Peripheral T-cell Research Articles

Alterations in peripheral T-cell and B-cell subsets in the ankylosing spondylitis patients with gut inflammation.

This study investigates changes in immune cell subsets in peripheral blood of ankylosing spondylitis (AS) patients with colitis or terminal ileitis. It aims to explore the connection between changes in lymphocyte subsets and gut inflammation, providing insights for early detection. Overall, 50 AS patients undergoing colonoscopy were enrolled. Flow cytometry was employed to analyze lymphocyte subsets, including T and B cells, in peripheral blood. Disease activity was assessed using CRP, ESR, BASDAI, ASDAS-CRP, and ASDAS-ESR. Compared to AS patients without gut inflammation, those with colorectal inflammation showed a significant increase in total T cells (p < .05), an increase in exhausted CD4+ T cells (p < .05), and a decrease in Th2 cells and total Tc cells (p < .05). Notably, in AS patients with terminal ileitis, there was an increase in total B cells and classic switched B cells (p < .05), with a decrease in double-positive T cells (p < .05). However, no significant differences were observed in the distribution of Tfh-cell subpopulations (Tfh1, Tfh2, Tfh17) and Tc-cell subpopulations (Tc1, Tc2, Tc17) between AS patients with either colorectal inflammation or terminal ileitis (p > .05). We explored the relationship between disease activity scores, ESR, CRP, and lymphocyte subsets, but found no statistically significant correlation between them. Distinct immune patterns may exist in AS with different types of intestinal inflammation. Colitis in AS is primarily characterized by a significant increase in exhausted CD4+ T cells, along with a decrease in Th2 cells. In contrast, terminal ileum inflammation in AS is marked by an increase in total B cells and classic switched B cells. These findings offer new insights for early detection and therapeutic intervention.

Clinical and Real-World Effectiveness of Mogamulizumab: A Narrative Review.

Mogamulizumab (MOG) is an antibody targeting the CCR4 receptor, authorized for relapsed or refractory peripheral T-cell (PTCL) and cutaneous T-cell lymphomas (CTCL). Its adoption in guidelines and endorsement by FDA and EMA established it as a systemic treatment, especially for advanced disease stages due to its comparatively lower toxicity. Clinical trials and real-world evidence have underscored its efficacy in advanced CTCLs, including mycosis fungoides and Sézary syndrome; PTCLs; and adult T-cell leukemia/lymphoma (ATLL), showcasing positive outcomes. Notably, the drug has demonstrated significant response rates, disease stability, and extended periods of progression-free survival, suggesting its applicability in cases with multiple treatment lines. Its safety profile is generally manageable, with adverse events (AEs) primarily related to the skin, infusion-related reactions, drug eruptions, autoimmune diseases, and skin disorders. The latter seem to appear as CCR4 can promote the skin-specific homing of lymphocytes, and MOG is directed against this receptor. While combination with immunostimulatory agents like interferon alpha and interleukin 12 has shown promising results, caution is urged when combining with PD1 inhibitors due to the heightened risk of immune-mediated AEs. The introduction of MOG as a systemic treatment implies a significant advancement in managing these diseases, supported by its favorable safety profile and complementary mechanisms.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: The Nordic Lymphoma Group (NLG) performed a dose-finding/expansion trial evaluating pixantrone, etoposide, bendamustine and, in CD20+ lymphomas, rituximab (P[R]EBEN) in patients (pts) with relapsed diffuse large B-cell (DLBCL) or peripheral T-cell (PTCL) lymphomas. The regimen was out-patient based, and applicable in frail, heavily pre-treated pts. Here we present a long-term follow-up of the trial and a per-protocol correlative biological analysis of pre-therapeutic tumour biopsies looking for gene expression profiles associated with long-term response. Methods: We enrolled 60 pts (37 DLBCL and 23 PTCL; age range: 39–84 yrs, median: 71 yrs). Time to event parameters were analyzed by Kaplan-Meier estimates, log-rank test and Cox regression models. Pre-therapeutic biopsies from 42 pts (25 DLBCL, 17 PTCL) were analyzed for gene expression by NanoString PanCancer Pathways and Immune profiling panels (altogether 1348 genes). Results: Of the original 60 patients, 22 were alive at the time of the present analysis. The median follow-up of surviving pts was 41 mo (range 26–76 mo). The 38 deaths were due to: lymphoma (24; 63%), infections (3; 8%) and other causes (11; 29%: 1 lung carcinoma, 5 acute myeloid leukemia-AML, 1 myelodysplasia-MDS, 1 lung embolism, 1 allotransplant related and 2 unknown). Of the 6 pts with AML/MDS, 5 were PTCL and 1 DLBCL. Of 58 evaluable pts, 38 (65%) had a complete (CR) and 1 (3%) a partial response (B: 51%; T: 70%). The median duration of response (DoR) of the 38 CR pts was 17 mo (range 0.5–55 mo; B: 18 mo; T: 17 mo). Four pts were bridged to allogeneic transplant. The overall 5-yr OS and PFS were 33% and 19%, respectively. The median OS for DLBCL was 16.0 mo [IQR 7.0>] and for PTCL 18.0 mo [IQR 8.0>]. The median PFS for DLBCL was 10.0 mo [IQR 4.0–32.0] and for PTCL 10.0 mo [IQR 5.0–26.0]. To better understand molecular alterations underlying the delay of relapse in this heavily pre-treated elderly population, we determined differentially expressed genes between short (<12 mo) and long term (>12 mo) responders. In DLBCL, 31 genes showed significant differential expression between short- and long-term responders (P < 0.05). The latter had higher expression of genes related to transcription factor activity. The gene most significantly associated with long-term response was Neutrophil Cytosolic Factor 4 (NCF4). In PTCL, 23 genes were differentially expressed between short- and long-term responders, the latter showing enrichment in Ras signaling pathway genes (e.g., Rac Family Small GTPase 3, RAC3, and phospholipase A2 group IIA, PLA2G2A). The research was funded by: Servier Laboratoires Keywords: Aggressive B-cell non-Hodgkin lymphoma, Aggressive T-cell non-Hodgkin lymphoma, Combination Therapies No conflicts of interests pertinent to the abstract.

A NOVEL CONDITIONING REGIMEN OF CHIDAMIDE, CLADRIBINE, GEMCITABINE, AND BUSULFAN (CHICGB) IN THE AUTOLOGOUS STEM CELL TRANSPLANTATION OF AGGRESSIVE T‐CELL LYMPHOMA

Background: The prognosis of patients with peripheral T-cell (PTCL) or lymphoblastic T-cell lymphoma (T-LBL) remains poor under current conditioning regimens before receiving autologous stem cell transplantation (ASCT). Methods: Patients with PTCL or T-LBL were enrolled to receiviing ASCT using the conditioning regimen of chidamide, cladribine, gemcitabine and busulfan (ChiCGB). Positron emission tomography-computed tomography (PET/CT) was used to evaluate the response to ASCT. Overall survival (OS) and progression-free survival (PFS) were employed to assess patient outcome, and adverse events were used to assess the safety of the regimen. The survival curve was estimated via the Kaplan-Meier method. Results: Twenty-five PTCL and 11 T-LBL patients were recruited. The median time to white blood cell (WBC) and platelet engraftments were 10 days (8–13 days) and 13 days (9–31 days), respectively. The 3-year PFS and OS were 81.3 ± 7.2% and 88.5 ± 5.4% for all patients; 92.0 ± 5.4% and 81.2 ± 8.8% for PTCL patients; and both 81.8 ± 11.6% for T-LBL patients, respectively. The 3-year PFS and OS were both 92.9 ± 4.9% for patients with complete response (CR), but 50.0 ± 17.7% and 75.0 ± 15.3% for patients with non-CR, respectively. Infection was the most common non-hematological toxicity, and all toxicities were mild and controllable. Conclusions: ChiCGB was a potentially effective and well-tolerated conditioning regimen to improve the prognosis of patients with aggressive T-cell lymphoma. Future randomized controlled trials are needed to further assess ChiCGB as conditioning regimen for ASCT. Keywords: Aggressive T-cell non-Hodgkin lymphoma, Chemotherapy, Stem Cell Transplant No conflicts of interests pertinent to the abstract.

A phase 2 trial of chemotherapy, pembrolizumab, and propranolol in patients with advanced esophageal/gastroesophageal junction adenocarcinoma (EGAC).

TPS4171 Background: Chronic stress suppresses the immune system and promotes cancer cell growth. In animal studies, we reported that this is mediated by the beta-adrenergic (BA) pathway. We further showed that concurrently blocking the BA pathway with β-blockers (BB) can improve immune checkpoint inhibitor (ICI) efficacy. Our retrospective data analysis revealed that esophageal cancer (EC) patients taking BB for other non-cancer reasons while receiving chemoradiation had significantly better survival and distant control than those not taking BB. We hypothesized that blocking the effects of adrenergic stress with a commonly used BB, propranolol, will improve response to therapies in EC patients. Methods: This is an open-label, non-randomized, phase 2 study of propranolol combined with pembrolizumab and standard chemotherapy in frontline unresectable/metastatic EGAC. Eligible patients must be treatment-naïve, have adequate organ function, have an ECOG performance status of 0 –1, and be able to swallow and retain oral medication. Patients with Her-2 positive cancer, active autoimmune disease, active HIV, Hepatitis B or C, or a history of non-infectious pneumonitis/interstitial lung disease that requires treatment, are ineligible. Patients who are on BB for various indications are also ineligible. Eligible patients will receive mFOLFOX6 every two weeks in combination with pembrolizumab 400 mg intravenously every six weeks and propranolol 30 mg orally twice daily. The mFOLFOX6 dosing regimen will consist of dl-LV 400 mg/m2 and oxaliplatin 85 mg/m2 followed by bolus 5-FU 400 mg/m2 and a 48-hour infusion of 5-FU 2400 mg/m2. The study will include an initial safety lead-in cohort of six patients. The primary endpoint is the overall response rate (ORR) determined by RECIST 1.1. Secondary endpoints include safety, progression-free survival (PFS), overall survival (OS), and ORR as determined by iRECIST. Correlative studies will assess baseline levels or changes in the levels of biomarkers, like, peripheral T-cell subsets/myeloid-derived suppressor cells (MDSC)/cytokines/ and perceived stress scale PSS/chronotropic effects of exercise with efficacy (ORR, PFS, OS). Assuming a historic ORR of 50% with standard treatment, 37 evaluable pts are needed to show a 20% increase in ORR with our proposed treatment with 80% power at a one-sided significance level of α = 0.1. In stage 1, n1= 23 evaluable pts will be enrolled. If there are 13 or more ORRs, an additional n2= 14 pts will be enrolled in stage 2. If 24 or more ORRs are observed in the total n = 37 evaluable pts, the proposed treatment regimen will be considered promising for further study. The study is currently open to enrollment. Clinical trial information: NCT05651594 .

Peripheral T cell profiling reveals downregulated exhaustion marker and increased diversity in lymphedema post-lymphatic venous anastomosis

Lymphedema is a progressive condition accompanying cellulitis and angiosarcoma, suggesting its association with immune dysfunction. Lymphatic venous anastomosis (LVA) can provide relief from cellulitis and angiosarcoma. However, the immune status of peripheral Tcells during lymphedema and post-LVA remains poorly understood. Using peripheral blood Tcells from lymphedema, post-LVA, and healthy controls (HCs), we compared the profile of Tcell subsets and Tcell receptor (TCR) diversity. PD-1+ Tim-3 + expression was downregulated in post-LVA compared with lymphedema. IFN-γ levels in CD4+PD-1+ Tcells and IL-17A levels in CD4+ Tcells were downregulated in post-LVA compared with lymphedema. TCR diversity was decreased in lymphedema compared with HCs; such TCR skewing was drastically improved in post-LVA. Tcells in lymphedema were associated with exhaustion, inflammation, and diminished diversity, which were relieved post-LVA. The results provide insights into the peripheral Tcell population in lymphedema and highlight the immune modulatory importance of LVA.

A novel conditioning regimen of chidamide, cladribine, gemcitabine, and busulfan in the autologous stem cell transplantation of aggressive T-cell lymphoma.

The prognosis of patients with peripheral T-cell (PTCL) or lymphoblastic T-cell lymphoma (T-LBL) remains poor under current conditioning regimens before receiving autologous stem cell transplantation (ASCT). Patients with PTCL or T-LBL were enrolled to receive ASCT using the conditioning regimen of chidamide, cladribine, gemcitabine, and busulfan (ChiCGB). Positron emission tomography-computed tomography (PET/CT) was used to evaluate the response to ASCT. Overall survival (OS) and progression-free survival (PFS) were employed to assess the patient outcome, and adverse events were used to assess the regimen's safety. The survival curve was estimated via the Kaplan-Meier method. Twenty-five PTCL and 11 T-LBL patients were recruited. The median time to neutrophile and platelet engraftments was 10 days (8-13 days) and 13 days (9-31 days), respectively. The 3-year PFS and OS were 81.3 ± 7.2% and 88.5 ± 5.4% for all patients; 92.0 ± 5.4% and 81.2 ± 8.8% for PTCL patients; and both 81.8 ± 11.6% for T-LBL patients, respectively. The 3-year PFS and OS were both 92.9 ± 4.9% for patients with complete response (CR) but 50.0 ± 17.7% and 75.0 ± 15.3% for patients with non-CR, respectively. Infection was the most common non-hematological toxicity, and all toxicities were mild and controllable. ChiCGB was a potentially effective and well-tolerated conditioning regimen to improve the prognosis of patients with aggressive T-cell lymphoma. Future randomized controlled trials are needed to assess ChiCGB as a conditioning regimen for ASCT.

Pharmacodynamic activity of BMS-986156, a glucocorticoid-induced TNF receptor-related protein agonist, alone or in combination with nivolumab in patients with advanced solid tumors

The success of immune checkpoint inhibitors has revolutionized cancer treatment options and triggered development of new complementary immunotherapeutic strategies, including T-cell co-stimulatory molecules, such as glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR). BMS-986156 is a fully agonistic human immunoglobulin G subclass 1 monoclonal antibody targeting GITR. We recently presented the clinical data for BMS-986156 with or without nivolumab, which demonstrated no compelling evidence of clinical activity in patients with advanced solid tumors. Here, we further report the pharmacodynamic (PD) biomarker data from this open-label, first-in-human, phase I/IIa study of BMS-986156 ± nivolumab in patients with advanced solid tumors (NCT02598960). We analyzed PD changes of circulating immune cell subsets and cytokines in peripheral blood or serum samples collected from a dataset of 292 patients with solid tumors before and during treatment with BMS-986156 ± nivolumab. PD changes in the tumor immune microenvironment were measured by immunohistochemistry and a targeted gene expression panel. BMS-986156+ nivolumab induced a significant increase in peripheral T-cell and natural killer (NK) cell proliferation and activation, accompanied by production of proinflammatory cytokines. However, no significant changes in expression of CD8A, programmed death-ligand 1, tumor necrosis factor receptor superfamily members, or key genes linked with functional parameters of T and NK cells were observed in tumor tissue upon treatment with BMS-986156. Despite the robust evidence of peripheral PD activity of BMS-986156, with or without nivolumab, limited evidence of T- or NK cell activation in the tumor microenvironment was observed. The data therefore explain, at least in part, the lack of clinical activity of BMS-986156 with or without nivolumab in unselected populations of cancer patients.

Sixteen-Week Vitamin D3 Supplementation Increases Peripheral T Cells in Overweight Black Individuals: Post hoc Analysis of a Randomized, Double-Blinded, Placebo-Controlled Trial.

Background: Vitamin D is considered to modulate T-cell function, which has been implicated in the treatment of inflammatory conditions. However, there is limited knowledge on the effects of vitamin D and its influences on circulating T-cell profiles in humans, particularly in overweight Black individuals who are more likely to be vitamin D insufficient (serum 25(OH)D concentrations of ≤20 ng/mL). Thus, this study tested the hypothesis that vitamin D supplementation modulates T-cell composition, which is in a dose-dependent manner. Methods: A 16-week randomized, double-blinded, placebo-controlled trial of vitamin D3 supplementation was undertaken in 70 overweight/obese Black people (mean age = 26 years, 82% female) with 25 hydroxyvitamin D ≤ 20 ng/mL at baseline. Subjects were randomly assigned a supervised monthly oral vitamin D3 equivalent to approximately 600 IU/day (n = 17), 2000 IU/day (n = 18), 4000 IU/day (n = 18), or a placebo (n = 17). Fresh peripheral whole blood was collected and CD3+, CD4+ and CD8+ cell counts and percentages were determined by flow cytometry at baseline and at 16 weeks, among 56 subjects who were included in the analyses. Results: A statistically significant increase in CD3+% in the 2000 IU/day vitamin D3 supplementation group, and increases in CD4+% in the 2000 IU/day and 4000 IU/day vitamin D3 supplementation groups were observed (p-values < 0.05) from the changes in baseline to 16 weeks. Further adjustments for age, sex and BMI showed that 2000 IU/day vitamin D3 supplementation increased in CD3+ count, CD3%, CD4 count, and CD4%, as compared to the placebo group (p-values < 0.05). Moreover, the highest serum 25(OH)D quantile group had the highest CD3% and CD4%. Conclusions: Sixteen-week vitamin D3 supplementation increases peripheral blood T-cell numbers and percentages in overweight/obese Black patients with vitamin D insufficiency. This resulting shift in circulating T-cell composition, particularly the increase in T helper cells (CD4+ cells), suggests that vitamin D supplementation may improve immune function in Black individuals.

Abstract 1984: Baseline T-cell function predicts response to SBRT and immunotherapy in NSCLC

Abstract It is increasingly understood that cancers can be recognized by the immune system and inflammation relates to response. Combining stereotactic radiotherapy (SBRT), to increase release of cancer cell antigens, with an anti-CTLA-4 and PD-L1 inhibitor may lead to increased response rates. No solid biomarker in predicting treatment response is available. We set out to study feasibility of combined durvalumab (anti-PD-L1) and tremelimumab (anti-CTLA-4) with SBRT on the primary tumor. The extend of tumor-infiltrating cells has shown to correlate to improved prognosis using checkpoint inhibitor (CPI) treatment of various cancers. We hypothesized that the peripheral blood T-cell activation status may be a predictor of response, progression free survival (PFS) and overall survival (OS). Methods: Three immunotherapy regimes were combined with SBRT in sequential cohorts as ≥2nd line in CPI naïve patients with NSCLC stage IIIB/IV. One week after the 1st dose of CPI, patients were irradiated on the primary tumor (1x 20Gy on 9cc). The 1st cohort (n=3) received durvalumab, the 2nd and 3rd cohort (both n=6) a combination of durvalumab and tremelimumab, followed by durvalumab monotherapy. Exploratory endpoint was the correlation of response with T-cell function. T-cell function was assessed using flow cytometric analysis of peripheral blood T-cells and compared to simultaneously included healthy controls. In short, whole blood was activated ex vivo using Staphylococcal enterotoxin B (SEB) after which the T-cell activation status was assesses by expression of CD69 and cytokines (IL-2, IFN-γ, TNF-α). Patients were divided in above (responders, R) and below median PFS (non-responders, NR). We compared intracellular cytokine production in both CD4+ and CD8+ T-lymphocytes between both groups and with healthy controls (HC) using the Mann-Whitney U test. Findings: Fifteen patients were included as described above. Median PFS was 2 months, OS 10 months (immature). Baseline characteristics of both groups were comparable. T-cell function was assessed in 14 patients and HC at baseline. Data are shown as median (P-value) for R vs NR below. R had a significant higher percentage activated CD8+ T-cells upon SEB stimulation than NR: CD8+CD69+ 15.8 vs 3.5 (0.008) and IL-2+CD8+CD69+ 8.8 vs 2.9 (0.02). There was a trend in TNF-α+CD8+CD69+ 19.8 vs 8.0 (0.11) and IFN-ɣ+CD8+CD69+ 21.4 vs 5.5 (0.22). These differences were all significant for R compared to HC (p&amp;lt;0.03), but not for NR (P&amp;gt;0.66). Interpretation: With a mild antigen independent stimulus (SEB), the CD8+ T-cells of responders were significantly more activated at baseline compared to non-responders and healthy controls. This suggests an elevated pre-treatment T-cell activation status in patients that responded to the treatment and the possibility to use this as a potential biomarker to predict the response to CPI treatment. Disclosure: This study was sponsored by a research grant from AstraZeneca. Citation Format: Hanneke Kievit, M. Benthe Muntinghe-Wagenaar, Lucie B.M. Hijmering-Kappelle, Birgitta I. Hiddinga, J Fred Ubbels, Robin Wijsman, Bart-Jan Kroesen, Marcel J. van der Leij, A. Rutgers, Harry J. Groen, Huib A. Kerstjens, Anthonie J. van de Wekken, T Jeroen Hiltermann. Baseline T-cell function predicts response to SBRT and immunotherapy in NSCLC [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 1984.

Peripheral blood T-cell receptor repertoire profiling of advanced non-small cell lung cancer patients receiving PD-1/PD-L1 treatment.

e21004 Background: Whether peripheral blood T cell receptor (TCR) repertoire profiling can serve as a biomarker to predict clinical benefit from anti-PD-1/PD-L1 checkpoint immunotherapy is not well understood. Moreover, it is not known which methods for TCR repertoire analysis are most clinically meaningful. To address this, we have performed TCR repertoire analysis of patients with advanced/metastatic non-small cell lung cancer (NSCLC) receiving PD-1/PD-L1 treatment. Methods: We analyzed 29 patients receiving PD-1/PD-L1 monotherapy or combination therapy in any line of treatment, excluding patients with EGFR mutations or Alk alterations. Genomic DNA was extracted from peripheral blood examples, and CDR3 regions of the rearranged TCR beta genes were amplified and sequenced using the immunoSEQ platform (Adaptive Biotechnologies, Seattle, WA). Libraries were sequenced using Illumina HiSeq 2500. TCR clonality, diversity, evenness, and the percentage of high-frequency clones (&gt; 0.1% of the repertoire) were calculated at pre-treatment and post-treatment (1 – 3 months) timepoints. Morisita’s overlap index was calculated for 25 paired pre- and post-treatment samples. Metrics were compared in patients with and without durable clinical response at 6 months using the Mann-Whitney test. Results: There were no statistically significant differences in TCR repertoire clonality, diversity, evenness, or high-frequency clones between patients with and without durable clinical benefit, either at pre-treatment or post-treatment time points (p &gt; 0.05). Conclusions: TCR repertoire metrics including clonality, diversity, evenness, percentage of high-frequency clones, and Morisita’s overlap index do not predict immunotherapy responses in this cohort of advanced/metastatic NSCLC patients receiving PD-1/PD-L1 monotherapy or combination therapy. Limitations of this study include sample size and heterogeneity of the patient cohort. This highlights the need for advanced TCR repertoire metrics, for example, considering shared TCR specificities and clonal identity. In ongoing work, we are defining TCR specificity groups and TCR clones in NSCLC as biomarkers of immunotherapy responsiveness.

Single-cell profiling of immune cells associated with response to neoadjuvant chemoimmunotherapy in IIIA non-small cell lung cancer (NSCLC).

e20584 Background: The combination of immune checkpoint inhibitors (ICIs) with chemotherapy (chemoimmunotherapy) in the neoadjuvant setting have achieved favorable clinical benefits in non-small cell lung cancer (NSCLC), but the underlying molecular mechanism has not been fully elucidated. Methods: To identify factors associated with the clinical outcome, single-cell RNA/TCR sequencing was performed using 10x Genomics on CD45+ immune cells isolated from tumor and multiple immune-relevant tissues and peripheral blood of four treatment-naïve and eight neoadjuvant chemoimmunotherapy treated IIIA NSCLC patients (responders versus non-responders). Bioinformatics analysis was used to screen immune cell factors associated with therapy response in single-cell sequencing profiles, and results were verified at protein level by flow cytometry, multiplex fluorescent immunohistochemistry (mIHC) and ELISA. Results: A total of 186,477 immune cells from 48 samples were acquired after quality-control filtering. The synergistic increase of B cells and CD4+ T cells are associated with positive therapeutic response of neoadjuvant chemoimmunotherapy. B cell class switching to IgG1 and IgG3 plays a critical role in anti-tumor immune response in tumor lesion, and this process is driven by increased IL-21 protein secreted by infiltrated T follicular helper (Tfh) cells after neoadjuvant chemoimmunotherapy. Several critical events lead to the positive clinical outcome during neoadjuvant chemoimmunotherapy, including the diminished activated TNFRSF4+ regulatory T cells (Tregs), increased LAMP3+ dendritic cells (DCs), high pretreatment peripheral blood T-cell diversity, and the expansion of intratumoral CD4+ T clones and peripheral CD8+ T clones. A validation cohort of 26 treatment-naïve and 30 neoadjuvant chemoimmunotherapy treated IIIA NSCLC patients verified these findings. Conclusions: Single-cell profiling of the immune response to neoadjuvant chemoimmunotherapy uncovered several critical factors of anti-tumor immune response and provided insight of potential novel predictive factors and therapeutic targets for improving the efficacy of treatment in NSCLC.

Long-Term GAD-alum Treatment Effect on Different T-Cell Subpopulations in Healthy Children Positive for Multiple Beta Cell Autoantibodies.

Objective The objective of this study was to explore whether recombinant GAD65 conjugated hydroxide (GAD-alum) treatment affected peripheral blood T-cell subpopulations in healthy children with multiple beta cell autoantibodies. Method The Diabetes Prevention–Immune Tolerance 2 (DiAPREV-IT 2) clinical trial enrolled 26 children between 4 and 13 years of age, positive for glutamic acid decarboxylase autoantibody (GADA) and at least one other autoantibody (insulin, insulinoma antigen-2, or zinc transporter 8 autoantibody (IAA, IA-2A, or ZnT8A)) at baseline. The children were randomized to two doses of subcutaneously administered GAD-alum treatment or placebo, 30 days apart. Complete blood count (CBC) and immunophenotyping of T-cell subpopulations by flow cytometry were performed regularly during the 24 months of follow-up posttreatment. Cross-sectional analyses were performed comparing lymphocyte and T-cell subpopulations between GAD-alum and placebo-treated subjects. Results GAD-alum-treated children had lower levels of lymphocytes (109 cells/L) (p = 0.006), T-cells (103 cells/μL) (p = 0.008), T-helper cells (103 cells/μL) (p = 0.014), and cytotoxic T-cells (103 cells/μL) (p = 0.023) compared to the placebo-treated children 18 months from first GAD-alum injection. This difference remained 24 months after the first treatment for lymphocytes (p = 0.027), T-cells (p = 0.022), T-helper cells (p = 0.048), and cytotoxic T-cells (p = 0.018). Conclusion Our findings suggest that levels of total T-cells and T-cell subpopulations declined 18 and 24 months after GAD-alum treatment in healthy children with multiple beta-cell autoantibodies including GADA.

Subtlety of Granulomatous Mycosis Fungoides: A Retrospective Case Series Study and Proposal of Helpful Multimodal Diagnostic Approach With Literature Review.

Granulomatous mycosis fungoides (GMF) harbors a worse prognosis compared with classic MF and remains a significant diagnostic dilemma. We analyzed clinicopathologic, immunophenotypic, and molecular characteristics of GMF to develop a diagnostic algorithm. Our methodology involved a retrospective case series study of patients with GMF from our database between 2014 and 2020. A total of 8 patients with 9 biopsies of GMF were identified. Skin manifestations had variable clinical phenotype. Histologically, all cases demonstrated atypical CD4 + T-cell infiltrate with scant in 50% (n = 4), focal 37.5% (n = 3), and absent 25% (n = 2) epidermotropism. Granuloma formation was seen in 77.8% biopsies (n = 7) with sarcoid-type granulomas in 57.1% (n = 4) and granuloma annulare-like type in 42.9% (n = 3). In 66.7% of biopsies (n = 6), the CD4:CD8 ratio was >4:1 and 66.6% (n = 6) of biopsies showed ≥50% loss of CD7 expression. T-cell receptor gene rearrangement studies performed on biopsy sections were positive in all biopsies (n = 6), whereas peripheral blood T-cell receptor gene rearrangement studies did not identify clonality. In conclusion, GMF has subtle or absent epidermotropism and variable granulomatous reaction; thus, the diagnosis requires a multimodal approach, and our proposed algorithm provides a framework to approach this diagnostic challenge.

What prognostic information does flow cytometry provide in canine B-cell lymphoma?

PICO question&#x0D; In dogs with B-cell lymphoma, does the use of flow cytometry provide useful prognostic information?&#x0D; &#x0D; Clinical bottom line&#x0D; Category of research question&#x0D; Prognosis&#x0D; The number and type of study designs reviewed&#x0D; Twelve papers were critically reviewed. All were cohort studies&#x0D; Strength of evidence&#x0D; Moderate&#x0D; Outcomes reported&#x0D; There are multiple potential prognostic indicators in canine B-cell lymphoma that flow cytometry can be used to evaluate, including lymphoma stage, survival time and time to progression. There is promising evidence for the use of percentage expression of CD25 and Ki67 cellular markers in providing prognostic information in canine B-cell lymphoma and these should be assessed further in clinical practice. Flow cytometry has also been shown to be useful in assessing bone marrow infiltration and providing prognostic information relating to this. There is also evidence for the prognostic value of measuring expression of class II MHC and CTLA-4 cellular markers, peripheral lymphocyte / monocyte ratio, nodal regulatory T-cell populations and the ratio between T and B lymphocytes in extranodal locations. Peripheral regulatory T-cell populations and cellular size were also assessed, however further investigations are required before confirming their prognostic value&#x0D; Conclusion&#x0D; Flow cytometric analysis offers useful measures of prognosis in canine B-cell lymphoma, although further validation is required before introducing their routine use. Percentage expression of CD25 and Ki67 cellular markers from lymph node aspirates of dogs with B-cell lymphoma appear to be promising prognostic indicators in clinical investigations, however this needs to be translated into clinical practice. While there is evidence for the prognostic value of bone marrow infiltration measured flow cytometrically, expression of class II MHC and CTLA-4, peripheral lymphocyte / monocyte ratio, nodal regulatory T-cell populations and the ratio between T and B lymphocytes in extranodal locations, these need to be further investigated before introducing into clinical practice. As new antibodies against cellular targets in dogs become available, it is likely that flow cytometry will become even more useful in providing prognostic information&#x0D; &#x0D; How to apply this evidence in practice&#x0D; The application of evidence into practice should take into account multiple factors, not limited to: individual clinical expertise, patient’s circumstances and owners’ values, country, location or clinic where you work, the individual case in front of you, the availability of therapies and resources.&#x0D; Knowledge Summaries are a resource to help reinforce or inform decision making. They do not override the responsibility or judgement of the practitioner to do what is best for the animal in their care.&#x0D; &#x0D;

The altered HLA-DQ expression in peripheral blood T cells of chronic hepatitis B patients characterizes the function of T cells.

This study aimed to clarify the expression of HLA-DQ and granulysin in peripheral blood T-cell subsets in patients with chronic hepatitis B virus (CHB) and to evaluate their significance in assisting CHB diagnosis and immune status assessment. Peripheral blood from 34 CHB patients, 36 inactive HBsAg carriers and 33healthy controls were collected, and HLA-DQ and granulysin in a series of T-cell subsets were analysed by flow cytometry. The ability to secrete IL-10 and IFN-γ and the functional T-cell subsets were measured in Treg and CD4 cells expressing HLA-DQ or not. Correlation analyses were further conducted between HLA-DQ/granulysin-related subsets and clinical indicators of HBV infection, and ROC curves were built to evaluate diagnosis efficiency of HLA-DQ-related subsets. HLA-DQ+ percentages in circulating CD4T cells were downregulated in CHB patients. The proportions of HLA-DQ+Tfh in CHB were upregulated while HLA-DQ+ percentages in Treg were decreased. In terms of function, the IFN-γ secretion ability of CD4+T cells and IL-10secretion in Tregs were stronger in HLA-DQ+ than HLA-DQ- subsets. HLA-DQ+CD4+T cells and HLA-DQ+Treg were negatively correlated with HBV-DNA, while HLA-DQ+Tfh and Tfc cells were positively correlated with HBV-DNA and ALT. HLA-DQ+Treg/Tfh/Tfc could help to distinguish CHB from inactive HBsAg carriers. HLA-DQ on T cells can characterize the function of T-cell subsets and analysis of HLA-DQ can help to evaluate immune status and assist in diagnosis of CHB.

SUMOylation of PDPK1 Is required to maintain glycolysis-dependent CD4 T-cell homeostasis

The immune system is finely tuned to fight against infections, eradicate neoplasms, and prevent autoimmunity. Protein posttranslational modification (PTM) constitutes a molecular layer of regulation to guarantee the proper intensity of immune response. Herein, we report that UBC9-mediated protein SUMOylation plays an essential role in peripheral CD4 T-cell proliferation, but without a perceptible impact on T-cell polarization. Both conventional T-cell (Tcon) and regulatory T-cell (Treg) maintenance are differentially affected, which was likely caused by a shared deficit in cell glycolytic metabolism. Mechanistically, PDPK1 (3-phosphoinositide-dependent protein-kinase 1) was identified as a novel SUMOylation substrate, which occurred predominantly at lysine 299 (K299) located within the protein-kinase domain. Loss of PDPK1 SUMOylation impeded its autophosphorylation at serine 241 (S241), thereby leading to hypoactivation of downstream mTORC1 signaling coupled with incompetence of cell proliferation. Altogether, our results revealed a novel regulatory mechanism in peripheral CD4 T-cell homeostatic proliferation, which involves SUMOylation regulation of PDPK1–mTORC1 signaling-mediated glycolytic process.

Multiplex Epstein-Barr Virus Genotyping PCR Detects High-Risk Variants in Plasma for Population-Level Nasopharyngeal Carcinoma Screening

<h3>Purpose/Objective(s)</h3> Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is unusually geographically and ethnically restricted. Without screening, most patients present at an advanced stage. Population-level screening in high-risk populations can detect most cases at an early stage, but existing serologic or molecular diagnostics are limited by low positive predictive value secondary to benign EBV reactivation. In contrast to human genome-wide association studies, recent EBV genome-wide association studies have identified polymorphisms with much greater attributable risk for NPC. We hypothesized that a noninvasive molecular diagnostic could detect high-risk EBV haplotypes in plasma to triage patients who screen positive for plasma EBV DNA. <h3>Materials/Methods</h3> We designed a multiplex allele-specific real-time polymerase chain reaction (qPCR) genotyping assay to detect three non-synonymous polymorphisms within the EBV BALF2 gene (162215C > A, 162476T > C, 163364C > T). Two dsDNA gene fragments served as either the high-risk or low-risk variant controls. Supernatant from the EBV-infected B95-8 cell line served as an additional wild-type whole-virus control. Positive clinical specimens sent for routine plasma EBV EBNA-1 testing were collected from patients with a variety of benign and neoplastic EBV-associated disorders. Three sets of conserved primers flanked the polymorphisms, with one allele-specific hydrolysis probe for each of the three high-risk alleles. A fourth allele-specific probe (162215-mt) served as an internal control. The 95% lower limit of detection (LLOD) was assessed in replicates of 20; linearity was assessed from 0.0-6.0 log10 cp/µL. Mutant allele frequencies from 0-100% were assessed by mixing dsDNA controls. <h3>Results</h3> The assay's limit of quantitation and 95% LLOD were 50 and 5 copies per well, respectively. At 100 copies per well, the assay could detect mutant allele frequencies as low as 5%, below the intra-host heterozygosity threshold. Wild-type B95-8 DNA confirmed assay specificity. Forty EBV-positive plasma specimens were genotyped from patients with non-transplant-associated viremia, post-transplant lymphoproliferative disorders, EBV-associated lymphomas (diffuse large-cell, Hodgkin, NK/T-cell, peripheral T-cell), and EBV-positive NPC. High-risk haplotypes were more common among patients with NPC (64% vs. 10%, <i>P</i> = 0.002). Compared to screening with plasma BamHI-W PCR alone, triaging screen-detected EBV DNA with this genotyping assay modestly reduced screening sensitivity (97% to 91%) but could decrease false positives and unnecessary nasoendoscopies by 63% in a modeled high-risk population. <h3>Conclusion</h3> Triaging screen-detected plasma EBV DNA with a multiplex BALF2 genotyping assay has the potential to decrease false positives and resource utilization in high-risk populations screened for NPC. This may be a low-cost and accessible alternative to higher-complexity sequencing assays in low- and middle-income countries.

Interim Results from a Phase 1 Study of ADCT-301 (Camidanlumab Tesirine) Show Promising Activity of a Novel Pyrrolobenzodiazepine-Based Antibody Drug Conjugate in Relapsed/Refractory Hodgkin/Non-Hodgkin Lymphoma

Interim Results from a Phase 1 Study of ADCT-301 (Camidanlumab Tesirine) Show Promising Activity of a Novel Pyrrolobenzodiazepine-Based Antibody Drug Conjugate in Relapsed/Refractory Hodgkin/Non-Hodgkin Lymphoma

Abstract 3189: Dynamics characteristics and prognostic significance of profiling the peripheral blood T-cell receptor repertoire during immune checkpoint blockade in cancer

Abstract Introduction: Immune checkpoint blockade (ICB) targeted to CTLA-4, PD-1, and PD-L1 can promote anti-tumor T cell immunity and clinical responses. Tracking the dynamic changes of the T Cell Repertoire (TCR) can reflect the evolution of the immune system in tumor immunotherapy patients, predict the immune response to ICB, and provide high-value information for clinical monitoring of immune function status of cancer patients to make effective clinical decisions. The purpose of this study was to explore the correlation between the peripheral blood TCR repertoire dynamic changes and the immune response of patients with anti-PD-1/PD-L1, and to accurately guide the individualized treatment of immunotherapy. Methods: To comprehensively profile the TCR repertoire, high-throughput sequencing was used to identify hypervariable rearrangements of complementarity determining region 3 (CDR3) of the TCR β chain in peripheral blood samples from 31 advanced solid tumors (most were non-small cell lung cancer) patients treated with immune checkpoint blockade. The blood samples were obtained at baseline of treatment with PD-1/PD-L1 antibody, one to two additional blood samples were obtained during treatment to assess dynamic changes. Shannon-Wiener index was used to identify the diversity of TCR receptor. D50 index was used to account for diversity from the cumulative 50% of the total CDR3s counted in the sample. Results:Shannon-Wiener index at baseline was higher in durable clinical benefit (CB) group than non-clinical benefit (NCB) group (p value = 0.0040) treated with anti-PD1/PD-L1, and significantly increased in CB group ( p value = 0.031). There is a significant correlation between D50 index and clinical benefit during treatment (p value = 0.025). The higher the D50 index, the more likely it is to have durable clinical benefit. Significant difference in progression-free survival (PFS) rate was identified between the D50 high and D50 low groups (p value = 0.037). We validated this result in an independent cohort of NSCLC patients treated with anti-PD1/PD-L1(N=12). Conclusions:This study revealed the dynamic changes of peripheral blood T cell receptor beta-chain repertoire in patients with solid tumors before and during anti-PD1/PD-L1 treatment. This study found that baseline TCR diversity was associated with clinical efficacy. Patients with durable clinical benefit had an increased overall diversity of TCR. The D50 index of the sample during treatment can reflect whether the patient could benefit from anti-PD1/PD-L1 treatment or not, and was a significant prognostic factor for progression-free (PFS). These analyses provide that the peripheral blood TCR repertoire parameters may be useful for prognosis prediction and rapid determination of therapeutic outcomes for patients treated with immune checkpoint inhibitors. Citation Format: Ye Li, Jiaqian Wang, Shengjie Sun, Liangliang Wu, YunXiao Zhang, Guoqing Zhang, Xiaoting Li, Shunchang Jiao. Dynamics characteristics and prognostic significance of profiling the peripheral blood T-cell receptor repertoire during immune checkpoint blockade in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3189.