Objective:To explore the stimulation conditions,optimal culture time and infection time of C57BL/6J mice CD3+T cells in vitro,so as to improve the infection efficiency of CD 19 chimeric antigen receptor T cells(mCD19 CAR-T).Methods:Purified C57BL/6J mice CD3+T cells were cultured in anti-CD3/CD28 coated,anti-CD3 coated+soluble anti-CD28 and anti-CD3 coated,respectively.The cells were stimulated in above three conditions for 12 h and 24 h,following with 24 h,48 h and 72 h incubation and then the number of cell clones was recorded.C57BL/6J mice CD3+T cells were stimulated for 12 h,24 h,and 36 h under the above three conditions,then interleukin(IL)-2(100 U/ml)was added.The number of cell clones was recorded under microscope at 24 h,48 h,and 72 h of culture.After 24 h of stimulation,CD3+T cells derived from C57BL/6J mice were infected with retrovirus for 48 h to establish mCD19 CAR-T cells,and the percentage of GFP+CAR-T cells was detected by flow cytometry.Results:The infection efficiency of mCD19 CAR-T cells derived from C57BL/6J mice was only 5.23％under the optimized conditions of mCD19 CAR-T cells derived from BALB/c mice.The number of clones of C57BL/6J mice CD3+T cells was the highest in anti-CD3 coated+soluble anti-CD28 group after stimulated for 24 h and followed cultured for 48 h.After 24 hours of stimulation under the above conditions and 48 hours of culture with IL-2,the number of T cell proliferating clones in the anti-CD3 coated+soluble anti-CD28 group was significantly increased compared with the same group without IL-2,and the infection efficiency of CAR-T cells in this group reached 17.63％±4.17％.Conclusion:The optimal conditions for constructing CAR-T cells from C57BL/6J mice CD3+T cells are different from those of BABL/c mice.T cells stimulated by anti-CD3 coated+soluble anti-CD28+IL-2 can obtain mCD19 CAR-T cells with the highest efficiency after retrovirus infection.

Background Despite continuous improvements in the new target and construction of chimeric antigen receptor (CAR)-T, relapse remains a significant challenge following CAR-T therapy. Tumor microenvironment (TME) strongly correlates with the efficacy of CAR-T therapy. V-domain Ig suppressor of T-cell activation (VISTA), which exerts a multifaceted and controversial role in regulating the TME, acts not only as a ligand on antigen-presenting cells but also functions as a receptor on T cells. However, the characteristics and underlying mechanisms governing endogenous T-cell activation by VISTA, which are pivotal for reshaping the TME, remain incompletely elucidated.Methods The immunocompetent B acute lymphoblastic leukemia (B-ALL), lymphoma, and melanoma murine models were employed to investigate the characteristics of endogenous T cells within the TME following CD19 and hCAIX CAR-T cell therapy, respectively. Furthermore, we examined the role of VISTA controlled by interferon (IFN)-γ signaling in regulating endogenous T-cell activation and functionality in B-ALL mice.Results We demonstrated that the administration of CD19 CAR-T or hCAIX CAR-T cell therapy elicited augmented immune responses of endogenous T cells within the TME of B-ALL, lymphoma, and melanoma mice, thereby substantiating the efficacy of CAR-T cell efficacy. However, in the TME lacking IFN-γ signaling, VISTA levels remained elevated, resulting in attenuated cytotoxicity of endogenous T cells and reduced B-ALL recipient survival. Mice treated with CD19 CAR-T cells exhibited increased proportions of endogenous memory T cells during prolonged remission, which possessed the tumor-responsive capabilities to protect against B-ALL re-challenge. Compared with wild-type (WT) CAR-T treated mice, the administration of IFN-γ−/− CAR-T to both WT and IFN-γ−/− recipients resulted in a reduction in the numbers of endogenous CD4+ and CD8+ effectors, while exhibiting increased populations of naïve-like CD4+ T and memory CD8+ T cells. VISTA expression consistently remained elevated in resting or memory CD4+ T cells, with distinct localization from programmed cell death protein-1 (PD-1) expressing T subsets. Blocking the VISTA signal enhanced dendritic cell-induced proliferation and cytokine production by syngeneic T cells.Conclusion Our findings confirm that endogenous T-cell activation and functionality are regulated by VISTA, which is associated with the therapeutic efficiency of CAR-T and provides a promising therapeutic strategy for relapse cases in CAR-T therapy.

Objective:To harvest the primary Philadelphia chromosome-positive (Ph+) cells of B-acute lymphoblastic leukemia (B-ALL) and to establish the B-ALL mouse model. Methods:The plasmid carrying BCR-ABL P210 fusion gene was transferred into the bone marrow(BM) cells of C57BL/6J mice by retrovirus. Syngeneic mice irradiated with 9 Gy of 60Co γ-ray were injected with the transfected BM cells as the first generation (G1),and then the primary cells from the spleen and BM of the diseased mice were obtained and frozen. Sublethal γ-ray irradiated C57BL/6J mice were inoculated with the first generation of Ph+cells for in vivo passage,which were named as the second generation (G2). The third and fourth generations (G3 and G4) of Ph+cells and B-ALL mouse model were established by successive passages. Flow cytometry,H&E staining and peripheral blood smear were used to analyze the immunophenotypes and detect the pathological changes of the model mice. Results:After infusion of P210-NGFR retrovirus infected BM cells,the mice exhibited significant symptoms including weight loss,lower limbs paralysis,and arched back. Primitive and immature lymphocytes were observed in peripheral blood smears of the leukemia mice. The results of H&E staining showed obvious infiltration of leukemic cells around the central vein of the hepatic lobule and at the edge of the liver in the diseased mice. The results of flow cytometry showed that the percentages of CD19+NGFR+cells in spleen of the model mice were gradually increased with passage,which was 19.0％,47.3％ and 61.0％ in G1,G2 and G3 mice,respectively. Immunophenotypic analysis indicated that Ph+cells were stably passaged in B lymphocytes,and the purity of Ph+B lymphocytes was obviously elevated with the increase of passage frequency. Conclusion:In the present study,the primary Ph+cells were successfully obtatined and passaged in vivo,and the B-ALL mouse model was successfully established.

Given its nearly ubiquitous expression on plasma cells and limited expression on essential normal tissue, the G protein-coupled receptor class C group 5 member D (GPRC5D) presents a promising opportunity for utilization as an immunotherapy target in multiple myeloma (MM). The therapeutic strategies targeting GPRC5D, such as bispecific antibodies (BsAbs), chimeric antigen receptor (CAR) T cells, and antibody–drug conjugates (ADCs), have been prominently emphasized in relapsed/refractory MM (R/R MM) in recent years. Further clinical trials are necessary to confirm the long-term efficacy of GPRC5D-targeting immunotherapies alone, explore their potentials co-targeting with other specific antigens, or investigate their combinations with existing treatments to overcome MM resistance. This review provides an overview of current research progress in GPRC5D, encompassing its biological characteristics and translational journey from laboratory to clinical application.

PURPOSE:The aim of this study was to develop a rat model of limbal stem cell deficiency (LSCD) by forcing eye-open at birth (FEOB).METHODS:A total of 200 Sprague-Dawley neonatal rats were randomly divided into the control group and the experimental group, which received eyelid open surgery on postnatal day 1 (P1). Observation time points were defined as P1, P5, P10, P15, and P30. Slit-lamp microscope and corneal confocal microscope were used to observe the clinical features of the model. The eyeballs were collected for hematoxylin and eosin staining and periodic acid-Schiff staining. Proliferating cell nuclear antigen, CD68/polymorphonuclear leukocytes, and cytokeratin 10/12/13 immunostaining were performed, while the ultrastructure of the cornea was observed by scanning electron microscopy. Real-time polymerase chain reactions (PCRs), western blot, and immunohistochemical staining of activin A receptor-like kinase-1/5 were used to analyze the possible pathogenesis.RESULTS:FEOB could successfully induce the typical manifestations of LSCD, including corneal neovascularization, severe inflammation, and corneal opacity. In the FEOB group, goblet cells could be detected in the corneal epithelium by periodic acid-Schiff staining. The expression of cytokeratins was also different between the 2 groups. Furthermore, proliferating cell nuclear antigen immunohistochemical staining revealed the weak proliferation and differentiation ability of limbal epithelial stem cells in the FEOB group. Real-time PCRs, western blot, and immunohistochemical staining of activin A receptor-like kinase-1/activin A receptor-like kinase-5 in the FEOB group showed different expression patterns than those of the control group.CONCLUSIONS:FEOB in rats induces ocular surface changes resembling LSCD in humans, representing a novel model of LSCD.

OBJECTIVE:To investigate the safety and efficacy of intense pulsed light (IPL) in the treatment of severe chronic ocular graft-versus-host disease (coGVHD). METHODS:A prospective cohort study. Seventeen patients with severe coGVHD were selected for inclusion in this study. All subjects were treated with IPL every fortnight together with conventional treatment, observation time points were pre-treatment (W0), 4 weeks post-treatment (W4), 8 weeks post-treatment (W8) and 12 weeks post-treatment (W12). Dry eye related examinations include Tear meniscus height (TMH), Non-invasive break-up time (NIBUT), Schirmer I test, Tear film lipid layer thickness (LLT), Ocular surface staining (OSS) and assessment of meibomian gland. Corneal epithelial cell morphology and inflammatory cell infiltration were analyzed by corneal confocal microscopy, while goblet cell density and squamous epithelial grade were assessed by conjunctival imprinted cytology. RESULTS:Patients did not experience any adverse reactions during the follow-up period. All subjects showed significant improvement in clinical symptoms and most signs after IPL treatment. The corneal confocal microscopy showed that the number of dendritic cells infiltrates in the corneal stroma was significantly reduced after IPL treatment (p < 0.001). Conjunctival blot cytology suggested an increase in the number of conjunctival goblet cells from 5.12 ± 2.71 cells/mm2 before treatment to 22.00 ± 4.58 cells/mm2 after treatment, with a statistically significant difference (p < 0.001). An improvement in conjunctival epithelial cell morphology and a decrease in squamous epithelial grade was also observed. CONCLUSIONS:IPL treatment can effectively increase tear film stability in patients with severe coGVHD without significant side effects.

Objective: To optimize the stimulation and activation system of mouse CD3(+) T cells in vitro and explore the optimal infection time of CD3(+) T cells to establish mouse CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and to also verify its killing effect in vivo and in vitro. Method: Splenic CD3(+)T cells were isolated and purified using magnetic beads, and the cells were cultured in Soluble anti-CD3/CD28, PMA+Ionomycin, and Plated anti-CD3/CD28. Cell activation and apoptosis were assessed by flow cytometry after 8, 24, 48, and 72 hours. ScFv plasmid of mouse CD19 antibody was transfected to plat-E cells to package retrovirus. Activated CD3(+) T cells were infected to construct mouse-specific CD19 chimeric antigen receptor T cells (mCD19 CAR-T) , and mCD19 CAR-T cells were co-cultured with B-cell lymphoma cell line A20 in vitro. The specific toxicity of A20 was detected by flow cytometry, and mCD19 CAR-T cells were infused into the lymphoma mouse model to detect its killing effect and distribution. Results: The activation effect of Plated anti-CD3/CD28 on CD3(+) T cells was superior, with the cells exhibiting good viability 24-48 hours after stimulation. Established mCD19 CAR-T cells with stable efficiency[ (32.27±7.56) % ] were specifically able to kill A20 tumor cells (The apoptosis rate was 24.3% at 48 h) . In vivo detection showed a non-significant decrease in the percentage[ (1.83±0.58) % ] of splenic CD19(+) cells 6 days after mCD19 CAR-T cell infusion. A marked clearance in bone marrow and spleen appeared on day 12 compared with the A20 group, and this difference was statistically significant[spleen: (0.36±0.04) % vs (47.00±13.46) % , P<0.001; bone marrow: (1.82±0.29) % vs (37.30±1.44) % , P<0.0001]. Moreover, mCD19 CAR-T cells were distributed in high proportions in the peripheral blood, spleen, and bone marrow[ (2.90±1.12) % , (4.96±0.80) % , (13.55±1.56) % ]. Conclusion: This study demonstrated an optimized activation system and the optimal infection time of CD3(+) T cells. Furthermore, stable constructed mCD19 CAR-T cells showed a remarkable killing ability in vitro and in vivo.

嵌合抗原受体T细胞(CAR-T细胞)的诞生是人类与肿 瘤的斗争中具有里程碑意义的事件。从首个人工改造TCR 嵌合T细胞的出现并验证其有特异性抗原识别活性,到嵌合 入特异性抗体单链可变区(scFv)片段和共刺激分子区域的 CAR-T细胞成为主流,再到CAR-T细胞被临床公认为一种 安全、有效、持续杀伤肿瘤细胞的免疫细胞疗法,其过程复杂 又艰辛。CAR-T细胞的演变历程是CAR-T细胞结构完善的 历史,亦是CAR-T细胞经历体外实验、动物体内实验最终进 入临床试验的发展史。本文我们就CAR-T细胞的结构演变 及CAR-T细胞的结构创新综述如下。 一、CAR-T细胞结构演变历程 为提高CAR-T细胞的杀伤效率、降低体内不良反应发 生率、杀伤肿瘤效果更持久,研究者们对CAR的结构不断进 行改进和完善,并根据CAR的结构特点对其命名,从最初的 第一代CAR-T细胞发展到目前第四代CAR-T细胞经历了近 30年的时间(图1)。 1. 第一代CAR-T细胞简介:第一代CAR-T细胞指嵌合 入经典的 scFv片段并由铰链区串联胞内信号CD3ζ结构域的 T细胞(图 1)。在将Fv嵌合入TCR恒定区赋予T细胞类抗 体识别特性后,Eshhar团队致力于改良嵌合受体的组成和结 构以提高T细胞的靶向杀伤能力。1993年该团队报道,其设 计了编码TNP抗体重链和轻链的 scFv,分别与TCR-CD3复 合体中胞内ζ信号转导片段和免疫球蛋白Fc受体γ链(FcγR IIIA)结合,构建 scFvRζ和 scFvRγ后转染到T细胞内,使T细 胞同时具备了抗体样直接识别和细胞免疫活性的双重特 征。至此,由 scFv-铰链区-CD3ζ组成的CAR-T细胞基本结 构被广泛研究应用。包含此结构的CAR-T细胞公认为第一 代CAR-T细胞。 2. 第二代CAR-T细胞结构:第一代CAR-T细胞在设计 中并未包含共刺激分子片段,致使其无法完全激活,表现为

NLRP3 is associated with multiple risks in graft-versus-host disease, though unifying principles for these findings remain largely unknown. To explore the effects and mechanisms of the absence of NLRP3 function on hepatic graft-versus-host-disease, we established an allogeneic hematopoietic cell transplantation mice model by infusing bone marrow mononuclear cells and spleno-T cells of the BALB/c mouse into either NLRP3 knockout (NLRP3-/- ) or wild-type C57BL/6 mice. Elevated inflammatory cell infiltration, liver fibrosis, and secretions of alanine aminotransferase (ALT) and aspartate transaminase (AST), together with weight loss, were observed in C57BL/6 recipients after transplantation. However, moderate injury pathology was detected in the liver of NLRP3-/- recipients at day 14, which gradually improved over time. Likewise, proinflammatory cytokine IL-1β, a downstream effecter of NLRP3 inflammasome activation, showed significantly lower expression (P < .05) in the liver of NLRP3-/- recipients relative to C57BL/6 recipients at day 7 and day 21. Moreover, compared with C57BL/6 recipients, the expression of both TNF-α and IL-1β were decreased 3-fold and 4.7-fold, respectively, at day 21 in NLRP3-/- recipients. Interestingly, NLRP1a was expressed at a significantly reduced level in the liver of NLRP3-/- recipients (P < .001). Furthermore, systemic inflammation was analyzed by measuring the concentration of IL-1β and adenosine triphosphate (ATP) in serum. The concentration of IL-1β achieved a maximum at day 14, then decreased at day 21 and day 28 in NLRP3-/- recipients. In contrast, the concentration of IL-1β in C57BL/6 recipients gradually increased from day 7 to day 28. ATP levels reduced from day 7 to day 28 in NLRP3-/- recipients, but were extremely high in C57BL/6 recipients from day 14 to day 28 (P < .01). The decreased levels of P2X7R were connected to less ATP in NLRP3-/- recipients at day 21 and day 28. In conclusion, NLRP3 knockout in recipients could significantly relieve liver injury after transplantation and block the NLRP3 inflammasome pathway, thus providing a promising strategy for the treatment of graft-versus-host disease prophylaxis.

Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) complicated by bone marrow necrosis (BMN) and acute renal insufficiency (ARI) is rare in clinical practice. The aim of the present study was to summarize the clinical characteristics and treatment methods of a case of Ph+ ALL complicated by BMN and ARI. A 23-year-old male patient presented with pyrexia and a 2-month history of bone pain, and was diagnosed with Ph+ ALL complicated with BMN and ARI on peripheral blood tests, blood biochemistry tests, BM smear and fluorescence in situ hybridization. The patient underwent repeated hemodialysis, imatinib combination and maintenance chemotherapy, followed by allogeneic hematopoietic stem-cell transplantation. Some of the clinical signs and symptoms were alleviated, whereas others disappeared, and renal function was restored to normal. BM aspiration confirmed absence of necrosis and null lymphoblasts. Therefore, timely accurate diagnosis and effective treatment are crucial for patients with Ph+ ALL exhibiting potentially fatal complications.

Dihydroartemisinin (DHA) is a derivative of artemisinin. The present study aimed to investigate whether DHA induces apoptosis in the THP-1 human acute monocytic leukemia cell line (AMoL), and to identify the relative molecular mechanisms. The results of the present study demonstrated that the viability of THP-1 cells were inhibited by DHA in a dose- and time-dependent manner, which was accompanied by morphological characteristics associated with apoptosis. After 24 h of 200 µM DHA treatment, the proportion of apoptotic cells was significantly increased compared with the untreated controls (P<0.01). In addition, DHA downregulated the levels of B-cell lymphoma (Bcl)-2, protein kinase B (Akt)1, Akt2 and Akt3 gene expression, and increased the expression of the Bcl-2-associated X protein apoptosis regulator. The protein expression of phospho-Akt and phospho-extracellular signal-regulated kinase (ERK) was also decreased, and the protein expression level of cleaved caspase-3 was increased following treatment with DHA. Therefore, DHA may induce apoptosis in the AMoL THP-1 cell line via currently unknown underlying molecular mechanisms, including the downregulation of ERK and Akt, and the activation of caspase-3.

The purpose of this report was to evaluate the effect of IL1RAP sepecifc CAR-T cell on inducing IL1RAP positive chronic myeloid leukemia cell apoptosis. Leukemic stem cells (LSCs) as the main initiating factor of leukemia relapse still cannot be eradicated thoroughly. Even though Tyrosine kinase inhibitors (TKI) revolutionized the therapy of chronic myeloid leukemia (CML), it alone does not cure this disease for the no response to quiescent LSCs. Human IL1RAP identified by Landberg N and we can be used as a specific surface marker and tumor burden indicator of LSC in CML. Therefore, IL1RAP chimeric antibody receptor (CAR) T cell specific targeting LSCs might be a novel strategy to CML therapy. Here, we demonstrated that IL1RAP CAR T cell showed more powerful cytotoxicity to kill IL1RAP positive CML cell lines than that to directly block IL1RAP expression by shRNA. Furthermore, compared with shRAN group and blank vector treated group, higher rate of apoptosis and lower proliferation of leukemia cells were showed in IL1RAP CAR T cell co-cultured group. In conclusion, in the present study a potential creative therapeutic target to eliminate LSCs and assistant strategy to cure CML was proved.

Objective To compare the efficacy and safety of standard or reduced doses of dexamethasone combined with bortezomib and adriamycin ( PAD) in the treatment of patients with multiple myeloma ( MM) .Methods A total of 96 newly diagnosed patients were intravenously injected with 40 mg (Group PAD1) or 20 mg (Group PAD2) of dexam-ethasone and 10 mg/m2 of adriamycin on Days 1-4, and subcutaneously injected with 1.3 mg/m2 of bortezomib on Days 1, 4, 8, and 11 for four to six courses .Then, the efficacy and adverse reaction were observed .Results After four cour-ses, no statistical difference was found in overall response rate (ORR) and very good partial response (VGPR) between both groups (P>0.05).During one-year follow-up visits, the event free survival (EFS) was 56.7%and 56.4%in Groups PAD1 and PAD2, respectively (P=0.959).The overall survival (OS) was 100%for each gruop.No statistical difference was seen in neutropenia , thrombocytopenia , anemia and bortezomib -induced peripheral neuropathy between the two groups (P>0.05).Patients in Group PAD1 presented markedly higher incidence of infection , abdominal disten-tion and hypokalemia than those in Group PAD2 (P<0.01).Conclusion Compared with a standard dose , a reduced dose of PAD can produce similar clinical efficacy in initially diagnosed MM patients , with better tolerance .

Chronic myeloid leukemia (CML) is a clonal disease from hematopoietic stem cells. Surviving leukemia stem cells (LSCs) and progenitor cells are a potential source for CML relapse and progression. Recent data reported that IL-1 receptor accessory protein (IL1RAP) gene was differentially expressed in CML versus normal stem and progenitor cells. However, whether the level of IL1RAP is associated with clinical phases of CML, and correlations between IL1RAP expression and detections of diagnosis is still unclear. Here we demonstrated that IL1RAP was up-regulated in CD34+ and CD34+ CD38-cells which highly enriched with stem cells. Furthermore, IL1RAP expression in CD34+ CD38-cells was tightly consistent with the generation of BCR-ABL fusion gene and Philadelphia chromosome. Importantly, we found that the level of IL1RAP increased with disease progression from chronic phase (CP) into accelerated phase (AP) and blast phase (BP), which was investigated not only in new diagnosed CML patients but also in patients treated with tyrosine kinase inhibitors (TKI) and hydroxyurea. Negative correlation was detected between IL1RAP expression and neutrophil (NE), whereas no relation was found in white blood cell (WBC), lymphocyte (LY), red blood cell (RBC), platelet (PLT), age or gender of CML patients. In conclusion, we identified IL1RAP as a surface marker of LSCs may be a potential indicator for CML clinical phases.