An orthogonal IL-2 and IL-2Rß system drives persistence and activation of CAR T cells and clearance of bulky lymphoma.

Chimeric antigen receptor (CAR) T cells induce durable responses in patients with refractory hematological tumors. However, low CAR T cell activity, poor engraftment, or short in-patient persistence can lead to tumor progression or relapse. Furthermore, excessive CAR T cell expansion and activation can result in life-threatening cytokine release syndrome (CRS). Thus, in-patient control of the CAR T cell population is essential. Interleukin-2 (IL-2) is a critical cytokine for T cell proliferation and effector function, but its clinical use is limited by immune-mediated toxicity. Here, we report on an orthogonal IL-2 receptor and ligand system that enables specific in vivo control of CAR T cell expansion and activation, wherein an orthogonal human IL-2 (STK-009) selectively pairs with an orthogonal human IL-2Rß (hoRb) expressed on CAR T cells. STK-009 expands hoRb-expressing CAR T cells in the presence and absence of tumor antigen and maintains the presence of stem cell memory T cells (TSCM) and effector T cells. In preclinical models of human CAR-refractory lymphoma, STK-009 treatment resulted in systemic and intratumoral expansion and activation of hoRb-expressing anti­CD19-CD28ζ CAR T cells (SYNCAR). The orthogonal IL-2 receptor/ligand system delivers complete responses in large subcutaneous lymphomas, even with substantially reduced CAR T cell doses, by selectively expanding and activating CAR T cells in vivo. STK-009 withdrawal allowed normal CAR T cell contraction, thereby limiting CRS induced by tumor antigen­specific T cell activation. These data suggest that the orthogonal IL-2 receptor/ligand system provides the in vivo control necessary to maximize efficacy of CAR T therapies.

Claudin-18 deficiency results in alveolar barrier dysfunction and impaired alveologenesis in mice.

Claudins are a family of transmembrane proteins that are required for tight junction formation. Claudin (CLDN)-18.1, the only known lung-specific tight junction protein, is the most abundant claudin in alveolar epithelial type (AT) 1 cells, and is regulated by lung maturational agonists and inflammatory mediators. To determine the function of CLDN18 in the alveolar epithelium, CLDN18 knockout (KO) mice were generated and studied by histological, biochemical, and physiological approaches, in addition to whole-genome microarray. Alveolar epithelial barrier function was assessed after knockdown of CLDN18 in isolated lung cells. CLDN18 levels were measured by quantitative PCR in lung samples from fetal and postnatal human infants. We found that CLDN18 deficiency impaired alveolar epithelial barrier function in vivo and in vitro, with evidence of increased paracellular permeability and architectural distortion at AT1-AT1 cell junctions. Although CLDN18 KO mice were born without evidence of a lung abnormality, histological and gene expression analysis at Postnatal Day 3 and Week 4 identified impaired alveolarization. CLDN18 KO mice also had evidence of postnatal lung injury, including acquired AT1 cell damage. Human fetal lungs at 23-24 weeks gestational age, the highest-risk period for developing bronchopulmonary dysplasia, a disease of impaired alveolarization, had significantly lower CLDN18 expression relative to postnatal lungs. Thus, CLDN18 deficiency results in epithelial barrier dysfunction, injury, and impaired alveolarization in mice. Low expression of CLDN18 in human fetal lungs supports further investigation into a role for this tight junction protein in bronchopulmonary dysplasia.

The C. elegans rab family: identification, classification and toolkit construction.

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).

Claudin-4 levels are associated with intact alveolar fluid clearance in human lungs.

The removal of edema from the air spaces is a critical function of the alveolar barrier requiring intact tight junctions. Alveolar fluid clearance contributes to graft function after transplantation and is associated with survival in patients with acute lung injury. Claudin-4 concentrations are known to increase during lung injury and the loss of claudin-4 decreases alveolar fluid clearance in mice. This study was therefore undertaken to evaluate whether differences in lung expression of the tight junction protein claudin-4 are associated with alveolar fluid clearance or clinical measures of lung function. Alveolar fluid clearance rates were measured in ex vivo perfused human lungs not used for transplantation and were compared with histological lung injury and clinical measures of lung injury in the donors. Claudin-4 staining demonstrated a positive correlation with alveolar fluid clearance (Spearman rank correlation [r(s)] = 0.71; P < 0.003); however, claudin-4 staining was not strongly associated with histological measures of lung injury. The expression of other tight junction proteins (including ZO-1) was not associated with alveolar fluid clearance or claudin-4 levels. Claudin-4 staining was lower in lungs from donors with greater impairment in respiratory physiology. These data suggest that claudin-4 may promote alveolar fluid clearance and demonstrate that the amount of claudin-4 expressed may provide specific information regarding alveolar epithelial barrier function that strengthens the link between histological changes and physiological impairment.

Keratinocyte growth factor enhances barrier function without altering claudin expression in primary alveolar epithelial cells.

Keratinocyte growth factor (KGF) has efficacy in several experimental models of lung injury; however, the mechanisms underlying KGF's protective effect remain incompletely understood. This study was undertaken to determine whether KGF augments barrier function in primary rat alveolar epithelial cells grown in culture, specifically whether KGF alters tight junction function via claudin expression. KGF significantly increased alveolar epithelial barrier function in culture as assessed by transepithelial electrical resistance (TER) and paracellular permeability. Fluorescence-activated cell sorting of freshly isolated type 1 (AT1) and type 2 (AT2) cells followed by quantitative real-time RT-PCR revealed that more than 97% of claudin mRNA transcripts in these cells were for claudins-3, -4, and -18. Using cultured AT2 cells, we then examined the effect of KGF on the protein levels of the claudins with the highest mRNA levels: -3, -4, -5, -7, -12, -15, and -18. KGF did not alter the levels of any of the claudins tested, nor of zona occludens-1 (ZO-1) or occludin. Moreover, localization of claudins-3, -4, -18, and ZO-1 was unchanged. KGF did induce a marked increase in the apical perijunctional F-actin ring. Actin depolymerization with cytochalasin D blocked the KGF-mediated increase in TER without significantly changing TER in control cells. Together, these data support a novel mechanism by which KGF enhances alveolar barrier function, modulation of the actin cytoskeleton. In addition, these data demonstrate the complete claudin expression profile for AT1 and AT2 cells and indicate that claudins-3, -4, and -18 are the primary claudins expressed in these cell types.