Peripheral Blood T-cell Research Articles

T-cell lymphopenia in frequent volunteer platelet donors.

In the United States, more than 2 000 000 apheresis platelet units are collected annually from volunteer donors. Platelet donors in the United States and elsewhere are permitted to donate up to 24 times per year. Recently, frequent apheresis platelet donation has been associated with severe T-cell lymphopenia. Several frequent platelet donors have been found to have peripheral blood CD4+ T-cell counts below 200 cells/µL, the threshold for AIDS in HIV-positive individuals. Independent risk factors for plateletpheresis-associated lymphopenia include lifetime donations, age, and donations on the Trima Accel instrument (Terumo BCT), which uses a leukoreduction system (LRS) chamber to trap white blood cells. Less often, severe lymphopenia can occur in donors collected on the Fenwal Amicus instrument (Fresenius Kabi), which has no LRS. For Trima Accel donors, lymphopenia can be partially mitigated by performing a plasma rinseback step at the end of collection. To date, there is no definitive evidence that plateletpheresis-associated lymphopenia is harmful. In a study of frequent platelet donors with lymphopenia who were administered COVID-19 messenger RNA vaccines, immune responses were normal. The homeostatic mechanisms responsible for maintaining a normal peripheral blood T-cell count remain obscure, as do the causal mechanisms underlying plateletpheresis-associated lymphopenia.

Investigating the Immune Basis of Green Tea Extract Induced Liver Injury in Healthy Donors Expressing HLA-B*35:01.

Epigallocatechin-3-O-gallate (EGCG) is the major component of green tea extract, commonly found in dietary supplements, and has been associated with immune-mediated liver injury. The purpose of this study was to investigate the immunogenicity of EGCG in healthy donors expressing HLA-B*35:01, and characterize EGCG responsive T-cell clones. We have shown that EGCG can prime peripheral blood mononuclear cells and T-cells from donors with and without the HLA-B*35:01 allele. T-cell clones were CD4+ve and capable of secreting Th1, Th2, and cytolytic molecules. These data demonstrate that EGCG can activate T-cells in vitro, suggesting a significant role in the pathogenesis of green tea extract induced liver injury.

АНАЛИЗ РАЗНООБРАЗИЯ Vβ-СЕГМЕНТОВ Т-КЛЕТОЧНОГО РЕЦЕПТОРА У ЖИТЕЛЕЙ ПРИБРЕЖНЫХ СЁЛ РЕКИ ТЕЧИ, ПОДВЕРГШИХСЯ ХРОНИЧЕСКОМУ РАДИАЦИОННОМУ ВОЗДЕЙСТВИЮ, В ОТДАЛЕННОМ ПЕРИОДЕ

Purpose: Study the repertoire of the T-cell receptor in persons chronically exposed to radiation in the long-term period. Material and methods: The study involved 48 people, who were divided into two groups: a group of exposed persons – 31 individuals with the average accumulated dose to red bone marrow (RBM) of 981±130 mGy, and a comparison group – 17 individuals, the average accumulated dose to RBM was 25.3±5.91 mGy. The study groups did not differ significantly in age, gender and ethnicity. The repertoire of Vβ-segments of the T-cell receptor of the peripheral blood T-lymphocytes of exposed persons was analyzed by flow cytometry method. 24 Vβ-segments of the T-cell receptor were studied. Statistical processing of the obtained data was carried out using the Wilcoxon signed-rank test, and a direct description of Vβ-segment repertoire of the T-cell receptor was performed using the Lorenz curve and the Gini-TCR index. Results: The study revealed a statistically significant increase in the number of Vβ3 and Vβ5.2 T-cell receptor segments in exposed individuals relative to the comparison group (p=0.03 and p=0.003, respectively). It was also shown that the distribution of the Vβ-segments of the T-cell receptor is uneven in both study groups. However, there was no significant difference between the repertoires of the T-cell receptor of the studied groups by the Gini-TCR index (p=0.14).

Evaluating Tumor-Intrinsic and Patient-Specific Mechanisms of Resistance to Teclistamab in Anti-BCMA Exposed and Naïve Multiple Myeloma

Background: Teclistamab (Tec) is a CD3 x BCMA bispecific antibody granted accelerated FDA approval for treating relapsed/refractory (RR) multiple myeloma (MM) based off the results of the MajesTEC-1 trial (Usmani S, et al. Lancet 2021, Moreau P, et al. N Engl J Med 2022). However, patients with prior exposure to anti-BCMA therapies were excluded from this trial and there is no published data evaluating Tec efficacy in this setting. Additionally, little is known about tumor-intrinsic and patient-specific immunologic predictors of response to Tec. Methods: We performed an IRB-approved analysis of clinical outcomes for 52 commercially treated Tec patients treated as of 07/28/2023 at our center. Results were correlated to pre-treatment BCMA expression measured by immunohistochemistry (IHC). We also profiled peripheral blood T-cells from pre-treatment peripheral blood mononuclear cell (PBMC) samples from a sub cohort of patients via high-dimensional spectral cytometry using a 37-color panel including lineage, exhaustion, and activation markers. Results: Our commercial cohort was older (median age 70 vs 64), more heavily pretreated (median prior lines of therapy 7 vs 5) and had higher incidence of both high-risk cytogenetic abnormalities (33% vs 26%) and extramedullary disease (35% vs 17%) than the MajesTEC-1 population. Additionally, 52% had prior exposure to anti-BCMA therapies, including belantamab mafodotin (31%), anti-BCMA CAR T-cell therapies (37%), and anti-BCMA bispecific antibody therapies (4%), with some having exposure to multiple prior anti-BCMA therapies (17%). Among response evaluable patients (90%), with a median follow-up time of 100 days, the overall response rate (ORR) to Tec was 64% (36% ≥VGPR). Progression free survival (PFS) in patients without prior BCMA therapy was higher than in the anti-BCMA exposed population (median PFS NR vs 102 days, p = 0.032, Figure Panel A). However, the anti-BCMA therapy exposed population was more heavily pretreated (8 vs 5 median prior lines of therapy) and had more patients with high-risk cytogenetic abnormalities (46% vs 19%) and extramedullary disease (42% vs 27%) than the anti-BCMA therapy naïve cohort. No major differences in BCMA expression were noted between responding and non-responding patients or between anti-BCMA exposed and anti-BCMA naïve patients. However, two patients had absent BCMA expression following prior treatment with other BCMA targeting agents and did not respond to Tec. Cytokine release syndrome (CRS) during step-up dosing was strongly associated with Tec efficacy (median PFS NR vs 27 days, p <0.0001), with a 100% ORR for patients with CRS. Immune profiling experiments (Figure Panel B) revealed that Tec responders had peripheral blood enriched for both CD8 + effector memory T-cells (TEM, >6-fold increase, p = 0.0499) and CD8 + effector memory T-cells re-expressing CD45RA (TEMRA, >5-fold increase, p = 0.012) when compared to Tec non-responders. Tec non-responders had peripheral blood enriched for TIGIT + regulatory T-cells (Tregs, 3-fold increase, p = 0.03) when compared to Tec responders. No differences in PD-1, CTLA-4, LAG-3 or TIM-3 expression were observed in Tec responders vs non-responders. Patients with recent relapse from non-BCMA targeting bispecific antibodies (including products targeting FcRH5 or GPRC5D) had peripheral blood enriched for γδ T-cells with high TIGIT expression. Conclusion: Tec is an effective therapy in RRMM with a slightly lower efficacy observed in patients with prior anti-BCMA therapy exposure. PFS reductions among these anti-BCMA exposed patients may be partly due to independent disease associated risk factors. A pre-Tec peripheral blood T-cell population enriched with highly cytotoxic effector T-cells was associated with response to therapy, while suppressive TIGIT +Tregs were associated with nonresponse, suggesting a potential therapeutic role for TIGIT blockade or CD25 + cell depletion to enhance the therapeutic efficacy of bispecific antibodies. Clinical and translational data from additional patients will be presented at the meeting.

No Discernable Dysfunction of Peripheral T-Cells and NK-Cells Despite Distinct Phenotypes in MYC Rearranged Versus Non Rearranged B-Cell Lymphoma Patients

Background: Patients with diffuse large B-cell lymphoma/high-grade B-cell lymphoma with MYC and BCL2 rearrangements (DLBCL/HGBL- MYC/BCL2, hereafter HGBL- MYC/BCL2) respond poorly to standard immunochemotherapy treatment compared with DLBCL not otherwise specified (LBCL-NOS) patients without a MYC rearrangement. This suggests a negative interaction between MYC and the immune system. As novel T-cell and NK-cell based immunotherapeutic approaches emerge as potential treatment options for aggressive lymphomas, we conducted comprehensive immune profiling to compare peripheral blood T-cells and NK-cells of HGBL- MYC/BCL2 with LBCL NOS patients. Methods: Peripheral blood mononuclear cells (PBMCs) were prospectively collected from HGBL- MYC/BCL2 patients (registered in the HOVON-152 trial, NCT03620578), LBCL-NOS (registered in the HOVON-902 cohort, NCT04139252) and age-matched healthy donors (HDs). Flow-cytometry-based immune profiling of T-cells and NK-cells was performed on baseline samples available from n=66 HGBL- MYC/BCL2 patients (median age 62 years), n=67 LBCL NOS patients (median age 69 years) and n=17 HDs (median age 64 years). The flow cytometry data were computationally analyzed using FlowSOM and UMAP. T-cells were also functionally assessed for intracellular cytokine secretion (ICS) after stimulation with PMA/ionomycin (4h) and proliferation after stimulation with CD3/CD28 beads (5 days) using CellTrace Violet. NK-cells were assessed for cytotoxicity against K562 and degranulation (CD107a/b upregulation) after 4h. Mann-Whitney's U tests were used to compare HGBL-MYC/BCL2 patients with LBCL-NOS patients (primary comparison), and for pairwise comparisons of the lymphoma groups with HDs. To correct for baseline characteristics (age), linear regression analysis was performed. Results: CD4 + helper T-cells did not significantly between the lymphoma subgroups, but were decreased in LBCL-NOS patients as compared to HDs. Frequencies of cytotoxic CD8 + and CD4 +CD8 + T-cells did not significantly between the lymphoma subgroups and were similar as in HDs. Regulatory T-cell (Treg) frequencies of HGBL- MYC/BCL2 patients were similar to that of HDs, but were decreased in LBCL NOS patients. The frequency of CD4 -CD8 - T-cells was increased in LBCL NOS patients. Previously activated (HLA-DR +) CD4 + and CD8 + T-cells, as well as recently activated CD25 +, CD38 + or CD127 + CD4 + T-cells (Figure 1A) or activated/exhausted (PD-1 +) CD4 + T-cells and PD1 + Tregs were increased in both lymphoma subtypes (Figure 1B), but only HGBL- MYC/BCL2 displayed an increase in PD1 + CD8 + T-cells (Figure 1B). Conversely, recently activated CD8 + T-cells were decreased exclusively in LBCL NOS. HGBL- MYC/BCL2 patients and HDs had similar percentages of T-cells with a senescent phenotype (CD28 -CD57 +KLRG1 +), but the frequency of T-cells with this senescent phenotype was increased in LBCL NOS patients (Figure 1A), also after adjustment for age. Despite all these phenotypic differences, functional in vitro assays detected no differences in proliferation or cytokine secretion of peripheral T-cells from HGBL- MYC/BCL2 patients, LBCL NOS patients and HDs. In the NK-cell compartment, both lymphoma subtypes contained lower frequencies of CD56 brightTim3 +, CD56 bright/dimDNAM-1 + NK-cells, but LBCL NOS patients expressed a higher amount of NK-cells expressing inhibitory receptor NKG2A compared with HDs. NK-cells of HGBL- MYC/BCL2 patients, LBCL NOS patients and HDs displayed similar cytotoxic activity, suggesting no apparent functional impairment. Conclusions: HGBL- MYC/BCL2 patients have a distinct peripheral T-cell and NK-cell phenotype from LBCL NOS patients without a MYC rearrangement, but show no apparent functional deficiencies in terms of proliferation, cytokine secretion or cytotoxic activity. These results are not contradicting the application of T-cell and NK-cell based immunotherapeutic approaches for patients with aggressive B-cell lymphoma. Simultaneously, it emphasize the need to investigate the potential immunomodulatory impact of lymphoma intrinsic MYC in the tumor microenvironment.

Effects of different anesthesia methods on perioperative immune function and long-term regression of patients undergoing thoracoscopic radical esophagectomy for esophageal cancer

ObjectiveThis study investigated the effects of different anesthetic methods on perioperative immune function and long-term regression of patients undergoing thoracoscopic radical surgery for esophageal cancer. MethodsData from 147 patients undergoing thoracoscopic radical surgery for esophageal cancer in our hospital from January 2017 to June 2019 were selected for retrospective analysis. Patients were divided into control (intravenous anesthesia combined with an epidural block, n = 83) and observation (intravenous anesthesia combined with a thoracic paravertebral nerve block, n = 64) groups. The use of relevant drugs during the surgery, indexes of the peripheral blood T-lymphocyte subpopulation at different time points (before induction of anesthesia, at the completion of surgery, 24 h postoperatively, and 48 h postoperatively), and 3-year postoperative survival were compared between the two groups. ResultsData on propofol and remifentanil use during surgery were not significantly different (P > 0.05) between the two groups. The proportion of vasoactive drug use was lower in the observation group than in the control group (P < 0.05). Differences in data for CD3+, CD4+, and CD4+/CD8+ cells between time points were significant (F = 13.256, 20.307, 18.035, P < 0.05), while those between the two groups were not (F = 1.005, 1.135, 1.204, P > 0.05). There was no interaction between data processing factors and time between the control and observation groups in CD3+, CD4+, and CD4+/CD8+ cells (F = 0.358, 0.778, 1.107, P > 0.05). The median overall survival time was 21.988 (95 % confidence interval: 18.436–25.541) and 26.978 (95 % confidence interval: 20.124–33.833) months in the control and observation groups, respectively, with a significant difference between the two groups (Log-rank χ2 = 3.925, P < 0.05). ConclusionsBoth anesthetic methods achieve satisfactory anesthetic results and reduce postoperative stress and immunosuppression. However intravenous anesthesia combined with an epidural block can increase the incidence of intraoperative hypotension in the patients, thoracic paravertebral nerve block combined with general anesthesia is more favorable for maintaining intraoperative blood pressure and heart rate stability, as well as for postoperative survival.

Prediction of frequent acute respiratory infections in the first year of life in children with cerebral ischemia, survivors of cytomegalovirus infection in the neonatal period

Objective: to develop prognostic criteria for frequent respiratory diseases in the first year of life in children with cerebral ischemia who had a cytomegalovirus infection in the neonatal period. Research methods: 73 children of the first year of life with cerebral ischemia, who underwent cytomegalovirus infection in the neonatal period, were deployed. All observed patients at the age of three months underwent a study of the population composition of peripheral blood T-lymphocytes using flow cytometry for the expression of membrane markers, taking into account the results on a Beckman Coulter Epics XL II laser flow cytometer. Typing of lymphocytes was carried out using monoclonal antibodies to differentiation clusters CD3+, CD3+CD69+, CD3+CD71+, CD3+CD95+ from Immunotech (France). The observation groups consisted of 30 children (41.1%) with frequent acute respiratory infections (4—5 episodes per year) in the first year of life and 43 people (58.9%) — children with no acute respiratory infection’sepisodes in the first-year life. Results. From the set of studied T-lymphocytes, statistically significant for the prognosis of frequent acute respiratory infections in the first year of life in children with cerebral ischemia who underwent cytomegalovirus infection in the neonatal period were found: CD3+ CD71+, CD3+ CD95+. It was revealed that in children with a reduced level of CD3+ CD71+ and an increased level of CD3+ CD95+ in blood serum at the age of 3 months, frequent acute respiratory infections occurred in the first year of life.

IMMU-46. MONITORING T-CELL DYNAMICS AND CHECKPOINT EXPRESSION IN THE IMMUNE MICROENVIRONMENT OF PEDIATRIC HIGH GRADE BRAIN TUMORS: THE MIMIC PROGRAM

Abstract BACKGROUND Pediatric high grade brain tumors (pHGBT) pose a significant challenge due to limited treatment options and poor prognosis. Understanding the immune landscape, in particular T cell profiles and functionality, is of high interest as numerous immunotherapeutic approaches focus on or utilize T-cells. This study aimed to characterize T-cell phenotypes and their potential for immunotherapeutic interventions in tumor fragments (obtained from cavitron ultrasonic surgical aspiration (CUSA)) and tumor tissue (biopsy), in comparison with peripheral blood, collected from pHGBT patients. METHODS Spectral flow cytometry was employed to characterize the presence of T-cell subsets and their functional phenotype. RESULTS T cells were detected in all materials with similar CD8/CD4 ratios. At diagnosis, T-cells in the tumor microenvironment exhibited significantly increased expression of PD1, LAG3 and TIM3 checkpoint receptors compared to peripheral blood. This difference was more pronounced in biopsy samples compared to CUSA fragments. T-cell composition and phenotype in peripheral blood were similar to healthy adult controls at diagnosis. However, during therapy, the T-cell compartment showed a shift towards a higher CD8/CD4 ratio, accompanied by increased activation markers (CD25, CD69) and checkpoint receptor expression . Notably, CD8+ T-cells in both peripheral blood and tumor microenvironment showed high production of Granzyme B. CONCLUSION Our findings demonstrate that despite the high expression of checkpoint receptors on CD8+ T-cells, these cells retain the capability to produce significant amounts of Granzyme B, indicating their potential for cytotoxic activity. Furthermore, variations in checkpoint receptor expression on T-cells were observed when comparing CUSA fragments to biopsy material. Functional assays will further validate these findings and elucidate the functional status of these cells. Understanding the function of T-cells within the tumor microenvironment is essential for designing novel combination therapies to enhance T-cell cytotoxicity against pHGBT.

Elucidating the Heterogeneity of Immunotherapy Response and Immune-Related Toxicities by Longitudinal ctDNA and Immune Cell Compartment Tracking in Lung Cancer.

Although immunotherapy is the mainstay of therapy for advanced non-small cell lung cancer (NSCLC), robust biomarkers of clinical response are lacking. The heterogeneity of clinical responses together with the limited value of radiographic response assessments to timely and accurately predict therapeutic effect-especially in the setting of stable disease-calls for the development of molecularly informed real-time minimally invasive approaches. In addition to capturing tumor regression, liquid biopsies may be informative in capturing immune-related adverse events (irAE). We investigated longitudinal changes in circulating tumor DNA (ctDNA) in patients with metastatic NSCLC who received immunotherapy-based regimens. Using ctDNA targeted error-correction sequencing together with matched sequencing of white blood cells and tumor tissue, we tracked serial changes in cell-free tumor load (cfTL) and determined molecular response. Peripheral T-cell repertoire dynamics were serially assessed and evaluated together with plasma protein expression profiles. Molecular response, defined as complete clearance of cfTL, was significantly associated with progression-free (log-rank P = 0.0003) and overall survival (log-rank P = 0.01) and was particularly informative in capturing differential survival outcomes among patients with radiographically stable disease. For patients who developed irAEs, on-treatment peripheral blood T-cell repertoire reshaping, assessed by significant T-cell receptor (TCR) clonotypic expansions and regressions, was identified on average 5 months prior to clinical diagnosis of an irAE. Molecular responses assist with the interpretation of heterogeneous clinical responses, especially for patients with stable disease. Our complementary assessment of the peripheral tumor and immune compartments provides an approach for monitoring of clinical benefits and irAEs during immunotherapy.

Molecular Biomarkers of Aging to Predict Clinical Frailty in Newly Diagnosed Hematologic Malignancy

Introduction: Older adults with hematologic malignancy (HM) are a growing demographic with wide-ranging therapeutic options. With aging, patient health and treatment decision-making are heterogenous. Treatment decisions are impacted by chronological age, performance status, and comorbidities. An approach to treating older adults based on objective measures of health may better personalize therapy by focusing on physiologic parameters. Here we examined the relationship of a blood-based aging biomarker, p16 INK4a (p16), with clinical frailty. Methods: Newly diagnosed patients with HM were enrolled in a prospective study capturing fitness measures and peripheral blood T lymphocyte (PBTL) p16 levels pre-treatment. Clinical phenotypes of fitness included 11 measures: ECOG performance status, Patient-Reported Outcome Measurement Information System (PROMIS) Global Health Scale Short Form v1, Older Americans Resources and Services (OARS) instrumental activities of daily living (IADL), Medical Outcomes Study (MOS) physical function, patient-reported Karnofsky Performance Status (KPS), social activity and social support (MOS) scores, Mental Health Index (MHI), and cognition (Blessed Orientation-Memory-Concentration). Physical function was measured using the Short Physical Performance Battery (SPPB). PBTL p16 was measured using the OSU_Senescence Nanostring CodeSet, which includes 74 additional T cell markers and five controls. PBTL were also examined for DNA methylation. PBTL were extracted, bisulfite converted, and methylation was quantified using the Illumina 850K EPIC methylation array at the TruDiagnostic core laboratory. Raw data was processed using the minfi pipeline, followed by normalization, batch correction, and KNN-based imputation using the GMQN and impute R packages. Principal-component (PC) PhenoAge measures were calculated using the processed methylation values. Results: Patients newly diagnosed with HM (n=33) (acute leukemia n=13, 39.5%, plasma cell dyscrasia n=13, 39.5%, lymphoma n=4, 12.1%, chronic lymphocytic leukemia=3, 9.1%) and complete p16 analysis with geriatric deficits were included. The mean p16 score was 32.6 (range 1.7-259.7). We quantified a composite deficit index defined as a frailty score higher than the third quartile using the aggregate score of the above fitness measures (range 0 to 11). Using a threshold for p16 ≥31.9, the model had 80% sensitivity and 79% specificity to identify frailty. The positive predictive value was 0.40, the negative predictive value was 0.96, and the accuracy was 0.76. The area under the ROC curve (AUC) for frailty versus p16 was 0.725. PBTL p16 was also explored in relation to other biomarkers of interest including epigenetic age (n=50). PBTL p16 correlated with PhenoAge r=0.38, p=0.0067. Conclusion: Aging biomarkers provide a novel means to quantify physiologic age in newly diagnosed HM. PBTL p16 levels showed a significant relationship with a composite metric of clinical fitness and accelerated aging in patients with different HM. PBTL p16 was both a sensitive and specific blood-based biomarker to identify clinical frailty in this preliminary analysis.

Preclinical and Translational Biomarker Analyses to Inform Clinical Development of Mezigdomide (CC-92480) in Combination with Dexamethasone and Daratumumab in Multiple Myeloma

Introduction: Mezigdomide (MEZI) is a novel, oral cereblon (CRBN) E3 ligase modulator (CELMoD) that induces rapid and potent degradation of lkaros and Aiolos. Reduction of these transcription factors results in direct tumoricidal and immunomodulatory effects in multiple myeloma (MM). MEZI showed promising efficacy and safety in combination with dexamethasone in the phase 1/2 CC-92480-MM-001 trial (NCT03374085) in Relapsed/Refractory MM. MEZI is currently being investigated in combination with daratumumab and dexamethasone (MEZI + DARA + DEX or MEZI-Dd) in the phase 1/2 CC-92480-MM-002 trial (NCT03989414). In MEZI-Dd cohort, MEZI is administered at three different schedules (B1 for 21/28 days; B2 for 14/21 days; B3 for 7/14 days), and at two different doses (0.3mg; 0.6mg). Here we report preclinical data and pharmacodynamic (PD) biomarker analyses from blood and bone marrow samples collected in CC-92480-MM-002 to support dose and schedule optimization of MEZI-Dd. Methods:Preclinical studies on the pro-apoptotic effects of MEZI on daratumumab-mediated complement-dependent cytotoxicity (CDC) and peripheral blood mononuclear cell-based antibody-dependent cell-mediated cytotoxicity (ADCC) were performed in MM cell lines treated with MEZI-D in comparison to single-agent MEZI or DARA. Clinical PD biomarker analyses included peripheral blood samples collected on treatment Cycle (C)1 Day (D)1 and mid C1-C3 for the expression of Aiolos in T-cells and immunomodulation by flow cytometry. Bone marrow samples were collected for immunohistochemistry at screening and mid C2. Serum free light chain (sFLC) and soluble B-cell maturation antigen (BCMA) were analyzed as biomarkers for tumor burden from C1-C6. Results: Preclinically, combinations of MEZI-D induced synergistic anti-tumor activity in MOLP-8 cells. The MEZI-D combination showed greater anti-MM activities across the dosing gradient than either single-agent DARA (~2-4.5 fold) or MEZI (~1.7-2.4 fold) in a CDC assay. MEZI-D also induced significantly more cell apoptosis than POM-D (p&amp;lt;0.05) in an ADCC assay. In the clinic, MEZI-Dd was sufficient to induce rapid Aiolos degradation in the peripheral blood T-cells for doses as low as 0.3mg MEZI, but 0.6mg MEZI was necessary to result in &amp;gt;50% substrate degradation, suggesting 0.6mg MEZI induced deeper substrate degradation. While 0.3mg MEZI-Dd decreased total CD56+/CD16+ NK-cell counts (Median -83.8%) by mid C1 via immunophenotyping analyses, the combination increased %Ki67+ proliferative NK-cell (Median 251.9%). 0.3mg MEZI-Dd also showed consistent trends of schedule-dependent PD immune effects by mid C3, including increased %Ki67+ proliferative CD3+ T-cell (Median B1: 113.1%, B2: 365.1%, B3: 37.4%), decreased CD45RA+CCR7+ naïve CD4+ T-cell (Median B1: -82.0%, B2: -62.3%, B3: -43.9%), increased CD45RO+CCR7- effector memory CD4+ T-cell (Median B1: 142.3%, B2: 82.9%, B3: 13.9%), and increased HLA-DR expressing CD4+ T-cell activation (Median B1: 149.3%, B2: 107.9%, B3: 17.1%). Thus, greater PD immune effects were observed with schedules of 21/28 and 14/21 days, compared to the 7/14 schedule. At 0.6mg MEZI-Dd, greater median changes of PD immune effects were induced with the 7/14 schedule, compared to 0.3mg MEZI-Dd. Conversely, at continuous dosing schedules longer than 7 days, MEZI-Dd PD immune effects of 0.6mg MEZI were similar to 0.3mg MEZI. In addition, greater reduction of tumor burden was observed at 0.6mg MEZI-Dd, compared to 0.3mg MEZI-Dd across different schedules, as assessed by sFLC and soluble BCMA analyses. Conclusions: Our data show dose- and schedule-dependent PD effects of MEZI-Dd. PD analyses at 0.3mg MEZI suggest at least two weeks of continuous dosing may be needed for greater PD activities in combination with daratumumab, compared to one week of dosing. For continuous dosing of one week, a higher dose at 0.6mg MEZI may be needed to achieve greater PD activities. Further investigation of doses at 0.6mg and 1mg MEZI is underway to determine the optimal dose. Moreover, MEZI induction of NK-cell proliferation and/or activation may provide mechanistic rationale for possible MEZI-D synergy in the clinic.

Novel Peptide-Based Plectin-1 CAR T-Cells Target Specifically Pancreatic Cancer Cells

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating cancers, and therapies to treat this malignancy have not evolved rapidly to fulfill the need of affected patients. PDAC, like many solid malignancies, is considered a “cold immunological tumor” where the lack of targetable tumor-associated antigens has hampered the development of successful immunotherapies. Despite newer treatment approaches, PDAC patients with advanced disease have a dismal prognosis. Therefore, there is an urgent need to develop new therapies. Plectin-1 is a high molecular weight protein (∼500 kDa) that participates in cytoskeleton network organization linking intermediate filaments to microtubules and microfilaments. Plectin-1 is aberrantly expressed in the PDAC cellular membrane, contributes to PDAC pathogenesis, and confers a poor prognosis. Plectin-1-targeting peptides (PTP) in mice and humans have been successfully explored for their use in PDAC imaging and targeted payload delivery (small molecules, radioisotopes, and chemotherapy). We sought to take advantage of the specific binding of PTP and develop a peptide-based plectin-1 chimeric antigen receptor (CAR), and test T-cell transduced with this construct in vitro against PDAC cell lines. Methods: Using in silico tools, we optimized different pepto-CAR constructs and selected one that showed the highest levels of cytotoxicity against plectin-1-expressing PDAC cell lines. A myc tag was incorporated in the hinge region for flow cytometry or western blot detection. The intracellular signaling domain of these CAR sequences included 4-1BB and the zeta chain of the CD3 molecule ( Figure 1A). Plasmids containing the CAR sequence were packaged on lentivirus particles using HEK293T cells. Purified plectin-1 CAR lentiviruses were used to transduce T-cells derived from human peripheral blood T-cells obtained and purified from healthy donor whole blood. The expanded CAR T-cells were used for cytotoxic experiments against different cell lines. Results: Expression of plectin-1 CAR was confirmed by flow cytometry using an anti-myc tag antibody ( Figure 1B). Plectin-1 expression was analyzed in different pancreatic cancer cell lines, and the BxPC-3 cells showed the highest expression level ( Figure 1B). Using a luciferase-based assay, we plated 50,000 BxPC-3 cells per well in duplicates with different effector-to-target (E:T) ratios. After 24 hours, luciferin was added, and the plate was read using a luminometer. As a negative control for effector cells, we use T-cells without a CAR ( Figure 1B), and for the target cells, we use the plectin-1 negative lung cancer cell line A549 ( Figure 1B). Plectin-1 CAR T-cells effectively killed BxPC-3 cells at different E:T ratios in a specific manner compared with controls using unmodified T-cells. Furthermore, no cytotoxicity was observed against plectin-1 negative A549 cells ( Figure 1A). Conclusion: Our results show that a novel peptide-based CAR construct design against plectin-1 can induce specific cytotoxicity on plectin-1 expressing PDAC cell line BxPC-3. Furthermore, these data demonstrate the feasibility of using a CAR with a peptide-based antigen binding domain instead of the traditional antibody-derived scFv. This novel peptide-based CAR design offers alternatives for cellular therapy bioengineering. Moreover, our results can significantly impact immune-oncology therapeutic applications, particularly in challenging diseases like pancreatic cancer. Figure 1 . A) In the above section, the Plectin-1 CAR construct is depicted. Cytotoxicity of unmodified T-cells and Plectin-1 CAR T-cells against BxPC-3 and A549 is shown below. B) Expression of Plectin-1 CAR in unmodified T-cells and Plectin-1 CAR T-cells is shown on the left. The expression of plectin-1 antigen on the surface of BxPC-3 cells and A549 cells is shown on the right. PTP: plectin-1 targeting peptide.

Distinct Patterns of CD4+ and CD8+ T-Cell Clonal Expansion Enable Broad Clinical Responses to Pembrolizumab + GVD in Patients with Relapsed Hodgkin Lymphoma

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by immune dysregulation, with recurrent amplifications of the 9p24 locus encoding several genes including CD274 (PD-L1), PDCD1LG2 (PD-L2), and JAK2, frequent genetic and epigenetic downregulation of major histocompatibility complex (MHC)-I and (MHC)-II, and a tumor microenvironment (TME) defined by T-cell infiltration and high rates of PD-L1 expression. PD-1 blockade is highly active both as single agent and in combination with chemotherapeutic agents in relapsed/refractory (RR) cHL. However, the mechanism by which PD-1 blockade promotes clinical responses in combination with chemotherapy, and whether the mechanism of action varies based on tumor or TME-specific expression of either MHC or PD-L1 remains unclear. Methods: We previously conducted a phase II study in patients with relapsed or refractory cHL evaluating pembrolizumab in combination with gemcitabine, vinorelbine and liposomal doxorubicin (P-GVD) before proceeding to high-dose therapy and autologous hematopoietic cell transplantation (AHCT) (Moskowitz AJ et al, JCO 2021). Treatment consisted of 2 to 4 cycles of P-GVD followed by AHCT. We performed combined single-cell surface protein identification, RNA sequencing, and (T cell receptor) TCR sequencing using the 10x Genomics Single Cell Immune Profiling Platform on paired C1D1 and C3D1 peripheral blood mononuclear cells (PBMCs) from 23 patients who received P-GVD and 5 control patients with relapsed cHL treated with standard chemotherapy regimens. Expression of MHC-I, MHC-II, and PD-L1 on both Hodgkin Reed Sternberg (HRS) cells and within the TME was assessed by immunohistochemistry. Results:Of 38 evaluable pts, the complete response (CR) rate after 2 or 4 cycles of P-GVD was 95%. After a median follow-up of 40 months (range: 2-56), only 1 pt experienced relapse 23 months after AHCT and another pt died 41 months after AHCT while in remission due to unrelated causes. The estimated 40-month progression-free survival (PFS) is 96% (95% CI: 91-100). CITE-Sequencing of PBMCs across 56 samples from 28 patients revealed predominantly CD4+ T, CD8+ T, NK, and myeloid cells within the peripheral blood, with smaller proportions of hematopoietic progenitors and plasmacytoid dendritic cells. Combined RNA and TCR analysis of the T-cell compartment revealed significant pre-treatment clonal expansion of both CD4+ and CD8+ T-cells exhibiting a cytotoxic phenotype, including expression of GZMH, KLRG1, and CCL5 by CD8+ T-cells and expression of PRF1, NKG7, GNLY, FGFBP2, and GZMH by CD4+ T-cells ( Fig. 1A). Correlation of pre-treatment clonal expansion with tumor-specific MHC status demonstrated that clonal expansion of CD8+ T-cells was more robust in patients whose tumors expressed either MHC-I or MHC-II; conversely, clonal expansion of CD4+ T-cells was most robust in tumors expressing MHC-II but lacking MHC-I, suggesting cytotoxic CD4+ T-cell responses may promote anti-tumor immunity in MHC-I-deficient tumors ( Fig. 1B). Both CD4+ and CD8+ T-cell clonal expansion was highest in patients whose tumors expressed high levels of PD-L1 on stromal cells. Treatment with P-GVD did not result in further clonal expansion of either CD4+ or CD8+ T-cells but did increase expression of cytotoxic genes in pre-treatment expanded CD4+ T-cells, including NKG7 and PLEK, while downregulating negative genes of T-cell function in pre-treatment expanded CD8+ T-cells, including TNFAIP3 and DUSP1, suggesting that P-GVD promotes anti-tumor immunity in cHL by enhancing the cytotoxicity of tumor-reactive expanded CD4+ and CD8+ T-cells. Conclusions: Treatment of relapsed cHL with P- GVD resulted in durable remissions in patients undergoing AHCT. Distinct patterns of peripheral blood T-cell expansion were observed based on MHC-I and MHC-II expression on tumor cells, with CD4+ T-cell clonal expansion uniquely expanding in patients with MHC-I-deficient tumors. Further analysis of the impact of P-GVD on T-cell differentiation and function is ongoing. AcknowledgementT: Supported in part by a research grant from Investigator-Initiated Studies Program of Merck Sharp &amp; Dohme LLC. The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp &amp; Dohme LLC.

Constitutive Interleukin-7 Cytokine Signaling Enhances the Persistence of Epstein-Barr Virus-Specific T-Cells.

The efficacy of therapeutic T-cells is limited by a lack of positive signals and excess inhibitory signaling in tumor microenvironments. We previously showed that a constitutively active IL7 receptor (C7R) enhanced the persistence, expansion, and anti-tumor activity of T-cells expressing chimeric antigen receptors (CARs), and C7R-modified GD2.CAR T-cells are currently undergoing clinical trials. To determine if the C7R could also enhance the activity of T-cells recognizing tumors via their native T-cell receptors (TCRs), we evaluated its effects in Epstein-Barr virus (EBV)-specific T-cells (EBVSTs) that have produced clinical benefits in patients with EBV-associated malignancies. EBVSTs were generated by stimulation of peripheral blood T-cells with overlapping peptide libraries spanning the EBV lymphoma antigens, LMP1, LMP2, and EBNA 1, followed by retroviral vector transduction to express the C7R. The C7R increased STAT5 signaling in EBVSTs and enhanced their expansion over 30 days of culture in the presence or absence of exogenous cytokines. C7R-EBVSTs maintained EBV antigen specificity but were dependent on TCR stimulation for continued expansion. C7R-EBVSTs produced more rapid lymphoma control in a murine xenograft model than unmodified EBVSTs and persisted for longer. The findings have led to a clinical trial, evaluating C7R-EBVSTs for the treatment of refractory or relapsed EBV-positive lymphoma (NCT04664179).

The effect of PD-1/PD-L1 signaling axis on the interaction between CD19+B cells and CD4+T cells in peripheral blood of patients with systemic lupus erythematosus

BackgroundThe defect of B cell self-tolerance and the continuous antigen presentation by T cells (TCs) mediated by autoreactive B cells (BCs) play a key role in the occurrence and development of systemic lupus erythematosus (SLE). PD-1/PD-L1 signaling axis negatively regulates the immune response of TCs after activation and maintains immune tolerance. However, the effect of PD-1/PD-L1 signaling axis on the interaction between CD19+B/CD4+TCs in the peripheral blood of patients with SLE has not been studied in detail.MethodsPD-1/PD-L1 and Ki-67 levels in peripheral blood (PB) of 50 SLE patients and 41 healthy controls (HCs) were detected through flow cytometry, and then the expression of PD-1+/−cells and PD-L1+/−cells Ki-67 was further analyzed. CD19+B/CD4+TCs were separated for cell culture and the supernatant was collected to determine proliferation and differentiation of TCs. IL-10 and IFN-γ secretion in the supernatant was also determined using ELISA.ResultsThe PD-1, PD-L1, and Ki-67 levels on CD19+B/CD4+TCs in patients with SLE were higher than HCs. In CD19+B/CD4+TCs of SLE patients, the proliferative activity of PD-L1+ cells was higher than that of PD-L1− cells, and the proliferative activity of PD-1+ cells was higher than that of PD-1− cells. In the system co-culturing CD19+B/CD4+TCs from HCs/SLE patients, activated BCs promoted TCs proliferation and PD-L1 expression among TCs. Addition of anti-PD-L1 to co-culture system restored the proliferation of TCs, and inhibited IL-10/IFN-γ level. The addition of anti-PD-L1 to co-culture system also restored Tfh and downregulated Treg in HCs.ConclusionsAxis of PD-1/PD-L1 on CD19+B/CD4+TCs in PB of SLE patients is abnormal, and cell proliferation is abnormal. In CD19+B/CD4+TCs of SLE patients, the proliferative activity of PD-L1+ and PD-1+ cells compared with PD-L1− and PD-1− cells in SLE patients, respectively. CD19+B/CD4+TCs in SLE patients can interact through PD-1/PD-L1.

Ultrasound-based radiomics combined with immune status to predict sentinel lymph node metastasis in primary breast cancer

In the past few years, the axillary lymph node dissection technique has been steadily replaced by sentinel lymph node biopsy for treating and diagnosing breast cancer, thereby minimizing the complications and sequelae of the patients. Nevertheless, sentinel lymph node biopsy still presents limitations, such as high operation requirements, prolonged surgical duration, and adverse reactions to tracer agents. This study developed a novel non-invasive method to predict sentinel lymph node metastasis in breast cancer by analyzing the ultrasound imaging characteristics of the primary tumor, combined with the analysis of peripheral blood T-cell subsets that reflect the immune status of the body. The radiomic features analyzed in this study were extracted from preoperative ultrasound images of 199 solitary breast cancer patients, who were undergoing surgery and were pathologically diagnosed at the Yancheng First People's Hospital. All cases were randomly categorized in a 4:1 ratio to the training (n = 159) and validation (n = 40) cohorts. The extracted radiomics features were subjected to dimensional reduction with the help of the least absolute shrinkage and selection operator technique, resulting in the inclusion of 19 radiomics features. Four classifiers, including naïve Bayesian, logistic regression, classification decision tree, and support vector machine, were utilized to model the radiomics features, conventional ultrasound features, and peripheral blood T cell subsets in the training dataset, and validated using the validation dataset. The best-performing model was chosen for constructing the combined model. The radiomics model constructed using the logistic regression showed the best performance, with the training and validation cohorts showing areas under the curve (AUCs) of 0.77 and 0.68, respectively. The conventional ultrasound and peripheral blood T cell models constructed using the classification decision tree showed the best performance, wherein the training cohort presented AUCs of 0.71 and 0.81, respectively, while the validation cohort presented AUCs of 0.68 and 0.69, respectively. The combined model constructed by logistic regression showed AUCs of 0.91 and 0.79 in the training and validation datasets, respectively. The resulting combined model can be considered a simple, non-invasive method with strong reproducibility and clinical significance. Thus, it can be utilized to predict sentinel lymph node metastasis in breast cancer. Furthermore, the combined model can be effectively used to guide clinical decisions related to the selection of surgical procedures in breast surgery.

Inborn errors of immunity underlying defective T-cell memory.

T-cell memory is a complex process not well understood involving specific steps, pathways and different T-cell subpopulations. Inborn errors of immunity (IEIs) represent unique models to decipher some of these requirements in humans. More than 500 different IEIs have been reported to date, and recently a subgroup of monogenic disorders characterized by memory T-cell defects has emerged, providing novel insights into the pathways of T-cell memory generation and maintenance, although this new knowledge is mostly restricted to peripheral blood T-cell memory populations. This review draws up an inventory of the main and recent IEIs associated with T-cell memory defects and their mice models, with a particular focus on the nuclear factor kappa B (NF-κB) signalling pathway, including the scaffold protein capping protein regulator and myosin 1 linker 2 (CARMIL2) and the T-cell co-stimulatory molecules CD28 and OX-40. Besides NF-κB, IKZF1 (IKAROS), a key transcription factor of haematopoiesis and STAT3-dependent interleukin-6 signals involving the transcription factor ZNF341 also appear to be important for the generation of T cell memory. Somatic reversion mosaicism in memory T cells is documented for several gene defects supporting the critical role of these factors in the development of memory T cells with a potential clinical benefit. Systematic examination of T-cell memory subsets could be helpful in the diagnosis of IEIs.

The role of inflammatory factors and T-cell subsets in the diagnosis of recurrence in epithelial ovarian cancer patients and the effect of olaparin treatment on them.

The aim of the study is to investigate the role of serum inflammatory factors and T-cell subsets in the diagnosis of recurrence in epithelial ovarian cancer patients and the effect of olaparib on inflammatory factor and T-lymphocyte subsets in patients with recurrent epithelial ovarian cancer. In this study, 100 patients diagnosed as recurrent epithelial ovarian cancer in our hospital and 100 patients without recurrent epithelial ovarian cancer in the same period were selected. According to the treatment plan, the recurrent patients were divided into conventional therapy group (Paclitaxel and Carboplatin) and combined therapy group (Paclitaxel, Carboplatin, and olaparib). The levels of serum inflammatory factors were evaluated by enzyme-linked immunosorbent assay. The peripheral blood T-lymphocyte subsets in each group were detected by flow cytometry. Compared with nonrecurrent patients, recurrent patients have higher serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels (p < .05), and lower interferon-γ (IFN-γ) level and the CD4+/CD8+ ratio. After adjusting for confounding factors, the results showed that the serum IL-6, IFN-γ, and TNF-α levels were influencing factors of recurrence in epithelial ovarian cancer patients. The area under the receiver operating curve and the sensitivity of serum TNF-α in predicting ovarian cancer recurrence were higher than those of IL-6 and IFN-γ. After secondary chemotherapy and/or olaparib maintenance treatment, the IL-6 (p < .001) and TNF-α (p < .001) levels in combined therapy group were lower than those in the conventional therapy, whereas the IFN-γ level (p < .001), the CD4+ T-cell proportion (p = .0069) and the CD4+/CD8+ ratio (p = .0201) were higher than those in the conventional therapy. The serum IL-6, TNF-α, and IFN-γ levels were closely related to the recurrence of ovarian cancer. Olaparib maintenance treatment can significantly decrease the IL-6 and TNF-α level, and increase IFN-γ level and the CD4+/CD8+ ratio in patients with recurrent ovarian cancer.

Supervised clustering of peripheral immune cells associated with clinical response to checkpoint inhibitor therapy in patients with advanced melanoma

Immune therapy with checkpoint inhibitors (CPIs) is a highly successful therapy in many cancers including metastatic melanoma. Still, many patients do not respond well to therapy and there are no blood-borne biomarkers available to assess the clinical outcome. To investigate cellular changes after CPI therapy, we carried out flow cytometry-based immune monitoring in a cohort of 90 metastatic melanoma patients before and after CPI therapy using the FlowSOM algorithm. To evaluate associations to the clinical outcome with therapy, we divided the patients based on progression-free survival. We found significant associations with CPI therapy in both peripheral blood mononuclear cell and T-cell subsets, but with the most pronounced effects in the latter. Particularly CD4+ effector memory T-cell subsets were associated with response with a positive correlation between CD27+HLA-DR+CD4+ effector memory T cells in a univariate (odds ratio: 1.07 [95% confidence interval 1.02-1.12]) and multivariate regression model (odds ratio: 1.08 [95% confidence interval 1.03-1.14]). We also found a trend towards stronger accumulation of CD57+CD8+ T cells in non-responding patients. Our results show significant associations between immune monitoring and clinical outcome of therapy that could be evaluated as biomarkers in a clinical setting.

Therapeutic cancer vaccination against mutant calreticulin in myeloproliferative neoplasms induces expansion of specific T cells in the periphery but specific T cells fail to enrich in the bone marrow.

Therapeutic cancer vaccination against mutant calreticulin (CALR) in patients with CALR-mutant (CALRmut) myeloproliferative neoplasms (MPN) induces strong T-cell responses against mutant CALR yet fails to demonstrate clinical activity. Infiltration of tumor specific T cells into the tumor microenvironment is needed to attain a clinical response to therapeutic cancer vaccination. Determine if CALRmut specific T cells isolated from vaccinated patients enrich in the bone marrow upon completion of vaccination and explore possible explanations for the lack of enrichment. CALRmut specific T cells from four of ten vaccinated patients were expanded, enriched, and analyzed by T-cell receptor sequencing (TCRSeq). The TCRs identified were used as fingerprints of CALRmut specific T cells. Bone marrow aspirations from the four patients were acquired at baseline and at the end of trial. T cells were enriched from the bone marrow aspirations and analyzed by TCRSeq to identify the presence and fraction of CALRmut specific T cells at the two different time points. In silico calculations were performed to calculate the ratio between transformed cells and effector cells in patients with CALRmut MPN. The fraction of CALRmut specific T cells in the bone marrow did not increase upon completion of the vaccination trial. In general, the T cell repertoire in the bone marrow remains relatively constant through the vaccination trial. The enriched and expanded CALRmut specific T cells recognize peripheral blood autologous CALRmut cells. In silico analyses demonstrate a high imbalance in the fraction of CALRmut cells and CALRmut specific effector T-cells in peripheral blood. CALRmut specific T cells do not enrich in the bone marrow after therapeutic cancer peptide vaccination against mutant CALR. The specific T cells recognize autologous peripheral blood derived CALRmut cells. In silico analyses demonstrate a high imbalance between the number of transformed cells and CALRmut specific effector T-cells in the periphery. We suggest that the high burden of transformed cells in the periphery compared to the number of effector cells could impact the ability of specific T cells to enrich in the bone marrow.