Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

Pseudomonas aeruginosa flagellar motility activates the phagocyte PI3K/Akt pathway to induce phagocytic engulfment.

Phagocytosis of the bacterial pathogen Pseudomonas aeruginosa is the primary means by which the host controls bacterially induced pneumonia during lung infection. Previous studies have identified flagellar swimming motility as a key pathogen-associated molecular pattern (PAMP) recognized by phagocytes to initiate engulfment. Correspondingly, loss of flagellar motility is observed during chronic pulmonary infection with P. aeruginosa, and this likely reflects a selection for bacteria resistant to phagocytic clearance. However, the mechanism underlying the preferential phagocytic response to motile bacteria is unknown. Here we have identified a cellular signaling pathway in alveolar macrophages and other phagocytes that is specifically activated by flagellar motility. Genetic and biochemical methods were employed to identify that phagocyte PI3K/Akt activation is required for bacterial uptake and, importantly, it is specifically activated in response to P. aeruginosa flagellar motility. Based on these observations, the second important finding that emerged from these studies is that titration of the bacterial flagellar motility results in a proportional activation state of Akt. Therefore, the Akt pathway is responsive to, and corresponds with, the degree of bacterial flagellar motility, is independent of the actin polymerization that facilitates phagocytosis, and determines the phagocytic fate of P. aeruginosa. These findings elucidate the mechanism behind motility-dependent phagocytosis of extracellular bacteria and support a model whereby phagocytic clearance exerts a selective pressure on P. aeruginosa populations in vivo, which contributes to changes in pathogenesis during infections.

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development.

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3varepsilon proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3zeta-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.

Beyond alphabeta/gammadelta lineage commitment: TCR signal strength regulates gammadelta T cell maturation and effector fate.

Signaling by the gammadelta T cell receptor (TCR) is required not only for alphabeta/gammadelta lineage commitment but also to activate and elicit effector functions in mature gammadelta T cells. Notably, at both of these stages, the signal delivered by the gammadeltaTCR is more robust than the one delivered by either the preTCR or the alphabetaTCR. Recent studies now provide evidence that signaling by the gammadeltaTCR is also required at other stages during gammadelta T cell development. Remarkably, the strength of the gammadeltaTCR signal also plays a role at these other stages, as evidenced by the findings that genetic manipulation of gammadeltaTCR signal strength affects gammadelta T cell maturation and effector fate. In this review, we discuss how a strong TCR signal is a recurring theme in gammadelta T cell development and activation.

Genetic requirements for the development and differentiation of interleukin-17-producing γδ T cells.

Most effector T cells are generated in the periphery following an encounter with a foreign antigen and exposure to soluble and membrane-bound mediators. There are, however, some T cell subsets, such as γδ T cells and natural killer T cells, that acquire their effector potential in the thymus before their emigration to the periphery. This developmental preprogramming enables these cells to differentiate rapidly into cytokine-producing effectors during the host immune response. This review focuses on murine interleukin (IL)-17-producing γδ T (γδ-17) cells, which have been shown, through their early production of IL-17, to have a critical role in multiple infectious and autoimmune diseases. Specifically, we discuss what is currently known about the genetic requirements for their generation and compare it with what is known about that of the more extensively studied IL-17-producing helper T (Thl7) cells. Based on this comparison, we propose a model for murine γδ-17 development and differentiation.

Stoichiometry of the murine gammadelta T cell receptor.

The T cell receptor for antigen (TCR) complex is organized into two functional domains: the antigen-binding clonotypic heterodimer and the signal-transducing invariant CD3 and TCRzeta chains. In most vertebrates, there are two different clonotypic heterodimers (TCRalphabeta and TCRgammadelta) that define the alphabeta and gammadelta T cell lineages, respectively. alphabeta- and gammadeltaTCRs also differ in their invariant chain subunit composition, in that alphabetaTCRs contain CD3gammaepsilon and CD3deltaepsilon dimers, whereas gammadeltaTCRs contain only CD3gammaepsilon dimers. This difference in subunit composition of the alphabeta- and gammadeltaTCRs raises the question of whether the stoichiometries of these receptor complexes are different. As the stoichiometry of the murine gammadeltaTCR has not been previously investigated, we used two quantitative immunofluorescent approaches to determine the valency of TCRgammadelta heterodimers and CD3gammaepsilon dimers in surface murine gammadeltaTCR complexes. Our results support a model of murine gammadeltaTCR stoichiometry in which there are two CD3gammaepsilon dimers for every TCRgammadelta heterodimer.

Blk haploinsufficiency impairs the development, but enhances the functional responses, of MZ B cells.

Blk was identified two decades ago as a B-cell-specific member of the Src family of tyrosine kinases. Recent studies, however, have discovered that Blk is expressed in many cell types outside of the B lineage, including early thymic precursors, interleukin-17-producing γδ T cells and pancreatic ß-cells. In light of these recent discoveries, we performed a more comprehensive analysis of Blk expression patterns in hematopoietic cells and found that Blk is differentially expressed in mature B-cell subsets, with marginal zone (MZ) B cells expressing high levels, B1 B cells expressing intermediate-to-high levels and follicular (FO) B cells expressing low levels of Blk. To determine whether these differences in Blk expression levels reflected differential requirements for Blk in MZ, B1 and FO B-cell development, we analyzed the effects of reducing and eliminating Blk expression on B-cell development. We report that both Blk haploinsufficiency and Blk deficiency impaired the generation of MZ B cells. Moreover, although there were fewer MZ B cells in Blk(+/-) and Blk(-/-) mice as compared with Blk(+/+) mice, Blk-mutant MZ B cells were hyper-responsive to B-cell receptor stimulation, both in vitro and in vivo. Thus, this study has revealed a previously unappreciated role for Blk in the development and activation of MZ B cells.

Unexpected role for the B cell-specific Src family kinase B lymphoid kinase in the development of IL-17-producing γδ T cells.

The Ag receptors on αß and γδ T cells differ not only in the nature of the ligands that they recognize but also in their signaling potential. We hypothesized that the differences in αß- and γδTCR signal transduction were due to differences in the intracellular signaling pathways coupled to these two TCRs. To investigate this, we used transcriptional profiling to identify genes encoding signaling molecules that are differentially expressed in mature αß and γδ T cell populations. Unexpectedly, we found that B lymphoid kinase (Blk), a Src family kinase expressed primarily in B cells, is expressed in γδ T cells but not in αß T cells. Analysis of Blk-deficient mice revealed that Blk is required for the development of IL-17-producing γδ T cells. Furthermore, Blk is expressed in lymphoid precursors and, in this capacity, plays a role in regulating thymus cellularity during ontogeny.

Allogeneic CD20-targeted γδ T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models.

OBJECTIVES: Autologous chimeric antigen receptor (CAR) αß T-cell therapies have demonstrated remarkable antitumor efficacy in patients with haematological malignancies; however, not all eligible cancer patients receive clinical benefit. Emerging strategies to improve patient access and clinical responses include using premanufactured products from healthy donors and alternative cytotoxic effectors possessing intrinsic tumoricidal activity as sources of CAR cell therapies. γδ T cells, which combine innate and adaptive mechanisms to recognise and kill malignant cells, are an attractive candidate platform for allogeneic CAR T-cell therapy. Here, we evaluated the manufacturability and functionality of allogeneic peripheral blood-derived CAR+ Vδ1 γδ T cells expressing a second-generation CAR targeting the B-cell-restricted CD20 antigen. METHODS: Donor-derived Vδ1 γδ T cells from peripheral blood were ex vivo-activated, expanded and engineered to express a novel anti-CD20 CAR. In vitro and in vivo assays were used to evaluate CAR-dependent and CAR-independent antitumor activities of CD20 CAR+ Vδ1 γδ T cells against B-cell tumors. RESULTS: Anti-CD20 CAR+ Vδ1 γδ T cells exhibited innate and adaptive antitumor activities, such as in vitro tumor cell killing and proinflammatory cytokine production, in addition to in vivo tumor growth inhibition of B-cell lymphoma xenografts in immunodeficient mice. Furthermore, CD20 CAR+ Vδ1 γδ T cells did not induce xenogeneic graft-versus-host disease in immunodeficient mice. CONCLUSION: These preclinical data support the clinical evaluation of ADI-001, an allogeneic CD20 CAR+ Vδ1 γδ T cell, and a phase 1 study has been initiated in patients with B-cell malignancies (NCT04735471).

Activation-induced modification in the CD3 complex of the gammadelta T cell receptor.

The T cell antigen receptor complexes expressed on alphabeta and gammadelta T cells differ not only in their respective clonotypic heterodimers but also in the subunit composition of their CD3 complexes. The gammadelta T cell receptors (TCRs) expressed on ex vivo gammadelta T cells lack CD3delta, whereas alphabeta TCRs contain CD3delta. While this result correlates with the phenotype of CD3delta(-/-) mice, in which gammadelta T cell development is unaffected, it is inconsistent with the results of previous studies reporting that CD3delta is a component of the gammadelta TCR. Since earlier studies examined the subunit composition of gammadelta TCRs expressed on activated and expanded peripheral gammadelta T cells or gammadelta TCR(+) intestinal intraepithelial lymphocytes, we hypothesized that activation and expansion may lead to changes in the CD3 subunit composition of the gammadelta TCR. Here, we report that activation and expansion do in fact result in the inclusion of a protein, comparable in mass and mobility to CD3delta, in the gammadelta TCR. Further analyses revealed that this protein is not CD3delta, but instead is a differentially glycosylated form of CD3gamma. These results provide further evidence for a major difference in the subunit composition of alphabeta- and gammadelta TCR complexes and raise the possibility that modification of CD3gamma may have important functional consequences in activated gammadelta T cells.

Reduced B lymphoid kinase (Blk) expression enhances proinflammatory cytokine production and induces nephrosis in C57BL/6-lpr/lpr mice.

BLK, which encodes B lymphoid kinase, was recently identified in genome wide association studies as a susceptibility gene for systemic lupus erythematosus (SLE), and risk alleles mapping to the BLK locus result in reduced gene expression. To determine whether BLK is indeed a bona fide susceptibility gene, we developed an experimental mouse model, namely the Blk+/-.lpr/lpr (Blk+/-.lpr) mouse, in which Blk expression levels are reduced to levels comparable to those in individuals carrying a risk allele. Here, we report that Blk is expressed not only in B cells, but also in IL-17-producing γδ and DN αß T cells and in plasmacytoid dendritic cells (pDCs). Moreover, we found that solely reducing Blk expression in C57BL/6-lpr/lpr mice enhanced proinflammatory cytokine production and accelerated the onset of lymphoproliferation, proteinuria, and kidney disease. Together, these findings suggest that BLK risk alleles confer susceptibility to SLE through the dysregulation of a proinflammatory cytokine network.

γδ T cells acquire effector fates in the thymus and differentiate into cytokine-producing effectors in a Listeria model of infection independently of CD28 costimulation.

Both antigen recognition and CD28 costimulation are required for the activation of naïve αß T cells and their subsequent differentiation into cytokine-producing or cytotoxic effectors. Notably, this two-signal paradigm holds true for all αß T cell subsets, regardless of whether they acquire their effector function in the periphery or the thymus. Because of contradictory results, however, it remains unresolved as to whether CD28 costimulation is necessary for γδ T cell activation and differentiation. Given that γδ T cells have been recently shown to acquire their effector fates in the thymus, it is conceivable that the contradictory results may be explained, in part, by a differential requirement for CD28 costimulation in the development or differentiation of each γδ T cell effector subset. To test this, we examined the role of CD28 in γδ T cell effector fate determination and function. We report that, although IFNγ-producing γδ T (γδ-IFNγ) cells express higher levels of CD28 than IL-17-producing γδ T (γδ-17) cells, CD28-deficiency had no effect on the thymic development of either subset. Also, following Listeria infection, we found that the expansion and differentiation of γδ-17 and γδ-IFNγ effectors were comparable between CD28(+/+) and CD28(-/-) mice. To understand why CD28 costimulation is dispensable for γδ T cell activation and differentiation, we assessed glucose uptake and utilization by γδ T cells, as CD28 costimulation is known to promote glycolysis in αß T cells. Importantly, we found that γδ T cells express higher surface levels of glucose transporters than αß T cells and, when activated, exhibit effector functions over a broader range of glucose concentrations than activated αß T cells. Together, these data not only demonstrate an enhanced glucose metabolism in γδ T cells but also provide an explanation for why γδ T cells are less dependent on CD28 costimulation than αß T cells.

Increased caspase activity primes human Lyme arthritis synovial γδ T cells for proliferation and death.

γδ T cells function between the innate and adaptive immune responses, promoting antigen-presenting cell function and manifesting cytolytic activity. Their numbers often increase during infections, such as human immunodeficiency virus, and at sites of chronic inflammation. However, the turnover dynamics of human γδ T cells are poorly understood. Here we observed that despite more rapid proliferation in vitro by human Lyme arthritis synovial γδ T cells of the Vδ1 subset, they have reduced surviving cell numbers compared with αß T cells because of increased cell death by the γδ T cells. Because caspases are involved in cell proliferation and death, and because signaling is more efficient through T cell receptor (TCR)-γδ than through TCR-αß, we examined the levels of active caspases during cell cycling and following TCR restimulation. We observed higher overall caspase activity in Borrelia-reactive γδ T cells than in comparable αß T cells. This was paralleled by greater spontaneous cell death and TCR restimulation-induced cell death of the γδ T cells, which was caspase dependent. Our current findings thus are consistent with a model in which human γδ T cells evolved to function quickly and transiently in an innate fashion.

ITAM-mediated signaling by the T-cell antigen receptor.

Signal transduction by the T-cell antigen receptor (TCR) is initiated by phosphorylation of conserved motifs (ITAMs) contained within the cytoplasmic domains of the invariant subunits. TCR complexes contain a total of 10 ITAMs and this unusual configuration has prompted studies of the role of specific ITAMs, or of ITAM multiplicity, in regulating TCR-directed developmental and effector responses. Here, we summarize data generated during the past two decades and discuss how these findings have in some cases resolved, and in others complicated, outstanding questions relating to the function of TCR ITAMs.

Roles of the Src tyrosine kinases Lck and Fyn in regulating gammadeltaTCR signal strength.

Lck and Fyn, members of the Src family of tyrosine kinases, are key components of the alphabetaTCR-coupled signaling pathway. While it is generally accepted that both Lck and Fyn positively regulate signal transduction by the alphabetaTCR, recent studies have shown that Lck and Fyn have distinct functions in this signaling pathway, with Lck being a positive regulator and Fyn being a negative regulator of alphabetaTCR signal transduction. To determine whether Lck and Fyn also differentially regulate gammadeltaTCR signal transduction, we analyzed gammadelta T cell development and function in mice with reduced Lck or Fyn expression levels. We found that reducing Lck or Fyn levels altered the strength of the gammadeltaTCR signaling response, with low levels of Lck weakening gammadeltaTCR signal strength and low levels of Fyn augmenting gammadeltaTCR signal strength. These alterations in gammadeltaTCR signal strength had profound effects not only on alphabeta/gammadelta lineage choice, but also on gammadelta thymocyte maturation and gammadelta T cell effector function. These results indicate that the cellular levels of Lck and Fyn play a role in regulating the strength of the gammadeltaTCR signaling response at different stages in the life of the gammadelta T cell.

Profiling of the early transcriptional response of murine gammadelta T cells following TCR stimulation.

Gammadelta T cells represent one of the three lineages of lymphocytes, along with alphabeta T cells and B cells, which express antigen receptors. Since their discovery over two decades ago, considerable effort has been made to understand their antigen specificity and their contribution to the immune response. From these studies, we have learned that gammadelta T cells recognize a different set of antigens than alphabeta T cells, acquire effector functions faster than alphabeta T cells, regulate the response of other immune cells during infection, and play distinct roles in immunity. The molecular basis for how gammadelta T cells manifest their unique functions, however, remains unknown. To address this, we profiled the genes upregulated soon after TCR stimulation in order to identify which gene networks associated with T cell effector function are induced in gammadelta T cells. Interestingly, most of the genes in this transcriptional profile were not unique to activated gammadelta T cells, as they were also expressed in activated alphabeta T cells. However, many of the genes within this profile were upregulated with faster kinetics and/or greater magnitude in activated gammadelta T cells than in activated alphabeta T cells. In addition, we found that the genes in the transcriptional profile of activated wild-type gammadelta T cells can be used as a standard to screen activated gammadelta T cells from mice with potential signaling defects for alterations in gammadelta TCR signal transduction. Thus, by defining the early transcriptional response of activated wild-type gammadelta T cells and by comparing their transcriptional profile to that of activated wild-type alphabeta T cells as well as to that of activated gammadelta T cells from signaling defective mice, we are able to gain important insights into the molecular basis for gammadelta T cell function.

Dynamics of CD3gammaepsilon and CD3deltaepsilon dimer expression during murine T cell development.

The preTCR, gammadeltaTCR, and alphabetaTCR are the three isoforms of the T cell antigen receptor that are expressed during thymocyte development. Signaling by these isoforms is required at different stages of T cell development for lineage commitment, thymocyte maturation, and repertoire selection. All three isoforms are multimeric complexes, which are dependent on invariant CD3 dimers (CD3gammaepsilon and CD3deltaepsilon) and TCRzetazeta dimers for their assembly, stable surface expression and signal transduction. Notably, differences have been reported regarding the requirement for CD3delta in the assembly, surface expression and signaling abilities of the three TCR isoforms. Specifically, it has been shown that both the preTCR and gammadeltaTCR do not require CD3delta to transduce signals, whereas the alphabetaTCR does. The differences noted between the murine alphabeta- and gammadeltaTCRs in their requirement for CD3delta can be easily explained by the fact that CD3delta is a component of the alphabetaTCR but not the gammadeltaTCR. However, it is not clear why the preTCR does not require CD3delta, considering that CD3delta has been reported to be a subunit of the preTCR. Because the preTCR can be expressed on thymocytes at the immature CD4(-)CD8(-) stage and, to a lesser extent, at the later CD4(+)CD8(+) stage, it is conceivable that CD3deltaepsilon dimer expression is developmentally regulated during early T cell development such that preTCRs expressed on immature CD4(-)CD8(-) thymocytes contain primarily CD3gammaepsilon dimers while those expressed on CD4(+)CD8(+) thymocytes express both CD3deltaepsilon and CD3gammaepsilon dimers. To investigate this, we determined whether the expression of CD3delta and CD3gamma are developmentally regulated and whether there are differences in the availability and/or stability of CD3deltaepsilon and CD3gammaepsilon dimers during early T cell development. We report that even though both CD3delta and CD3gamma were expressed at relatively high levels in immature CD4(-)CD8(-) thymocytes, CD3gammaepsilon dimers predominated over CD3deltaepsilon dimers at this early stage. However, expression of CD3deltaepsilon dimers was rescued when pTalpha, TCRbeta and TCRalpha chains were also expressed at the CD4(-)CD8(-) stage, indicating that the relative amounts of pTalpha, TCRbeta and TCRalpha chains during early thymocyte development control the stability and, therefore, availability of CD3deltaepsilon dimers.

A retrospective on the requirements for gammadelta T-cell development.

Since the discovery of gammadelta T cells two decades ago, considerable effort has been made to understand their developmental program, their antigen specificity, and their contribution to the immune response. In this review, we focus on what is known about gammadelta T-cell development and on the advances that have been made in determining which genes are required. In addition, we compare the genetic requirements for alphabeta and gammadelta T-cell development with the hope of gaining a better picture of the signaling pathways that govern the development of gammadelta lineage cells.

Strength of signal: a fundamental mechanism for cell fate specification.

How equipotent cells develop into complex tissues containing many diverse cell types is still a mystery. However, evidence is accumulating from different tissue systems in multiple organisms that many of the specific receptor families known to regulate cell fate decisions target conserved signaling pathways. A mechanism for preserving specificity in the cellular response that has emerged from these studies is one in which quantitative differences in receptor signaling regulate the cell fate decision. A signal strength model has recently gained support as a means to explain alphabeta/gammadelta lineage commitment. In this review, we compare the alphabeta/gammadelta fate decision with other cell fate decisions that occur outside of the lymphoid system to attain a better picture of the quantitative signaling mechanism for cell fate specification.

Premature expression of chemokine receptor CCR9 impairs T cell development.

During thymocyte development, CCR9 is expressed on late CD4-CD8- (double-negative (DN)) and CD4+CD8+ (double-positive) cells, but is subsequently down-regulated as cells transition to the mature CD4+ or CD8+ (single-positive (SP)) stage. This pattern of expression has led to speculation that CCR9 may regulate thymocyte trafficking and/or export. In this study, we generated transgenic mice in which CCR9 surface expression was maintained throughout T cell development. Significantly, forced expression of CCR9 on mature SP thymocytes did not inhibit their export from the thymus, indicating that CCR9 down-regulation is not essential for thymocyte emigration. CCR9 was also expressed prematurely on immature DN thymocytes in CCR9 transgenic mice. Early expression of CCR9 resulted in a partial block of development at the DN stage and a marked reduction in the numbers of double-positive and SP thymocytes. Moreover, in CCR9-transgenic mice, CD25high DN cells were scattered throughout the cortex rather than confined to the subcapsular region of the thymus. Together, these results suggest that regulated expression of CCR9 is critical for normal development of immature thymocytes, but that down-regulation of CCR9 is not a prerequisite for thymocyte emigration.