Renee M. Laird

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 αβ 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 αβ- 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 αβ 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 αβ 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.

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 β‐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.

Beyond αβ/γδ lineage commitment: TCR signal strength regulates γδ 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.

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 αβ 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.

Roles of the Src Tyrosine Kinases Lck and Fyn in Regulating γδTCR Signal Strength

Lck and Fyn, members of the Src family of tyrosine kinases, are key components of the αβTCR-coupled signaling pathway. While it is generally accepted that both Lck and Fyn positively regulate signal transduction by the αβTCR, 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 αβTCR signal transduction. To determine whether Lck and Fyn also differentially regulate γδTCR signal transduction, we analyzed γδ 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 γδTCR signaling response, with low levels of Lck weakening γδTCR signal strength and low levels of Fyn augmenting γδTCR signal strength. These alterations in γδTCR signal strength had profound effects not only on αβ/γδ lineage choice, but also on γδ thymocyte maturation and γδ T cell effector function. These results indicate that the cellular levels of Lck and Fyn play a role in regulating the strength of the γδTCR signaling response at different stages in the life of the γδ T cell.

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 αβ T cells and their subsequent differentiation into cytokine-producing or cytotoxic effectors. Notably, this two-signal paradigm holds true for all αβ 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 αβ T cells. Importantly, we found that γδ T cells express higher surface levels of glucose transporters than αβ T cells and, when activated, exhibit effector functions over a broader range of glucose concentrations than activated αβ 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 αβ T cells.

Profiling of the early transcriptional response of murine γδ 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 CD3γɛ and CD3δɛ 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.