Cell banking, disease modeling, and cell therapy applications have placed increasing demands on hiPSC technology. Specifically, the high-throughput derivation of footprint-free hiPSCs and their ...expansion in systems that allow scaled production remains technically challenging. Here, we describe a platform for the rapid, parallel generation, selection, and expansion of hiPSCs using small molecule pathway inhibitors in stage-specific media compositions. The platform supported efficient and expedited episomal reprogramming using just OCT4/SOX2/SV40LT combination (0.5%–4.0%, between days 12 and 16) in a completely feeder-free environment. The resulting hiPSCs are transgene-free, readily cultured, and expanded as single cells while maintaining a homogeneous and genomically stable pluripotent population. hiPSCs generated or maintained in the media compositions described exhibit properties associated with the ground state of pluripotency. The simplicity and robustness of the system allow for the high-throughput generation and rapid expansion of a uniform hiPSC product that is applicable to industrial and clinical-grade use.
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•FRM supports highly efficient minimal factor episomal reprogramming•Clonal derivation of hiPSCs through high-resolution single-cell sorting•FMM supports long-term maintenance of genomically stable, transgene-free hiPSCs•Maintenance of homogeneous pluripotent culture resembling the ground state
Cell therapy and banking applications have placed increasing demands on hiPSC technology. Here, Flynn and colleagues demonstrate a simple multiplex selection and maintenance platform utilizing stage-specific small molecule media additives and flow cytometry sorting to derive minimal factor episomal hiPSCs that are genomically stable and resemble ground state pluripotent stem cells. This platform may serve as a path to generate clinically relevant hiPSCs.
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ProTmune is an ex vivo pharmacologically-modulated, next-generation mobilized peripheral blood (mPB) cell graft with FDA fast track designation for the reduction of incidence and severity of acute ...graft versus host disease (GvHD) in patients undergoing allogeneic hematopoietic cell transplantation (HCT).
Here we report the 1-year safety and efficacy data from the Phase 1 stage of PROTECT, an ongoing Phase 1 open-label / Phase 2 blinded, randomized and controlled trial of ProTmune in adult subjects with hematologic malignancy undergoing matched unrelated donor HCT following myeloablative conditioning and receiving standard GvHD prophylaxis of methotrexate and tacrolimus.
Seven subjects were administered ProTmune in the Phase 1 stage of PROTECT. Underlying hematologic diseases included ALL (n=3), AML (n=3) and MDS (n=1). Subjects were predominantly female (n=5) with a median age of 56 (range 34 to 69) and a median weight of 79 kg (range 53 to 133). As of July 23, 2018, five subjects remain on study with a median follow up of 390 days (range 342 to 490 days).
ProTmune was manufactured on-site on the day of HCT by pharmacologically-modulating a conventional mobilized peripheral blood (mPB) cell graft ex vivo with 16,16-dimethyl prostaglandin E2 and dexamethasone, to enhance the biologic properties and therapeutic function of the graft. All ProTmune units were manufactured successfully with a mean of 6.6 (range 3.0 to 11.0) x106 CD34+ cells/kg and 2.3 (range 1.2 to 3.1) x108 CD3+ cells/kg.
ProTmune was well tolerated. ProTmune-related AEs were all mild to moderate and consisted of nausea, vomiting and non-cardiac chest pain on the day of administration. No ProTmune-related SAEs have been reported. All subjects engrafted with no primary or secondary graft failure. Average time to neutrophil engraftment was 17 days (range 14 to 22).
Day 100 acute GvHD assessment revealed three of the seven subjects had steroid responsive acute CIBMTR Grade 2-4 GvHD with median time of seven days range 5 to 8 days to resolution of the maximum GvHD grade.
Since therapeutic approaches intended to reduce acute GvHD may increase disease relapse risk, composite endpoints are being explored by the HCT community to more thoroughly define transplant success. 1-year current GvHD-free, relapse-free survival (CGRFS) is one endpoint under study measuring proportion of subjects that are alive without relapse and without active moderate or severe chronic GvHD per NIH consensus criteria at Day 365.
We intend to present final 1-year CGRFS on the seven Phase 1 subjects at the ASH Annual Meeting. To date, non-relapse mortality, deemed not attributable to ProTmune, occurred in two subjects (Subject 1 on Day 228 from pulmonary edema; Subject 3 on Day 151 from cardiac arrhythmia). There has been no disease relapse, and no moderate / severe chronic GvHD at Day 365. Two subjects are pending the 1-year assessment.
The Phase 2 stage of PROTECT is currently enrolling a planned 60 subjects at 15 U.S. centers. Subjects with hematologic malignancies undergoing matched unrelated donor HCT following myeloablative conditioning are randomized 1:1 to receive in a blinded manner, either ProTmune or a conventional matched unrelated donor mPB unit as the cell graft.
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Maziarz:Athersys, Inc.: Patents & Royalties; Incyte: Consultancy, Honoraria; Kite Therapeutics: Honoraria; Juno Therapeutics: Consultancy, Honoraria; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Saad:Actinium: Consultancy. Diaz:Fate Therapeutics, Inc.: Employment. Medcalf:Fate Therapeutics, Inc.: Employment. Storgard:Fate Therapeutics, Inc.: Employment, Equity Ownership. Deol:Kite Pharmaceuticals: Consultancy; Novartis: Consultancy.
ProTmune, an ex vivo modulated mobilized peripheral blood (mPB) graft, has FDA fast track designation for the reduction of incidence and severity of acute graft-versus-host disease (GvHD) in patients ...undergoing allogeneic hematopoietic cell transplantation (HCT). Despite the use of standard protocols to prevent its occurrence, 50 to 60% of patients receiving an unrelated allogeneic HCT experience acute GvHD within 60 days post-HCT.
Here we report early Phase 1 safety and efficacy data from PROTECT, the ongoing Phase 1 open-label / Phase 2 blinded and randomized controlled trial (RCT) of ProTmune in adult subjects with hematologic malignancy undergoing HCT following myeloablative conditioning using a matched unrelated donor and receiving standard GvHD prophylaxis of methotrexate and tacrolimus.
As of July 31, 2017, five subjects were administered ProTmune in the Phase 1 portion of PROTECT. Underlying hematologic diseases included ALL, AML and MDS. Subjects were predominantly female (80%), with mean age of 54 (range 34 to 66) and mean weight of 75.2 +/- 14.7 kg. All subjects remain on study as of July 31, 2017 with a median follow-up of 53 days (range 33 to 224 days).
ProTmune was manufactured on-site the day of HCT (Day 0) by pharmacologically modulating a mPB graft ex vivo with two small molecules (FT1050 and FT4145) to enhance the biologic properties and therapeutic function of the graft. All manufacturing runs have been successful, maintaining high cell viability (87.9 +/- 10.9%) and CD34+ cell recovery (87.7 +/- 13.5%). Successful pharmacologic modulation was confirmed by the marked increase in the percentage of cells positive for the potency marker CXCR4 (5.0 +/- 0.1% pre-manufacture to 67.3 +/- 7.9% post-manufacture). ProTmune, containing on average 6.8 +/- 3.6 x106 CD34+ cells/kg and 2.5 +/- 0.5 x108 CD3+ cells/kg, was administered on Day 0.
ProTmune has been well tolerated in these five subjects. ProTmune-related AEs were limited to two cases of Grade 1 vomiting on the day of administration. No ProTmune-related SAEs have been reported. All subjects engrafted with no reports of secondary graft failure. Median time to neutrophil engraftment was Day 15 (range Day 14 to Day 21). There have been no disease relapse or deaths.
The primary efficacy endpoint is incidence of acute GvHD through Day 100. In Phase 1, one subject was reported to have an acute GvHD event by Day 100 (limited to skin with complete recovery on methylprednisolone).
The Phase 1 portion of PROTECT is projected to enroll 6-10 subjects. By the 2017 ASH meeting, enrollment will be complete with at least six subjects projected to have reached Day 100+, and the safety and efficacy results will be updated.
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Maziarz:Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria; Juno Therapeutics: Honoraria; Kite Therapeutics: Honoraria; Athersys, Inc: Patents & Royalties. Saad:Spectrum: Honoraria; Actinium: Consultancy. Diaz:Fate Therapeutics, Inc: Employment. Medcalf:Fate Therapeutics, Inc: Employment, Equity Ownership. Fremgen:Fate Therapeutics, Inc.: Employment. Storgard:Fate Therapeutics, Inc: Employment.
Abstract
Adoptive transfer of autologous T cells expressing chimeric antigen receptor (CAR) has shown great promise in the treatment of blood malignancies. Challenges for the application of current ...CAR T cell therapies to broader and more diverse patient populations include inherent variability, cost of manufacture, and the requirement for precise genetic engineering to generate a highly homogenous and consistent CAR T cell product. We have previously reported pre-clinical data supporting the development of FT819, a first-of-kind off-the-shelf CAR T cell product candidate. FT819 is generated from a renewable clonal master human induced pluripotent stem cell (hiPSC) line derived from a single cell engineered to contain bi-allelic disruption of the T cell receptor (TCR) and a novel CD19 CAR targeted into the T cell receptor α constant (TRAC) locus to provide antigen specificity and enhanced efficacy while eliminating the possibility of graft versus host disease. For the manufacture of a clinical-grade FT819 clonal master hiPSC line, we sourced peripheral blood mononuclear cells from a fully consented and eligible donor with protocol overseen by an independent Institutional Review Board. Sourced T cells were enriched (>98%) through positive selection for TCRαβ, and cryopreserved cells were confirmed to have stable genome by karyotyping. Using our proprietary non-integrating cellular reprogramming platform, αβ T cells were reprogrammed into hiPSCs. Concurrently with the reprogramming process, reprogrammed cells received nuclease and donor template to mediate targeting of CD19 CAR into the TRAC locus with bi-allelic knockout of the TCR. To generate clonal lines, engineered cells were sorted by flow cytometry for various markers and single cells were seeded into individual wells of feeder-free 96-well plates. hiPSC clones were screened for bi-allelic integration of CAR into the TRAC locus by amplifying the genomic DNA flanking the homologous recombination site and confirmed by a SNP phasing assay. Clones were further screened for random integration of donor template by quantitative PCR (qPCR), and the CAR copy number was confirmed by droplet digital PCR. Out of 545 hiPSC clones screened, 27 clones (5%) had bi-allelic TRAC targeting with no detectable random integration. Maintenance of pluripotency was confirmed in 19 out of the 27 engineered hiPSC clones (70%). Seventeen clones were further tested and were confirmed to be footprint-free of transgenic reprogramming factors. Of the 18 clones tested for genomic stability, 12 clones had normal karyotypes (67%). Validated, TRAC-targeted hiPSC clones were cryopreserved (~150 vials per clone) and are currently being assessed for off-target editing, differentiation propensity into highly-functional T cells, genomic stability, clone identity, sterility and lack of mycoplasma detection. In summary, using our novel iPSC technology platform for reprogramming, single cell engineering and multiplex high-throughput screening of hiPSCs, we have generated clinical-grade clonal master hiPSC lines in support of our first-of-kind clinical trials evaluating FT819 allogenic off-the-shelf hiPSC-derived TCR-less TRAC-CAR19 T cells for the treatment of blood malignancies.
Citation Format: Ramzey Abujarour, Yi-Shin Lai, Mochtar Pribadi, Tom Lee, Megan Robinson, Chelsea Ruller, Sjoukje Van der Stegen, Xiuyan Wang, Jolanta Stefanski, Juan Zhen, Jason Dinella, Greg Bonello, Janel Huffman, Helen Chu, Raedun Clarke, Alec Witty, Amanda Medcalf, Jaeger Davis, Stacey Moreno, Pieter Lindenbergh, Isabelle Riviere, Michel Sadelain, Bahram Valamehr. Generation of novel single cell-derived engineered master pluripotent cell line as a renewable source for off-the-shelf TCR-less CAR T cells in support of first-of-kind clinical trial abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-073.
Umbilical cord blood (UCB) offers many potential advantages as a source of hematopoietic stem cells (HSCs) for allogeneic transplantation, including ease of collection, rapid availability, ...flexibility of HLA-matching, lower rates of GvHD and potentially lower relapse rates. However, the low HSC content of UCB compared to other graft sources results in a prolonged time to engraftment, and higher rates of graft failure and early mortality.
Pulse ex vivo exposure of HSCs to 16,16-dimethyl PGE2 (FT1050) has been demonstrated to enhance HSC engraftment potential, which could benefit clinical UCB transplant. FT1050 modulation promotes multiple mechanisms, including increased proliferation, reduced apoptosis, and improved migration and homing North 2007&2009; Hoggatt 2009. Improved HSC homing is mediated by induction of CXCR4 gene expression leading to increased cell surface CXCR4. Further optimization of the UCB modulation process demonstrated that incubation with 10µM FT1050 for 2 hrs at 37C resulted in a maximal biological response of the FT1050-UCB (ProHema®).
A Phase 1 trial was performed to evaluate the safety of FT1050-UCB paired with an unmanipulated UCB unit in reduced-intensity double UCBT (dUCBT) Cutler 2013. We observed durable, multi-lineage engraftment of FT1050-UCB with acceptable safety. Earlier neutrophil engraftment was observed relative to historical controls (median 17.5 vs. 21 days (historical control), p=0.045), coupled with preferential engraftment of the FT1050-UCB unit in 10 of 12 subjects. A Phase 2 multi-center clinical trial of FT1050-UCB in adult patients undergoing dUCBT for hematologic malignancies was then initiated. Subjects are randomized 2:1 to FT1050-UCB-containing vs. standard dUCBT after high-dose conditioning. The primary endpoint is a categorical analysis of neutrophil engraftment using a pre-specified control median. Data on the initial 11 subjects, of which 8 were randomized to receive FT1050-UCB, continue to demonstrate acceptable safety with adverse events attributed to FT1050-UCB limited primarily to common infusion-related side effects. Of the 8 FT1050-UCB subjects, 1 died prior to neutrophil engraftment, with the remaining 7 subjects engrafting at a median of 28 days vs. 31 days for the 3 control subjects. With median overall follow-up of 16.1 months, 4 of 8 subjects on the FT1050-UCB arm are alive with a median survival not reached (> 11.0 months). 1 of 3 control subjects is alive with median survival of 6.0 months.
During the clinical translation process, the media used during FT1050 modulation of UCB was identified as a key variable. Standard UCB washing media, consisting of a nutrient-free saline solution of low molecular weight dextran and human serum albumin (LMD/HSA), is used clinically to stabilize fragile cells post-thaw by reducing lysis. This media was used in the Phase 1 trial and to initiate Phase 2. Early during the Phase 2 trial, we identified a novel cell-stabilizing nutrient-rich formulation (NRM), containing glucose, amino acids and other HSC-supporting nutrients that promoted full FT1050 modulation of UCB and increased cell viability. The expression of key FT1050-pathway genes was significantly higher with NRM compared to intermediate levels observed with LMD/HSA. Modulation of human CD34+ (hCD34+) cells with FT1050 in NRM led to an 8-fold increase over LMD/HSA in induced CXCR4 gene expression (20-fold total), which translated to significantly increased surface CXCR4 protein. In vivo homing models demonstrated that UCB CD34+ cells modulated with FT1050 in NRM resulted in a 2.2-fold homing increase relative to vehicle (p < 0.001) compared to a 1.6-fold increase with LMD/HSA (p = 0.002), with a significant difference between the two media conditions (p = 0.04). A xenotransplantation study in NSG mice with hCD34+ cells modulated with FT1050 in either NRM or LMD/HSA demonstrated a 2-fold increase in circulating hCD45+ cells 12-weeks post-transplant with NRM (p = 0.007; unpaired t-test). These findings supported the incorporation of NRM into the FT1050-UCB manufacturing process in order to further improve its clinical engraftment potential. Enrollment of a 60-patient Phase 2 trial has been initiated that incorporates this manufacturing change.
Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics: Employment. Guerrettaz:Fate Therapeutics: Employment. Robbins:Fate Therapeutics: Employment. Medcalf:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment, Equity Ownership. Ferraro:Fate Therapeutics: Employment. Multani:Fate Therapeutics: Employment.
Rho-kinase (ROCK) inhibition, broadly utilised in cardiovascular disease, may protect the blood-brain barrier (BBB) during thrombolysis from rt-PA-induced damage. While the use of nonselective ROCK ...inhibitors like fasudil together with rt-PA may be hindered by possible hypotensive side-effects and inadequate capacity to block detrimental rt-PA activity in brain endothelial cells (BECs), selective ROCK-2 inhibition may overcome these limitations. Here, we examined ROCK-2 expression in major brain cells and compared the ability of fasudil and KD025, a selective ROCK-2 inhibitor, to attenuate rt-PA-induced BBB impairment in an in vitro human model. ROCK-2 was highly expressed relative to ROCK-1 in all human and mouse brain cell types and particularly enriched in rodent brain endothelial cells and astrocytes compared to neurons. KD025 was more potent than fasudil in attenuation of rt-PA- and plasminogen-induced BBB permeation under normoxia, but especially under stroke-like conditions. Importantly, only KD025, but not fasudil, was able to block rt-PA-dependent permeability increases, morphology changes and tight junction degradation in isolated BECs. Selective ROCK-2 inhibition further diminished rt-PA-triggered myosin phosphorylation, shape alterations and matrix metalloprotease activation in astrocytes. These findings highlight ROCK-2 as the key isoform driving BBB impairment and brain endothelial damage by rt-PA and the potential of KD025 to optimally protect the BBB during thrombolysis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Traumatic brain injury (TBI) leaves many survivors with long-term disabilities. A prolonged immune response in the brain may cause neurodegeneration, resulting in chronic neurological disturbances. ...In this study, using a TBI mouse model, we correlate changes in the local immune response with neurodegeneration/neurological dysfunction over an 8-month period. Flow cytometric analysis reveals a protracted increase in effector/memory CD8+ T cells (expressing granzyme B) in the injured brain. This precedes interleukin-17+CD4+ T cell infiltration and is associated with progressive neurological/motor impairment, increased circulating brain-specific autoantibodies, and myelin-related pathology. Genetic deficiency or pharmacological depletion of CD8+ T cells, but not depletion of CD4+ T cells, improves neurological outcomes and produces a neuroprotective Th2/Th17 immunological shift, indicating a persistent detrimental role for cytotoxic T cells post-TBI. B cell deficiency results in severe neurological dysfunction and a heightened immune reaction. Targeting these adaptive immune cells offers a promising approach to improve recovery following TBI.
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•Granzyme B+ CD8+ T cells accumulate in the brain after traumatic brain injury (TBI)•Brain CD8+ T cells contribute to chronic motor deficits and myelin pathology•Deficiency/depletion of CD8+ T cells promotes neurological recovery following TBI•B cells and autoreactive antibodies appear to play a regulatory role in TBI
Daglas et al. show that granzyme B+CD8+ T cells accumulate in the brain after traumatic brain injury, triggering chronic neurological/motor impairment and myelin pathology. Genetic deficiency/pharmacological depletion of CD8+ T cells, but not B cells, promotes recovery post-injury and offers a therapeutic option to minimize long-term disabilities in trauma patients.
Removal of dead cells in the absence of concomitant immune stimulation is essential for tissue homeostasis. We recently identified an injury-induced protein misfolding event that orchestrates the ...plasmin-dependent proteolytic degradation of necrotic cells. As impaired clearance of dead cells by the innate immune system predisposes to autoimmunity, we determined whether plasmin could influence endocytosis and immune cell stimulation by dendritic cells - a critical cell that links the innate and adaptive immune systems. We find that plasmin generated on the surface of necrotic cells enhances their phagocytic removal by human monocyte-derived dendritic cells. Plasmin also promoted phagocytosis of protease-resistant microparticles by diverse mouse dendritic cell sub-types both in vitro and in vivo. Together with an increased phagocytic capacity, plasmin-treated dendritic cells maintain an immature phenotype, exhibit reduced migration to lymph nodes, increase their expression/release of the immunosuppressive cytokine TGF-β, and lose their capacity to mount an allogeneic response. Collectively, our findings support a novel role for plasmin formed on dead cells and other phagocytic targets in maintaining tissue homeostasis by increasing the phagocytic function of dendritic cells while simultaneously decreasing their immunostimulatory capacity consistent with producing an immunosuppressive state.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cellular injury causes a myriad of processes that affect proteostasis. We describe nucleocytoplasmic coagulation (NCC), an intracellular disulfide-dependent protein crosslinking event occurring upon ...late-stage cell death that orchestrates the proteolytic removal of misfolded proteins. In vitro and in vivo models of neuronal injury show that NCC involves conversion of soluble intracellular proteins, including tubulin, into insoluble oligomers. These oligomers, also seen in human brain tissue following neurotrauma, act as a cofactor and substrate for the plasminogen-activating system. In plasminogen−/− mice, levels of misfolded β-tubulin were elevated and its clearance delayed following neurotrauma, demonstrating a requirement for plasminogen in the removal of NCC constituents. While additional in vivo studies will further dissect this phenomenon, our study clearly shows that NCC, a process analogous to the formation of thrombi, generates an aggregated protein scaffold that limits release of cellular components and recruits clearance mechanisms to the site of injury.
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► Cell death triggers the process of nucleocytoplasmic coagulation (NCC) ► NCC is the abrupt misfolding and disulfide crosslinking of intracellular proteins ► NCC-aggregated proteins facilitate plasmin formation and subsequent proteolysis ► Thus, NCC limits release and promotes proteolytic clearance of dead cell debris
During cell death, intracellular proteins become exposed to the extracellular environment, undergo conformational change, and are ultimately cleared. Samson, Medcalf, and colleagues now describe a protein aggregation event during the late stage of cell death referred to as nucleocytoplasmic coagulation (NCC). NCC results in the formation of insoluble oligomers that are dependent upon disulfide crosslinking. NCC is shown to provide a template mechanism for plasmin-dependent removal of NCC constituents in a process analogous to the proteolytic removal of blood clots.
Background
Traumatic brain injury (TBI) is known to promote immunosuppression, making patients more susceptible to infection, yet potentially exerting protective effects by inhibiting central nervous ...system (CNS) reactivity. Plasmin, the effector protease of the fibrinolytic system, is now recognized for its involvement in modulating immune function.
Objective
To evaluate the effects of plasmin and tranexamic acid (TXA) on the immune response in wild‐type and plasminogen‐deficient (plg−/−) mice subjected to TBI.
Methods
Leukocyte subsets in lymph nodes and the brain in mice post TBI were evaluated by flow cytometry and in blood with a hemocytometer. Immune responsiveness to CNS antigens was determined by Enzyme‐linked Immunosorbent Spot (ELISpot) assay. Fibrinolysis was determined by thromboelastography and measuring D‐dimer and plasmin‐antiplasmin complex levels.
Results
Plg−/− mice, but not plg+/+ mice displayed increases in both the number and activation of various antigen‐presenting cells and T cells in the cLN 1 week post TBI. Wild‐type mice treated with TXA also displayed increased cellularity of the cLN 1 week post TBI together with increases in innate and adaptive immune cells. These changes occurred despite the absence of systemic hyperfibrinolysis or coagulopathy in this model of TBI. Importantly, neither plg deficiency nor TXA treatment enhanced the autoreactivity within the CNS.
Conclusion
In the absence of systemic hyperfibrinolysis, plasmin deficiency or blockade with TXA increases migration and proliferation of conventional dendritic cells (cDCs) and various antigen‐presenting cells and T cells in the draining cervical lymph node (cLN) post TBI. Tranexamic acid might also be clinically beneficial in modulating the inflammatory and immune response after TBI, but without promoting CNS autoreactivity.
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