DX5/CD49b-positive T cells are not synonymous with CD1d-dependent NKT cells

NKT cells are typically defined as CD1d-dependent T cells that carry an invariant TCR alpha-chain and produce high levels of cytokines. Traditionally, these cells were defined as NK1.1+ T cells, although only a few mouse strains express the NK1.1 molecule. A popular alternative marker for NKT cells has been DX5, an Ab that detects the CD49b integrin, expressed by most NK cells and a subset of T cells that resemble NKT cells. Interpretation of studies using DX5 as an NKT cell marker depends on how well DX5 defines NKT cells. Using a range of DX5 and other anti-CD49b Abs, we reveal major differences in reactivity depending on which Ab and which fluorochrome are used. The brightest, PE-conjugated reagents revealed that while most CD1d-dependent NKT cells expressed CD49b, they represented only a minority of CD49b+ T cells. Furthermore, CD49b+ T cell numbers were near normal in CD1d-/- mice that are completely deficient for NKT cells. CD1d tetramer- CD49b+ T cells differ from NKT cells by their activation and memory marker expression, tissue distribution, and CD4/CD8 coreceptor profile. Interestingly, both NKT cells and CD1d tetramer- CD49b+ T cells produce cytokines, but the latter are clearly biased toward Th1-type cytokines, in contrast to NKT cells that produce both Th1 and Th2 cytokines. Finally, we demonstrate that expression of CD49b by NKT cells does not dramatically alter with age, contrasting with earlier reports proposing DX5 as a maturation marker for NKT cells. In summary, our data demonstrate that DX5/CD49b is a poor marker for identifying CD1d-dependent NKT cells.     

A subset of NKT cells that lacks the NK1.1 marker, expresses CD1d molecules, and autopresents the alpha-galactosylceramide antigen

In the present report, we characterize a novel T cell subset that shares with the NKT cell lineage both CD1d-restriction and high reactivity in vivo and in vitro to the alpha-galactosylceramide (alpha-GalCer) glycolipid. These cells preferentially use the canonical Valpha14-Jalpha281 TCR-alpha-chain and Vbeta8 TCR-beta segments, and are stimulated by alpha-GalCer in a CD1d-dependent fashion. However, in contrast to classical NKT cells, they lack the NK1.1 marker and express high surface levels of CD1d molecules. In addition, this NK1.1(-) CD1d(high) T subset, further referred to as CD1d(high) NKT cells, can be distinguished by its unique functional features. Although NK1.1(+) NKT cells require exogenous CD1d-presenting cells to make them responsive to alpha-GalCer, CD1d(high) NKT cells can engage their own surface CD1d in an autocrine and/or paracrine manner. Furthermore, in response to alpha-GalCer, CD1d(high) NKT cells produce high amounts of IL-4 and moderate amounts of IFN-gamma, a cytokine profile more consistent with a Th2-like phenotype rather than the Th0-like phenotype typical of NK1.1(+) NKT cells. Our work reveals a far greater level of complexity within the NKT cell population than previously recognized and provides the first evidence for T cells that can be activated upon TCR ligation by CD1d-restricted recognition of their ligand in the absence of conventional APCs.     

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.     

CD44 differentially activates mouse NK T cells and conventional T cells

NK T (NKT) cells are an important component of the innate immune system and recognize the MHC class I-like CD1d molecule. NKT cells possess significant immunoregulatory activity due to their rapid secretion of large quantities of pro- and anti-inflammatory cytokines following CD1d-dependent stimulation. Because the innate immune system is programmed to respond to a multitude of diverse stimuli and must be able to quickly differentiate between pathogenic and endogenous signals, we hypothesized that, apart from stimulation via the TCR (e.g., CD1d-dependent activation), there must be multiple activation pathways that can be triggered through other cell surface receptors on NKT cells. Therefore, we analyzed the ability of CD44, a structurally diverse cell surface receptor expressed on most cells, to stimulate murine NKT cells, compared with conventional T cells. Stimulation of CD44 through Ab cross-linking or binding to its natural ligands hyaluronan and osteopontin induced NKT cells to secrete cytokines, up-regulate activation markers, undergo morphological changes, and resist activation-induced cell death, whereas conventional T cells only exhibited changes in morphology and protection from activation-induced cell death. This CD44-specific stimulation of NKT cells correlated with their ability to bind hyaluronan. Thus, fundamental differences in CD44 function between these lymphocyte subsets suggest an important biological role for CD44 in the innate immune response.     

Direct regulatory role of NKT cells in allogeneic graft survival is dependent on the quantitative strength of antigenicity

The role of NKT cells during immune responses is diverse, ranging from antiviral and antitumor activity to the regulation of autoimmune diseases; however, the regulatory function of CD1d-dependent NKT cells in rejection responses against allogeneic graft is uncertain. In this study, we demonstrated the direct regulatory effects of CD1d-dependent NKT cells using an allogeneic skin transplantation model. H-Y-mismatched skin graft survival was shortened in CD1d-/- recipients compared with wild-type recipients. Adoptive transfer of syngeneic NKT cells via splenocytes or hepatic mononuclear cells into CD1d-/- recipients restored graft survival times to those of wild-type recipients. alpha-Galactosylceramide, a specific activator of NKT cells, further prolonged graft survival. Although CD1d-dependent NKT cells did not extend skin graft survival in either major or complete minor histocompatibility-mismatched models, these cells affected graft survival in minor Ag mismatch models according to the magnitude of the antigenic difference. The afferent arm of NKT cell activation during transplantation required CD1d molecules expressed on host APCs and the migration of CD1d-dependent NKT cells into grafts. Moreover, the regulatory effects of CD1d-dependent NKT cells against alloantigen were primarily IL-10 dependent. Taken together, we concluded that CD1d-dependent NKT cells may directly affect the outcome of allogeneic skin graft through an IL-10-dependent regulatory mechanism.     

Differential requirement for the SAP-Fyn interaction during NK T cell development and function

The adaptor molecule SAP (signaling lymphocytic activation molecule-associated protein) plays a critical role during NK T (NKT) cell development in humans and mice. In CD4(+) T cells, SAP interacts with the tyrosine kinase Fyn to deliver signals required for TCR-induced Th2-type cytokine production. To determine whether the SAP-dependent signals controlling NKT cell ontogeny rely on its binding to Fyn, we used the OP9-DL1 system to initiate structure function studies of SAP in murine NKT cell development. In cultures containing wild-type (WT) hematopoietic progenitors, we noted the transient emergence of cells that reacted with the NKT cell-specific agonist alpha-galactosyl ceramide and its analog PBS57. Sap(-/-) cells failed to give rise to NKT cells in vitro; however, their development could be rescued by re-expression of WT SAP. Emergence of NKT cells was also restored by a mutant version of SAP (SAP R78A) that cannot bind to Fyn, but with less efficiency than WT SAP. This finding was accentuated in vivo in Sap(R78A) knock-in mice as well as Sap(R78A) competitive bone marrow chimeras, which retained NKT cells but at significantly reduced numbers compared with controls. Unlike Sap(R78A) CD4(+) T cells, which produce reduced levels of IL-4 following TCR ligation, alpha-galactosyl ceramide-stimulated NKT cells from the livers and spleens of Sap(R78A) mice produced Th2 cytokines and activated NK cells in a manner mimicking WT cells. Thus, SAP appears to use differential signaling mechanisms in NKT cells, with optimal ontogeny requiring Fyn binding, while functional responses occur independently of this interaction.     

Protein tyrosine phosphatase alpha regulates Fyn activity and Cbp/PAG phosphorylation in thymocyte lipid rafts

A role for the receptor protein tyrosine phosphatase alpha (PTPalpha) in immune cell function and regulation of Src family kinases was investigated using thymocytes from PTPalpha-deficient mice. PTPalpha-null thymocytes develop normally, but unstimulated PTPalpha-/- cells exhibit increased tyrosine phosphorylation of specific proteins, increased Fyn activity, and hyperphosphorylation of Cbp/PAG that promotes its association with C-terminal Src kinase. Elevated Fyn activity in the absence of PTPalpha is due to enhanced phosphorylation of Fyn tyrosines 528 and 417. Some PTPalpha is localized in lipid rafts of thymocytes, and raft-associated Fyn is specifically activated in PTPalpha-/- cells. PTPalpha is not a Cbp/PAG phosphatase, because it is not required for Cbp/PAG dephosphorylation in unstimulated or anti-CD3-stimulated thymocytes. Together, our results indicate that PTPalpha, likely located in lipid rafts, regulates the activity of raft Fyn. In the absence of PTPalpha this population of Fyn is activated and phosphorylates Cbp/PAG to enhance association with C-terminal Src kinase. Although TCR-mediated tyrosine phosphorylation was apparently unaffected by the absence of PTPalpha, the long-term proliferative response of PTPalpha-/- thymocytes was reduced. These findings indicate that PTPalpha is a component of the complex Src family tyrosine kinase regulatory network in thymocytes and is required to suppress Fyn activity in unstimulated cells in a manner that is not compensated for by the major T cell PTP and SFK regulator, CD45.     

Targeted expression of human CD1d in transgenic mice reveals independent roles for thymocytes and thymic APCs in positive and negative selection of Valpha14i NKT cells

CD1d-dependent invariant Valpha14 (Valpha14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Valpha14-Jalpha18 TCR alpha-chain paired mostly with Vbeta8.2 and Vbeta7. The cellular requirements for thymic positive and negative selection of Valpha14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Valpha14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Valpha14i NKT cells that proved functional when activated ex vivo with the Ag alpha-galactosyl ceramide. Valpha14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vbeta8.2. The low number of Vbeta8.2+ Valpha14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vbeta7+ Valpha14i NKT cells. Vbeta8.2+, but not Vbeta7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Valpha14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.     

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.     

Differential function of PTPalpha and PTPalpha Y789F in T cells and regulation of PTPalpha phosphorylation at Tyr-789 by CD45

CD45 is a major membrane protein tyrosine phosphatase (PTP) expressed in T cells where it regulates the activity of Lck, a Src family kinase important for T cell receptor-mediated activation. PTPalpha is a more widely expressed transmembrane PTP that has been shown to regulate the Src family kinases, Src and Fyn, and is also present in T cells. Here, PTPalpha was phosphorylated at Tyr-789 in CD45(-) T cells but not in CD45(+) T cells suggesting that CD45 could regulate the phosphorylation of PTPalpha at this site. Furthermore, CD45 could directly dephosphorylate PTPalpha in vitro. Expression of PTPalpha and PTPalpha-Y789F in T cells revealed that the mutant had a reduced ability to decrease Fyn and Cbp phosphorylation, to regulate the kinase activity of Fyn, and to restore T cell receptor-induced signaling events when compared with PTPalpha. Conversely, this mutant had an increased ability to prevent Pyk2 phosphorylation and CD44-mediated cell spreading when compared with PTPalpha. These data demonstrate distinct activities of PTPalpha and PTPalpha-Y789F in T cells and identify CD45 as a regulator of PTPalpha phosphorylation at tyrosine 789 in T cells.     

Defective thymocyte maturation by transgenic expression of a truncated form of the T lymphocyte adapter molecule and Fyn substrate,Sin

Adapter molecules that promote protein-protein interactions play a central role in T lymphocyte differentiation and activation. In this study, we examined the role of the T lymphocyte-expressed adapter protein and Src kinase substrate, Sin, on thymocyte function using transgenic mice expressing an activated, truncated allele of Sin (SinDeltaC). We found that SinDeltaC expression led to reduced numbers of CD4(+) and CD8(+) single-positive cells and reduced thymic cellularity due to increased thymocyte apoptosis. Because the adapter properties of Sin are mediated by tyrosine-based motifs and given that Sin is a substrate for Src tyrosine kinases, we examined the involvement of these kinases in the inhibitory effects of SinDeltaC. We found that in transgenic thymocytes, SinDeltaC was constitutively phosphorylated by the Src kinase Fyn, but not by the related kinase Lck. Using SinDeltaC and fyn(-/-) animals, we also found that the expression of Fyn was required for the inhibitory effect of SinDeltaC on thymocyte apoptosis but not for SinDeltaC-mediated inhibition of T cell maturation. The inhibitory effect of SinDeltaC on thymocyte maturation correlated with defective activation of the mitogen-activated protein kinase extracellular signal-regulated kinase. Our results suggest that the Sin mutant inhibits thymocyte differentiation through Fyn-dependent and -independent mechanisms and that endogenous Sin may be an important regulator of thymocyte development.     

Cutting edge: influence of the TCR Vbeta domain on the selection of semi-invariant NKT cells by endogenous ligands

Invariant Valpha14 (Valpha14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a Valpha14-Jalpha18 rearrangement and expression of mostly Vbeta8.2 and Vbeta7. Whereas the TCR Vbeta domain influences the binding avidity of the Valpha14i TCR for CD1d-alpha-galactosylceramide complexes, with Vbeta8.2 conferring higher avidity binding than Vbeta7, a possible impact of the TCR Vbeta domain on Valpha14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vbeta7(+), but not Vbeta8.2(+), Valpha14i NKT cells is favored in situations where endogenous ligand concentration or TCRalpha-chain avidity are suboptimal. Furthermore, thymic Vbeta7(+) Valpha14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vbeta domain influences the selection of Valpha14i NKT cells by endogenous ligands, presumably because Vbeta7 confers higher avidity binding.     

Natural killer T cells restricted by the monomorphic MHC class 1b CD1d1 molecules behave like inflammatory cells.

Murine Valpha14(inv)T cells (NKT cells), restricted by the CD1d1 MHC 1b molecules, are a distinctive subset of T cells endowed with pleiotropic functions. CD1d1-restricted NKT cells infiltrate the granulomas induced by the s.c. injection of mycobacterial phosphatidylinositoldimannoside (PIM(2)) but not of its deacylated derivative. NKT cells are detectable as early as 6 hours following the injection. Although the molecular structure of PIM(2) meets the requirements for presentation by CD1d1, Ab blocking and adoptive transfer experiments of wild-type NKT cells into CD1d1(-/-) mice show that CD1d1 expression is not required for the early recruitment of NKT cells to the injection site. This conclusion was confirmed by the finding that IL-12Rbeta(-/-) and CD40(-/-) mice were able to recruit NKT cells after PIM(2) challenge. Moreover, the injection of alpha-galactosylceramide, an NKT cell ligand that is recognized in the context of CD1d1, promoted only a minor recruitment of NKT cells. By contrast, injection of beta-galactosylceramide, a synthetic glycolipid that binds to CD1d1 but does not activate the CD1d/TCR pathway, resulted in the development of large granulomas rich in NKT cells. Finally, local injection of TNF-alpha mimics the effect of glycolipids. It is concluded that NKT cells migrate to and accumulate at inflammatory sites in the same way as other cells of the innate immune system and that migration to and accumulation at inflammatory sites are processes independent of the CD1d1 molecule.     

CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45

CD44 is a cell adhesion molecule implicated in leukocyte adhesion and migration, co-stimulation of T cells, and tumor metastasis. CD45 is a leukocyte-specific protein tyrosine phosphatase that dephosphorylates the Src family kinases, Lck and Fyn, in T cells. Positive regulation of Lck by CD45 is required for its effective participation in T cell receptor signaling events. Here, immobilized CD44 antibody induced a distinctive cell spreading in CD45(-), but not CD45(+), T cells, and this correlated with the induction of tyrosine-phosphorylated proteins. Two focal adhesion family kinases, Pyk2 and, to a lesser extent, FAK were inducibly phosphorylated, as was a potential substrate, Cas. CD44-mediated cell spreading and induced tyrosine phosphorylation were prevented by the Src family kinase inhibitor, PP2. Furthermore, 2-fold more Lck associated with CD44 in the low density sucrose fraction from CD45(-) T cells compared with CD45(+) T cells, suggesting that CD45 may regulate the association of Lck with CD44 in this fraction. Therefore, in CD45(-) T cells, CD44 signaling is mediated by Src family kinases, and this leads to Pyk2 phosphorylation, cytoskeletal changes, and cell spreading. This implicates CD45 in the negative regulation of Src family kinase-mediated CD44 signaling leading to T cell spreading.     

The effect of intracellular trafficking of CD1d on the formation of TCR repertoire of NKT cells

CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to αβ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of αβ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant Vα14-Jα18 TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire. [BMB Reports 2014; 47(5): 241-248]     

Temporal differences in the dependency on phosphoinositide-dependent kinase 1 distinguish the development of invariant Valpha14 NKT cells and conventional T cells

This study uses two independent genetic strategies to explore the requirement for phosphoinositide-dependent kinase-1 (PDK1) in the development of mature T cell populations from CD4/CD8 double-positive thymocytes. The data show that CD4/CD8 double-positive thymocytes that do not express PDK1 or express a catalytically inactive PDK1 mutant fail to produce mature invariant Vα14 NKT cells but can differentiate to conventional CD4, CD8, or regulatory T cell subsets in the thymus. The PDK1 requirement for Vα14 NKT cell development reflects that these cells require the PDK1 substrate protein kinase B to meet the metabolic demands for proliferative expansion in response to IL-15 or AgR stimulation. There is also constitutive PDK1 signaling in conventional α/β T cells that is not required for lineage commitment of these cells but fine-tunes the expression of coreceptors and adhesion molecules. Also, although PDK1 is dispensable for thymic development of conventional α/β T cells, peripheral cells are reduced substantially. This reflects a PDK1 requirement for lymphopenia-induced proliferation, a process necessary for initial population of the peripheral T cell niche in neonatal mice. PDK1 is thus indispensable for T cell developmental programs, but the timing of the PDK1 requirement is unique to different T cell subpopulations.     

Quantitative and qualitative differences in the in vivo response of NKT cells to distinct alpha- and beta-anomeric glycolipids

NKT cells represent a unique subset of immunoregulatory T cells that recognize glycolipid Ags presented by the MHC class I-like molecule CD1d. Because of their immunoregulatory properties, NKT cells are attractive targets for the development of immunotherapies. The prototypical NKT cell ligand alpha-galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, has potent immunomodulatory activities in mice, demonstrating therapeutic efficacy against metastatic tumors, infections, and autoimmune diseases, but also has a number of adverse side effects. In vivo administration of alpha-GalCer to mice results in the rapid activation of NKT cells, which is characterized by cytokine secretion, surface receptor down-regulation, expansion, and secondary activation of a variety of innate and adaptive immune system cells. In this study, we have evaluated the in vivo immune response of mice to a set of structural analogues of alpha-GalCer. Our results show that, contrary to current thinking, beta-anomeric GalCer can induce CD1d-dependent biological activities in mice, albeit at lower potency than alpha-anomeric GalCer. In addition, we show that the response of NKT cells to distinct GalCer differs not only quantitatively, but also qualitatively. These findings indicate that NKT cells can fine-tune their immune responses to distinct glycolipid Ags in vivo, a property that may be exploited for the development of effective and safe NKT cell-based immunotherapies.     

Fyn Kinase Is Required for Optimal Humoral Responses

The generation of antigen-specific antibodies and the development of immunological memory require collaboration between B and T cells. T cell-secreted IL-4 is important for B cell survival, isotype switch to IgG1 and IgE, affinity maturation, and the development of germinal centers (GC). Fyn, a member of the Src family tyrosine kinase, is widely expressed in many cell types, including lymphocytes. This kinase is known to interact with both the B cell and T cell receptor (BCR and TCR, respectively). While Fyn deletion does not impair the development of immature T cells and B cells, TCR signaling is altered in mature T cells. The current study demonstrates that Fyn deficient (KO) B cells have impaired IL-4 signaling. Fyn KO mice displayed low basal levels of IgG1, IgE and IgG2c, and delayed antigen-specific IgG1 and IgG2b production, with a dramatic decrease in antigen-specific IgG2c following immunization with a T-dependent antigen. Defects in antibody production correlated with significantly reduced numbers of GC B cells, follicular T helper cells (T_FH), and splenic plasma cells (PC). Taken together, our data demonstrate that Fyn kinase is required for optimal humoral responses.     

Normal development and activation but altered cytokine production of Fyn-deficient CD4+ T cells

The Src family kinase Fyn is expressed in T cells and has been shown to phosphorylate proteins involved in TCR signaling, cytoskeletal reorganization, and IL-4 production. Fyn-deficient mice have greatly decreased numbers of NKT cells and have thymocytes and T cells with compromised responses following Ab crosslinking of their TCRs. Herein we have addressed the role of Fyn in peptide/MHC class II-induced CD4(+) T cell responses. In Fyn-deficient mice, CD4(+) T cells expressing the DO11.10 TCR transgene developed normally, and the number and phenotype of naive and regulatory DO11.10(+)CD4(+) T cells in the periphery were comparable with their wild-type counterparts. Conjugation with chicken OVA peptide 323-339-loaded APCs, and the subsequent proliferation in vitro or in vivo of DO11.10(+) Fyn-deficient CD4(+) T cells, was virtually indistinguishable from the response of DO11.10(+) wild-type CD4(+) T cells. Proliferation of Fyn-deficient T cells was not more dependent on costimulation through CD28. Additionally, we have found that differentiation, in vitro or in vivo, of transgenic CD4(+) Fyn-deficient T cells into IL-4-secreting effector cells was unimpaired, and under certain conditions DO11.10(+) Fyn-deficient CD4(+) T cells were more potent cytokine-producing cells than DO11.10(+) wild-type CD4(+) T cells. These data demonstrate that ablation of Fyn expression does not alter most Ag-driven CD4(+) T cell responses, with the exception of cytokine production, which under some circumstances is enhanced in Fyn-deficient CD4(+) T cells.