Changes In Immune Cell Subsets Research Articles

Microwave ablation enhances the systemic immune response in patients with lung cancer.

Microwave ablation (MWA) is a key alternative therapy to conventional surgery for the treatment of lung cancer. In addition to eliminating local tumors, MWA may promote antitumor immunological responses, such as abscopal effects in distant lesions. However, the intensity of MWA is limited and the underlying mechanisms are not well-defined. The present study assessed the impact of MWA on immune cell subsets and cytokines in patients with lung cancer. A total of 45 patients with lung cancer who underwent percutaneous lung tumor MWA were enrolled. Peripheral blood samples were collected before and 24 h after MWA and changes in immune cell subsets [lymphocytes, CD3+, CD4+ and CD8+ T cells, B cells and natural killer (NK) cells] and serum cytokine levels (IL-1β, IL-2, IL-4-6, IL-8, IL-10, IL-12p70, IL-17A and F, IL-22, TNF-α, TNF-β and IFN-γ) were assessed by flow cytometry and ELISA. The number of total lymphocytes, CD4+ T and NK cells in the peripheral blood significantly decreased 24 h after MWA, while number of CD8+ T cells remained stable, leading to a higher proportion of CD8+ T cells. In addition, the serum levels of IL-2, IL-1β, IL-6, IL-12p70, IL-22, TNF-α and IFN-γ were significantly increased 24 h after MWA, indicating a T helper 1 type immune response. The immune response in patients with advanced stage disease was comparable with patients in the early stage group; however, the number of total lymphocytes and CD3+ T cells significantly decreased and the ratio of CD4/CD8 and IL-2 levels significantly increased. The early immune response after MWA may contribute to systemic antitumor immunity in patients with both early and advanced disease. Thus, MWA may exhibit potential as a local therapy and trigger abscopal effects in distant lesions in patients with lung cancer.

Maternal urban particulate matter (SRM 1648a) exposure disrupted the cellular immune homeostasis during early life: The potential attribution of altered placental transcriptome profile

Ambient fine particular matter (PM2.5) exposure has been associated with numerous adverse effects including triggering functional disorders of the placenta and inducing immune imbalance in offspring. However, how maternal PM2.5 exposure impacts immune development during early life is not fully understood. In the current study, we exposed mice with low-, middle-, and high-dose PM2.5 during pregnancy to investigate the potential link between the transcriptional changes in the placenta and immune imbalance in mice offspring induced by PM2.5 exposures. Using flow cytometry, we found that the proportions of B cells, CD3+CD4+ T cells, CD3+CD8+ T cells, and macrophage (Mφ) cells were altered in the blood of PM2.5-exposed mice pups but not dendritic cells (DCs) and natural killer cells (NKs). Using bulk RNA sequencing, we found that PM2.5 exposure altered the transcriptional profile which indicated an inhibition of the complement and coagulation cascades in the placenta. Weighted gene co-expression network analysis (WGCNA) revealed the potential crosstalk between the perturbation of placental gene expression and the changes of immune cell subsets in pups on postnatal day 10 (PND10). Specifically, WGCNA identified a cluster of genes including Defb15, Defb20, Defb25, Cst8, Cst12, and Adam7 that might regulate the core immune cell types in PND10 pups. Although the underlying mechanisms of how maternal PM2.5 exposure induces peripheral lymphocyte disturbance in offspring still remain much unknown, our findings here shed light on the potential role of placental dysfunction in these adverse effects.

Potent induction of antitumor immunity by combining cryo-thermal ablation with immune checkpoint inhibitors in hepatocellular carcinoma.

The low response rate of immune checkpoint inhibitors (ICIs) prompts the exploration of novel combination therapies for patients with hepatocellular carcinoma (HCC). Here, we aimed to examine the efficiency and potential mechanism of cryo-thermal ablation (Cryo-A) combined with anti-programmed death protein 1 (αPD1) and/or cytotoxic T-lymphocyte antigen 4 (αCTLA4) inhibitors in a murine hepatoma model. Immunocompetent C57BL/6 mice inoculated with unilateral or bilateral H22 hepatic tumour cells were treated with Cryo-A and/or ICIs (αPD1 and/or αCTLA4). Flow cytometry, immunohistochemistry, ELISpot assay, time-of-flight cytometry, tumour rechallenging, and T-cell depletion assay were used to assess the dynamic changes of immune cell subsets following therapy. We found Cryo-A resulted in immunogenic cell death of tumour cells, activation of dendritic cells, and enhancement of antitumor immunity. Cryo-A alone was insufficient to extend survival, combining Cryo-A with αPD1 and αCTLA4 further modulated the tumour microenvironment, inducing a durable antitumor immune response by tumour-reactive CD8+ T cells and significantly prolonged survival. Time-of-flight cytometry (CyTOF) data revealed that combination therapies reshaped the tumour microenvironment by the increase of intratumoral CD8+ T cells expressed higher levels of cytotoxic markers and immune checkpoint molecules, and by downregulation of intratumoral granulocytes. The combination also resulted in the eradication of remote unablated tumours (abscopal effect). These findings suggested that Cryo-A turned HCC from "cold" tumours to "hot" tumours and the combination of Cryo-A with αPD1 and αCTLA4 may be a promising approach to improve the prognosis of HCC.

Roginolisib a Highly Selective Allosteric Modulator of the Phosphoinositide 3-Kinase Delta (PI3Kδ) in Patients with Refractory/Relapsed Follicular Lymphoma

Introduction PI3Kδ inhibitors are active in patients (pts) with lymphomas, but their clinical development has been hampered by toxicity (Brown et al, Lancet Oncol 2022). Roginolisib (IOA-244) exerts an allosteric modulation of the PI3Kδ C-terminus and functions as a non-ATP competitive PI3Kδ inhibitor, delivering a selective inhibition without off-target effects typically seen with other PI3K inhibitors (Johnson et al, Cancer Res Commun 2023). Using the recommended phase 2 dose (RP2D) of 80 mg QD established in solid tumors patients, patients with refractory/relapsed (r/r) follicular lymphoma (FL) were treated to investigate its safety profile in pts with hematologic malignancies (NCT04328844). Methods In Part A of the FiH dose escalation study, roginolisib was investigated at 20 and 80 mg QD in pts with r/r FL. Primary objective: safety of the RP2D established in solid tumor pts. Secondary objectives: PK; PD (e.g., changes in immune cell subsets in peripheral blood); Lugano-based responses; PFS and OS. Results In Part A for pts with r/r FL, there were two cohorts: (a) Cohort 1: 20 mg QD daily (4/4; 2 female, 2 male); (b) Cohort 2: 80 mg QD (4/4; 3 female, 1 male). There were no DLT observed and no dose modifications were needed. The mean time on treatment was 1.9 months (20 mg QD) and 4.3 months (80 mg QD), with one patient at the 80 mg receiving Roginolisib for 9 mo. More than 4 prior lines of treatment were in 3/8 pts. Transient (lasting less than 48 hrs) platelet reduction (G3) and AST/ALT elevation (G3) were observed in 2/8 pts, which improved while pts continued without a treatment intervention, such as dose modification or medical treatment with steroids. Lugano-based PRs were observed in 2/4 pts at the 80 mg QD dose and none at the 20 mg QD. At the 80 mg QD dose a third pt had a clinical response manifested by improvement in night sweats and fever, reduction in cervical palpable lymph nodes and relief from chronic pleural effusion. This clinical improvement lasted less than 5 weeks. While Overall Survival (OS) is still ongoing, the median has not been reached in this study and is currently at 14.3 mo. Subsequent to ending Roginolisib treatment, patients received at least one additional therapy (6/8), one patient was able to undergo autologous transplant with a complete metabolic response (CMR). Biomarker evaluation using flow cytometry showed low counts of T regulatory cells (Treg) in peripheral blood in all 8 r/r FL patients compared to solid tumor patients at baseline and throughout the treatment. Myeloid derived suppressor cells in peripheral blood remained unchanged during the course of treatment with Roginolisib. Conclusions The safety profile of roginolisib in pts with r/r FL appears to match that in pts with solid malignancies. In contrast to other PI3Kδ inhibitors, roginolisib is highly selective for binding to PI3Kδ, favoring an inactive confirmation of PI3Kd and functions as a non-ATP competitive inhibitor.

Immune cells are associated with mortality: the Health and Retirement Study.

Age-related immunosenescence is characterized by changes in immune cell subsets and is associated with mortality. However, since immunosenescence is associated with other concurrent age-related changes such as inflammation and multi-organ dysfunction, it is unclear whether the association between age-related immunosenescence and mortality is independent of other concurrent age-related changes. To address these limitations, we evaluated the independent association between immune cell subsets and mortality after adjustment for age-related inflammation and biologic age. Data for this study was obtained from the 2016 interview of the Health and Retirement Study (N=6802). Cox proportional hazards regression models were used to estimate the association between 25 immune cell subsets (11 T-cell subsets, 4 B-cell subsets, 3 monocyte subsets, 3 natural killer cell subsets, 3 dendritic cell subsets, and neutrophils) and 4-year mortality adjusting for covariates such as the Klemera-Doubal algorithm biological age, chronological age, gender, race/ethnicity, BMI, smoking status, comorbidity index, CMV seropositivity, and inflammatory latent variable comprising C-reactive protein, and 4 cytokines (interleukin-10, interleukin-1 receptor antagonist, interleukin-6, and soluble tumor necrosis factor). Four hundred and seventy-six participants died during the study period with an overall median follow up time of 2.5 years. After controlling for covariates and adjustment for sample-weights, total T cells [HR: 0.86, p=0.004], NK CD56LO cells [HR: 0.88, p=0.005], and neutrophils [HR: 1.22, p=0.004] were significantly associated with mortality. These findings support the idea that an aging immune system is associated with short-term mortality independent of age-related inflammation or other age-related measures of physiological dysfunction. If replicated in other external cohorts, these findings could identify novel targets for both monitoring and intervention to reduce the age-related mortality.

First-in-human (FIH) phase I dose escalation study (part A) of the first oral allosteric modulator of phosphoinositide 3-kinase inhibitor delta (PI3Kδ) roginolisib in patients with advanced cancer and dose confirmation in uveal melanoma (part B).

3110 Background: The highly selective oral allosteric modulator of PI3Kδ, roginolisib (IOA-244), blocks the activity of PI3Kδ -dependent signalling, in both tumour cells and Tregs. Methods: Part A explored the continuous daily dosing of roginolisib at 10, 20, 40 and 80 mg in patients (pts) with solid tumours and Follicular Lymphoma (FL). Part B consists of dose confirmation cohorts, including uveal melanoma (UM) pts. Primary objective: safety of the anticipated biologically effective dose (BED), or the recommended phase 2 dose (RP2D). Secondary objectives: PK; PD (e.g., inhibition of CD63 expression on basophils, changes in immune cell subsets in peripheral blood); RECIST 1.1. or Lugano-based responses; PFS and OS. Exploratory studies: phenotypic changes in circulating immune cells by Cytometry by Time of Flight (CyTOF); response assessments by radiomics; analysis of circulating protein signatures via Olink proteomic. Results: Part A Solid Tumour (completed): Sixteen pts were treated in 4 cohorts, each with 4 pts. Pts characteristics: uveal melanoma (9/16; 56%), cutaneous melanoma (5/16; 31%) and pleural mesothelioma (2/16; 13%). Only Grade 1 and 2 AEs by CTCAE v5 were observed, including 2 cases of transient (lasting < 24 hrs) diarrhoea and 2 of AST/ALT elevation. Part A FL Cohort (completed): 8 pts were treated at 20 mg (n=4) and 80 mg (n=4). Grade 3 toxicities were transient and observed in 2/8 (25%) pts. 2/4 pts (50%) at the 80 mg had PR as per Lugano criteria. Neither in the solid tumour nor in the haematology pts treatment-emergent adverse events (TEAE) led to study drug discontinuation, immune related toxicity, or a Dose Limiting Toxicity. Subgroup evaluation: UM pts (Part A and Part B): 24 pts (Part A: 9 pts; Part B ongoing: 15 pts), of which 18 pts treated at 80 mg QD (RP2D). Mean time on treatment: 8.6 mo (range: 1.0-27.9 mo). ORR (RECIST 1.1): PR: 1/20 (5%); SD: 16/20 (80%). Median OS for Part A: 20.8 mo (5/9 with 4 pts alive); Part B: not determined; 1-year OS rate: Part A 66.7%; Part B: ongoing. Exploratory Investigations: Radiomics-based evaluation of CT images in pts with solid tumors assessed each metastatic organ, detecting mixed responses; peripheral proteomic evaluation detected changes in several cytokines and chemokines; CyTOF detected reduction in circulating Tregs associated with increased activated effector CD8+ T cells. Conclusions: Roginolisib is well tolerated at the 80 mg dose, shows signs of efficacy and induces phenotypic changes in the circulating immune cells. As best response at the RP2D, the most robust anti-tumour activity was observed in FL (50% PR) and UM pts (5% PR; 80% SD). Clinical trial information: NCT04328844 .

The role of innate immune system in modulating CHK1 inhibitor (CHK1i) response in BRCA wild-type (BRCAwt), platinum-resistant high-grade serous ovarian cancer (PR-HGSOC): Exploratory analysis from a phase II study of CHK1i prexasertib.

5557 Background: Immune checkpoint blockade, e.g., anti-PD-1/PD-L1, demonstrated limited efficacy in HGSOC despite high levels of tumor-infiltrating lymphocytes, suggesting the innate, rather than adaptive, immune system may play a prominent role in anticancer response. Preclinical studies suggest CHK1i modulates the innate immune milieu. We previously showed clinical activity of prexasertib in BRCAwt, PR-HGSOC patients (pts). Here, we investigated the association of innate immunogenic features with CHK1i response. Methods: Prexasertib was given at 105 mg/m2 IV every 14 days in a 28-day cycle. Baseline (BL) fresh core biopsies were collected from pts enrolled in the biopsy (Bi) cohort for RNA sequencing. Computational immunogenomic analysis (CIBERSORT) was used for immune cell fractions quantification. Blood samples (BS) were obtained at BL and cycle 1 day 15 (C1D15) from pts enrolled in both Bi and non-Bi (nBi) cohorts for multiparametric flow cytometry (FC) analysis to evaluate dynamic changes in immune cell subsets. Mann-Whitney and Wilcoxon tests were used to compare unpaired and paired data, respectively. Results: 49 BRCAwt, PR-HGSOC pts were enrolled in the Bi cohort (n=25) and nBi cohort (n=24). 39 were evaluable per RECIST v1.1. 18/39 pts (46.2%) had a clinical benefit (CB), defined as progression-free survival≥6 months. Transcriptomic profiles of BL biopsies (n=15) met the quality control and assurance (Gene Similarity Index, Principal Component Analysis) for further in silico analysis. CIBERSORT analysis did not exhibit any differences in the immune cell fractions between CB and non-CB (NCB) groups at BL. FC analysis on paired BS (BL and C1D15) showed an increase of immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSCs, p<0.001) and classical monocytes (CM, p=0.003) in the NCB group (18 paired BS). In the CB group (19 paired BS), CD141+ and CD1c+ dendritic cells (DCs) express higher rates of CD83, a marker of maturity and functionality (p<0.001 and p<0.001, respectively), despite no increase in the absolute number of DCs. At C1D15, a higher rate of M-MDSCs (median 7.8% vs. 5.52%; p=0.03) and CM (median 40.7% vs. 31.7%; p=0.03) were found in the NCB group (n=18) compared to the CB group (n=19); also, CM showed lower expression of HLA-DR in the NCB group (median 19.25% vs. 27.8%; p=0.02). Conclusions: Collectively, our data suggest the possible involvement of innate immunity in modulating CHK1i response. Increasing peripheral immunosuppressive cells and lower immunocompetence of the innate immune system may contribute to CHK1i resistance, whereas the functionality of DCs may be involved in the response. Enhancing innate immunity may represent a strategy to increase CHK1i response in this hard-to-treat population. Clinical trial information: NCT02203513 .

Immune-Modifying Microparticles for Treatment of Acute Graft-Versus-Host-Disease

Abstract Background: Acute Graft versus host disease (aGVHD) is one of the most common complication of allogeneic hematopoietic stem cell transplant. We have previously shown that infused immune-modifying particles (IMPs) can reduce clinical symptoms and improve survival in inflammatory conditions. IMPs are taken up by inflammatory monocytes (θIM) via the macrophage receptor with collagenous structure (MARCO) and then undergo splenic sequestration. Given these findings, IMPs may be an effective method of reducing inflammation in aGVHD. Methods: We tested the efficacy of IMP in an established aGVHD murine model. We also explored the changes in immune cell subsets and serum cytokines. To assess impact of IMP on graft-versus-tumor (GVT), we infused host mice with A20 lymphoma cells. Results: We demonstrated that treatment with IMPs effectively rescues mice in a lethal aGVHD model. Treatment with IMPs also significantly improved clinical and target organ histopathological scores. IMP treatment resulted in increased splenic and intestinal CD4 +CD25 +Foxp3 +Tregs. IMP treated mice had lower numbers of colonic CD11b +Ly6c hiθIM and a lower peak of serum inflammatory cytokines. Maintenance of GVT effect was demonstrated by comparable survival rates in host mice with co-infusion of A20 lymphoma cells. Conclusion: Systemic IMP was effective at rescuing mice in this lethal aGVHD model. This demonstrates that targeting θIMs with IMPs could be an effective strategy to treat aGVHD. Further investigation of the mechanisms by which IMPs ameliorate aGVHD is warranted, as is investigation of the use of IMPs to treat aGVHD in the clinical setting. Supported by Nanoparticle Tolerance Research Fund

Immunophenotypic Alterations in Adult Patients with Steroid-Dependent and Frequently Relapsing Nephrotic Syndrome.

Immune dysregulation plays a key role in the pathogenesis of steroid-dependent/frequently relapsing nephrotic syndrome (SDNS/FRNS). However, in contrast with evidence from the pediatric series, no major B- or T-cell alterations have been described for adults. In these patients, treatment with rituximab allows safe discontinuation of steroids, but long-term efficacy is variable, and some patients experience NS relapses after B cell reconstitution. In this study, we aimed to determine disease-associated changes in the B and T cell phenotype of adult patients with SDND/FRNS after steroid-induced remission. We also investigated whether any of these changes in immune cell subsets could discriminate between patients who developed NS relapses after steroid-sparing treatment with rituximab from those who did not. Lymphocyte subsets in SDNS/FRNS patients (n = 18) were compared to those from patients with steroid-resistant NS (SRNS, n = 7) and healthy volunteers (HV, n = 15). Before rituximab, SDND/FRNS patients showed increased frequencies of total and memory B cells, mainly with a CD38-negative phenotype. Within the T-cell compartment, significantly lower levels of FOXP3+ regulatory T cells (Tregs) were found, mostly due to a reduction in CD45RO+ memory Tregs compared to both SRNS and HV. The levels of CD45RO+ Tregs were significantly lower at baseline in patients who relapsed after rituximab (n = 9) compared to patients who did not (n = 9). In conclusion, patients with SDND/FRNS displayed expansion of memory B cells and reduced memory Tregs. Treg levels at baseline may help identify patients who will achieve sustained remission following rituximab infusion from those who will experience NS relapses.

Noninvasive ultrasound stimulation to treat myocarditis through splenic neuro-immune regulation

BackgroundThe cholinergic anti-inflammatory pathway (CAP) has been widely studied to modulate the immune response. Current stimulating strategies are invasive or imprecise. Noninvasive low-intensity pulsed ultrasound (LIPUS) has become increasingly appreciated for targeted neuronal modulation. However, its mechanisms and physiological role on myocarditis remain poorly defined.MethodsThe mouse model of experimental autoimmune myocarditis was established. Low-intensity pulsed ultrasound was targeted at the spleen to stimulate the spleen nerve. Under different ultrasound parameters, histological tests and molecular biology were performed to observe inflammatory lesions and changes in immune cell subsets in the spleen and heart. In addition, we evaluated the dependence of the spleen nerve and cholinergic anti-inflammatory pathway of low-intensity pulsed ultrasound in treating autoimmune myocarditis in mice through different control groups.ResultsThe echocardiography and flow cytometry of splenic or heart infiltrating immune cells revealed that splenic ultrasound could alleviate the immune response, regulate the proportion and function of CD4+ Treg and macrophages by activating cholinergic anti-inflammatory pathway, and finally reduce heart inflammatory injury and improve cardiac remodeling, which is as effective as an acetylcholine receptor agonists GTS-21. Transcriptome sequencing showed significant differential expressed genes due to ultrasound modulation.ConclusionsIt is worth noting that the ultrasound therapeutic efficacy depends greatly on acoustic pressure and exposure duration, and the effective targeting organ was the spleen but not the heart. This study provides novel insight into the therapeutic potentials of LIPUS, which are essential for its future application.Graphical

A Lesson of Immunosuppression in Renal Transplant: Retreat or Hold?

Background: Aiming at understanding whether there are cases of near-tolerance among long-term surviving kidney transplant recipients in our center, or even operant tolerance can be attempted based on their immune status, we analyzed changes of immune cell subsets and cytokines in various groups, and evaluated immune status of long-term survival recipients. Methods: A real-world, observational, retrospective cohort study was conducted in our hospital. Twenty-eight long-term recipients were selected as study subjects, 15 recent postoperative stable recipients, and 15 healthy subjects as controls. T and B lymphocyte subsets, MDSCs, and cytokines were detected and analyzed. Results: Treg/CD4 T cells, total B and B10 cells in long-term and recent renal recipients were lower than healthy controls (HC). The level of IFN-γ and IL-17A in long-term survival patients was obviously higher than that in recent postoperative stable recipients and HC, while TGF-β1 level was significantly lower in long-term survival group than in short-term postoperative group and HC. Notably, compared with short-term recipients, it has been found that the IL-6 level in both positive and negative HLA groups were obviously lower (all P<0.05). In the long-term survival group, 43% of recipients were positive for urinary protein and 50% were positive for HLA antibody. Conclusion: This "real-world" study validates the findings of real status of long-term survival recipients observed in clinical trials. Contrary to a state of proper tolerance as expected, the group recipients in long-term survival were accompanied by the increased indicators of immune response, while those related to immune tolerance were not significantly increased. Long-term survival recipients with stable renal function may be in an immune equilibrium state where immunosuppression and rejection coexist under the action of low-intensity immune agents. If immunosuppressive agents are reduced or even removed, rejection may occur.

The combination of oxaliplatin and anti-PD-1 inhibitor promotes immune cells infiltration and enhances anti-tumor effect of PD-1 blockade in bladder cancer.

Bladder cancer (BLCA) is a highly malignant tumor of the urinary system, but the prognosis and survival rates have little improvement based on current therapeutic strategy. Immune checkpoint inhibitors (ICIs) therapy revolutionized the treatment of BLCA, but the clinical application of ICIs is limited by low response rate. Oxaliplatin (OXP), a second line chemotherapy drug for BLCA, may reshape the tumor immune microenvironment (TIME) via recruiting immune cells. Here, we conducted the study of oxaliplatin combined with anti-PD-1 inhibitor in BLCA mice models. The 6-8 weeks old female C57BL/6J mice were used to establish subcutaneous model of bladder tumor. After tumors developed, mice were given tail vein injections of PBS or oxaliplatin (2.5 mg/kg) and/or anti-PD-1 antibody (10 mg/kg). Tumor tissue samples and peripheral blood mononuclear cell (PBMC) were collected to systemically evaluate the efficiency and safety of combination OXP and anti-PD-1 inhibitor. The change of immune cells populations and the corresponding phenotypic diversity in TIME and PBMC were analysed by flow cytometry. Tumor growth experiments clarified that the combination therapy was more efficient than medication alone. Flow cytometry analysis of tumor samples showed significant differences between untreated and treated mice. Oxaliplatin influences the TIME by increasing immune cells infiltration, including CD3+ T cells, CD4+ T cells, CD8+ T cells, dendritic cells (DC cells) and natural killer cells (NK cells). As for infiltrating cells, oxaliplatin upregulated the expression of CD134 and downregulated TIM-3 of CD4+ T cells, downregulated the PD-L1 expression of DC cells, which contributed to improve the anti-tumor effect and the treatment response of ICIs. Additionally, the evaluation of PBMC found that there were no significant changes in immune cell subsets and phenotypes, which validated the safety of the combination therapy. These results show the therapeutic potential for the combination of OXP and anti-PD-1 inhibitor in BLCA. OXP could increase the infiltration of immune cells in TIME to promote the anti-tumor activity of anti-PD-1 inhibitor. The present research provided an appropriate rationale of combination chemotherapy and immunotherapy therapy for BLCA.

Immunogenic change after percutaneous microwave ablation in pulmonary malignancies: Variation in immune cell subsets and cytokines in peripheral blood.

To investigate immunogenic changes after percutaneous microwave ablation (MWA) in pulmonary malignancies. Twenty-two consecutive patients with pulmonary malignancies who underwent percutaneous lung tumor MWA were prospectively enrolled in this study. Peripheral blood samples were collected on the day before (D0) and one month (M1) after MWA. Changes in immune cell subsets (CD3+, CD4+, and CD8+ T cells, and B, natural killer, regulatory T (Treg), and CD3-CD20+ cells) and cytokines (interleukin [IL]-2, 4, 6, 10, 17A, tumor necrosis factor [TNF]-α, and interferon-γ) were noted and compared. Progression-free survival (PFS) and potentially related factors were analyzed. The proportion of CD8+ T cells increased from 22.95 ± 7.38% (D0) to 25.95 ± 9.16% (M1) (p = 0.031). The proportion of Treg cells decreased from 10.82 ± 4.52% (D0) to 8.77 ± 2.05% (M1) (p = 0.049). The IL-2 concentration was also decreased from 1.58 ± 0.46 pg/mL (D0) to 1.26 ± 0.60 pg/mL (M1) (p = 0.028). The reduction in Treg cells predicted PFS independently of clinical prognostic features in multivariate analysis (hazard ratio = 4.97, 95% confidence interval: 1.32-18.66, p = 0.018). A reduction in the proportion of Treg cells was observed in 15 patients (68.2%) and the average of the reduction was 2.05 ± 4.60%. Those patients with a reduction in the proportion of Treg cells that was more than average showed a significantly longer median PFS time than those with a reduction that was less than average (16 months vs. 8.5 months, p = 0.025). Percutaneous MWA of pulmonary malignancies leads to immunogenic changes. The reduction in the proportion of Treg cells was independently associated with PFS.

Association of Immune Dynamics and Treatment Outcome in Patients (pts) with Relapsed/Refractory Non-Hodgkin Lymphoma (R/R NHL) Treated with the Novel Cereblon (CRBN) E3 Ligase Modulator (CELMoD) Agent CC-99282

Introduction Compounds inducing CRBN-mediated degradation of Ikaros/Aiolos have demonstrated antitumor activity in pts with NHL. The pharmacodynamic (PD) effects of these agents include T-cell activation, increased trafficking of macrophages, and increased number of regulatory T cells (Tregs) in peripheral blood (PB). Furthermore, high baseline immune infiltration in tumors has been correlated with improved outcomes. CC-99282 (BMS-986369) is a novel CELMoD® agent that has enhanced antiproliferative, apoptotic, and immune-stimulatory effects compared with other immunomodulatory agents in various preclinical NHL models. Part A of the CC-99282-NHL-001 study (NCT03930953) showed CC-99282 monotherapy has a predictable and manageable safety profile, dominated by the class effect of neutropenia, and promising efficacy (objective response rate 42%) in heavily pretreated pts with R/R NHL. Methods CC-99282-mediated immune effects and the dependency of response on immune changes were evaluated using biomarker data from part A of CC-99282-NHL-001, including longitudinal substrate degradation, circulating tumor DNA (ctDNA), ex vivo cytokine secretion, immunophenotyping, and baseline tumor RNA-seq data. Results At steady state, CC-99282 induced deep (> 90%) and sustained degradation of Ikaros/Aiolos in monocytes, B cells, and T cells that was independent of response. PD effects were also confirmed in tumor cells obtained at screening and on treatment. PB T cells from pts dosed with CC-99282 showed increased secretion of IL-2, TNFα, INFγ, and GM-CSF on ex vivo stimulation with anti-CD3. Tumor and PB biomarkers were analyzed for correlation to response. ctDNA data available for 39/50 pts treated in part A showed high tumor burden (> 2.5 log10 of tumor-specific mutant sequence [Kurtz, et al. J Clin Oncol 2018]) in 28/39 pts (72%) at study entry, and yet 13/28 pts (43%) still achieved a partial or complete response. B-cell content estimation in screening biopsies, calculated from bulk RNA-seq data using cell-type-specific markers (Bindea, et al. Immunity 2013), confirmed similar B-cell content in responders and non-responders prior to CC-99282 treatment. In contrast to treatment with other CELMoD agents, this analysis showed that durable responses to CC-99282 were observed in pts with R/R NHL despite their high tumor burden and low immune infiltration. Baseline PB immunophenotype was not significantly associated with response. However, unsupervised clustering of changes in immune cell subsets in pts between baseline and cycle (C) 1 day (D) 15, revealed 5 clusters (Figure 1). Cluster 5, enriched in diffuse large B-cell lymphoma (DLBCL) non-responders, showed upregulation of peripheral Tregs without changes in T-cell activation. Clusters 1 and 4 also showed upregulation of Tregs, with cluster 1 showing additional immune activation. Cluster 2, enriched for follicular lymphoma (FL), only showed T-cell activation. Cluster 3 was enriched in responders without changes in immune subsets upon CC-99282 treatment, suggesting a stronger direct effect on tumor cells in the early stages of treatment. Baseline tumor biopsies from pts in all clusters had similar Treg levels, independent of their immune changes upon treatment. Further, longitudinal analysis of ctDNA during treatment showed that all clusters of responders had a similar decrease in ctDNA that was maintained until the end of treatment despite increases in Tregs. Immunophenotyping at C2D1 and C3D1 showed that immune activation was maintained in responders and increases in Tregs progressively extended to all pts independently of response. The implications of these differences remain unclear. The only previous systematic analyses of the prognostic role of Tregs in lymphoma concluded that increase in Tregs correlated with longer overall survival for pts with DLBCL, but found no significant association in pts with FL (Glowala-Kosinska, et al. Eur J Haematol 2013; Peng, et al. J Cancer Res Clin Oncol 2020). Conclusion Taken together, these results indicate a link between patterns of immune activation and response to CC-99282, but whether this link is causative, or correlative, remains to be elucidated. Responder subsets were identified based on effects on Tregs and immune activation; however, all cases led to durable responses. Further study and validation of these data could provide rationale for pt selection and combination strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy and Safety of Low-Dose Interleukin 2 for Primary Sjögren Syndrome

Primary Sjögren syndrome (pSS) is a systemic autoimmune disease associated with dysregulated immune cells, with no efficient therapy. There is a need to study potential therapeutic approaches. To investigate the efficacy, safety, and immune response of low-dose interleukin 2 (LD-IL-2) in the treatment of pSS. A double-blind, placebo-controlled randomized clinical trial was conducted with a 2-group superiority design from June 2015 to August 2017. Sixty patients, aged 18 to 70 years, were recruited from Peking University People's Hospital. Efficacy analyses were based on the intention-to-treat (ITT) principle. Data were analyzed from December 2018 to March 2020. Patients with pSS were treated with LD-IL-2 or placebo for 12 weeks and accompanied by 12 weeks of follow-up. The primary end point was defined as a 3-point or greater improvement on the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (ESSDAI) by week 24. The secondary end points included other clinical responses, safety, and changes of immune cell subsets at week 12 and 24. Sixty patients with pSS were recruited, with 30 in the LD-IL-2 group (mean [SD] age, 47.6 [12.8] years; 30 [100%] women) and 30 in the placebo group (mean [SD] age, 51.0 [11.9] years; 30 [100%] women), and 57 completed the trial. More patients in the LD-IL-2 group (20 [66.7%]) achieved ESSDAI score reduction of at least 3 points than in the placebo group (8 [26.7%]) at week 24 (P = .004). There were greater resolutions of dryness, pain, and fatigue in the LD-IL-2 group than placebo group at week 12 (dryness: difference, -18.33 points; 95% CI, -28.46 to -8.21 points; P = .001; pain: difference, -10.33 points; 95% CI, -19.38 to -1.29 points; P = .03; fatigue: difference, -11.67 points; 95% CI, -20.65 to -2.68 points; P = .01). No severe adverse events were observed in either group. In addition, the LD-IL-2 group showed a significant decrease in infection compared with the placebo group (1 [3.3%] vs 9 [30.0%]; P = .006). Immunological analysis revealed that LD-IL-2 promoted an expansion of regulatory T cells and regulatory CD24highCD27+ B cells. In this randomized clinical trial, LD-IL-2 was effective and well tolerated in patients with pSS, and it restored immune balance, with enhanced regulatory T cells and CD24highCD27+ B cells. ClinicalTrials.gov Identifier: NCT02464319.

Single-cell mechanistic studies of radiation-mediated bystander effects.

Ionizing radiation (IR) has been widely used in the diagnosis and treatment of clinical diseases, with radiation therapy (RT) being particularly rapid, but it can induce "bystander effects" that lead to biological responses in non-target cells after their neighboring cells have been irradiated. To help clarify how radiotherapy induces these effects, To help clarify how radiotherapy induces these effects, we analyzed single-cell RNA sequencing data from irradiated intestinal tissues on day 1 (T1 state), day 3 (T3 state), day 7 (T7 state), and day 14 (T14 state) after irradiation, as well as from healthy intestinal tissues (T0 state), to reveal the cellular level, molecular level, and involvement of different time irradiated mouse intestinal tissues in biological signaling pathways. In addition, changes in immune cell subpopulations and myeloid cell subpopulations after different radiation times were further explored, and gene regulatory networks (GRNs) of these cell subpopulations were constructed. Cellular communication between radiation-specific immune cells was explored by cell-to-cell communication events. The results suggest that radiotherapy trigger changes in immune cell subsets, which then reprogram the immune ecosystem and mediate systemic bystander effects. These radiation-specific immune cells participate in a wide range of cell-to-cell communication events. In particular, radiation-specific CD8+T cells appear to be at the core of communication and appear to persist in the body after recovery from radiotherapy, with enrichment analysis showing that radiation-specific CD8+ T cells are associated with ferroptosis. Thus, radiation-specific CD8+ T cells may be involved in cellular ferroptosis-mediated adverse effects caused by RT.

Immune cell subset profiling in multiple sclerosis after fingolimod initiation and continued treatment: The FLUENT study.

BackgroundFingolimod is a sphingosine 1-phosphate receptor modulator approved for relapsing MS. Long-term effects on the immunological profile are not fully understood.ObjectiveInvestigate fingolimod's temporal effects on immune cell subsets, and safety outcomes.MethodsIn FLUENT, a 12-month, prospective, non-randomized, open-label, phase IV study, adult participants received fingolimod 0.5 mg/day. Changes in immune cell subsets, anti-John Cunningham virus (JCV) antibody index, and serum neurofilament levels were assessed.Results165 fingolimod-naive and 217 participants treated for 2–12 years in routine clinical practice were enrolled. Levels of all monitored peripheral lymphocyte subsets were reduced from month 3 in fingolimod-naive participants. Greatest reductions occurred in naive and central memory CD4+ and CD8+ T cells, and in naive and memory B cells. Most lymphocyte subset levels remained stable in the continuous fingolimod group. Components of the innate immune system remained within reference ranges. No increase in JCV seropositivity was observed. No single cellular subset correlated with anti-JCV antibody index at any time point. Neurofilament levels remained within healthy adult reference limits throughout. No opportunistic infections were reported; no new or unexpected safety signals were observed.ConclusionFLUENT provides insights into the utility of immunological profiling to evaluate therapy response and potential infection risk.

P1116: SAFETY, PHARMACOKINETIC (PK), PHARMACODYNAMIC (PD) AND ACTIVITY OF THE HIGHLY SELECTIVE PHOSPHOINOSITIDE 3-KINASE INHIBITOR DELTA (PI3KΔ) INHIBITOR IOA-244 IN PATIENTS WITH FOLLICULAR LYMPHOMA (FL)

Background: IOA-244 is a highly selective inhibitor of PI3Kδ, and has a favourable safety and ADME profile in patients (pts) with solid malignancies. In pts with uveal melanoma, IOA-244 also reduced circulating Tregs counts at the biologically effective dose (BED) range. Aims: As part of the First-in-human (FiH) dose escalation study, pts with FL received IOA-244 at the BED range established in patients with solid malignancies to confirm its favourable safety and PK profile in pts with haematologic malignancies. Methods: IOA-244 was investigated in a two-part FIH study. Part A explored the continuous daily dosing of IOA-244 at 10, 20, 40 and 80 mg in pts with solid malignancies and at 20 mg and 80 mg in pts with FL. Part B consists of expansion cohorts of specific tumour indications, including pts with lymphoma. Primary objective: safety of the anticipated BED, or the recommended phase 2 dose (RP2D). Secondary objectives: PK; PD (e.g., inhibition of CD63 expression on basophils, changes in immune cell subsets in peripheral blood); Lugano-based responses; PFS and OS Results: While Part A for solid malignancies have previously been reported (Di Giacomo et al 2021), we here report for the first time the safety, PK, PD and activity of pts with FL. This part of the study is still ongoing in patients with FL and is anticipated to be completed in 2022. Pts at the first cohort of 20 mg QD daily (4/4; 2 female and 2 males) had no DLT and no dose interruptions. Transient platelet reduction (G3) and AST/ALT elevation (G2) were observed in 1/4 pts, which improved while pt was on therapy. The PK and ADME profile was consistent with the ones observed in pts with solid malignancies. One patient had FL and DLBCL as prior co-primary malignancy with 7 prior lines of therapy. The other 3 pts had 1-3 prior lines of therapy. The best response was SD (1/3 pts) and all pts progressed after completing 2 cycles. LDH was initially elevated in 2/4 pts which subsequently decreased on therapy. 3/4 pts had low Treg counts at baseline. Additional pts are being recruited at the RP2D for solid malignancies, at 80 mg QD. Reference: Di Giacomo et al. Annals of Oncology (2021) 32 (suppl_7): S1428-S1457. 10.1016/annonc/annonc787 Summary/Conclusion: The PK and ADME profile of IOA-244 in pts with FL appears to match that in pts with solid malignancies. Additional pts at the 80 mg QD dose are expected to match the safety observed in pts with solid malignancies. In contrast to other PI3Kd inhibitors, IOA-244 is highly selective and may therefore target Tregs in pts with FL without off-target effects induced by inhibition of other PI3K isoenzymes.

Abstract 3908: A streamlined and automated approach to high-content cytometric immunophenotyping with CyTOF XT

Abstract High-parameter immune profiling is crucial in translational and clinical research to quantify changes in immune cell populations over time. CyTOF® mass cytometry is a high-plex, single-cell analysis platform that uses isotopically pure metal-labeled antibodies. The major advantage of CyTOF is its ability to resolve 40-plus markers in a single panel without compensation, making mass cytometry ideal for routine immunophenotyping. The autosampler module of CyTOF XT™ provides significant time savings by allowing automated sample acquisition. Tubes of pelleted stained samples are loaded into the Autosampler carousel, and the samples are resuspended with EQ™ Calibration Beads for acquisition. User input is only required during instrument startup, tuning, and batch setup. The added automation of CyTOF XT provides a streamlined workflow for suspension mass cytometry. Testing was performed to ensure that the data obtained on CyTOF XT was comparable to manual acquisition systems. The performance of CyTOF XT was tested in parallel with its predecessor, Helios™. Several workflows and applications for suspension mass cytometry including sample barcoding with the Cell-ID™ 20-Plex Pd Barcoding Kit, and surface, cytoplasmic, and nuclear staining and phosphostaining were evaluated on human PBMC. Manual gating analysis was performed to assess population frequencies and median intensities for each marker. Resolution index was calculated to assess how well positive and negative populations separated from each other. There was no significant difference between population frequencies analyzed between the two CyTOF systems. Moreover, samples acquired on CyTOF XT, on average, resulted in greater signal resolution between positive and negative populations compared to Helios. The Maxpar Direct Immune Profiling System was also compared on CyTOF XT and Helios using human whole blood and PBMC. The Maxpar® Direct™ Immune Profiling Assay™ and Maxpar Pathsetter™ software were developed as a sample-to-answer system for human immune profiling using CyTOF. The Maxpar Direct Immune Profiling Assay includes an optimized panel of 30 unique markers in a dry, single-tube format. Maxpar Pathsetter is an automated software used to report population statistics, stain assessments, and relevant data plots for the panel. The automated staining assessment in Maxpar Pathsetter was compared between files acquired on CyTOF XT and Helios. Comparable population frequencies were obtained between the two acquisition systems, and improved staining assessment was observed on CyTOF XT. Overall, these studies demonstrate that CyTOF XT generates better signal resolution as compared to Helios. The automated acquisition of CyTOF XT enables researchers to streamline immunophenotyping of human samples while accurately and reproducibly monitoring changes in immune cell subsets. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Thiru Selvanantham, Stephen K.H. Li, Nick Zabinyakov, Alexandre Bouzekri, Raymond Jong, Michael Sullivan, Alexander Laboda, Daniel Majonis, Christina Loh. A streamlined and automated approach to high-content cytometric immunophenotyping with CyTOF XT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3908.

The ratio of PD1+CD8+T cells in stromal area of tumor tissue is associated with the effect of neoadjuvant chemotherapy in HER2 negative breast cancer patients.

e12626 Background: For patients diagnosed with early breast cancer, surgery remains the standard of care. Early patients achieving pathological complete response (pCR) after neoadjuvant therapy have been proved to be associated with improved survival. However, the accurate prediction of pCR in a clinical setting remains challenging. Tumor infiltrating lymphocyte (TIL) levels have recently been identified as an independent predictor of pCR in breast cancer. However, due to extremely complex tumor microenvironment within tumor, the role of different immune cell subtypes in neoadjuvant chemotherapy (NAC) is inconclusive. This study aimed to explore the potentially predictive role and dynamic changes of immune cell subsets in patients with HER2 negative breast cancer receiving NAC. Methods: Since January 2020, tumor tissue samples of 12 HER2 negative breast cancer patients were collected at baseline and after NAC. Several kinds of immune cells including CD3+T cells, CD4+T cells, CD8+T cells, PD-1+CD8+T cells, Tregs, M1/M2 tumor associated macrophages(TAM), PD-L1+CD68+TAM, B cells, CD56bright NK cells and CD56dim NK cells in the tumor tissue were evaluated by multiple fluorescence immunohistochemistry (mIHC) and counted in intratumoral and stromal area for density and ratio. T test was used for the comparison of immune cells and statistical significance was set at p = 0.05. Results: We examined immune cell subtypes and PD-L1/PD-1 expression of 12 HER2 negative breast cancer patients. According to achieving pCR or not, these 12 patients were divided into two groups, pCR (n = 3) and non-pCR group (n = 9). The results showed that the ratio of PD1+CD8+T cells in stromal area of baseline tumor tissue of pCR group was significantly higher than non-pCR group (median ratio, pCR group vs. non-pCR group = 17.3% vs. 3.6%, p = 0.029).In addition, the ratio of PD1+CD8+T cells in stromal area of pCR group was still significantly higher than that of non pCR patients after NAC (median ratio, pCR group vs. non-pCR group = 20.7% vs. 2.6%, p = 0.0003). Conclusions: Whether at baseline or after NAC, the ratio of PD1+CD8+T cells in stromal area is associated with the effect of NAC in HER2 negative breast cancer.