Rac-King up New Roles in Haematopoiesis

HIGHLIGHTS

numbers of pro-B, pre-B and imma- SIGNALLING IN BRIEF ture B cells in the bone marrow, but reduced numbers of B cells at later Rac-king up developmental stages in the spleen NATURAL KILLER CELLS and lymph nodes. In the absence of new roles in both Rac1 and Rac2, B-cell develop- The mature activating natural killer cell immunologic ment seems to be blocked at the synapse is formed in distinct stages. haematopoiesis transitional type 1 B-cell stage in the Orange, J. S. et al. Proc. Natl Acad. Sci. USA 100, 14151–14156 (2003) spleen. Rac1 and Rac2 guanosine triphos- Recent studies have shown activa- The interface between a natural killer (NK) cell and its target cell phatases (GTPases) are crucial sig- tion of Rac1 after B-cell receptor — the activating NK-cell immunological synapse — is highly nalling regulators in eukaryotic (BCR) stimulation, and signals from organized. In this study, Orange et al. show that CD2 and CD11b, cells, acting downstream of various the BCR are known to be crucial for similar to CD11a, co-localize with filamentous actin in the cellular receptors. Two studies pub- B-cell development, so are BCR sig- peripheral supramolecular activation cluster (pSMAC), whereas lished in Science have used condi- nalling defects responsible for the perforin accumulates in the central SMAC (cSMAC). Polarization tional gene targeting to clarify the observed phenotype of Rac1BRac2–/– of CD2, CD11a and CD11b to the pSMAC was dependent on roles of Rac1 and Rac2 in particular mice? Rac2–/– and Rac1–/+Rac2–/– actin polymerization and Wiskott-Aldrich syndrome protein aspects of haematopoietic-cell mature B cells stimulated with an (WASP), but independent of microtubule function. By contrast, differentiation. IgM-specific antibody had decreased perforin accumulation in the cSMAC was dependent on actin Gu et al. looked at the differential survival compared with wild-type polymerization, WASP and microtubule function. In addition, activity of Rac1 and Rac2 in B cells, which correlated with the rate of cSMAC perforin accumulation was slower than the haematopoietic stem/progenitor cells decreased induction of the anti- recruitment of receptors to the pSMAC, indicating that formation of the NK-cell immunological synapse is a highly regulated, (HSC/Ps). As Rac1 deficiency is apoptotic protein Bcl-XL.These embryonic lethal, they generated B cells also proliferated less than sequential process. conditional Rac1-knockout mice. wild-type cells. This was shown to be Deficiency of Rac1 inhibited the due to decreased induction of the MUCOSAL IMMUNOLOGY ability of HSC/Ps to reconstitute cell-cycle regulator cyclin D2 after haematopoiesis in an engraftment stimulation. So, Rac1 and Rac2 Spheniscins: avian β-defensins in preserved stomach model (which requires adhesion and transduce BCR signals for B-cell sur- contents of the king penguin, Aptenodytes proliferation in the bone marrow), vival and entry into the cell cycle. patagonicus. and the absence of both Rac1 and Finally, in contrast to wild-type cells, Thouzeau, C. et al. J. Biol. Chem. 2 October 2003 (doi:10.1074/jbc.M306839200) Rac2 led to the movement of progen- BAFF could not increase the survival In this paper, Thouzeau and colleagues have identified two new itor cells out of the bone marrow and of Rac1BRac2–/– immature B cells, avian antimicrobial peptides in the stomach of the male king into the peripheral circulation, as a and this was probably the result of penguin. These peptides, known as spheniscin 1 and 2, belong result of decreased adhesion to decreased expression of BAFF recep- to the β-defensin family and have broad antimicrobial activity fibronectin. Rac1 and Rac2 were also tor (BAFFR). Rac1 and Rac2 were against pathogenic bacteria and fungi. The levels of spheniscin shown to have roles in regulating the shown to transduce BCR signals lead- 1 and 2 are higher during periods of food storage than when cytoskeletal changes that are required ing to the upregulation of expression the birds are digesting, and interestingly, a drop in spheniscin for engraftment and mobilization. of BAFFR messenger RNA. levels correlated with a change from food storage to digestion. Rac1–/–, and to a greater extent These two studies show that in These antimicrobial peptides act to protect the surface of the Rac1–/–Rac2–/–, HSC/Ps had reduced both B cells and HSC/Ps, Rac1 and bird’s gastrointestinal tract from damage or invasion, and proliferation in response to growth Rac2 have essential roles in control- might be important for the long-term preservation of stored factors in vitro,correlating with ling proliferation and survival. food, which can be important for chick survival. decreased levels of cyclin D1 and Furthermore, despite their sequence phosphorylation of extracellular similarity, the two GTPases seem to signal-regulated kinase (ERK), and have partially non-redundant physi- increased levels of the cyclin- ological functions. Gu et al.con- dependent kinase inhibitor KIP1. By firmed this functional distinction in contrast, Rac2–/– cells were more sus- neutrophils, in which Rac2 seems to ceptible to apoptosis after growth- be the main GTPase regulating factor stimulation, associated with directed migration and superoxide reduced Akt activation. Therefore, it generation, whereas Rac1 regulates seems that Rac1 regulates entry into neutrophil shape. the cell cycle, whereas Rac2 mainly Kirsty Minton regulates cell survival. Walmsley et al.looked further References and links ORIGINAL RESEARCH PAPERS Gu, Y. et al. downstream at B-cell development Hematopoietic cell regulation by Rac1 and Rac2 by generating, for the first time, mice guanosine triphosphatases. Science 302, with a conditional deletion of Rac1 445–449 (2003) | Walmsley, M. J. et al. Critical B roles for Rac1 and Rac2 GTPases in B cell in the B-cell lineage (Rac1 mice). development and signaling. Science 302, Rac1BRac2–/– mice had normal 459–462 (2003)

NATURE REVIEWS | IMMUNOLOGY VOLUME 3 | DECEMBER 2003 | 925 HIGHLIGHTS

LYMPHOCYTE MIGRATION The ectodomain of the adhesion mol- evidence that naive T-cell trafficking is ecule L-selectin — which is crucial for not regulated by L-selectin shedding. T-cell migration from the bloodstream The authors further investigated Restricted entry into lymph nodes into peripheral lymph nodes (PLNs) the importance of L-selectin shedding — is proteolytically shed from the cell in T-cell rolling in vitro,using a hydro- surface after cross-linking. A new dynamic flow model, and in T-cell study by Galkina et al. has now defined trafficking in vivo after T-cell transfer that L-selectin shedding shapes the to normal or recombinase-activating migration pattern of T cells after anti- gene 1 (Rag1)-deficient mice. In vitro, gen activation, but that it is not the number of rolling L∆P and WT T required for naive T-cell trafficking. cells, as well as their rolling velocity L-selectin mediates tethering and and behaviour, was the same. In vivo, rolling of naive T cells in high transferred WT and L∆P naive T cells endothelial venules (HEVs) prior to migrated in equal numbers to the their migration to the PLNs. To inves- PLNs. Together, these studies indicate tigate the importance of L-selectin clearly that naive T-cell homing to the shedding in this process, Ann Ager PLNs is independent of L-selectin and colleagues generated endogenous shedding. L-selectin-deficient mice transgenic After activation, T cells downregu- for either wild-type L-selectin (WT) late the expression of L-selectin and or uncleavable L-selectin (L∆P) in T their migration patterns change. cells. The percentage of T cells in the Antigen-specific L-selectin downregu- PLNs of these animals was not statisti- lation was shown to be mediated by cally different, providing preliminary L-selectin shedding using offspring of

T-CELL SIGNALLING Stimulation of human peripheral blood T cells with CD4-specific antibodies induced tyrosine phosphorylation of LIME by protein tyrosine kinases, including LCK. A new adaptor in the LIME-light Phosphorylation of distinct tyrosine residues mediated the association of LIME Several transmembrane adaptor proteins Several signalling molecules, including with LCK and the negative regulator of SRC found in lipid rafts have been shown to the p85 regulatory subunit of kinases, CSK kinase, indicating that LIME influence the outcome of immunoreceptor phosphatidylinositol 3-kinase (PI3K) and phosphorylation can direct LCK and CSK signalling in lymphocytes. Two studies in growth factor receptor-bound protein 2 recruitment to lipid rafts. The Journal of Experimental Medicine have (GRB2), were found to associate with LIME, LIME-associated LCK was phosphorylated now defined a new transmembrane adaptor and LIME-specific signals were shown to at both the negative and positive regulatory molecule — LIME (LCK-interacting induce the phosphorylation of JUN tyrosine residues, and overexpression of membrane molecule) — which is involved in N-terminal kinase (JNK) and extracellular LIME induced an enhanced calcium regulating LCK-transduced signals in T cells. signal-regulated kinase 1 (ERK1)/ERK2. response to TCR triggering by CD3-specific Using a tyrosine phosphorylation- Together, the results presented by Yun and antibodies. These observations led Horejˇ ˇsí dependent yeast two-hybrid system, Hur et al. colleagues indicate that through its association and colleagues to suggest that after TCR and identified LIME as a protein that associates with LCK, LIME has a role in several signalling CD4 co-ligation, LIME might potentiate with the tyrosine kinase LCK. This association pathways that are activated after TCR TCR-mediated signals by binding LCK and was confirmed in immunoprecipitation stimulation. so recruiting it to the lipid rafts and blocking studies and was shown to require LCK- In a separate study, Brdickoˇ vá et al. its switch to the inactive form. mediated phosphorylation of LIME. identified mouse LIME by database searching These two studies have identified a new In mice, LIME was detected in resting for molecules that contain the domains transmembrane adaptor protein — LIME — peripheral T cells and its expression levels characteristic of lipid-raft-associated that regulates T-cell signalling through the were upregulated after stimulation with transmembrane adaptor proteins, and then tyrosine kinase LCK; however, further CD3-specific and CD28-specific antibodies. cloned the human homologue.As in mice, studies will be required to define its role Confocal microscopy showed that LIME was LIME protein was shown to be expressed by fully. distributed throughout the plasma membrane human peripheral blood T cells, in which it Karen Honey of resting T cells and that it relocated to the was palmitoylated and present in lipid rafts. immunological synapse after T-cell receptor However, a decrease in the expression level References and links ORIGINAL RESEARCH PAPERS Brdiˇcková, N. et al. LIME: (TCR) recognition of antigen. These data of LIME was detected after CD3-specific a new membrane raft-associated adaptor protein involved indicate that LIME is a component of lipid antibody stimulation, and removal of the TCR in CD4 and CD8 coreceptor signaling. J. Exp. Med. rafts and this cellular localization was shown signal induced LIME re-expression, indicating 10 November 2003 (doi: 10.1084/jem.20031484) | Hur, E.M. et al. LIME, a novel transmembrane adaptor protein to be a result of palmitoylation of the that TCR signals regulate LIME expression by associates with p56lck and mediates T cell activation. J. Exp. membrane-proximal region of LIME. human peripheral blood T cells. Med. 10 November 2003 (doi: 10.1084/jem.20030232)

926 | DECEMBER 2003 | VOLUME 3 www.nature.com/reviews/immunol WT and L∆P mice crossed with animals expressing a T-cell receptor specific for an influenza-virus-derived peptide (F5). When transferred to Rag1-deficient mice, activated F5/L∆P T cells were more efficient at entering the PLNs than F5/WT T cells exposed to antigen, indicating that L-selectin shedding prevents the re-entry of activated T cells into the PLNs. These studies show that the physi- ological role of L-selectin shedding is to shape the migration pattern of activated T cells and prevent them from re-entering PLNs. Karen Honey References and links ORIGINAL RESEARCH PAPER Galkina, E. et al. L-selectin shedding does not regulate constitutive T cell trafficking but controls the migration pathways of antigen-activated T lymphocytes. IMMUNE RESPONSES J. Exp. Med. 198, 1323–1335 (2003). FURTHER READING Grabovsky, V., Dwir, O. & Alon, R. Endothelial chemokines destabilize L-selectin-mediated lymphocyte rolling without inducing selectin shedding. J. Biol. Chem. 277, Mast cells act by remote control 20640–20650 (2002).

Swollen ‘glands’ are often one of the first signs bacteria into the footpad of mice also resulted that you have picked up a bacterial infection. in considerable lymph-node hypertrophy. This lymph-node hypertrophy results from the A closer investigation of the site of infection accumulation of circulating lymphocytes in the showed that although the overall number of draining lymph nodes, which can interact with mast cells had not increased 4 hours after antigen-presenting cells (APCs) that are loaded bacterial innoculation, the percentage of with microbial antigen and so initiate an degranulated mast cells had. This probably adaptive immune response. Bacterial products indicates that mast cells act remotely to induce at the site of infection are thought to induce the nodal hypertrophy and, instead of leaving the emigration and maturation of APCs, but the site of infection and migrating to the lymph signals that control T-cell recruitment to the nodes themselves, they release a product on lymph nodes are unknown. Work now degranulation that drains into the lymph node published in Nature Immunology shows a and signals for hypertrophy to occur. central role for mast cells in this process. Which mast-cell product might be involved? Mast cells are important components of the TNF, but not other inflammatory mediators innate immune response against bacteria, that mast cells release, was shown to have through degranulation and the release of potent hypertrophic effects, and 3 hours after inflammatory mediators, including tumour infection (or mast-cell activation with 48/80), necrosis factor (TNF). Here, McLachlan et al. the level of TNF in the draining lymph nodes used a mouse model of localized infection increased markedly. In addition, mast-cell to investigate if these cells are involved in activation resulted in a threefold increase in the controlling bacteria-triggered nodal numbers of T cells recruited to the lymph hypertrophy and so have a role in the induction nodes. of adaptive immune defence. This study indicates that, through the release In wild-type mice, injection of bacteria of TNF, mast cells provide an essential signal into the footpad resulted in lymph-node early in infection to trigger, by remote control, hypertrophy within 24 hours. However, lymph- the hypertrophy of draining lymph nodes and node swelling was markedly reduced in mast- the initiation of an adaptive immune response. cell-deficient mice (W/Wv mice). Injection of Jenny Buckland mast cells into the footpads of W/Wv mice before bacterial challenge resulted in a similar References and links ORIGINAL RESEARCH PAPER McLachlan, J. B. et al. Mast cell- level of lymph-node hypertrophy as seen in derived tumor necrosis factor induces hypertrophy of draining lymph wild-type mice, indicating an important role nodes during infection. Nature Immunol. 2 November 2003 for mast cells in this process. The authors (doi:1038/ni1005). WEB SITE confirmed this by using a specific mast-cell Soman N. Abraham’s lab: http://pathology.mc. activator (48/80), which if injected instead of duke.edu/research/postabraham.html

NATURE REVIEWS | IMMUNOLOGY VOLUME 3 | DECEMBER 2003 | 927 HIGHLIGHTS

T-CELL DEVELOPMENT could migrate in response to CXCL12. Trans- well migration assays using CXCL12 as a chemoattractant showed that all populations of progenitors that were tested migrated towards Directional cues from CXCR4 CXCL12. To confirm the role of CXCR4 signalling in this directional movement, the in vivo The CXC-chemokine receptor 4 (CXCR4) pro- As thymocytes mature in the post-natal thy- migration of CXCR4-deficient progenitor thy- vides essential signals that guide thymocytes on mus, they migrate from their entry point near the mocytes was assessed. T-cell numbers were low their developmental journey through the thy- cortico-medullary junction (CMJ), outwards and thymocyte differentiation was blocked at an mus, according to a recent study in The Journal across the cortex towards the capsule, and then early stage in mice reconstituted with bone of Immunology. back again across the cortex towards the medulla. marrow that lacked CXCR4 expression. Fur- This migration enables these developing progeni- thermore, thymocytes derived from CXCR4- tor cells to interact with thymic stromal cells that deficient bone marrow accumulated at the CMJ provide signals that are required for commit- and did not migrate efficiently into the cortex. ment to the T-cell lineage, and for efficient This study highlights the essential and non- thymocyte differentiation and proliferation. redundant role for CXCR4–CXCL12 signalling How is this cortical migration controlled? in controlling the migration of thymocyte prog- Petrie and colleagues investigated the role of enitors across the cortex — a process that is chemokines in this process by first asking which required for the development of mature T cells chemokine receptors were expressed by thymo- in the post-natal thymus. cyte progenitors. Of all of the known chemo- Jenny Buckland kine receptors, CXCR4 was the most abundant, being expressed by all thymocyte prognenitors. References and links ORIGINAL RESEARCH PAPER Plotkin, J. et al. Critical role Further experiments showed that the ligand for for CXCR4 signaling in progenitor localization and T cell this receptor, CXCL12, is produced by cortical differentiation in the postnatal thymus. J. Immunol. 171, stromal cells, indicating that signals through 4521–4527 (2003) FURTHER READING Petrie, H. T. Cell migration and the control CXCR4–CXCL12 could potentially be involved of post-natal T-cell lymphopoiesis in the thymus. Nature Rev. in guiding progenitors into the cortex. Immunol. 3, 859–866 (2003) WEB SITE To investigate this possibility, the authors Howard Petrie’s lab: http://www.mskcc.org/ next measured whether thymocyte progenitors mskcc/html/11136.cfm

γ REGULATORY TCELLS mouse CTLA4 induced IFN- production, upregulation of IDO expression and tryptophan degradation by mouse DCs. IDO — influencing They then showed that regulatory T cells also induced IFN-γ production and dendritic-cell options tryptophan degradation by DCs and that this was inhibited by CTLA4-specific CD4+CD25+ regulatory T cells express neutralizing antibodies. Activation of cytotoxic T lymphocyte-associated regulatory T cells with CD3-specific antigen 4 (CTLA4) constitutively, and antibodies enhanced their ability to induce although this has been shown in several IFN-γ production and tryptophan settings to be important for regulatory degradation by DCs and this depended on T-cell function, the mechanisms by which their increased expression of CTLA4. CTLA4 induces suppression have remained Does tryptophan catabolism alter the unclear. Now, a study in Nature capacity of DCs to induce immune Immunology shows that CTLA4 can responses in vivo? The ability of CD8– DCs modulate dendritic cells (DCs), initiating loaded with a synthetic peptide (NRP-A7) the immunosuppressive pathway of to induce a persistent immune response tryptophan catabolism. in mice was eliminated by pre-exposure Interferon-γ (IFN-γ) regulates of the DCs to regulatory T cells activated transcription of the gene encoding with CD3-specific antibody. However, the indoleamine 2,3-dioxygenase (IDO) — the ability of peptide-loaded DCs to initiate enzyme that catalyses the initial step of an immune response was restored if an DCs to initiate IFN-γ production and, tryptophan degradation. IDO activity has an inhibitor of IDO was present during thereby, IDO activity and tryptophan immunosuppressive effect that has been DC exposure to the activated regulatory catabolism. linked with tolerance induction during T cells. Karen Honey pregnancy, transplantation and These studies identify a CTLA4- autoimmunity. dependent mechanism by which References and links + + ORIGINAL RESEARCH PAPER Fallarino, F. et al. Fallarino et al. observed that human CD4 CD25 regulatory T cells induce Modulation of tryptophan catabolism by regulatory T cells Jurkat T cells that were transfected with immunosuppression — modulation of Nature Immunol. 26 October 2003 (doi:10.1038/ni1003)

928 | DECEMBER 2003 | VOLUME 3 www.nature.com/reviews/immunol HIGHLIGHTS

IN BRIEF

CYTOKINES

IL-23 produced by CNS-resident cells controls T-cell encephalitogenicity during the effector phase of experimental autoimmune encephalomyelitis. Becher, B., Durell, B. G. & Noelle, R. J. J. Clin. Invest. 112, 1186–1191 (2003) Interleukin-23 (IL-23) consists of a unique p19 subunit and the p40 subunit of IL-12, and is crucial for the development of experimental autoimmune encephalomyelitis in mice. Becher et al. use p40-deficient mice to generate irradiation bone-marrow chimaeras and show that in the absence of p40 expression by irradiation-resistant cells, previously activated antigen-reactive exposure to TD antigen. So, Blimp1 is B-CELL DEVELOPMENT cells infiltrate the central nervous system (CNS), but do not required for the development of both induce encephalitogenicity. In addition, disease induced by plasma cells and pre-plasma memory immunization is markedly diminished when p40 is absent from B cells during both a primary and Plasma-cell irradiation-resistant cells and this is associated with a bias towards memory response. the production of T helper 2-type cytokines by infiltrating cells. differentiation To investigate the mechanisms by The authors, therefore, suggest that p40 expression by CNS- which Blimp1 regulates immuno- resident cells contributes to T-cell polarization and thereby requires Blimp1 globulin secretion and plasma-cell modulates disease pathogenesis. differentiation, Shapiro-Shelef et al. Although the transcriptional repres- analysed Blimp1-deficient B cells TRANSPLANTATION sor B-lymphocyte-induced matur- that were exposed to lipopoly- ation protein 1 (Blimp1) has been saccharide (LPS) ex vivo.In the Prevention of organ allograft rejection by a specific previously shown to induce B-cell dif- absence of Blimp1, switching from Janus kinase 3 inhibitor. ferentiation into immunoglobulin- the membrane-associated form of producing plasma cells, until now, it the immunoglobulin heavy chain Changelian, P. S. et al. Science 302, 875–878 (2003) µ µ has not been identified as non-redun- ( M) to the secreted form ( S) was The development of effective immunosuppressive agents that are dant and essential for this process. markedly impaired, as was the targeted specifically to immune cells and do not produce serious µ Mice lacking the gene encoding induction of S messenger RNA. In side effects remains a pressing need for the prevention and Blimp1, Prdm1,die in utero, so to addition, the transcription factor treatment of transplant rejection. This study reports pre-clinical study the dependence of B-cell differ- X-box binding protein 1 (XBP1) trials of a new immunosuppressive agent (CP-690,550) that entiation and function on Blimp1, was not induced by LPS stimulation prolonged survival in both a mouse model of heart transplantation –/– Shapiro-Shelef et al. generated mice of Prdm1 B cells, identifying it and a cynomolgus monkey model of kidney transplantation, in which Prdm1 was specifically as a downstream target of Blimp1. but that did not result in the dose-limiting side effects that are eliminated in mature B cells, However, retrovirus-mediated over- associated with present therapies. CP-690,550 inhibits Janus kinase 3 flox/flox cre/+ Prdm1 CD19 mice. B-cell expression of XBP1 by Blimp1- (JAK3), which is crucial for signalling through cytokine receptors development was normal in these deficient B cells was insufficient to that use the common γ-chain and, therefore, for development and animals but serum immunoglobulin rescue plasma-cell development, homeostasis of immune cells. It is the first JAK3 inhibitor to have was markedly reduced, even in indicating that Blimp1 must target shown efficacy in non-human primates. unimmunized mice. many factors to induce plasma-cell After immunization with either a differentiation. IMMUNOTHERAPY thymus-independent (TI) or thymus- These studies using a conditional dependent (TD) antigen, the gene-deletion strategy define Blimp1 A critical role for OX40 in T cell-mediated immunoglobulin response generated as essential for the differentiation of immunopathology during lung viral infection. in Prdm1flox/floxCD19cre/+ mice was sub- plasma cells and immunoglobulin Humphreys, I. R. et al. J. Exp. Med. 198, 1237–1242 (2003) stantially diminished when compared secretion, and indicate that this with control animals, as was immuno- transcription factor also has a key Respiratory infections are an important example of how an over- globulin secretion after re-challenge role in pre-plasma memory B-cell active T-cell response can be more of a hindrance than a help, with TD antigen. This decrease in development. with T-cell responses resulting in airway occlusion and pathology. immunoglobulin production corre- Karen Honey Previous T-cell-targeted therapies have affected both bystander lated with a marked reduction in the References and links and antigen-specific T cells, which might result in dangerous number of antigen-specific immuno- ORIGINAL RESEARCH PAPER Shapiro-Shelef, immunosuppression. These authors used an OX40–immuno- M. et al. Blimp-1 is required for the formation of globulin fusion protein to target recently activated T cells globulin-secreting cells and consistent immunoglobulin secreting plasma cells and pre- with this, few plasma cells or pre- plasma memory cells. Immunity 19, 607–620 specifically. OX40 signalling prevents the death of activated plasma memory B cells could be (2003) T cells, but it is not expressed by naive T cells. Interference with flox/flox cre/+ WEB SITE detected in Prdm1 CD19 mice Kathryn Calame’s lab: http://cpmcnet.columbia. this T-cell survival signal in a mouse influenza model reduced after either primary or secondary edu/dept/gsas/biochem/faculty/calame.html immunopathology without preventing virus clearance.

NATURE REVIEWS | IMMUNOLOGY VOLUME 3 | DECEMBER 2003 | 929