T-cell counts in peripheral blood at leukapheresis predict responses to subsequent CAR-T cell therapy

CAR-T細胞の「原料の質」が治療効果と相関 --細胞採取時のリンパ球数がCAR-T細胞の体内での増殖と治療効果を予測する--. 京都大学プレスリリース. 2022-11-22.Prediction of responses to chimeric antigen receptor (CAR)-T cell therapies is essential to maximize their therapeutic efficacy for diffuse large B-cell lymphoma (DLBCL). While several tumor-intrinsic risk factors of resistance and/or early relapse have been identified, clinically useful markers that determine potential activity of CAR-T cells have not been fully investigated. T-cell property at the time of leukapheresis may serve as such a marker. Therefore, we evaluated the clinical impact of CD3⁺ cell count in peripheral blood at leukapheresis on clinical outcomes of CAR-T cell therapy. In total, 44 patients with relapsed or refractory (r/r) DLBCL who received tisagenlecleucel at Kyoto University Hospital were included. According to CD3⁺ cell counts, patients were categorized into CD3[LOW] and CD3[HIGH] groups with a threshold of 553/μL, based on receiver operating characteristic curve analysis. 1-year progression-free survival was significantly higher in the CD3[HIGH] group than the CD3[LOW] group (68.3% vs. 17.3%; adjusted hazard ratio [aHR], 0.37; p = 0.042). Overall survival was also superior in the CD3[HIGH] group (aHR, 0.24; p = 0.043). Moreover, higher CD3⁺ cell counts at leukapheresis were associated with significantly higher lymphocyte counts in peripheral blood at day 7 after CAR-T cell infusion (median 860 vs. 420/μL, P = 0.021), suggesting more extensive expansion of infused CAR-T cells in vivo. In conclusion, we demonstrated that the CD3⁺ cell count at leukapheresis predicts both expansion of CAR-T cells after infusion and outcomes of CAR-T cell therapy, and are useful for building comprehensive therapeutic strategies at the time of leukapheresis

H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner.

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body's iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin

Clinical Significance of Serum Hepcidin Levels on Early Infectious Complications in Allogeneic Hematopoietic Stem Cell Transplantation

The association of iron overload with complications of allogeneic hematopoietic stem cell transplantation (HSCT) has been suggested in previous studies. Because hepcidin plays a central role in the regulation of iron homeostasis, we analyzed the association between pretransplant serum hepcidin-25 levels and early infectious complications after allogeneic HSCT. We studied 55 consecutive adult patients with a median age of 47 years (range: 20-64 years) who underwent allogeneic HSCT for hematologic malignancies at our institution. Thirty-two patients had myelogenous malignancies; the remaining 23 had lymphogenous malignancies. The median pretransplant serum hepcidin level of patients in the study was 21.6 ng/mL (range: 1.4-371 ng/mL), which was comparable to that of healthy volunteers (median: 19.1 ng/mL [range: 2.3-37 ng/mL]; n = 17). When cumulative incidences of documented bacterial and cytomegalovirus (CMV) infections at day 100 were compared according to pretransplant hepcidin-25 levels, the incidence of bacterial, but not CMV, infection, was significantly higher in the high-hepcidin group (?50 ng/mL; n = 17) than in the low-hepcidin group (<50 ng/mL; n = 38) (65% [95% confidence interval, 38%-82%] versus 11% [3%-23%]; P < .001). This finding was confirmed by multivariate Cox analysis adjusted for confounders, including pretransplant ferritin and C-reactive protein (CRP) levels. No fungal infection was documented in either group. These results suggest that the pretransplant serum hepcidin-25 level may be a useful marker for predicting the risk of early bacterial complications after allogeneic HSCT. Larger prospective studies are, however, warranted to confirm our findings

Risk analysis of fluctuating hypercalcemia after leukapheresis in cellular therapy

Abstract Optimized management of citrate-induced hypocalcemia is required to provide safe leukapheresis. We prospectively analyzed subjects who underwent leukapheresis for cytotherapy, and evaluated serum ionized (iCa) concentrations before, at the end of, and 1 h after leukapheresis. During leukapheresis, calcium gluconate solution was continuously supplemented intravenously with hourly measurement of iCa. 76 patients including 49 lymphapheresis for chimeric antigen receptor T-cell therapy and 27 stem cell collections were enrolled. Median processing blood volume was 10 L (range, 6–15 L). Fluctuating hypercalcemia, in which the iCa concentration rose above its upper limit 1 h after leukapheresis, was observed in 58 subjects (76.3%). Multivariate analysis revealed that higher ratios of processing blood volume to body weight, more rapid calcium supplementation, and lower iCa concentration at the end of leukapheresis significantly increased elevation of serum iCa concentration by 1 h after leukapheresis. Based on multivariate analyses, we developed a formula and a diagram that accurately estimates serum iCa concentration 1 h post-leukapheresis. This suggests optimal targets for iCa concentration and calcium supplementation rates. In cases with high ratios of processing blood volume to body weight, slowing the rate of blood processing, rather than increasing calcium supplementation should safely alleviate hypocalcemia during leukapheresis without inducing hypercalcemia thereafter

An AKI biomarker lipocalin 2 in the blood derives from the kidney in renal injury but from neutrophils in normal and infected conditions.

First online: 06 March 2014[Background]Lipocalin 2 (LCN2 or neutrophil gelatinase-associated lipocalin) is a secretory protein discovered from neutrophils, which accumulates in the blood and urine during acute kidney injury (AKI) and in the blood by bacterial infection. Little is known about the tissue source and molecular forms of this protein under normal and pathophysiologic conditions. [Methods]By sandwich ELISA, serum and urinary LCN2 levels were measured in 36 patients with hematologic malignancies who transiently became neutropenic by stem cell transplantation (SCT). To evaluate contribution of neutrophil-derived LCN2 in the physiologic blood LCN2 concentrations, we examined CCAAT/enhancer-binding protein ε (C/EBPε) knockout mice, which lack mature neutrophils. [Results]In patients without AKI and bacterial infection, at 1 week after SCT, the median blood neutrophil counts became zero and serum LCN2 levels were decreased by 76 ± 6 % (p 100 kDa), while urinary LCN2 was mainly in low molecular weight forms. [Conclusion]Our findings suggest that neutrophils are the major source of circulating LCN2 in normal and infected conditions, whereas blood and urinary LCN2 mainly derive from the kidney during AKI, and that the molecular forms and regulation of blood and urinary LCN2 are clearly distinct

Relative hypercoagulation induced by suppressed fibrinolysis after tisagenlecleucel infusion in malignant lymphoma

キメラ抗原受容体T細胞療法による血液凝固と線溶の変動を解析 --サイトカイン放出症候群に伴う凝固障害の病態解析にむけて--. 京都大学プレスリリース. 2022-06-20.Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy has facilitated progress in treatment of refractory/relapsed diffuse large B-cell lymphoma (DLBCL). A well-known adverse event after CAR-T therapy is cytokine release syndrome(CRS). However, the etiology and pathophysiology of CRS-related coagulopathy remain unknown. Therefore, we conducted a prospective cohort study to comprehensively analyze coagulation/ fibrinolysis parameters present in peripheral blood of adult DLBCL patients treated with tisagenlecleucel in a single institution. Samples were collected from 25 patients at 3 time points: before lymphocyte-depletion chemotherapy and on days 3 and 13 after CAR-T infusion. After infusion, all patients except 1 experienced CRS, and 13 required the administration of tocilizumab. A significant elevation in the plasma level of total plasminogen activator inhibitor 1 (PAI-1), which promotes the initial step of coagulopathy (mean, 22.5 ng/mL before lymphocyte-depletion and 41.0 on day 3, P = .02), was observed at the onset of CRS. Moreover, this suppressed fibrinolysis-induced relatively hypercoagulable state was gradually resolved after CRS remission with normalization of total PAI-1 to preinfusion levels without any organ damage (mean values of soluble fibrin: 3.16 µg/mL at baseline, 8.04 on day 3, and 9.16 on day 13, P < .01; and mean PAI-1: 25.1 ng/mL on day 13). In conclusion, a hypofibrinolytic and relatively hypercoagulable state concomitant with significant total PAI-1 elevation was observed at the onset of CRS even in DLBCL patients with mild CRS. Our results will facilitate understanding of CRS-related coagulopathy, and they emphasize the importance of monitoring sequential coagulation/fibrinolysis parameters during CAR-T therapy

HFt uptake by erythroid cells in various iron statuses.

<p>(A, B) UT7/EPO and HEL cells were incubated with indicated concentrations of FAC or DFO for 24 h before flow cytometry analysis of CD71 (TFR1) expression (upper panels) and HFt (HFt) uptake (lower panels). MFI values relative to those of controls were calculated for each experiment; the results represent the mean ± standard error of at least three independent experiments. *P <0.05, **P <0.01; n.s., not significant.</p

TFR1 expression levels required for H-ferritin uptake.

<p>(A) AF488-labeled H-ferritin (HFt) and holo-transferrin (Tf) uptake was evaluated in CHO-TRVb cells lacking endogenous TFR1 (control) and CHO-TRVb cells expressing either wild-type or mutant human TFR1 (R646H and R646H/647A) or wild-type human TFR2. Shaded areas represent isotype control experiments (CD71 expression) and untreated control experiments (Tf and HFt uptake). (B) AF488-labeled holo-Tf and HFt uptake by CHO-TRVb cells expressing moderate or high levels of human TFR1. Representative histograms are shown (left panels). Uptake of AF488-labeled ligand (solid lines), competition experiments using a 20-fold excess of unlabeled ligand (broken lines), and controls (shaded areas) are shown. Data shown in the right panel represent the mean ± standard error. (C) Relationship between CD71 expression and HFt uptake by CHO-TRVb cells expressing wild-type or mutant forms of human TFR1. Data from two cell lines expressing wild-type TFR1 and two expressing a mutated form of TFR1 (R646H/647A) are shown. Right panels show a schematic representation of two different patterns of ligand uptake.</p