Pilar Delgado

CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection.

Thymocytes from mice lacking the CD3δ chain of the T-cell receptor (TCR), unlike those of other CD3-deficient mice, progress from a CD4-CD8- double-negative to a CD4 +CD8+ double-positive stage. However, CD3δ -/- double-positive cells fail to undergo positive selection, by which double-positive cells differentiate into more mature thymocytes. Positive selection is also impaired in mice expressing inactive components of the Ras/mitogen activated protein (MAP) kinase signalling pathway. Here we show that CD3δ-/- thymocytes are defective in the induction of extracellular signal-regulated protein kinase (ERK) MAP kinases upon TCR engagement, whereas activation of other MAP kinases is unaffected. The requirement for CD3δ maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3δ rescues both ERK activation and positive selection in CD3δ-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3ζ chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3δ-/- thymocytes may derive from defective tyrosine phosphorylation of CD3ζ in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.

Initiation of TCR signaling: regulation within CD3 dimers

The number of possible T cell activation outcomes resulting from T cell receptor (TCR) engagement suggests that the TCR is able to differentially activate a myriad of signaling pathways depending on the nature of the stimulus. The complex structural organization of the TCR itself could underlie this diversity of responses. Assembly and stoichiometric studies have helped us to shed some light on the initiation of TCR signaling. The TCR is composed of TCR and CD3 dimers. Changes in the interaction between CD3 subunits within the CD3 dimers and in the interaction of these dimers with the TCR heterodimer could be the triggering mechanism that initiates the first activation events. One of the hallmarks of these early changes in TCR conformation is the induced recruitment of the adapter protein Nck to a proline‐rich sequence of the cytoplasmic tail of CD3ε, but there may be others. According to our most recent observations, the TCR is organized in pre‐existing clusters within plasma membrane microdomains, exhibiting a complexity above and beyond that of dimer composition complexity. How the presence of TCR in clusters influences TCR avidity and propagation of TCR signals is something that has yet to be investigated.

Essential function for the GTPase TC21 in homeostatic antigen receptor signaling

T cell antigen receptors (TCRs) and B cell antigen receptors (BCRs) transmit low-grade signals necessary for the survival and maintenance of mature cell pools. We show here that TC21, a small GTPase encoded by Rras2, interacted constitutively with both kinds of receptors. Expression of a dominant negative TC21 mutant in T cells produced a rapid decrease in cell viability, and Rras2−/− mice were lymphopenic, possibly as a result of diminished homeostatic proliferation and impaired T cell and B cell survival. In contrast, TC21 was overexpressed in several human lymphoid malignancies. Finally, the p110δ catalytic subunit of phosphatidylinositol-3-OH kinase (PI(3)K) was recruited to the TCR and BCR in a TC21-dependent way. Consequently, we propose TC21 directly links antigen receptors to PI(3)K-mediated survival pathways.

Rho regulates T cell receptor ITAM-induced lymphocyte spreading in an integrin-independent manner

T cell receptor (TCR) engagement increases integrin‐mediated adhesion to APC, resulting in the stabilization of the T cell : APC interaction and the close apposition of the two cell membranes. Here we show that engagement of either the TCR or CD3 chimeras with immobilized antibodies causes the rapid spreading of T cells in an integrin‐independent fashion. This effect concurs with the polymerization of the actin cytoskeleton and is dependent on the integrity of the immunoreceptor tyrosine‐based activation motifs of the CD3 subunits. Expression of a dominant negative mutant of RhoA, as well as the Rho‐specific inhibitor C3 toxin, abolished TCR‐induced spreading. In contrast, constitutively active or dominant negative forms of Rac and Cdc42 did not affect cell spreading. We conclude that signals emanating from the TCR can directly induce T cell spreading, independently of integrins, and via a Rho‐dependent reorganization of the actin cytoskeleton.

The Rho Exchange Factors Vav2 and Vav3 Favor Skin Tumor Initiation and Promotion by Engaging Extracellular Signaling Loops

Rho GEFs Vav2 and Vav3 regulate pro-tumorigenic autocrine/paracrine signals in keratinocytes and are dispensable for skin homeostasis.

An orderly inactivation of intracellular retention signals controls surface expression of the T cell antigen receptor

Exit from the endoplasmic reticulum (ER) is an important checkpoint for proper assembly of multimeric plasma membrane receptors. The six subunits of the T cell receptor (TCR; TCRα, TCRβ, CD3γ, CD3δ, CD3ɛ, and CD3ζ) are each endowed with ER retention/retrieval signals, and regulation of its targeting to the plasma membrane is therefore especially intriguing. We have studied the importance of the distinct ER retention signals at different stages of TCR intracellular assembly. To this end, we have characterized first the presence of ER retention signals in CD3γ. Despite the presence of multiple ER retention signals in CD3γ, ɛγ dimers reach the cell surface when the single CD3ɛ ER retention signal is deleted. Furthermore, inclusion of this CD3ɛ mutant promoted plasma membrane expression of incomplete αβγɛ and αβδɛ complexes without CD3ζ. It therefore appears that the CD3ɛ ER retention signal is dominant and that it is only overridden upon the incorporation of CD3ζ. We propose that the stepwise assembly of the TCR complex guarantees that all assembly intermediates have at least one functional ER retention signal and that only a full signaling-competent TCR complex is expressed on the cell surface.

UNG shapes the specificity of AID-induced somatic hypermutation

UNG activity repairs activation-induced deaminase-generated U:G mismatches via error-prone or error-free repair, depending on the sequence context of the deaminated cytosine.

Role of chimaerins, a group of Rac-specific GTPase activating proteins, in T-cell receptor signaling

Chimaerins are GTPase-activating proteins that inactivate the GTP-hydrolase Rac1 in a diacylglycerol-dependent manner. To date, the study of chimaerins has been done mostly in neuronal cells. Here, we show that alpha2- and beta2-chimaerin are expressed at different levels in T-cells and that they participate in T-cell receptor signaling. In agreement with this, we have observed that alpha2- and beta2-chimaerins translocate to the T-cell/B-cell immune synapse and, using both gain- and loss-of-function approaches, demonstrated that their catalytic activity is important for the inhibition of the T-cell receptor- and Vav1-dependent stimulation of the transcriptional factor NF-AT. Mutagenesis-based approaches have revealed the molecular determinants that contribute to the biological program of chimaerins during T-cell responses. Unexpectedly, we have found that the translocation of chimaerins to the T-cell/B-cell immune synapse does not rely on the canonical binding of diacylglycerol to the C1 region of these GTPase-activating proteins. Taken together, these results identify chimaerins as candidates for the downmodulation of Rac1 in T-lymphocytes and, in addition, uncover a novel regulatory mechanism that mediates their activation in T-cells.

Inactivation of nuclear GSK3β by Ser 389 phosphorylation promotes lymphocyte fitness during DNA double-strand break response

Variable, diversity and joining (V(D)J) recombination and immunoglobulin class switch recombination (CSR) are key processes in adaptive immune responses that naturally generate DNA double-strand breaks (DSBs) and trigger a DNA repair response. It is unclear whether this response is associated with distinct survival signals that protect T and B cells. Glycogen synthase kinase 3β (GSK3β) is a constitutively active kinase known to promote cell death. Here we show that phosphorylation of GSK3β on Ser389 by p38 MAPK (mitogen-activated protein kinase) is induced selectively by DSBs through ATM (ataxia telangiectasia mutated) as a unique mechanism to attenuate the activity of nuclear GSK3β and promote survival of cells undergoing DSBs. Inability to inactivate GSK3β through Ser389 phosphorylation in Ser389Ala knockin mice causes a decrease in the fitness of cells undergoing V(D)J recombination and CSR. Preselection-Tcrβ repertoire is impaired and antigen-specific IgG antibody responses following immunization are blunted in Ser389GSK3β knockin mice. Thus, GSK3β emerges as an important modulator of the adaptive immune response.

R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes

Metabolic reprogramming of germinal center B cells requires the activity of R-Ras2. R-Ras2 revs replication Germinal centers are specialized lymphoid structures that facilitate the development of high-affinity antibody responses by B cells, a process that requires the guanosine triphosphate hydrolase activity of Ras family members. Mendoza et al. characterized germinal center formation in mice lacking R-Ras2 or other related family members and found that Ras proteins are functionally specialized. Only B cell–intrinsic R-Ras2 activity was necessary for cellular proliferation and germinal center development. R-Ras2 deficiency specifically reduced the expression of mitochondrial tRNAs necessary for mitochondrial replication and glucose metabolism in B cells. These data suggest that R-Ras2 signaling is essential for the metabolic reprograming of germinal center B cells to supply the energy required to generate effective antibody responses. Upon antigen recognition within peripheral lymphoid organs, B cells interact with T cells and other immune cells to transiently form morphological structures called germinal centers (GCs), which are required for B cell clonal expansion, immunoglobulin class switching, and affinity maturation. This process, known as the GC response, is an energetically demanding process that requires the metabolic reprogramming of B cells. We showed that the Ras-related guanosine triphosphate hydrolase (GTPase) R-Ras2 (also known as TC21) plays an essential, nonredundant, and B cell–intrinsic role in the GC response. Both the conversion of B cells into GC B cells and their expansion were impaired in mice lacking R-Ras2, but not in those lacking a highly related R-Ras subfamily member or both the classic H-Ras and N-Ras GTPases. In the absence of R-Ras2, activated B cells did not exhibit increased oxidative phosphorylation or aerobic glycolysis. We showed that R-Ras2 was an effector of both the B cell receptor (BCR) and CD40 and that, in its absence, B cells exhibited impaired activation of the PI3K-Akt-mTORC1 pathway, reduced mitochondrial DNA replication, and decreased expression of genes involved in glucose metabolism. Because most human B cell lymphomas originate from GC B cells or B cells that have undergone the GC response, our data suggest that R-Ras2 may also regulate metabolism in B cell malignancies.