T cell–derived exosomes induced macrophage inflammatory protein‐1α/β drive the trafficking of CD8+ T cells in oral lichen planus

Oral lichen planus (OLP) is a T cell–mediated chronic inflammatory disease with uncertain aetiology. Exosomes are nanosized particles with biological capacities. Here, we aimed to study the effects of T cell–derived exosomes (T‐exos) on the pathogenesis of OLP and its mechanism. T‐exos were incubated with Jurkat cells for 48 hours, and 26 cytokines in the supernatant were measured by luminex assay. The expression of macrophage inflammatory protein (MIP)‐1α/β was detected using immunohistochemistry and ELISA; that of CCR1/3/5 on peripheral T cells was determined by flow cytometry. Transwell assay was performed to investigate the chemotactic effect of MIP‐1α/β, and cells in the lower chambers were examinated by flow cytometry. As a result, OLP T‐exos elevated the production of MIP‐1α/β, which were highly expressed in OLP tissues and plasma. CCR1/5 were markedly expressed on OLP peripheral T cells, and the majority of CCR1/5⁺ T cells were CD8⁺ T cells. Besides, MIP‐1α/β promoted the migration of OLP mononuclear cells, while inhibiting CCR1/5 significantly decreased the trafficking of mononuclear cells, especially that of CD8⁺ T cells. Conclusively, OLP T‐exos‐induced MIP‐1α/β may drive the trafficking of CD8⁺ T cells after binding with CCR1/5 in OLP, contributing to the development of OLP.     

ATM Influences the Efficiency of TCRβ Rearrangement,Subsequent TCRβ-Dependent T Cell Development,and Generation of the Pre-Selection TCRβ CDR3 Repertoire

Generation and resolution of DNA double-strand breaks is required to assemble antigen-specific receptors from the genes encoding V, D, and J gene segments during recombination. The present report investigates the requirement for ataxia telangiectasia-mutated (ATM) kinase, a component of DNA double-strand break repair, during TCRβ recombination and in subsequent TCRβ-dependent repertoire generation and thymocyte development. CD4⁻CD8⁻ double negative stage 2/3 thymocytes from ATM-deficient mice have both an increased frequency of cells with DNA break foci at TCRβ loci and reduced Vβ-DJβ rearrangement. Sequencing of TCRβ complementarity-determining region 3 demonstrates that ATM-deficient CD4⁺CD8⁺ double positive thymocytes and peripheral T cells have altered processing of coding ends for both in-frame and out-of-frame TCRβ rearrangements, providing the unique demonstration that ATM deficiency alters the expressed TCRβ repertoire by a selection-independent mechanism. ATMKO thymi exhibit a partial developmental block in DN cells as they negotiate the β-selection checkpoint to become double negative stage 4 and CD4⁺CD8⁺ thymocytes, resulting in reduced numbers of CD4⁺CD8⁺ cells. Importantly, expression of a rearranged TCRβ transgene substantially reverses this defect in CD4⁺CD8⁺ cells, directly linking a requirement for ATM during endogenous TCRβ rearrangement to subsequent TCRβ-dependent stages of development. These results demonstrate that ATM plays an important role in TCRβ rearrangement, generation of the TCRβ CDR3 repertoire, and efficient TCRβ-dependent T cell development.     

Calcium channel α2δ1 subunit is a functional marker and therapeutic target for tumor-initiating cells in non-small cell lung cancer

It is hypothesized that tumor-initiating cells (TICs) with stem cell-like properties constitute a sustaining force to drive tumor growth and renew fully established malignancy. However, the identification of such a population in non-small cell lung carcinoma (NSCLC) has been hindered by the lacking of reliable surface markers, and very few of the currently available surface markers are of functional significance. Here, we demonstrate that a subpopulation of TICs could be specifically defined by the voltage-gated calcium channel α2δ1 subunit from non-small cell lung carcinoma (NSCLC) cell lines and clinical specimens. The α2δ1⁺ NSCLC TICs are refractory to conventional chemotherapy, and own stem cell-like properties such as self-renewal, and the ability to generate heterogeneous tumors in NOD/SCID mice. Moreover, α2δ1⁺ NSCLC cells are more enriched for TICs than CD133⁺, or CD166⁺ cells. Interestingly, α2δ1 is functionally sufficient and indispensable to promote TIC properties by mediating Ca²⁺ influx into cells, which subsequently activate Calcineurin/NFATc2 signaling that directly activates the expression of NOTCH3, ABCG2. Importantly, a specific antibody against α2δ1 has remarkably therapeutic effects on NSCLC xenografts by eradicating TICs. Hence, targeting α2δ1 to prevent calcium influx provides a novel strategy for targeted therapy against TICs of NSCLC.Subject terms: Cancer stem cells, Predictive markers     

Induction of γδT cells from HSC‐enriched BMCs co‐cultured with iPSC‐derived thymic epithelial cells

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co‐culturing HSC‐enriched bone marrow cells (HSC‐eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC‐eBMCs and iTECs cultured with IL‐2 + IL‐7 for up to 21 days induced CD45.1⁺ γδT cells that expressed a broad repertoire of Vγ and Vδ T‐cell receptors. Notably, the induced lymphocytes contained few or no αβT cells, NK1.1⁺ natural killer cells, or B220⁺ B cells. Adoptive transfer of the induced γδT cells to leukemia‐bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.     

The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.     

Ribosylation of the CD8αβ heterodimer permits binding of the nonclassical major histocompatibility molecule,H2-Q10

The CD8αβ heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD⁺, the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or β chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD⁺. These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses.     

Antigen-derived peptides engage the ER stress sensor IRE1α to curb dendritic cell cross-presentation

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8⁺ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER), bind to major histocompatibility type I (MHC-I) protein complexes, and are transported to the cell surface for cross-presentation. DCs can exhibit activation of the ER stress sensor IRE1α without ER stress, but the underlying mechanism remains obscure. Here, we show that antigen-derived hydrophobic peptides can directly engage ER-resident IRE1α, masquerading as unfolded proteins. IRE1α activation depletes MHC-I heavy-chain mRNAs through regulated IRE1α-dependent decay (RIDD), curtailing antigen cross-presentation. In tumor-bearing mice, IRE1α disruption increased MHC-I expression on tumor-infiltrating DCs and enhanced recruitment and activation of CD8⁺ T cells. Moreover, IRE1α inhibition synergized with anti–PD-L1 antibody treatment to cause tumor regression. Our findings identify an unexpected cell-biological mechanism of antigen-driven IRE1α activation in DCs, revealing translational potential for cancer immunotherapy.     

Expression of the Mucosal Homing Receptor α4β7 Correlates with the Ability of CD8+ Memory T Cells To Clear Rotavirus Infection

The integrin α₄β₇ plays an important role in lymphocyte homing to mucosal lymphoid tissues and has been shown to define a subpopulation of memory T cells capable of homing to intestinal sites. Here we have used a well-characterized intestinal virus, murine rotavirus, to investigate whether memory/effector function for an intestinal pathogen is associated with α₄β₇ expression. α₄β₇^hi memory phenotype (CD44^hi), α₄β₇⁻ memory phenotype, and presumptively naive (CD44^lo) CD8⁺ T lymphocytes from rotavirus-infected mice were sorted and transferred into Rag-2 (T- and B-cell-deficient) recipients that were chronically infected with murine rotavirus. α₄β₇^hi memory phenotype CD8⁺ cells were highly efficient at clearing rotavirus infection, α₄β₇⁻ memory cells were inefficient or ineffective, depending on the cell numbers transferred, and CD44^lo cells were completely unable to clear chronic rotavirus infection. These data demonstrate that functional memory for rotavirus resides primarily in memory phenotype cells that display the mucosal homing receptor α₄β₇.     

Evidence that Distinct States of the Integrin α6β1 Interact with Laminin and an ADAM

Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin α6β1, on mouse eggs and on α6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin β). In the present study we tested the hypothesis that different states of α6β1 interact with fertilin and laminin, an extracellular matrix ligand for α6β1. Using α6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl₂) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin β or with the laminin E8 fragment to bind to eggs. In Ca²⁺-containing media, fertilin β beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin β and by the α6 integrin subunit. In Ca²⁺-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn²⁺ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (α6β1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).     

Loss of the orphan nuclear receptor NR2F6 enhances CD8+ T-cell memory via IFN-γ

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127⁻KLRG1⁺) or memory precursors cells (MPECs, CD127⁺KLRG1⁻) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8⁺ memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-γ, TNFα, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6^−/− OT-I T-cells showed that the augmented memory formation is CD8⁺ T-cell intrinsic. Although the relative difference between the Nr2f6^+/+ and Nr2f6^−/− OT-I memory compartment declines over time, Nr2f6-deficient OT-I memory T cells mount significantly enhanced IFN-γ responses upon reinfection with increased clonal expansion and improved host antigen-specific CD8⁺ T-cell responses. Following a secondary adoptive transfer into naïve congenic mice, Nr2f6-deficient OT-I memory T cells are superior in clearing LmOVA infection. Finally, we show that the commitment to enhanced memory within Nr2f6-deficient OT-I T cells is established in the early phases of the antibacterial immune response and is IFN-γ mediated. IFN-γ blocking normalized MPEC formation of Nr2f6-deficient OT-I T cells. Thus, deletion or pharmacological inhibition of NR2F6 in antigen-specific CD8⁺ T cells may have therapeutic potential for enhancing early IFN-γ production and consequently the functionality of memory CD8⁺ T cells in vivo.Subject terms: Interferons, Bacterial infection     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

Microbial Conversion of α-Ionone, α-Methylionone, and α-Isomethylionone

α-Ionone, α-methylionone, and α-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from α-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-α-ionone, trans-3-hydroxy-α-ionone, and 3-oxo-α-ionone. 2,3-Dehydro-α-ionone, 3,4-dehydro-β-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from α-methylionone and α-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for α-ionones and elucidation of stereochemical features of the bioconversion.     

High-Throughput Sequencing of Islet-Infiltrating Memory CD4+ T Cells Reveals a Similar Pattern of TCR Vβ Usage in Prediabetic and Diabetic NOD Mice

Autoreactive memory CD4⁺ T cells play a critical role in the development of type 1 diabetes, but it is not yet known how the clonotypic composition and TCRβ repertoire of the memory CD4⁺ T cell compartment changes during the transition from prediabetes to diabetes. In this study, we used high-throughput sequencing to analyze the TCRβ repertoire of sorted islet-infiltrating memory CD4⁺CD44^high T cells in 10-week-old prediabetic and recently diabetic NOD mice. We show that most clonotypes of islet-infiltrating CD4⁺CD44^high T cells were rare, but high-frequency clonotypes were significantly more common in diabetic than in prediabetic mice. Moreover, although the CD4⁺CD44^high TCRβ repertoires were highly diverse at both stages of disease development, dominant use of TRBV1 (Vβ2), TRBV13-3 (Vβ8.1), and TRBV19 (Vβ6) was evident in both prediabetic and diabetic mice. Our findings strongly suggest that therapeutic targeting of cells specifically expressing the dominant TCRβ might reduce pancreatic infiltration in prediabetic mice and attenuate the progression to diabetes.     

Reducing TGF‐β1 cooperated with StemRegenin 1 promoted the expansion ex vivo of cord blood CD34+ cells by inhibiting AhR signalling

ObjectiveAs an inhibitor of the AhR signalling pathway, StemRegenin 1 (SR1) not only promotes the expansion of CD34⁺ cells but also increases CD34⁻ cell numbers. These CD34⁻ cells influenced the ex vivo expansion of CD34⁺ cells. In this work, the effects of periodically removing CD34⁻ cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated.Materials and methodsCD34⁻ cells were removed periodically with SR1 addition to investigate cell subpopulations, cell expansion, biological functions, expanded cell division mode and supernatant TGF‐β1 contents.ResultsAfter 10‐day culture, the expansion of CD34⁺ cells in the CD34⁻ cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover, periodically removing CD34⁻ cells with SR1 addition improved the biological function of expanded CD34⁺ cells and significantly increased the percentage of self‐renewal symmetric division of CD34⁺ cells. In addition, the concentration of total TGF‐β1 and activated TGF‐β1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT‐qPCR results showed that the periodic removal of CD34⁻ cells with cooperation from SR1 further reduced the expression of AhR‐related genes.ConclusionsPeriodic removal of CD34⁻ cells plus cooperation with SR1 improved the expansion of CD34⁺ cells, maintained better biological function of expanded CD34⁺ cells and reduced the TGF‐β1 contents by downregulating AhR signalling.

SR1 increased self‐renewal symmetric division of cord blood CD34⁺ cells. Removal of CD34⁻ cells cooperated with SR1 increased ex vivo expansion of cord blood CD34⁺ cells. Removal of CD34⁻ cells cooperated with SR1 further downregulated AhR signalling     

Endogenous Production of β-Chemokines by CD4+, but Not CD8+, T-Cell Clones Correlates with the Clinical State of Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and May Be Responsible for Blocking Infection with NonSyncytium-Inducing HIV-1 In Vitro

Recent studies have demonstrated that the β-chemokines RANTES, MIP-1α, and MIP-1β suppress human immunodeficiency virus type 1 (HIV-1) replication in vitro and may play an important role in protecting exposed but uninfected individuals from HIV-1 infection. However, levels of β-chemokines in AIDS patients are comparable to and can exceed levels in nonprogressing individuals, indicating that global β-chemokine production may have little effect on HIV-1 disease progression. We sought to clarify the role of β-chemokines in nonprogressors and AIDS patients by examination of β-chemokine production and HIV-1 infection in patient T-lymphocyte clones established by herpesvirus saimiri immortalization. Both CD4⁺ and CD8⁺ clones were established, and they resembled primary T cells in their phenotypes and expression of activated T-cell markers. CD4⁺ T-cell clones from all patients had normal levels of mRNA-encoding CCR5, a coreceptor for non-syncytium-inducing (NSI) HIV-1. CD4⁺ clones from nonprogressors and CD8⁺ clones from AIDS patients secreted high levels of RANTES, MIP1α, and MIP-1β. In contrast, CD4⁺ clones from AIDS patients produced no RANTES and little or no MIP-1α or MIP-1β. The infection of CD4⁺ clones with the NSI HIV-1 strain ADA revealed an inverse correlation to β-chemokine production; clones from nonprogressors were poorly susceptible to ADA replication, but clones from AIDS patients were highly infectable. The resistance to ADA infection in CD4⁺ clones from nonprogressors could be partially reversed by treatment with anti-β-chemokine antibodies. These results indicate that CD4⁺ cells can be protected against NSI-HIV-1 infection in culture through endogenously produced factors, including β-chemokines, and that β-chemokine production by CD4⁺, but not CD8⁺, T cells may constitute one mechanism of disease-free survival for HIV-1-infected individuals.     

Construction of a Stable α-Galactosidase-Producing Baker's Yeast Strain

Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of α-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel⁻. In this study we integrated the yeast MEL1 gene, which codes for α-galactosidase, into a commercial mel⁰ baker's yeast strain. The Mel⁺ phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel⁺ baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The α-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more α-galactosidase than did a wild-type Mel⁺ strain and may prove useful for commercial production of α-galactosidase.     

Intestinal Intraepithelial Lymphocytes Are Primed for Gamma Interferon and MIP-1β Expression and Display Antiviral Cytotoxic Activity despite Severe CD4+ T-Cell Depletion in Primary Simian Immunodeficiency Virus Infection

Intraepithelial lymphocytes (IEL) are a critical effector component of the gut-associated lymphoid tissue (GALT) and play an important role in mucosal immunity as well as in the maintenance of the epithelial cell integrity and barrier function. The objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of rhesus macaques would cause alterations in the immunophenotypic profiles of IEL and their mitogen-specific cytokine (gamma interferon [IFN-γ] and MIP-1β) responses (by flow cytometry) and virus-specific cytotoxic T-cell (CTL) activity (by the chromium release assay). Virally infected IEL were detected through the entire course of SIV infection by in situ hybridization. Severe depletion of CD4⁺ single-positive and CD4⁺CD8⁺ double-positive T cells occurred early in primary SIV infection, which was coincident with an increased prevalence of CD8⁺ T cells. This was in contrast to a gradual depletion of CD4⁺ T cells in peripheral blood. The CD8⁺ IEL were the primary producers of IFN-γ and MIP-1β and were found to retain their potential to produce both IFN-γ and MIP-1β through the entire course of SIV infection. SIV-specific CTL activity was detected in primary IEL at 1, 2, and 4 weeks post-SIV infection. These results demonstrated that IEL may be involved in generating antiviral immune responses early in SIV infection and in suppressing viral infection thereafter. Alterations in homeostasis in epithelia due to severe CD4⁺ T-cell depletion accompanied by changes in the cytokine and chemokine production by IEL may play a role in the enteropathogenesis of SIV infection.     

Subcellular dynamics and functional activity of the cleaved intracellular domain of the Na+ channel β1 subunit

The voltage-gated Na⁺ channel β1 subunit, encoded by SCN1B, regulates cell surface expression and gating of α subunits and participates in cell adhesion. β1 is cleaved by α/β and γ-secretases, releasing an extracellular domain and intracellular domain (ICD), respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on β1 function in breast cancer cells. Using a series of GFP-tagged β1 constructs, we show that β1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal β1-GFP levels occurred following γ-secretase inhibition, implicating endosomes and/or the preceding plasma membrane as important sites for secretase processing. Using live-cell imaging, we also report β1ICD-GFP accumulation in the nucleus. Furthermore, β1-GFP and β1ICD-GFP both increased Na⁺ current, whereas β1STOP-GFP, which lacks the ICD, did not, thus highlighting that the β1-ICD is necessary and sufficient to increase Na⁺ current measured at the plasma membrane. Importantly, although the endogenous Na⁺ current expressed in MDA-MB-231 cells is tetrodotoxin (TTX)-resistant (carried by Na_v1.5), the Na⁺ current increased by β1-GFP or β1ICD-GFP was TTX-sensitive. Finally, we found β1-GFP increased mRNA levels of the TTX-sensitive α subunits SCN1A/Na_v1.1 and SCN9A/Na_v1.7. Taken together, this work suggests that the β1-ICD is a critical regulator of α subunit function in cancer cells. Our data further highlight that γ-secretase may play a key role in regulating β1 function in breast cancer.