Mean Fluorescence Intensity Ratio Research Articles

B-053 Four years summary of flow cytometry live cell based assay for detecting serum anti-aquaporin 4 IgG and anti-myelin oligodendrocyte glycoprotein IgG in a Korean reference laboratory

Abstract Background In the diagnosis of neuromyelitis optica spectrum disorder (NMOSD), it is important to sensitively detect anti-aquaporin 4 (AQP4) IgG and anti-myelin oligodenrocyte glycoprotein (MOG) IgG in patients' serum. NMOSD is a disease that shows 3-7 times more common prevalence in Far East Asia than in the West. According to the IPND diagnostic criteria, it is recommended to diagnose as a cell based assay using full length recombinant protein. In this study, we would like to review 4 years results of AQP4, MOG requested to Eone laboratories in Korea performed with flow cytometry live cell based assay (FC-CBA). Methods FC-CBA was performed using piRES2 cell transfected with AQP4, MOG and stained with DsRed2. Subcultured cells were counted with 10^6, and reacted with patients' serum for 1 hour in a refrigerated state. Thereafter, reaction with FITC anti-human IgG for 2nd antibody for 30 minutes, and 10,000 cells were read from Navius (Beckman Coulter). The mean fluorescence intensity (MFI) ratio , which is the ratio obtained by dividing the MFI value of both FITC and DsRed2 positive cells and DsRed2 positive cells to negative cells, was used to determine positive, borderline, and negative for the serum sample. AQP4 reviewed cases during the period from November 2018 to January 2024, and MOG reviewed cases during the period from December 2019 to January 2024. Indirect immunofluorescence assay using formalin fixed cells is commercially available and compared to the FC-CBA of this institution. Results During the study period, 4,733 cases of AQP4 were performed, and 23,540 cases of MOG were performed by FC-CBA. Among them, AQP4 was positive in 405 cases (8.5%) and borderline in 71 cases (1.5%), and all others were negative. MOG was 2,597 positive (11%), borderline in 1,144 cases (4.8%), and all others were negative. The positive and borderline cutoffs for MFI ratio were changed during the study period, and the positive rate was significantly changed. Conclusions Eone laboratories are the only institution in Korea that performs live cell based assay using flow cytometry for AQP4 and MOG IgG, and receives samples from patients suspected of NMOSD at university hospitals. When reading with MFI ratio, it is important to properly set the cutoff to determine the positivity rate.

Characterization of Non-Small Cell Lung Cancer Tissue by Quantitative Assessment of Class III β-Tubulin Expression

Background. The cytoskeletal protein β-tubulin class III (Tubb3) is associated with tumor resistance to taxanes and vinca alkaloids, as well as with the metastatic potential of neoplasm, however, data from immunohistochemical analysis of Tubb3 expression in non-small cell lung cancer (NSCLC) tissue are few and contradictory. Purpose. Characterization of the level and intensity of Tubb3 expression in NSCLC tissue and analysis of the identified parameters correlation with clinically significant characteristics of the disease. Methods. Quantitative assessment of the level and intensity of Tubb3 expression in 120 surgical samples of NSCLC was carried out by immunofluorescence method associated with flow cytometry. Primary rabbit monoclonal antibodies specific to Tubb3 and secondary anti-rabbit antibodies conjugated with fluorescent dye DyLight650 (ab98510, UK) were used. The expression of the marker was assessed by two parameters: the level of expression measured as the percentage of the cells expressing Tubb3 and the intensity of expression in conventional units (CU) represented as the ratio of the geometric mean fluorescence intensity in the experimental and control samples (cells incubated with secondary antibodies only). Results. 1. Tubb3 expression was detected in all NSCLC samples studied. The median level and intensity of Tubb3 expression was 30.5% and 2.0 CU with significant differences (up to 10 times) in the quantitative values of both parameters in different patients. 2. The distribution of the studied tumors in terms of the level and intensity of Tubb3 expression differs from normal (P<0.001), the associative relationship between the assessed parameters is very strong (Spearman's rank correlation coefficient was 0.91; P<0.0001). 3. Statistical analysis did not reveal correlations between the level of Tubb3 expression and the gender and smoking status of the patients, with the degree of tumor differentiation, as well as with the stage of NSCLC. 4. In the group of lung adenocarcinomas, the median level of Tubb3 expression is higher compared with squamous cell lung cancer in male and female patients (P=0.01). Conclusion. High heterogeneity of Tubb3 expression level in NSCLC tissue in the patients and differences in the parameters between the tumors of various histotypes indicate the importance of further correlation analysis of Tubb3 expression level with the patients' life span in order to identify the prognostic value of the marker.

4-Color Flow Cytometric Crossmatch Using Whole Blood Lysis

BackgroundIn lymphocyte crossmatch using flow cytometry (flow cytometric crossmatch, FCXM), the conventional tricolor FCXM protocol requires a mononuclear cell isolation step. To develop a new, more streamlined protocol, we introduced whole blood lysis (WBL) and CD45 fluorescence-triggered acquisition using 4-color flow cytometry. MethodsA total of 186 donor/recipient pairs for transplantation were classified into donor-specific human leukocyte antigen (HLA) alloantibody-positive (DSA+, n = 78) and DSA-negative (DSA−, n = 108) groups. The latter group was reclassified into blood group ABO-incompatible (ABOi, n = 56) and ABO-compatible (n = 52) subgroups. The WBL FCXM protocol with CD45 V500-C was optimized using a FACSLyric cytometer (BD Biosciences) with 3 lasers. Measurements for T cells or B cells were calculated as a mean fluorescence intensity (MFI) ratio (test divided by control). WBL FCXM was compared with conventional FCXM in each group. ResultsWBL FCXM showed no difference quantitatively compared with conventional FCXM, except for the B cell FCXM in the DSA− group (B cell MFI ratio: 1.06 ± 0.44 and 0.92 ± 0.41, respectively [P = .0001]). There was no ABO antibody interference in the ABOi subgroup. Similar results were observed in the qualitative determinations of FCXM as follows: 1) In the DSA+ group, the sensitivity of B cell WBL FCXM (96.2%) showed no difference compared with that of conventional FCXM (91.0%, P = .2188) and 2) In the DSA− group, the specificity of T cell WBL FCXM (96.3%) showed no difference compared with that of conventional FCXM (98.1%, P = .6250). WBL FCXM reduced the turnaround time by 50 min compared with that by conventional FCXM. ConclusionsWBL FCXM demonstrated comparable assay performance to that of conventional FCXM. Because this new FCXM protocol is simple and does not compromise assay sensitivity, it has the potential to replace the conventional method in histocompatibility laboratory settings.

#2806 Role of non-HLA antibodies in pre-transplant evaluation of antibody-mediated damage risk

Abstract Background and Aims Donor-specific HLA antibodies (HLA-DSA) contribute to antibody-mediated rejection (ABMR) after kidney transplantation (KT) and worse graft-survival. Non-HLA antibodies may play a role in the development of ABMR in the presence of HLA-DSA or in the development of microvascular inflammation (MVI) in the absence of HLA-DSA. Considering both antibodies in potential recipients before or after KT could enhance the prediction of post-KT ABMR/MVI. Method Retrospective study of a cohort of 169 KT recipients with: a) pre- and post-KT sera; b) follow-up HLA antibody monitoring; and c) follow-up biopsies. We determined the presence of 60 non-HLA antibodies in sera with a multiplex test: the Single Non-HLA Beads kit (LIFECODES®) on a Luminex® platform. We obtained an absolute number of positive antibodies and a sample MFI ratio sum according to which we stratified results into quartiles. Results Pre-KT, we detected a 15.4% of KT recipients with high HLA sensitization (cPRA >50%), being 8.3% HLA-DSA, and non-HLA antibodies in 85% of KT recipients, with a median of 3 (2-5) antibodies and a median MFI ratio sum of 10.1 (2.9-18.5). During a median follow-up of 60 (18.5-89) months 73 patients developed ABMR/MVI. We observed that a high burden of pre-KT non-HLA antibodies was associated with a higher risk of ABMR/MVI, regardless of HLA sensitization and pre-KT HLA-DSA. Notably, 57% of patients with ABMR/MVI had a high non-HLA burden before KT and 24.6% had a HLA cPRA >50%. Altogether, 68.5% of these patients showed either a high HLA sensitization or a high non-HLA burden before KT (Figure). Conclusion A high non-HLA preTR non-HLA antibody burden detected with a multiplex panel allows identification of patients more likely to develop ABMR/MVI, irrespective of the presence of anti-HLA antibodies.

Abstract LB448: VBC103: An innovative Trop2/Nectin4 targeted bispecific antibody drug conjugate (ADC) in bladder urothelial carcinoma (UC), triple-negative breast cancer (TNBC) and beyond

Abstract Trop2 and Nectin4 are transmembrane proteins involved in cancer pathogenesis. The therapeutic potential of targeting Trop2 or Nectin4 has been demonstrated through the approval of Trodelvy™ (Sacituzumab govitecan; SG) and Padcev™ (Enfortumab Vedotin; EV). However, the clinical use of these agents is accompanied by safety concerns. Trodelvy carries Boxed Warnings for severe or life-threatening neutropenia and severe diarrhea, while Padcev is associated with serious skin reactions. These safety issues underscore the importance of improving the drugs' safety profiles. It should be noted that the co-expression of Trop2 and Nectin4 is high in solid tumors while limited or moderate in normal tissues, presents an opportunity for more tumor-specific targeting and supports the bispecific design that could potentially address these safety issues. VBC103 is a bispecific antibody-drug conjugate (ADC) that selectively targets both Trop2 and Nectin4, which are highly co-expressed in UC, TNBC and other tumors. It delivers a TOPOi payload with a strong bystander effect to tumor cells. Our unique design maximizes efficacy while minimizing safety concerns. VBC103 has the following features to differentiate itself from other drugs targeting Trop2 or Nectin4 separately in clinical development and on the market. 1. Modulated affinity and valency. VBC103 incorporates a moderate affinity arm that targets Trop2 and a moderate affinity but high avidity arm that targets Nectin4, allowing for enhanced binding avidity, internalization, and cytotoxicity compared to its parental molecules (anti-Nectin4 parental antibody) or approved drug like EV. Additionally, the moderate affinity of both Trop2 and Nectin4 in VBC103 helps to reduce target-driven toxicities in normal tissues. 2. Innovative format of VHH-Fc fusion protein (MW 90kDa) demonstrated the superior tumor penetration, accumulation (MFI ratio in tumor: 280 VS 174) and distribution (MFI ratio in liver: 10 VS 18) compared to the IgG1 mAb- enfortumab (MW 150kDa). 3. Stronger bystander cell killing effect of the TOPOi payload vs. that of the payload used in EV. 4. Superior in vivo efficacy. In the UC CDX model, TGI 95% [VBC103, 5 mpk] vs 77% [EV, 8 mpk] vs 70% [EV+SG, 4+4 mpk] (Q3W X 3; ADC molecular equivalence) at Day 42 post injection. In the TNBC CDX model, VBC103 demonstrated continued superior efficacy compared to EV (maximum tolerated dose) and SG when administered as a single dose treatment at Day 21 post injection. 5. Wide therapeutic widow (TW). Preliminary toxicity studies in cynomolgus monkeys demonstrated good tolerability of repeated doses (18 and 36 mpk) of VBC103, which is molecular equivalent to 30 and 60 mpk of IgG1 ADC. These findings could establish a TW of >10 (HNSTD/MED). 6. Good developability. VBC103 also exhibited excellent developability based on plasma stability and other stress test data, which suggests it a good candidate in future CMC development. In summary, VBC103, with its unique bispecific design and differentiated features in affinity, valency, linker-payload, has shown promising potential as a first-in-class ADC candidate. Discovery studies have revealed favorable efficacy and toxicity profiles, supporting advancing VBC103 into clinical trials. Citation Format: Wei Wang, Jing Li, Lingyu Guan, Man Xu, Qun Yin. VBC103: An innovative Trop2/Nectin4 targeted bispecific antibody drug conjugate (ADC) in bladder urothelial carcinoma (UC), triple-negative breast cancer (TNBC) and beyond [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB448.

Establishing and verifying the threshold value of HLA mixed antigen reagent screening test results

Objective: To establish the threshold value of human leukocyte antigen (HLA) mixed antigen reagent screening test results, and to verify it by HLA single antigen reagent confirmation test results. Methods: The results of 2 255 serum samples tested for HLA antibodies by HLA mixed antigen reagent in the department of HLA Laboratory, the First Affiliated Hospital of Soochow University from October 2017 to December 2021 were retrospectively analyzed. Among them, 1 139 samples were also tested by single antigen HLA Class-Ⅰ reagent and 1 116 samples were also tested by single antigen HLA Class-Ⅱ reagent. Based on the same antigens coated with both reagents, the Mean Fluorescence Intensity (MFI) and Nomalized Background ratio (NBG ratio) of 12 HLA Class-Ⅰ beads and 5 HLA Class-Ⅱ beads in the HLA mixed antigen reagent and the MFI of 77 anti-HLA class-Ⅰ antibodies and 35 anti-HLA class-Ⅱ antibodies detected by HLA single antigen reagent were recorded. The MFI and NBG ratio of HLA mixed antigen reagent beads in 1 139 or 1 116 samples were segmented according to the positive rate of antibodyies detected by the single antigen reagent corresponding to the antigens coated with each HLA mixed antigen reagent bead, and the results of the HLA mixed antigen screening test were verified by the HLA single antigen reagent confirmation test. Results: The threshold values of MFI and NBG ratio of HLA mixed antigen reagent's 17 beads were established. The MFI of No. 1 to No. 17 beads of HLA mixed antigen reagent ranged from 26.86 to 21 925.58, and the NBG ratio ranged from 0 to 434.65. According to the positive detection rate of HLA single antigen reagent corresponding to the coated antigens, the MFI and NBG ratio of the beads of HLA mixed antigen reagent were divided into positive interval, suspicious positive interval, suspicious negative interval and negative interval. The positive rates of anti-HLA class-Ⅰ antibodies by HLA mixed antigen reagent and single antigen HLA Class-Ⅰ reagent were 87.5% (997/1 139) and 66.3% (755/1 139). The positive rates of anti-HLA class-Ⅱ antibodies were 63.4% (707/1 116) and 44.9% (501/1 116). In the samples with suspicious negative, suspicious positive and positive results of HLA class-Ⅰ、Ⅱ antibodies detected by HLA mixed antigen reagent, the positive detection rates of single antigen HLA Class-Ⅰ reagent were 14.9% (17/114), 41.3% (145/351) and 91.3% (590/646), respectively. The positive detection rates of single antigen HLA Class-Ⅱ reagent were 15.5% (58/375), 26.5% (81/306) and 88.8% (356/401), respectively. Conclusions: In this study, the threshold values of MFI and NBG ratio of HLA mixed antigen reagent screening test are established, and the threshold values are verified by the results of HLA single antigen reagent confirmation test. HLA mixed reagent screening test can be used for screening of HLA antibodies, and if necessary, it should be combined with HLA single antigen confirmatory test for clinical detection of HLA antibodies.

Exploring the dynamics of Programmed Death-Ligand 1 in canine lymphoma: unraveling mRNA amount, surface membrane expression and plasmatic levels.

Programmed Death-Ligand 1 is a well-known immune checkpoint molecule. Recent studies evaluated its expression in different canine cancer types through different laboratory techniques. The present study aims to evaluate the surface membrane protein expression (mPD-L1) by means of flow cytometry (FC) in different canine lymphoma immunophenotypes. Furthermore, in a subset of cases, mRNA and plasmatic soluble protein (sPD-L1) have been assessed in the same patient, and correlations among results from the three analyses investigated. Samples obtained for diagnostic purpose from untreated dogs with a confirmed lymphoma immunophenotype were included: surface protein was assessed via FC and quantified with median fluorescence index ratio (MFI ratio), gene expression was evaluated by real time quantitative polymerase chain reaction (RT-qPCR) and plasmatic concentration of soluble protein (sPD-L1) measured with ELISA. Statistical analyses were performed to investigate any difference among FC immunophenotypes, updated Kiel cytological classes, and in the presence of blood infiltration. Considering FC, most B-cell lymphomas (BCL) were positive, with higher MFI ratios than other subtypes (81%, median MFI ratio among positive samples = 1.50, IQR 1.21-2.03, range 1.01-3.47). Aggressive T-cell lymphomas had a lower percentage of positive samples (56%) and showed low expression (median MFI ratio in positive samples = 1.14, IQR 1.07-1.32, range 1.02-2.19), while T-zone lymphomas (TZL) were frequently positive (80%) but with low expression (median MFI ratio in positive samples = 1.19, IQR 1.03-1.46, range 1.02-6.03). Cellular transcript and sPD-L1 were detected in all samples, without differences among immunophenotypes. No correlation between results from different techniques was detected, but sPD-L1 resulted significantly increased in FC-negative lymphomas (p = 0.023). PD-L1 molecule is involved in canine lymphoma pathogenesis, with differences among immunophenotypes detected by FC. Specifically, BCL have the highest expression and aggressive T-cell lymphomas the lowest, whereas TZL need further investigations.

Beyond Mitigating Immune-Related Adverse Events: Glucocorticoids Ameliorate Bispecific Antibody-Mediated T-Cell Exhaustion I n Vitro

Following the approval of blinatumomab in 2015 T-cell recruiting bispecific antibodies (BsAbs) became the established standard-of-care for relapsed or refractory (r/r) and minimal residual disease positive B-cell precursor acute lymphoblastic leukemia. More recently, there has been a notable increase in the approval of additional BsAb constructs for malignancies such as r/r follicular lymphoma, r/r large B-cell lymphoma, and advanced multiple myeloma (MM), with further approvals anticipated. Despite the clinical success, administration of BsAbs is commonly linked to dose-limiting immune-related adverse events (IrAEs), such as cytokine release syndrome, possibly compromising treatment efficacy. Hence, current BsAb as well as CAR T approvals integrate prophylactic and acute glucocorticoid (GC) application upon occurrence of IrAEs. Surprisingly, despite their immunosuppressive nature, GC administration had no negative impact on response or survival of MM patients receiving CAR T-cell therapy (Wang 2022). Based on these data, we hypothesize that the GC-mediated downregulation of BsAb-induced T-cell function has a dual effect: mitigating IrAEs and ameliorating T-cell exhaustion. Here we tested the effect of simultaneous administration of the GC dexamethasone (dexa) and a CD3xCD19 targeting BsAb on T-cell function and exhaustion. We first evaluated the short-term effect of dexa exposure on BsAb-mediated cytotoxicity by co-culturing healthy donor (HD) T cells with CD19 + cell lines and stimulating with a CD3xCD19 BsAb alone or in combination with 300nM dexa for 3-5 days. In fact, the addition of dexa decreased BsAb-mediated tumor cell lysis against OCI-Ly1 cells after 3 days, but not after 5 days. Surprisingly, however, T-cells showed a significantly increased expansion upon dexa exposure after 3 and 5 days (day 3: Ki-67 MFI ratio BsAb=337.2, +dexa=676.0, p=0.008; day 5: CD2 +-fold change to day 0 BsAb only=7.1; +dexa=11.6; p=0.01). In cytotoxicity assays against murine Ba/F3 cells transduced with human CD19 with or without the co-stimulatory molecule CD86, T cells showed significantly reduced BsAb-mediated cytotoxicity and proliferation upon dexa exposure in the absence of CD86. Intriguingly, however, T-cell function was rescued upon CD86 co-expression. To evaluate long-term effects of GCs on T-cell function and exhaustion, we next used our established long-term in vitro model system to mimic continuous stimulation of T cells with a BsAb ± GCs. HD T cells were co-cultured with CD19 + OCI-Ly1 cells for 28 days and continuously stimulated with a CD3xCD19 BsAb alone or in combination with dexa. T-cell fitness was assessed longitudinally. Surprisingly, but in line with our short-term data, we found increased CD8 + T-cell expansion upon BsAb +dexa co-exposure in long-term cultures (abs. CD8 + cell count day 21 BsAb=1.613, +dexa=34.583). Furthermore, immunophenotyping revealed similar upregulation of the checkpoint inhibitors PD-1, Tim-3, Lag-3 after 7 days. On day 21, however, Lag-3 expression on dexa exposed CD4 + and CD8 + T cells was decreased (MFI ratio BsAb=10.5, +dexa=1.7, p=0.02). Inflammatory cytokine (IL-2, IFN-γ, TNF-α) levels were decreased in the supernatant of dexa-exposed vs BsAb only co-cultures. However, low levels of IFN-γ were maintained in the dexa co-exposed condition over 24 days (mean level IFN-γ day 24 BsAb=53pg/ml, +dexa=1756 pg/ml). Finally, T cells were isolated from cultures every 7 days and tested in 72h cytotoxicity assays, without further exposure to dexa. Intriguingly, T cells previously exposed to dexa maintained a significantly higher cytotoxicity over 28 days (mean % specific lysis day 21 BsAb=34.2, +dexa=90.3, p=0.004). Taken together, our data demonstrate a positive impact of GCs on BsAb-mediated T-cell expansion in short- and long-term setting. Characterization of T cells upon long term BsAb exposure demonstrated an ameliorating effect of GCs on BsAb-induced T-cell exhaustion. Our data suggest that dampening of T-cell activity by GC administration in BsAb-treated patients could serve two purposes: managing IrAEs while simultaneously ameliorating T-cell exhaustion and improving T-cell persistence. In future studies we will further dissect the effect of dexa on the transcriptional landscape of BsAb-stimulated T cells by performing RNA sequencing and also explore the effect of GCs on CAR T-cell function.

CO-RELATION OF P-GLYCOPROTEIN AND PLASMA IMATINIB IN IMATINIB RESPONDERS AND NON-RESPONDERS PATIENTS WITH CHRONIC MYELOID LEUKAEMIA

Objective: To measure and compare P-glycoprotein (P-gp) expressions in imatinib responders and non-responders with chronic myeloid leukemia-chronic phase (CML-CP) and correlate with plasma imatinib levels. Methods: Patients were classified into two groups based on their haematological and cytogenetic responses to imatinib: responders and non-responders. Liquid chromatography-mass spectrometry was used to measure plasma imatinib levels, while flow cytometry was used to evaluate leucocyte P-gp expression. Results: The median plasma imatinib trough levels in non-responders were 496 (217-3150) ng/ml compared to 2245 (454-4270) ng/ml in the responders, which was statistically significant (p=0.0003). The proportion of patients expressing P-gp in granulocytes was higher in the non-responder group than in the responder group (75% vs. 62.5%). The ratio of mean fluorescence intensity (RFI) revealed that non-responders had higher median P-gp expression than did respondents MFI (1.16(1.06-1.50) and 1.12(1.01-1.38), respectively; p = 0. 2307). In both groups, there was a negative correlation between P-gp expression and plasma imatinib trough levels (-0.4384 vs.-0.2848). Conclusion: Imatinib median plasma trough levels in non-responders were considerably lower. This was highly supported by P-gp expression in granulocytes, which is inversely related to imatinib plasma trough levels; however, the difference was not statistically significant, which could be attributed to the small number of patients. This could be the cause of imatinib resistance in non-responder CML-CP patients, and P-gp levels should be evaluated to optimize treatment in patients who do not achieve hematologic or cytogenetic response.

Accomplices in Cure: Blinatumomab + Dasatinib Reduces TCR Signaling and Effector Function Stronger Than Ponatinib and Ameliorates T-Cell Exhaustion

The tyrosine kinase inhibitors (TKIs) dasatinib (dasa) and ponatinib (pona) are being tested in clinical trials in philadelphia chromosome-positive (Ph +) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients in combination with the CD19xCD3 bispecific blinatumomab (blina). Pona but not dasa is able to bind the BCR-ABL T315I mutation, which frequently occurs in BCP-ALL patients. Therefore, clinical studies are moving away from dasa and favor pona. Nevertheless, both TKIs in combination with blina have led to striking remission rates in Ph + BCP-ALL (complete response dasa: 98%, Foá 2020; pona: 92%, Jabbour 2023). Interestingly, increased lymphocyte frequencies were reported in patients under blina + dasa therapy (Puzzolo 2021), indicating that the effect of dasa is not limited to toxicity towards ALL cells conferred by BCR-ABL inhibition. Indeed, dasa modulates T-cell function by interfering with TCR signaling (Mestermann 2019). Also, we have shown that resting of T cells by high-dose dasa treatment ameliorates bispecific-induced T-cell exhaustion (Philipp 2022). In contrast, less is known about the effect of pona on T-cell function. Based on these data, we hypothesize that TKIs fine-tune TCR signaling, which ameliorates T-cell exhaustion during continuous blina infusion. Here, we systematically compared the influence of dasa and pona on blina-mediated TCR signaling and T-cell function. Inhibition of T-cell activation and function by dasa or pona at 0.3 - 100 nM was tested in vitro over 3 days: (1) CD69 expression, (2) blina-mediated killing as specific lysis of CD19 + tumor cells (3) T-cell expansion analyzed by dye dilution or CD2+ fold change (FC), (4) cytokine secretion measured by cytokine catch or cytometric bead array. Inhibition of TCR signaling was evaluated using a Jurkat-NFAT T-cell line stimulated for 6h with blina and CD19 + tumor cells. Phosphoflow of pAKT and pZAP70 was performed on CD3/CD28 activated primary human T cells. Toxicity of the TKIs against BCR-ABL +/- cell lines was evaluated after 3 days. T-cell exhaustion upon continuous blina + dasa stimulation was assessed in an in vitro long-term culture system for continuous bispecific exposure (Philipp 2022). Functional assays as described in (1)-(4) were performed every 7 days with isolated T cells from long-term cultures. Both TKIs inhibited T-cell function in a dose-dependent manner. Surprisingly, dasa was more potent than pona at low-intermediate doses in reducing T-cell function, shown by CD69 expression (mean % at TKI = 3 nM: basal = 70, dasa = 25, pona = 64, p<0.0001), cytotoxic function (mean % specific lysis at TKI = 12.5 nM: basal = 55, dasa = 0, pona = 54, p<0.0001), T-cell proliferation (mean % proliferated at TKI = 1.5 nM: basal = 62, dasa = 25, pona = 51, p<0.0001), as well as IFN secretion (normalized mean fluorescence intensity (MFI) at TKI = 12.5 nM: basal = 655, dasa = 0, pona = 924, p<0.0001). We also observed that NFAT engagement was significantly lower in dasa-treated Jurkat-NFAT cells (relative luminescence units at TKI 12.5 nM: basal = 1620, dasa = 392, pona = 996, p<0.001). Similarly, phosphoflow analyses revealed lower levels of pAKT and pZAP70 upon CD3/CD28 + dasa versus pona treatment (MFI of pAKT at TKI = 25 nM: basal = 2347, dasa = 1642, pona = 2045). Importantly, both TKIs induced cell death of BCR-ABL + but not BCR-ABL - cell lines at concentrations >3 nM. Interestingly, 14 days continuously stimulated T cells with blina + dasa (12.5 nM) did not lead to co-expression of inhibitory checkpoints (mean % of PD-1 +Tim-3 +LAG-3 + T cells on day 14: blina = 31, +dasa = 0.6, p<0.01). Furthermore, when isolated from long-term cultures and tested in functional assays, blina + dasa treated T cells maintained high cytokine secretion (mean IL-2 in pg/ml on day 14: blina = 730, blina + dasa = 5638, p<0.01), granzyme B expression (mean MFI ratio of CD8 + on day 14: blina = 96, blina + dasa = 335, p<0.01) and cytotoxic activity (mean % specific lysis on day 14: blina = 61, blina + dasa = 80). Together, these findings indicate that while both TKIs confer comparable toxicity to BCR-ABL + leukemia cells, dasa dampens TCR signaling and T-cell effector function more potently than pona. Strikingly, dasa can ameliorate T-cell exhaustion upon continuous blina exposure. Our data suggest that low-dose TKIs can fine-tune T-cell activity and might play a considerable role for the clinical success of combinatorial bispecific and TKI therapy in Ph + BCP-ALL.

Preclinical development and evaluation of nanobody-based CD70-specific CAR T cells for the treatment of acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) treatment remains challenging. CD70 was reported as a promising AML-specific antigen. Preclinically, CAR T-cell with single-chain-variable fragment (scFv) or truncated CD27 targeting CD70 has been reported to treat AML. However, various disadvantages including spontaneous exhaustion, proteinase-mediated loss of functional receptors, and high immunogenicity, limited its further application to clinical settings. Alternatively, the single-variable domain on heavy chain (VHH), also known as nanobodies, with comparable binding ability and specificity, provides an optional solution.MethodWe generated CD70 knocked-out novel nanobody-based anti-CD70-CAR T-cells (nb70CAR-T) with two different VHHs for antigen detection. Next, we detected the CD70 expression on primary AML blasts by flow cytometry and associated the efficacy of nb70CAR-T with the target antigen density. Finally, epigenetic modulators were investigated to regulate the CD70 expression on AML cells to promote the functionality of nb70CAR-T.ResultsOur nb70CAR-T exhibited expected tumoricidal functionality against CD70-expressed cell lines and primary AML blasts. However, CD70 expression in primary AML blasts was not consistently high and nb70CAR-T potently respond to an estimated 40.4% of AML patients when the CD70 expression level was over a threshold of 1.6 (MFI ratio). Epigenetic modulators, Decitabine and Chidamide can up-regulate CD70 expression on AML cells, enhancing the treatment efficacy of nb70CAR-T.ConclusionCD70 expression in AML blasts was not fully supportive of its role in AML targeted therapy as reported. The combinational use of Chidamide and Decitabine with nb70CAR-T could provide a new potential for the treatment of AML.

Manufacturing and validation of Good Manufacturing Practice–compliant regulatory dendritic cells for infusion into organ transplant recipients

Background aimsRegulatory (or “tolerogenic”) dendritic cells (DCregs) are a highly promising, innovative cell therapy for the induction or restoration of antigen-specific tolerance in immune-mediated inflammatory disorders. These conditions include organ allograft rejection, graft-versus-host disease following bone marrow transplantation and various autoimmune disorders. DCregs generated for adoptive transfer have potential to reduce patients’ dependence on non-specific immunosuppressive drugs that can induce serious side effects and enhance the risk of infection and certain types of cancer. Here, our aim was to provide a detailed account of our experience manufacturing and validating comparatively large numbers of Good Manufacturing Practice–grade DCregs for systemic (intravenous) infusion into 28 organ (liver) transplant recipients and to discuss factors that influence the satisfaction of release criteria and attainment of target cell numbers. ResultsDCregs were generated in granulocyte-macrophage colony stimulating factor and interleukin (IL)-4 from elutriated monocyte fractions isolated from non-mobilized leukapheresis products of consenting healthy adult prospective liver transplant donors. Vitamin D3 was added on day 0 and 4 and IL-10 on day 4 during the 7-day culture period. Release and post-release criteria included cell viability, purity, phenotype, sterility and functional assessment. The overall conversion rate of monocytes to DCregs was 28 ± 8.2%, with 94 ± 5.1% product viability. The mean cell surface T-cell co-inhibitory to co-stimulatory molecule (programmed death ligand-1:CD86) mean fluorescence intensity ratio was 3.9 ± 2.2, and the mean ratio of anti-inflammatory:pro-inflammatory cytokine product (IL-10:IL-12p70) secreted upon CD40 ligation was 60 ± 63 (median = 40). The mean total number of DCregs generated from a single leukapheresis product (n = 25 donors) and from two leukapheresis products (n = 3 donors) was 489 ± 223 × 106 (n = 28). The mean total number of DCregs infused was 5.9 ± 2.8 × 106 per kg body weight. DCreg numbers within a target cell range of 2.5–10 × 106/kg were achieved for 25 of 27 (92.6%) of products generated. ConclusionsHigh-purity DCregs meeting a range of quality criteria were readily generated from circulating blood monocytes under Good Manufacturing Practice conditions to meet target cell numbers for infusion into prospective organ transplant recipients.

2072. Of Mice and Men: HIV-induced CD38 expression on CD8+ T lymphocytes contributes to mitochondrial dysfunction and chronic inflammation despite antiretroviral therapy

Abstract Background Antiretroviral treatment (ART) has increased the life expectancy of people with HIV (PWH), however, comorbidities are higher in PWH than HIV-negative individuals. This is partly due to accelerated cellular aging, a consequence of CD38 expression, mitochondrial dysfunction, and inflammation. However, the role of CD38-expressing T cells on accelerated cellular aging in PWH is not completely understood. Methods The proportion of cytotoxic CD38+ CD8+ T lymphocytes from peripheral blood mononuclear cells (PBMCs) of PWH and HIV negative-individuals was measured. PBMCs from PWH on ART were cultured with or without gag-specific peptide stimulation and cytokine production was detected by flow cytometry. Mitochondrial function from T cells of PWH on ART and healthy donors was compared. Last, we generated a humanized (NSG) mouse model and infected mice with HIV (Bal strain) to determine the proportion of CD38+ T cells and cytokine production. Results Mean CD38 expression on CD8+ T cells was significantly different between PWH on ART and HIV-negative individuals (Figure 1). In PWH, gag-specific peptide stimulation of CD8+ T cells significantly increased CD38 expression (Figure 2A) and augmented secretion of IFNγ+ and TNFα+ (Figure 2B). There was a higher expression of CD38 among CD8+ T cells that produced IFNγ and TNFα (Figures 2C). T cells in PWH have altered mitochondrial function (Figures 2D-2F). Finally, HIV-infected NSG mice had T cell changes consistent with HIV infection (Figure 3A), a higher proportion of CD38+ T cells with an activated phenotype (Figures 3B and 3C), and significantly higher levels of inflammatory cytokines (Figure 3D). Figure 1.CD38 expression in PWH and HIV-negative individuals.Mean %CD38+CD8+ T cells in people with HIV (PWH) on and off antiretroviral therapy (ART) and HIV-negative individuals. Statistical significance was tested by Kruskal-Wallis test with post hoc analysis (Dunn’s Multiple comparison test). ****, P value< 0.0001.Figure 2.Cytokine production in response to gag stimulation from CD38+CD8+ T cells and mitochondrial function in T cells of PWH. Figure 2. (A) Frequency of CD38+ cells in gag-peptide stimulated CD8+ T cells and unstimulated cells. (B) Frequency of IFNγ and TNFα producing CD8+ T cells in gag-stimulated and unstimulated cells. Proportion of CD38+ cells among (C) IFNγ-/IFNγ+ and TNFα-/TNFα+ gag-peptide stimulated CD8+ T cells. Statistical significance was tested by paired t test. (D) Mitochondrial mass was determined by measuring the mean fluorescence intensity (MFI) of mitochondria-specific dye mitotracker green (MTG) among CD8+ T lymphocytes. (D) Frequency of mitochondrial superoxide positive lymphocytes and level of mitochondrial superoxide (MFI) were determined by staining the CD8+ T lymphocytes with mitosox red dye. (F) Mitochondrial membrane depolarization was determined by the red to green ratio of MFIs of mitochondrial membrane potential-dependent dye JC-1 among the CD8+ T lymphocytes. Statistical significance was tested by Mann-Whitney test. *, P value< 0.05; **, P value< 0.01; ***, P value< 0.001; ns – not significant. PWH=People with HIV. HD= Healthy Donors. Figure 3.Lymphocyte changes, CD38 expression, and cytokine production in HIV-infected NSG mice.(A) Frequency of CD4+ and CD8+ T lymphocytes and comparison of ratio of the frequencies of CD4+ to CD8+ T cells between control and HIV infected humanized mice. Statistical significance determined by Mann-Whitney test. (B) Frequency of CD38+ and CD38+HLADR CD4+ (A) and CD8+ (B) T cells in HIV- infected humanized mice compared to control. Statistical significance determined by Mann-Whitney test. (D)Cytokine measurements of HIV-infected humanized mice and controls. Statistical significance was tested by independent t test. *, P value< 0.05; **, P value< 0.01; ns – not significant. HLADR= Human Leukocyte Antigen – DR isotype, IFN=Interferon, IP=Inducible Protein, MCP=Monocyte Chemoattractant Protein, TNF=Tumor Necrosis Factor, IL=Interleukin. Conclusion ART decreases CD38 expression on CD8+ T cells, but not to the levels of HIV-negative individuals. In vitro, T cells of PWH have altered mitochondrial function, and HIV-specific stimulation augments CD38 expression on CD8+ T cells and contributes to a proinflammatory response. These findings translate to a humanized mouse model, where HIV infection upregulates CD38 expression and cytokine production. Together, these data suggest that CD38 is a potential therapeutic target for mitigating the mitochondrial dysfunction and chronic inflammation that drive cellular aging and comorbidities in PWH. Disclosures Poonam Mathur, DO, MPH, American Academy of HIV Medicine: Honoraria|Med Learning Group: Speaker/presenter|NACCME: Advisor/Consultant|NKGP Pharma: Advisor/Consultant Shyam Kottilil, MD, PhD, Arbutus Pharmaceuticals: Grant/Research Support|Gilead: Grant/Research Support|Merck: Grant/Research Support|Regeneron Pharmaceuticals: Advisor/Consultant|Silverback Therapeutics: Advisor/Consultant|The Liver Company: Advisor/Consultant|Yufan Biotechnologies: Advisor/Consultant.

Evaluation of Analysis Strategy for Cytoplasmic Lineage-Associated Markers in Mixed-Phenotype Acute Leukemia (MPAL) As Devised By Who Classification 2022

Background: Conventional rating of flow cytometrically determined expression of cytoplasmic lineage-associated markers MPO, CD3 and CD79a has defined positivity by ≥10% leukemic cells being positive compared to internal negative controls. WHO classification 2022 has devised a new approach defining positivity by part of leukemic population exceeding 50% of expression level of normal counterpart populations used as positive controls. Aim: Evaluate interpretation of cytoplasmic expression of MPO, CD3 and CD79a in MPAL and other acute leukemias. Methods: 277 acute leukemia cases were analyzed (MPAL-BM, n=18; MPAL-TM, 20; AML M0, 42; AML M1, 40; AML M2, 39; APL, 39; BCP-ALL, 40; T-ALL, 39). Following WHO 2022 positivity for cytoplasmic MPO, CD3 and CD79a was determined setting a cutoff at 50% of mean fluorescence intensity (MFI) of granulocytes, mature T-cells and mature B-cells, respectively. An alternative refined approach was evaluated calculating the MFI ratio between leukemic populations and positive controls. Results of MFI ratios are given as log differences, i.e. 0 representing identical MFIs, -1 meaning 1 log lower expression in leukemic population compared to controls. Log difference of -1.5 was set as cutoff. Results: Applying WHO 2022 majority of cases were rated positive for expected markers, i.e. AML, MPAL-BM and MPAL-TM for MPO, T-ALL and MPAL-TM for CD3 and BCP-ALL and MPAL-BM for CD79a (figure, A). However, there were cases rated positive in other groups although in general only few leukemic cells were causing positive rating, indicating limited specificity of this approach. In detail, MPO was positive in 69% of M0, 95% of M1, 92% of M2, 92% of APL, 61% of MPAL-BM and 70% of MPAL-TM, but also in other groups (BCP-ALL, 35%; T-ALL, 51%).Applying WHO 2022, diagnosis would have changed to BCP-ALL in 39% of MPAL-BM and to T-ALL in 30% of MPAL-TM cases. CD3 was positive in 92% T-ALL and 95% MPAL-TM cases, but also in other groups (M0, 29%; M1, 25%; M2, 15%, APL, 49%; BCP-ALL, 13%; MPAL-BM, 56%). Applying WHO 2022, diagnosis would have changed to AML in 5% of MPAL-TM cases. CD79a was positive in 100% of BCP-ALL and 94% of MPAL-BM, but also in other groups (M0, 55%; M1, 43%; M2, 51%, APL, 49%; T-ALL, 33%; MPAL-TM, 75%). Applying WHO 2022, diagnosis would have changed to AML in 6% of MPAL-BM cases. In order to refine the WHO 2022 approach and to improve specificity MFI ratio between leukemic cells and positive controls was applied. Similarly to the WHO 2022 approach the MFI ratio approach resulted in expected differences but also significant overlaps (figure, B), although to a lower degree for MPO. In detail, MPO was positive in 31% of M0, 75% of M1, 77% of M2, 85% of APL, 72% of MPAL-BM and 50% of MPAL-TM, but also in other groups (BCP-ALL, 13%; T-ALL, 23%). Applying the MFI ratio approach, diagnosis would have changed to BCP-ALL in 28% of MPAL-BM and to T-ALL in 50% of MPAL-TM cases. CD3 was positive in 92% of T-ALL and 100% of MPAL-TM cases, but also in other groups (M0, 14%; M1, 23%; M2, 18%, APL, 85%; BCP-ALL, 13%; MPAL-BM, 72%). Applying the MFI ratio approach there would have been no changes in MPAL-TM diagnosis. CD79a was positive in 98% of BCP-ALL and 94% of MPAL-BM, but also in other groups (M0, 50%; M1, 65%; M2, 46%, APL, 87%; T-ALL, 56%; MPAL-TM, 85%). Applying the MFI ratio approach, diagnosis would have changed to AML in 6% of MPAL-BM. In 6 and 7 cases analyzed by WHO 2022 and MFI ratio, respectively, changes in diagnosis from MPAL to ALL were not supported by NGS yielding mutations known to be associated with myeloid neoplasms while changes were supported by NGS in a minority of cases. Discussion: The WHO 2022 definition, as compared to conventional analysis, changed diagnosis in a significant number of MPAL cases, in particular due to rating MPO negative. At the same time, application of WHO 2022 approach to cases with AML, BCP-ALL and T-ALL suggests limited specificity with significant rates of positivity outside the lineage determined by conventional approach. The MFI ratio approach also changed diagnosis in a significant number of MPAL cases in comparison to the conventional approach, however, data suggests a better specificity for MPO. Further, MFI ratio considers the whole leukemic population and thus may result in higher reproducibility. Overall this data argues in favor of considering multiple lineage-associated markers instead of single markers. In addition, classification based on genetic markers should be maximized also in MPAL.

Frequency of myelin oligodendrocyte glycoprotein antibodies in pediatric onset multiple sclerosis

BackgroundMyelin oligodendrocyte glycoprotein antibodies (MOG-Ab) are associated with acute demyelinating syndromes and only rarely detected in multiple sclerosis (MS). As MOG-Ab associated disease is common in childhood, we speculated young patients might be more likely to produce MOG-Ab and investigated the frequency of MOG-Ab seropositivity in pediatric onset MS (POMS). Material and methodsPatients who experienced their first acute demyelinating event before age 18 years and were diagnosed with MS during follow-up were included in this single-center study. Patient data were retrieved from clinical records. Serum samples obtained and frozen at clinical visits were analyzed for MOG-Ab by a live cell-based assay (CBA) measuring delta mean fluorescence intensity (MFI) and MFI ratio. The control group consisted of patients referred to pediatric neurology for headache or vertigo and who had no neurological disorder (n = 48). Another control group consisted of patients with systemic inflammatory disorders systemic lupus erythematosus (n = 17) and juvenile idiopathic arthritis (n = 13) diagnosed in the rheumatology clinic. ResultsThe patient group (n = 122, F/M: 90/32, mean age 17.8 ± 2.6 years) were initially diagnosed as: MS, 62/122 (50.8%), clinically isolated syndrome, 43/122 (35.2%), radiologically isolated syndrome, 9/122 (7.3%), and acute disseminated encephalomyelitis 8/122 (6.5%). All received the final diagnosis of POMS. Serum was sampled 22.4 ± 29.2 (0–132) months after the first episode. None of the control groups had MOG-Ab positivity while 2/122 (1.6%) POMS cases had MOG-Abs, and a third patient had positive MFI and a MFI ratio slightly below the cut-off. These three patients’ initial and final diagnoses were MS, their annualized relapsing rates (ARRs) were 0.4–0.6, and most recent Expanded Disability Status Scale was 0. ConclusionLow titers of MOG-Ab can be detected in a small number of POMS patients at similar frequency with adult MS. Our POMS cases with MOG-Abs presented brainstem-cerebellar findings or seizures and had low ARR. Further series and longer follow-up will define whether these cases differ significantly from MOG-Ab negative POMS cases.

Seroprevalence of SARS-CoV-2 antibodies and associated risk factors in children less than 6 years of age in the canton of Fribourg, Switzerland (COVPED study): a population-based cross-sectional study.

To date, few data are available about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence in young children and the role of early-life childcare arrangements in transmission of the virus. In this study, we assessed the SARS-CoV-2 seroprevalence in children less than 6 years of age in the canton of Fribourg and identified risk factors associated with seropositivity. The COVPED study is a population-based cross-sectional study in children less than 6 years of age living in the canton of Fribourg, Switzerland, who presented to a private paediatrician or the paediatric emergency department of the Fribourg Hospital during a 9-week period between 11 January and 14 March 2021. Immunoglobulin G antibodies against SARS-CoV-2 trimeric spike protein were measured in capillary blood samples using an in-house Luminex assay. A mean fluorescence intensity ratio of above 6 was considered as positive. Metadata was collected through electronic questionnaires. Logistic regression analysis was performed to assess the risk of seropositivity and associated factors. A total of 871 children, with a median age of 33 months (range 6 days to 5 years 11 months) were included; 412 (47%) were female. Overall, 180 (21%, 95% confidence interval [CI] 18-24%) children were seropositive. Age as continuous variable was not associated with seropositivity risk, apart from a higher rate in children less than 3 months of age. Univariable analysis showed that female sex was associated with a lower seropositivity risk (unadjusted odds ratio [OR] 0.69, 95% CI 0.49-0.96; p = 0.03). Day-care attendance was also associated with a lower seropositivity risk (OR 0.67, 95% CI 0.47-0.95; p = 0.03), whereas all other childcare arrangements were not associated with seropositivity. No association was found between the number of children and adults present in extra-familial care and seropositivity. Multivariable analysis identified the number of household members above the age of 12 years being positive for SARS-CoV-2 as the main risk factor for seropositivity in children (adjusted odds ratio [aOR] 7.80, 95% CI 4.65-13.07; p <0.001 for one household member, aOR 22.07, 95% CI 13.49-36.11; p <0.001 for two household members and aOR 32.20, 95% CI 9.30-111.55; p <0.001 for three or more household members). The number of household members tested positive for SARS-CoV-2 (PCR test) is the main exposure risk to seropositivity for children less than 6 years of age. But the family size is not associated with an increased risk of infection. In young children, extra-familial care does not increase the risk of becoming SARS-CoV-2 seropositive, neither does the number of contacts present in extra-familial care. As adults and children will be vaccinated and new virus variants will be circulating the risk of exposure for young children will likely change and needs further monitoring.

A Novel Multicolor Flow Cytometry Scoring System for the Diagnosis and Prognostic Stratification of Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are a class of myeloid neoplasms featuring inefficient maturation and differentiation of hematopoietic cells, blood cytopenia, and a high risk of leukemia onset. The diagnosis of MDS remains a challenging task owing to its complexity, heterogeneity, and the lack of specific characteristics. To look for an easy and inexpensive diagnostic method for MDS, we tried to establish an FCM scoring systems (FCSS) with a combination of antibodies for diagnosis and prognostic stratification of MDS. This FCSS adopted four parameters; i.e., the frequency of myeloblasts in nucleated cells, the ratio between pro-B cells and CD117+ cells, the ratio of CD45 mean fluorescence intensity between lymphocytes and myeloblasts, and the ratio of SSC peak values between mature granulocytes and lymphocytes. We tested the correlation between the total FCSS score with conventional IPSS-R. Additionally, the correlation between the score of each FCSS parameter and IPSS-R was also evaluated. We found that total FCSS score had a positive correlation with IPSS-R, while FCSS parameter 1 and 4 were also correlated with IPSS-R. Furthermore, this FCSS had a sound sensitivity and specificity in the diagnosis of MDS. The FCSS represents a convenient and affordable approach for the diagnosis and prognostic stratification of MDS.

Expression characteristic of 4Ig B7-H3 and 2Ig B7-H3 in acute myeloid leukemia

ABSTRACT 4IgB7-H3 (4Ig) and 2IgB7-H3 (2Ig) expression characteristics in acute myeloid leukemia (AML) remain unknown. This study investigated mRNA and membrane protein expression of two B7-H3 isoforms in AML cell lines and de novo patients by using RT-PCR and flow cytometry, and analyzed the B7-H3 promoter methylation state by utilizing RQ-MSP. 4Ig was the dominant isoform. 2Ig mRNA expression rate and abundance were elevated in AML in comparison with controls (P = 0.000 and 0.000), while no significant difference in 4Ig (P = 0.802, P = 0.398). Membrane protein levels of B7-H3 isoforms in AML was higher than controls, detected by total B7-H3 expression rate (P = 0.002), total B7-H3 mean fluorescence intensity (MFI) ratio of blast cells and lymphocytes (MFI ratio) (P = 0.000), and 4Ig B7-H3 MFI ratio (P = 0.005). Compared with 2Ig low group, 2Ig high patients had older age, lower NPM1 mutation, higher FLT3-ITD mutation, and declining complete remission (CR) rates (P = 0.026, 0.012, 0.047, and 0.028). In B7-H3 high group, there was a trend toward older age, M4 and M5, worse karyotype, and lower CR rates, although with no marked difference (P > 0.05). The overall survival (OS) of 2Ig high and B7-H3 high groups were shorter than that of 2Ig low and B7-H3 low groups in the whole and non-M3 AML cohorts (P = 0.006 and 0.046; P = 0.003 and 0.032). Besides, an unmethylated B7-H3 promoter was identified. In conclusion, 2Ig mRNA and total B7-H3 membrane protein tended to have potential diagnostic value for AML. Specifically, high 2Ig mRNA and total B7-H3 membrane protein expression indicate worse OS. 4Ig and 2Ig expression are methylation-independent.

Altered expressions of CXCR4 and CD26 on T-helper lymphocytes in hereditary hemorrhagic telangiectasia

BackgroundHereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease characterized by a deregulated neo-angiogenesis. Besides a mainly vascular phenotype (muco-cutaneous telangiectases, arteriovenous malformations), a specific risk of infection is suggested by case series of severe and atypical infections as well as by reports of decreased T and natural killer (NK) lymphocyte counts. As some evidence supports a dysregulation of the CXCR4/CXCL12 chemotactic axis of HHT endothelial cells, we hypothesized that a similar phenomenon could occur on lymphocytes.MethodsEighteen HHT patients with history of severe infection (HSI) were matched in age and sex with 18 HHT without HSI and 18 healthy control subjects (HC). We assessed the cell count and the surface expression of CXCR4 and CD26 (CXCL12 inactivating peptidase) of circulating T-helper and T-cytotoxic lymphocytes (including naive, memory and activated subsets) and NK cells.ResultsThe overall HHT group of 36 patients exhibited a reduction of circulating T-helper lymphocytes compared to HC (median: 517 vs. 1026 cells/mm3, p < 0.0001), correlated with age (r = − 0.46, p = 0.005), requirement of intravenous iron or blood transfusions (median: 291 vs. 627 cells/mm3, p = 0.03) and CXCR4 surface expression (r = 0.353, p = 0.0345). CXCR4 and CD26 membrane expression were both decreased on HHT T-helper lymphocytes (median MFI ratio: 4.49 vs. 5.74 for CXCR4 and 3.21 vs. 4.33 for CD26, p = 0.03 and 0.0018 respectively) with an unchanged CXCR4/CD26 ratio. The HHT group with HSI had a higher CXCR4/CD26 ratio on the total T-lymphocyte population, as well as on the T-helper population and its naive subset (median on naive T-helper cells: 2.34 vs. 1.32, p = 0.0002).ConclusionsOur findings support a dysregulation of the CXCL12/CXCR4 chemotaxis of T-helper lymphocytes in HHT patients, potentially linked to their T-helper lymphopenia and susceptibility to infection.

Augmenting Efficacy of T-Cell Bispecific Antibodies in AML through a Tumor Stroma-Targeted 4-1BB Agonist

Bispecific antibodies represent a promising treatment option for acute myeloid leukemia (AML). We have recently described a novel T-cell bispecific antibody (TCB) targeting the intracellular tumor antigen Wilms tumor 1 (WT1) in the context of HLA-A*02 (Augsberger et al. Blood 2021). Based on these findings a multicenter first-in-human clinical trial was initiated in relapse/refractory AML (NCT04580121). Possible immune escape mechanisms against T-cell based immunotherapy are provided by the tumor microenvironment (TME) of the bone marrow by co-inhibition of T cells or stromal cells shielding leukemic cells from immune effector cells. To overcome the immunosuppressive effect of the TME and to enhance T-cell responses, we evaluated the combination of the WT1-TCB with an antibody fusion protein that targets a stromal antigen (Fibroblast-activation protein; FAP) and provides a positive costimulatory signal (4-1BBL) to T cells. FAP is upregulated on cancer-associated fibroblasts after remodulation of the bone marrow niche by leukemic cells, and the FAP specificity of the molecule therefore provides T-cell co-stimulation tightly restricted to the tumor niche.Efficacy of the combination (WT1-TCB + FAP-4-1BBL antibody fusion protein) was evaluated in co-culture assays over 4 days with primary HLA-A*02 + AML cells, healthy donor (HD) T cells and three NIH-3T3 fibroblast cell lines. NIH-3T3 cell lines were genetically modified to express low and high levels of FAP, respectively. Wild-type NIH-3T3 cells were included as control. Additionally, a control (Ctrl)-TCB and a Ctrl-4-1BBL antibody fusion protein recognizing a non-tumor target derived from the human germline repertoire were included. Enhancement of T-cell mediated cytotoxicity by the FAP-4-1BBL antibody fusion protein was evaluated by (1) specific lysis of primary AML cells, (2) upregulation of the T-cell activation markers CD25 and 4-1BB, (3) T-cell expansion calculated as fold change compared to day 0, and (4) Granzyme B-expression which was evaluated by intracellular staining.After 4 days of co-culture, with an E:T ratio of 1:2, we observed a mean specific lysis of 55.1±8.2% (±SEM; n=4) of primary AML cells mediated by HD T cells and WT1-TCB. Notably, this was reduced to 19.4±5.9% (±SEM; n=4) in the presence of NIH-3T3 cells. However, AML cell lysis was restored by the addition of the FAP-4-1BBL antibody fusion protein in the presence of high FAP expressing NIH-3T3 cells (mean specific lysis: 62.8±7.3%; ±SEM; n=4). Concomitantly, the FAP-4-1BBL antibody fusion protein led to increased expression of the activation molecules CD25 (MFI ratio: 22.1±5.3 vs. 10.4±1.3; ±SEM; n=4) and 4-1BB (MFI ratio: 10.4±6.0 vs. 2.1±0.3; ±SEM; n=4) on CD3 + T cells. Furthermore, lysis was accompanied by increased frequencies of granzyme B expressing T cells (45.0±2.5% vs. 16.1±5.3%; n=3). Importantly, the FAP-4-1BBL antibody fusion protein led to improved T-cell proliferation, especially of CD8 + T cells (fold change on day 4 vs day 0: 5.7±2.2 vs. 1.0±0.3; ±SEM; n=4). Overall similar observations were made in the presence of low FAP expressing NIH-3T3 cells.Taken together, we have established an in vitro model system mimicking the immunoprotective bone marrow TME using NIH-3T3 cells resulting in impaired AML cell lysis. Providing additional T-cell co-stimulation by a tumor-stroma targeted 4-1BB agonist, however, restored WT1-TCB-mediated cytotoxicity of primary AML cells in the presence of FAP expressing cell lines. Importantly, the combination overcame the immunosuppressive effect of the NIH-3T3 cells on T cells as further demonstrated by improved T-cell activation and expansion. The tumor-stroma targeted 4-1BB agonist therefore represents a promising combinatorial approach to enhance T-cell activity at the local tumor site and warrants further investigations in an in vivo model system. DisclosuresPulko: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Claus: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Buecklein: Pfizer: Consultancy, Honoraria, Speakers Bureau; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Miltenyi: Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Umana: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Klein: Roche: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; Klinikum der Universität München: Current Employment; Roche: Research Funding; Seattle Genetics: Consultancy, Research Funding; Pfizer: Consultancy, Speakers Bureau; Janssen: Consultancy; Takeda: Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.