Anthony R. Lanfranco

CTLA-4 and PD-1 Receptors Inhibit T-Cell Activation by Distinct Mechanisms

ABSTRACT CTLA-4 and PD-1 are receptors that negatively regulate T-cell activation. Ligation of both CTLA-4 and PD-1 blocked CD3/CD28-mediated upregulation of glucose metabolism and Akt activity, but each accomplished this regulation using separate mechanisms. CTLA-4-mediated inhibition of Akt phosphorylation is sensitive to okadaic acid, providing direct evidence that PP2A plays a prominent role in mediating CTLA-4 suppression of T-cell activation. In contrast, PD-1 signaling inhibits Akt phosphorylation by preventing CD28-mediated activation of phosphatidylinositol 3-kinase (PI3K). The ability of PD-1 to suppress PI3K/AKT activation was dependent upon the immunoreceptor tyrosine-based switch motif located in its cytoplasmic tail, adding further importance to this domain in mediating PD-1 signal transduction. Lastly, PD-1 ligation is more effective in suppressing CD3/CD28-induced changes in the T-cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct cellular phenotypes. Together, these data suggest that CTLA-4 and PD-1 inhibit T-cell activation through distinct and potentially synergistic mechanisms.

B and T Lymphocyte Attenuator-Mediated Signal Transduction Provides a Potent Inhibitory Signal to Primary Human CD4 T Cells That Can Be Initiated by Multiple Phosphotyrosine Motifs

The B and T lymphocyte attenuator (BTLA) is a recently identified member of the CD28 family of cell receptors. Initial reports demonstrated that mice deficient in BTLA expression were more susceptible to experimental autoimmune encephalomyelitis, indicating that BTLA was likely to function as a negative regulator of T cell activation. However, cross-linking of BTLA only resulted in a 2-fold reduction of IL-2 production, questioning the potency with which BTLA engagement blocks T cell activation. We established a model in which BTLA signaling could be studied in primary human CD4 T cells. We observed that cross-linking of a chimeric receptor consisting of the murine CD28 extracellular domain and human BTLA cytoplasmic tail potently inhibits IL-2 production and completely suppresses T cell expansion. Mutation of any BTLA tyrosine motifs had no effect on the ability of BTLA to block T cell activation. Only mutation of all four tyrosines rendered the BTLA cytoplasmic tail nonfunctional. We performed structure-function studies to determine which factors recruited to the BTLA cytoplasmic tail correlated with BTLA function. Using pervanadate as a means to phosphorylate the BTLA cytoplasmic tail, we observed both Src homology protein (SHP)-1 and SHP-2 recruitment. However, upon receptor engagement, we observed only SHP-1 recruitment, and mutations that abrogated SHP-1 recruitment did not impair BTLA function. These studies question whether SHP-1 or SHP-2 have any role in BTLA function and caution against the use of pervanadate as means to initiate signal transduction cascades in primary cells.

High Complication Rate after Introduction of Transbronchial Cryobiopsy into Clinical Practice at an Academic Medical Center

Rationale: Transbronchial cryobiopsy is an emerging technique for obtaining biopsies of lung parenchyma. Despite limited evidence of its safety and efficacy in direct comparison with other available biopsy procedures, pulmonologists are integrating this technique into clinical practice with the hope of avoiding the risks of surgical lung biopsy. Objectives: To report the rate of severe complications and diagnostic outcomes immediately after introduction of transbronchial cryobiopsy into the clinical practice of a single‐center, high‐volume, interventional pulmonary group at a large academic medical center in the United States. Methods: We conducted a retrospective review of a case series. Results: Twenty‐five consecutive patients underwent transbronchial cryobiopsy for a variety of indications over a period of 14 weeks. In the absence of a strict protocol, a variety of techniques were employed by four attending interventional pulmonologists and one advanced interventional pulmonology fellow to plan and complete the procedures. Three patients (12%) experienced serious hemorrhage immediately after biopsy, including one patient who survived a life‐threatening bleed. Two procedures were complicated by an iatrogenic pneumothorax. One patient experienced hypercapnic respiratory failure shortly after the procedure. A definitive diagnosis was made with 14 cryobiopsies (56%). Another five biopsies (20%) contributed to a presumptive diagnosis achieved by multidisciplinary consensus. Conclusions: Transbronchial cryobiopsy may have diagnostic and safety limitations that are not yet well appreciated, given the state of the published medical literature. Major questions remain regarding the safest procedural protocol to be used when performing transbronchial cryobiopsy. Thorough planning and a high degree of caution are encouraged on first introduction of this technique into a clinical practice.

Bronchoscopic Cryotherapy. Clinical Applications of the Cryoprobe, Cryospray, and Cryoadhesion

Cryotherapy is an evolving therapeutic and diagnostic tool used during bronchoscopy. Through rapid freeze-thaw cycles, cryotherapy causes cell death and tissue necrosis or tissue adherence that can be used via the flexible or rigid bronchoscope. This extreme cold can be used through the working channel of the bronchoscope via a specialized cryoprobe or directly with the use of spray cryotherapy. These properties allow for multiple bronchoscopic techniques, each with its own equipment and procedural, safety, and efficacy considerations. Bronchoscopic cryotherapy can be used in a variety of clinical scenarios, including the treatment of malignant and benign central airway obstruction and low-grade airway malignancy, foreign body removal or cryoextraction, endobronchial biopsy, and transbronchial biopsy. The bulk of the experience with bronchoscopic cryotherapy consists of uncontrolled case series of malignant central airway obstruction. There are also controlled data supporting the use of cryoadhesion for endobronchial biopsies, albeit with an increased risk of controllable bleeding. The use of cryoadhesion for transbronchial biopsies is an active area of investigation with limited controlled data. In addition, there are promising future directions using bronchoscopic cryotherapy, including chemosensitizing malignancy with cryotherapy and capitalizing on the synergy between cryotherapy and radiation.

Dynamic Contrast-Enhanced Magnetic Resonance Lymphangiography and Percutaneous Lymphatic Embolization for the Diagnosis and Treatment of Recurrent Chyloptysis

Chyloptysis, or the expectoration of triglyceride-rich sputum, is rare and typically treated with diet modification and thoracic duct ligation. This article describes 2 patients with prolonged histories of chyloptysis who failed conservative treatment and thoracic duct ligation. Dynamic contrast-enhanced magnetic resonance imaging delineated the lymphatic anatomy and identified the abnormal pulmonary lymphatic perfusion pathways in both patients. This imaging provided guidance for successful percutaneous lymphatic embolization which resulted in resolution of symptoms in both patients.

Blue Bronchoscopy: Confirmation of Aberrant Pulmonary Lymphatic Perfusion During Lymphangiography

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