Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV.

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Via next generation sequencing on a cohort of patients with severe atopic dermatitis, some with comorbid infections, we found 8 individuals from 4 families with novel heterozygous mutations in CARD11, a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant expression constructs into T cell lines demonstrated both loss of function and dominant interfering activity upon antigen receptor-induced NF-κB and mTORC1 activation. Patient T-cells had similar defects, as well as diminished IFN-γ cytokine production. The mTORC1 and IFN-γ production defects could be partially rescued by supplementing with glutamine, which requires CARD11 for import into T cells. Our findings indicate a single hypomorphic gene mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

The caspase recruitment domain family member 11 (CARD11 or CARMA1)—B cell CLL/lymphoma 10 (BCL10)—MALT1 paracaspase (MALT1) [CBM] signalosome complex serves as a molecular bridge between cell surface antigen receptor signaling and the activation of the NF-κB, JNK, and mTORC1 signaling axes. This positions the CBM complex as a critical regulator of lymphocyte activation, proliferation, survival, and metabolism. Inborn errors in each of the CBM components have now been linked to a diverse group of human primary immunodeficiency diseases termed “CBM-opathies.” Clinical manifestations range from severe combined immunodeficiency to selective B cell lymphocytosis, atopic disease, and specific humoral defects. This surprisingly broad spectrum of phenotypes underscores the importance of “tuning” CBM signaling to preserve immune homeostasis. Here, we review the distinct clinical and immunological phenotypes associated with human CBM complex mutations and introduce new avenues for targeted therapeutic intervention.

These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in human subjects and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity.

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells.

CARD11 is a lymphocyte-specific scaffold molecule required for proper activation of B- and T-cells in response to antigen. Germline gain-of-function (GOF) mutations in the CARD11 gene cause a unique B cell lymphoproliferative disorder known as B cell Expansion with NF-κB and T cell Anergy (BENTA). In contrast, patients carrying loss-of-function (LOF), dominant negative (DN) CARD11 mutations present with severe atopic disease. Interestingly, both GOF and DN CARD11 variants cause primary immunodeficiency, with recurrent bacterial and viral infections, likely resulting from impaired adaptive immune responses. This report describes a unique four-generation family harboring a novel heterozygous germline indel mutation in CARD11 (c.701-713delinsT), leading to one altered amino acid and a deletion of 4 others (p.His234_Lys238delinsLeu). Strikingly, affected members exhibit both moderate B cell lymphocytosis and atopic dermatitis/allergies. Ectopic expression of this CARD11 variant stimulated constitutive NF-κB activity in T cell lines, similar to other BENTA patient mutations. However, unlike other GOF mutants, this variant significantly impeded the ability of wild-type CARD11 to induce NF-κB activation following antigen receptor ligation. Patient lymphocytes display marked intrinsic defects in B cell differentiation and reduced T cell responsiveness in vitro. Collectively, these data imply that a single heterozygous CARD11 mutation can convey both GOF and DN signaling effects, manifesting in a blended BENTA phenotype with atopic features. Our findings further emphasize the importance of balanced CARD11 signaling for normal immune responses.

Nestin, an intermediate filament protein and a stem cell marker is expressed in several tumors. Until recently, little was known about the expression levels and the role of Nestin in endometrial cancer. Compared to the immortalized endometrial epithelial cell line EM-E6/E7-TERT, endometrial cancer cell lines express high to moderate levels of Nestin. Furthermore, endometrial tumors and tumor cell lines have a cancer stem-like cell subpopulation expressing CD133. Among the cancer lines, AN3CA and KLE cells exhibited both a significantly higher number of CD133+ cells and expressed Nestin at higher levels than Ishikawa cells. Knockdown of Nestin in AN3CA and KLE increased cells in G0/G1 phase of the cell cycle, whereas overexpression in Ishikawa decreased cells in G0/G1 phase and increased cells in S-phase. Nestin knockdown cells showed increased p21, p27, and PNCA levels and decreased expression of cyclin-D1 and D3. In contrast, Nestin overexpression revealed an inverse expression pattern of cell cycle regulatory proteins. Nestin knockdown inhibited cancer cell growth and invasive potential by downregulating TGF-β signaling components, MMP-2, MMP-9, vimentin, SNAIL, SLUG, Twist, N-cadherin, and upregulating the epithelial cell marker E-cadherin whereas the opposite was observed with Nestin overexpressing Ishikawa cells. Nestin knockdown also inhibited, while overexpression promoted invadopodia formation and pFAK expression. Knockdown of Nestin significantly reduced tumor volume in vivo. Finally, progesterone inhibited Nestin expression in endometrial cancer cells. These results suggest that Nestin can be a therapeutic target for cancer treatment.

Background Characterizing the longevity and quality of cellular immune responses to SARS-CoV-2 enhances understanding of COVID-19 immunity that influences clinical outcomes. Prior studies suggest SARS-CoV-2-specific T cells are present in peripheral blood 10 months after infection. Further analysis of the function, durability, and diversity of the cellular response long after natural infection, over a wider range of ages and disease phenotypes, is needed to further identify preventative and therapeutic interventions. Methods We identified participants in our multi-site longitudinal, prospective cohort study 12-months post SARS-CoV-2 infection representing a range of disease severity. We investigated the function, phenotypes, and frequency of T cells specific for SARS-CoV-2 using intracellular cytokine staining and spectral flow cytometry. In parallel, the magnitude of SARS-CoV-2-specific antibodies was compared. Results SARS-CoV-2-specific antibodies and T cells were detected at 12-months post-infection. Severity of acute illness was associated with higher frequencies of SARS-CoV-2-specific CD4 T cells and antibodies at 12-months. In contrast, polyfunctional and cytotoxic T cells responsive to SARS-CoV-2 were identified in participants over a wide spectrum of disease severity. Conclusions Our data show that SARS-CoV-2 infection induces polyfunctional memory T cells detectable at 12-months post-infection, with higher frequency noted in those who originally experienced severe disease.