The limited ability of cytotoxic T cells to infiltrate solid tumors hampers immune surveillance and the efficacy of immunotherapies in cancer. Adenosine accumulates in solid tumors and inhibits tumor-specific T cells. Adenosine inhibits T cell motility through the A2A receptor (A2AR) and suppression of KCa3.1 channels. Herein, we conducted 3-dimensional chemotaxis experiments to elucidate the effect of adenosine on the migration of peripheral blood CD8+ T cells from head and neck squamous cell carcinoma (HNSCC) patients. The chemotaxis of HNSCC CD8+ T cells was reduced in the presence of adenosine, and the effect was greater on HNSCC CD8+ T cells than on healthy donor (HD) CD8+ T cells. This response correlated with the inability of CD8+ T cells to infiltrate tumors. The effect of adenosine was mimicked by an A2AR agonist and prevented by an A2AR antagonist. We found no differences in A2AR expression, cAMP abundance, or protein kinase A1 activity between HNSCC and HD CD8+ T cells. We instead detected a decrease in KCa3.1 channel activity, but not expression, in HNSCC CD8+ T cells. Activation of KCa3.1 channels by 1-EBIO restored the ability of HNSCC CD8+ T cells to chemotax in the presence of adenosine. Our data highlight the mechanism underlying the increased sensitivity of HNSCC CD8+ T cells to adenosine and the potential therapeutic benefit of KCa3.1 channel activators, which could increase infiltration of these T cells into tumors.

Objectives: The aim of this study was to examine the success rate of less invasive surfactant administration (LISA), to identify early predictive factors for the outcome of LISA, and to compare neonatal outcomes between the LISA failure group and the group of infants who were successfully treated with LISA.Design: A retrospective cohort study.Patients: Infants born at less than 33 weeks of gestation (n = 158) and treated with LISA for respiratory distress syndrome.Results: LISA was successful in 86 cases (54.4%); 72 preterm infants (45.6%) needed additional surfactant therapy and/or mechanical ventilation in the first 72 h (LISA failure). In a multivariate logistic regression analysis, six independent predictors of LISA success were identified: core temperature at the time of admission (adjusted odds ratio (OR): 3.56), dose of poractant alfa (<200 mg/kg; adjusted OR: 0.254), elevated C-reactive protein (>10 mg/L) at 24 h of life (adjusted OR: 0.28), highest respiratory severity score (RSS) during the first hour of life or at the time of LISA (adjusted OR: 0.463), maternal age (adjusted OR: 0.923), and birth weight (adjusted OR: 1.003). The receiver operating curve created by using the identified factors indicates good predictive power with an area under the curve of 0.85. LISA failure was associated with a substantially higher risk of complications. Conclusion:LISA success can be predicted by variables available before the intervention.Failure of LISA is relatively frequent event in very preterm infants and is associated with adverse outcomes. Prevention of hypothermia during early stabilization and appropriate dosing of surfactant may increase LISA success rates and improve patient outcome.

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In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.

Ion channels are crucially important for the activation and proliferation of T lymphocytes, and thus, for the function of the immune system. Previous studies on the effects of channel blockers on T cell proliferation reported variable effectiveness due to differing experimental systems. Therefore our aim was to investigate how the strength of the mitogenic stimulation influences the efficiency of cation channel blockers in inhibiting activation, cytokine secretion and proliferation of T cells under standardized conditions. Human peripheral blood lymphocytes were activated via monoclonal antibodies targeting the TCR-CD3 complex and the co-stimulator CD28. We applied the blockers of Kv1.3 (Anuroctoxin), KCa3.1 (TRAM-34) and CRAC (2-Apb) channels of T cells either alone or in combination with rapamycin, the inhibitor of the mammalian target of rapamycin (mTOR). Five days after the stimulation ELISA and flow cytometric measurements were performed to determine IL-10 and IFN-γ secretion, cellular viability and proliferation. Our results showed that ion channel blockers and rapamycin inhibit IL-10 and IFN-γ secretion and cell division in a dose-dependent manner. Simultaneous application of the blockers for each channel along with rapamycin was the most effective, indicating synergy among the various activation pathways. Upon increasing the extent of mitogenic stimulation the anti-proliferative effect of the ion channel blockers diminished. This phenomenon may be important in understanding the fine-tuning of T cell activation.

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Kv1.3 channels are expressed in several cell types including immune cells, such as T lymphocytes. The targeting of Kv1.3 to the plasma membrane is essential for T cell clonal expansion and assumed to be guided by the C-terminus of the channel. Using two point mutants of Kv1.3 with remarkably different features compared to the wild-type Kv1.3 (A413V and H399K having fast inactivation kinetics and tetraethylammonium-insensitivity, respectively) we showed that both Kv1.3 channel variants target to the membrane when the C-terminus was truncated right after the conserved HRET sequence and produce currents identical to those with a full-length C-terminus. The truncation before the HRET sequence (NOHRET channels) resulted in reduced membrane-targeting but non-functional phenotypes. NOHRET channels did not display gating currents, and coexpression with wild-type Kv1.3 did not rescue the NOHRET-A413V phenotype, no heteromeric current was observed. Interestingly, mutants of wild-type Kv1.3 lacking HRET(E) (deletion) or substituted with five alanines for the HRET(E) motif expressed current indistinguishable from the wild-type. These results demonstrate that the C-terminal region of Kv1.3 immediately proximal to the S6 helix is required for the activation gating and conduction, whereas the presence of the distal region of the C-terminus is not exclusively required for trafficking of Kv1.3 to the plasma membrane.

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