the clinical pathological data and paired tissue specimens before and after NACT were collected. Immunohistochemistry and immunofluorescence were used to detect the protein levels of STING, PD-L1 and IFN-β, and the infiltration density of CD3+ TILs and CD8+TILs. The correlation between the expression of STING, PD-L1, IFN-β and the infiltration density of CD3+ TILs and CD8+ TILs as well as the clinicopathological characteristics before and after NACT was analyzed. The relationship between the related indexes, clinicopathological features and prognosis was also discussed. NACT increased the expression of STING, IFN-β and PD-L1 in tumor cells, and the infiltration of CD3+ and CD8+ TILs. In addition, ypTNM stage, ypN stage, changes in CD3+ TILs and in PD-L1 were associated with DFS (disease-free survival). CD3+ TILs changes and ypN stage were associated with OS (overall survival). Notably, ypN stage and CD3+ TILs changes were independent prognostic factors for DFS and OS. NACT stimulates STING/IFN-β pathway, promotes infiltration of CD3+ and CD8+ TILs, triggers innate and adaptive immunity, and also upregulates PD-L1, which complemented the rationale for neoadjuvant chemotherapy in combination with immunotherapy. In addition, DFS was longer in patients with ypTNM I, ypN0-1, and elevated CD3+TILs after NACT. Patients with ypN0 and elevated CD3+ TILs after NACT had better OS benefits.