Ccrk-Mak/Ick signaling is a ciliary transport regulator essential for retinal photoreceptor survival.

Primary cilia are microtubule-based sensory organelles whose dysfunction causes ciliopathies in humans. The formation, function, and maintenance of primary cilia depend crucially on intraflagellar transport (IFT); however, the regulatory mechanisms of IFT at ciliary tips are poorly understood. Here, we identified that the ciliopathy kinase Mak is a ciliary tip-localized IFT regulator that cooperatively acts with the ciliopathy kinase Ick, an IFT regulator. Simultaneous disruption of Mak and Ick resulted in loss of photoreceptor ciliary axonemes and severe retinal degeneration. Gene delivery of Ick and pharmacological inhibition of FGF receptors, Ick negative regulators, ameliorated retinal degeneration in Mak -/- mice. We also identified that Ccrk kinase is an upstream activator of Mak and Ick in retinal photoreceptor cells. Furthermore, the overexpression of Mak, Ick, and Ccrk and pharmacological inhibition of FGF receptors suppressed ciliopathy-related phenotypes caused by cytoplasmic dynein inhibition in cultured cells. Collectively, our results show that the Ccrk-Mak/Ick axis is an IFT regulator essential for retinal photoreceptor maintenance and present activation of Ick as a potential therapeutic approach for retinitis pigmentosa caused by MAK mutations.

A case of bilateral absent pulmonary arteries.

Herein, we present the case of a 17-month-old boy with asplenia, bilaterally absent pulmonary arteries, and bilateral patent ductus arteriosus, who underwent a successful Fontan operation. The central pulmonary artery was created using a pericardial roll, which was initially oversized due to the elevated pressure from the systemic-to-pulmonary shunt. The size of the roll was reduced through the process of pressure reduction by bidirectional Glenn and Fontan operations. This case provides an example of blood source-associated size transition of the pericardial roll used in the pulmonary position. Recognizing this phenomenon is vital for the successful management of this patient group.

NR4A ablation improves mitochondrial fitness for long persistence in human CAR-T cells against solid tumors.

BACKGROUND: Antitumor effect of chimeric antigen receptor (CAR)-T cells against solid tumors is limited due to various factors, such as low infiltration rate, poor expansion capacity, and exhaustion of T cells within the tumor. NR4A transcription factors have been shown to play important roles in T-cell exhaustion in mice. However, the precise contribution of each NR4a factor to human T-cell differentiation remains to be clarified. METHODS: In this study, we deleted NR4A family factors, NR4A1, NR4A2, and NR4A3, in human CAR-T cells recognizing human epidermal growth factor receptor type 2 (HER2) by using the CRISPR/Cas9 system. We induced T-cell exhaustion in these cells in vitro through repeated co-culturing of CAR-T cells with Her2+A549 lung adenocarcinoma cells and evaluated cell surface markers such as memory and exhaustion phenotypes, proliferative capacity, cytokine production and metabolic activity. We validated the antitumor toxicity of NR4A1/2/3 triple knockout (TKO) CAR-T cells in vivo by transferring CAR-T cells into A549 tumor-bearing immunodeficient mice. RESULTS: Human NR4A-TKO CAR-T cells were resistant against exhaustion induced by repeated antigen stimulation in vitro, and maintained higher tumor-killing activity both in vitro and in vivo compared with control CAR-T cells. A comparison of the effectiveness of NR4A single, double, and TKOs demonstrated that triple KO was the most effective in avoiding exhaustion. Furthermore, a strong enhancement of antitumor effects by NR4A TKO was also observed in T cells from various donors including aged persons. Mechanistically, NR4A TKO CAR-T cells showed enhanced mitochondrial oxidative phosphorylation, therefore could persist for longer periods within the tumors. CONCLUSIONS: NR4A factors regulate CAR-T cell persistence and stemness through mitochondrial gene expression, therefore NR4A is a highly promising target for the generation of superior CAR-T cells against solid tumors.

Secondary ileal lymph node metastases from rectal cancer: a case report.

BACKGROUND: Colorectal cancer can invade adjacent organs, but rarely metastasizes to the regional lymph nodes (LNs) of the invaded organ. Herein, we report a case of rectal cancer invading the ileum and metastasized to the regional ileal LNs. CASE PRESENTATION: A 77-year-old male presented abdominal pain and anorexia, diagnosed with rectal cancer invading the small intestine and concurrently metastasized to the regional LN of the intestine and liver. High anterior resection and partial resection of the small intestine was performed, then, the patient was referred to our hospital for chemotherapy. We performed 17 cycles of systemic chemotherapy that achieved a partial reduction in size of the LN, followed by an ileocecal resection with ileal mesentery resection for regional LNs removal. Histopathological analysis of the resected ileal LNs and six liver lesions revealed a moderately differentiated tubular adenocarcinoma. The patient was discharged on postoperative day 18. Cancer recurrences developed in the lungs 5 months after the surgery, then to the liver and peritoneum, and further surgery and chemotherapy were performed. Despite the challenging presentation, the patient survived for 40 months after the first surgery. CONCLUSIONS: We report a rare case of a surgical resection of a secondary ileal LN metastasis from rectal cancer. The patient survives for a relatively long time after surgical resection. When colorectal cancer invades the small intestine, clinicians should consider the possibility of secondary LN metastasis in the invaded site.

Internal thoracic artery as a growing conduit for pulmonary shunt.

Studies suggest the internal thoracic artery as a shunt option due to its growth potential. However, long-term data are lacking. Here, a patient with a failing single ventricle shunt had an enlarged internal thoracic artery. We followed the patient for 12 years after converting this artery into a Blalock-Taussig shunt, analysing its growth to assess its effectiveness.

A phase 1 study of the irreversible FLT3 inhibitor FF-10101 in relapsed or refractory acute myeloid leukemia.

ABSTRACT: FLT3 tyrosine kinase inhibitors (TKIs) have clinical efficacy for patients with FLT3-mutated AML (acute myeloid leukemia), but their impact is limited by resistance in the setting of monotherapy and by tolerability problems when used in combination therapies. FF-10101 is a novel compound that covalently binds to a cysteine residue near the active site of FLT3, irreversibly inhibiting receptor signaling. It is effective against most FLT3 activating mutations, and, unlike other inhibitors, is minimally vulnerable to resistance induced by FLT3 ligand. We conducted a phase 1 dose escalation study of oral FF-10101 in patients with relapsed and/or refractory AML, the majority of whom harbored FLT3-activating mutations and/or had prior exposure to FLT3 inhibitors. Fifty-four participants enrolled in cohorts receiving doses ranging from 10 to 225 mg per day and 50 to 100 mg twice daily (BID). The dose limiting toxicities were diarrhea and QT prolongation. Among 40 response-evaluable participants, the composite complete response rate was 10%, and the overall response rate (including partial responses) was 12.5%, including patients who had progressed on gilteritinib. Overall, 56% of participants had prior exposure to FLT3 inhibitors. The recommended phase 2 dose was 75 mg BID. FF-10101 potentially represents a next-generation advance in the management of FLT3-mutated AML. This trial was registered at www.ClinicalTrials.gov as #NCT03194685.

NR4a1/2 deletion promotes accumulation of TCF1+ stem-like precursors of exhausted CD8+ T cells in the tumor microenvironment.

T cell exhaustion impairs tumor immunity and contributes to resistance against immune checkpoint inhibitors. The nuclear receptor subfamily 4 group A (NR4a) family of nuclear receptors plays a crucial role in driving T cell exhaustion. In this study, we observe that NR4a1 and NR4a2 deficiency in CD8+ tumor-infiltrating lymphocytes (TILs) results in potent tumor eradication and exhibits not only reduced exhaustion characteristics but also an increase in the precursors/progenitors of exhausted T (Pre-Tex) cell fraction. Serial transfers of NR4a1-/-NR4a2-/-CD8+ TILs into tumor-bearing mice result in the expansion of TCF1+ (Tcf7+) stem-like Pre-Tex cells, whereas wild-type TILs are depleted upon secondary transfer. NR4a1/2-deficient CD8+ T cells express higher levels of stemness/memory-related genes and illustrate potent mitochondrial oxidative phosphorylation. Collectively, these findings suggest that inhibiting NR4a in tumors represents a potent immuno-oncotherapy strategy by increasing stem-like Pre-Tex cells and reducing exhaustion of CD8+ T cells.

External Esophageal Stenting Technique in Palliation for Tracheal Agenesis in a Case of Esophageal Lung: A Lesson Learned from the Experience for Tracheomalacia.

Tracheal agenesis (TA) is a rare congenital anomaly with an incidence of 1 per 50,000 newborns. It appears at birth with severe respiratory distress, cyanosis, and inaudible crying. Prompt esophageal intubation and long-term management of the esophageal airway are essential to overcome this catastrophic condition. In the long-term management, external stenting of the esophageal airway has been reported as promising to support the fragile esophageal wall; this technique was taken from the surgery for tracheomalacia. We experienced a case of an infant with tracheal agenesis whose respiratory status was stabilized after external esophageal stenting. The stenting was performed based on a lesson learned in the extensive experience in the surgical treatment for tracheomalacia, and the surgical techniques for successful stenting are herein described.

The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina.

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen-induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokinesignaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis.

SOCS3 deletion in effector T cells confers an anti-tumorigenic role of IL-6 to the pro-tumorigenic cytokine.

Interleukin (IL)-6 is abundantly expressed in the tumor microenvironment and is associated with poor patient outcomes. Here, we demonstrate that the deletion of the suppressor of cytokine signaling 3 (SOCS3) in T cells potentiates anti-tumor immune responses by conferring the anti-tumorigenic function of IL-6 in mouse and human models. In Socs3-deficient CD8+ T cells, IL-6 upregulates the expression of type I interferon (IFN)-regulated genes and enhances the anti-tumor effector function of T cells, while also modifying mitochondrial fitness to increase mitochondrial membrane potential and reactive oxygen species (ROS) levels and to promote metabolic glycolysis in the energy state. Furthermore, Socs3 deficiency reduces regulatory T cells and increases T helper 1 (Th1) cells. SOCS3 knockdown in human chimeric antigen receptor T (CAR-T) cells exhibits a strong anti-tumor response in humanized mice. Thus, genetic disruption of SOCS3 offers an avenue to improve the therapeutic efficacy of adoptive T cell therapy.

Memory B Cells and Memory T Cells Induced by SARS-CoV-2 Booster Vaccination or Infection Show Different Dynamics and Responsiveness to the Omicron Variant.

Although the immunological memory produced by BNT162b2 vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been well studied and established, further information using different racial cohorts is necessary to understand the overall immunological response to vaccination. We evaluated memory B and T cell responses to the severe acute respiratory syndrome coronavirus 2 spike protein before and after the third booster using a Japanese cohort. Although the Ab titer against the spike receptor-binding domain (RBD) decreased significantly 8 mo after the second vaccination, the number of memory B cells continued to increase, whereas the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the Ab titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, whereas memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR6- circulating follicular Th1 was positively correlated with RBD-specific Ab-secreting B cells. For the response to variant RBDs, although 60-80% of memory B cells could bind to the omicron RBD, their avidity was low, whereas memory T cells show an equal response to the omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate Ab production and T cell responses after omicron strain infection, which prevents severe illness and death due to coronavirus disease 2019.

A protein kinase D inhibitor suppresses AKT on T cells and antagonizes cancer immunotherapy by anti-PD-1.

Antibodies that block the interaction between PD-1 and PD-1 ligands (anti-PD-1) are in clinical use for the treatment of cancer, yet their efficacy is limited. Pre-approved therapies that enhance the effect of anti-PD-1 in combination are beneficial. Small-molecule inhibitors that attenuate T cell receptor signaling are reported to prevent T cell exhaustion and induce memory T cells with stem cell potential, resulting in a durable effector T cell response in combination with anti-PD-1. In search of such targets, we focused on protein kinase D (PKD), which is suggested to be suppressive in both tumor growth and TCR signaling. We report that CRT0066101, a PKD inhibitor (PKDi), suppressed the growth of mouse tumors at a sub-micromolar concentration in vitro. Despite its inhibitory effects on tumors, a single treatment of tumor-bearing mice with PKDi did not inhibit, but rather accelerated tumor growth, and reversed the therapeutic effect of anti-PD-1. Mice treated with PKDi showed reduced T cell infiltration and defects in the generation of effector T cells, compared to those treated with anti-PD-1, suggesting that PKDi inhibited ongoing antitumor responses. Mechanistically, PKDi inhibited phosphorylation of AKT, a primary checkpoint that is reactivated by anti-PD-1. In conclusion, PKD is fundamentally required for T cell reactivation by anti-PD-1; therefore, inhibition of PKD is not appropriate for combination therapy with anti-PD-1. On the other hand, a single dose of PKDi was shown to strongly suppress experimental autoimmunity in mice, indicating that PKDi could be useful for the treatment of immune-related adverse events that are frequently reported in anti-PD-1 therapy.

The Irreversible FLT3 Inhibitor FF-10101 Is Active Against a Diversity of FLT3 Inhibitor Resistance Mechanisms.

Small-molecule FLT3 inhibitors have recently improved clinical outcomes for patients with FLT3-mutant acute myeloid leukemia (AML) after many years of development, but resistance remains an important clinical problem. FF-10101 is the first irreversible, covalent inhibitor of FLT3 which has previously shown activity against FLT3 tyrosine kinase inhibitor resistance-causing FLT3 F691L and D835 mutations. We report that FF-10101 is also active against an expanded panel of clinically identified FLT3 mutations associated with resistance to other FLT3 inhibitors. We also demonstrate that FF-10101 can potentially address resistance mechanisms associated with growth factors present in the bone marrow microenvironment but is vulnerable to mutation at C695, the amino acid required for covalent FLT3 binding. These data suggest that FF-10101 possesses a favorable resistance profile that may contribute to improved single-agent efficacy when used in patients with FLT3-mutant AML.

SOCS: negative regulators of cytokine signaling for immune tolerance.

Cytokines are important intercellular communication tools for immunity. Many cytokines promote gene transcription and proliferation through the JAK/STAT (Janus kinase/signal transducers and activators of transcription) and the Ras/ERK (GDP/GTP-binding rat sarcoma protein/extracellular signal-regulated kinase) pathways, and these signaling pathways are tightly regulated. The SOCS (suppressor of cytokine signaling) family members are representative negative regulators of JAK/STAT-mediated cytokine signaling and regulate the differentiation and function of T cells, thus being involved in immune tolerance. Human genetic analysis has shown that SOCS family members are strongly associated with autoimmune diseases, allergy and tumorigenesis. SOCS family proteins also function as immune-checkpoint molecules that contribute to the unresponsiveness of T cells to cytokines.

[Impending Paradoxical Embolization in a Patient with a Thrombus Straddling the Patent Foramen Ovale].

The patent foramen ovale (PFO) is known as a risk of paradoxical embolism in patients with deep venous thromboses. However, PFO is usually found after systemic embolic symptoms become apparent. A 60-year-old male had complained of dyspnea for two weeks. Ultrasound echocardiography showed a thrombus straddling PFO, and venous echography showed blood clots in the right popliteal and soleus veins. Contrast computed tomography revealed multiple pulmonary embolisms and a thrombus in the right atrium expanding to the left atrium through the atrial septum. The straddling thrombus in the atrium and pulmonary thrombi were extirpated under circulatory arrest with deep hypothermia. An inferior vena cava filter was inserted intravenously four days after surgery. The patient was discharged on the 19th postoperative day without any signs of thromboembolism. Prompt surgery is considered important to prevent thromboembolism in the case of impending paradoxical embolism.

Rejuvenating Effector/Exhausted CAR T Cells to Stem Cell Memory-Like CAR T Cells By Resting Them in the Presence of CXCL12 and the NOTCH Ligand.

T cells with a stem cell memory (TSCM) phenotype provide long-term and potent antitumor effects for T-cell transfer therapies. Although various methods for the induction of TSCM-like cells in vitro have been reported, few methods generate TSCM-like cells from effector/exhausted T cells. We have reported that coculture with the Notch ligand-expressing OP9 stromal cells induces TSCM-like (iTSCM) cells. Here, we established a feeder-free culture system to improve iTSCM cell generation from expanded chimeric antigen receptor (CAR)-expressing T cells; culturing CAR T cells in the presence of IL7, CXCL12, IGF-I, and the Notch ligand, hDLL1. Feeder-free CAR-iTSCM cells showed the expression of cell surface markers and genes similar to that of OP9-hDLL1 feeder cell-induced CAR-iTSCM cells, including the elevated expression of SCM-associated genes, TCF7, LEF1, and BCL6, and reduced expression of exhaustion-associated genes like LAG3, TOX, and NR4A1. Feeder-free CAR-iTSCM cells showed higher proliferative capacity depending on oxidative phosphorylation and exhibited higher IL2 production and stronger antitumor activity in vivo than feeder cell-induced CAR-iTSCM cells. Our feeder-free culture system represents a way to rejuvenate effector/exhausted CAR T cells to SCM-like CAR T cells. Significance: Resting CAR T cells with our defined factors reprograms exhausted state to SCM-like state and enables development of improved CAR T-cell therapy.

CD19-targeted CAR regulatory T cells suppress B cell pathology without GvHD.

Regulatory T cells (Tregs) play essential roles in maintaining immunological self-tolerance and preventing autoimmunity. The adoptive transfer of antigen-specific Tregs has been expected to be a potent therapeutic method for autoimmune diseases, severe allergy, and rejection in organ transplantation. However, effective Treg therapy has not yet been established because of the difficulty in preparing a limited number of antigen-specific Tregs. Chimeric antigen receptor (CAR) T cells have been shown to be a powerful therapeutic method for treating B cell lymphomas, but application of CAR to Treg-mediated therapy has not yet been established. Here, we generated CD19-targeted CAR (CD19-CAR) Tregs from human PBMCs (hPBMCs) and optimized the fraction of the Treg source as CD4+CD25+CD127loCD45RA+CD45RO-. CD19-CAR Tregs could be expanded in vitro while maintaining Treg properties, including high expression of the latent form of TGF-ß. CD19-CAR Tregs suppressed IgG antibody production and differentiation of B cells via a TGF-ß-dependent mechanism. Unlike conventional CD19-CAR CD8+ T cells, CD19-CAR Tregs suppressed antibody production in immunodeficient mice that were reconstituted with hPBMCs, reducing the risk of graft-versus-host disease. Therefore, the adoptive transfer of CD19-CAR Tregs may provide a novel therapeutic method for treating autoantibody-mediated autoimmune diseases.

Vascular compression of the left bronchus in a patient with funnel chest.

We present a case of 5 year-old female with funnel chest, by which cardiovascular structures were displaced into the left chest cavity. This caused left bronchial compression by the pulmonary artery and the descending aorta, for which an external stenting was successfully performed. The patient had features suggestive of skeletal dysplasia with tracheobronchial malacia. One possible mechanism of vascular compression could be an association of fragility of the thoracic cavity and inspiratory dyspnea, causing thoracic deformity.

Surgical repair in a patient with Berry syndrome.

Berry syndrome is a trilogy involving an aorto-pulmonary window, right pulmonary artery from ascending aorta and an interrupted aortic arch. Surgical repair for this anomaly is associated with a high mortality rate and incidence of right pulmonary arterial stenosis. Herein, we present the case of a patient with Berry syndrome, who underwent a rapid two-stage repair involving bilateral pulmonary arterial banding and complete repair. Postoperative computed tomography scan showed well-repaired great vessels free of obstruction.

In Vitro Generation of Stem Cell Memory-Like T Cells from Activated T Cells.

Adoptive T-cell therapy is an attractive strategy for cancer immunotherapy. The transfer of in vitro expanded tumor-associated antigen (TAA)-specific T cells from patients may effectively fight against the original tumor cells. The chimeric antigen receptor-engineered T (CAR-T) cells are also shown to be a promising therapy for hematologic malignancies. However, one of the limitations of these T-cell-based therapies is a rapid acquisition of tolerant (anergy, deletion, dysfunctional and/or exhausted) phenotypes of T cells during activation in vitro and/or after transfer in vivo. We and others found that stem cell memory T (TSCM) cells are strongly resistant against such tolerance, showing strong expansion and persistence in vivo, and provide long-lasting antitumor effects. Here we describe a protocol for the generation of phenotypically TSCM-like cells (iTSCM cells), which can be induced by simple co-culture of activated T cells with OP9 stroma cells expressing a Notch ligand. We also showed the methods of cancer immunotherapy by using NSG mice.