Kinase P56lck Research Articles

Involvement of CD4+ and CD8+ T-lymphocytes in the modulation of nociceptive processing evoked by CCL4 in mice

AimsTo explore the mechanisms involved in the transformation of analgesia produced by low doses of CCL4 (pg/kg) to hyperalgesia when higher doses (ng/kg) are administered to mice. Main methodsThe unilateral hot plate test was used to assess thermal nociception. CD3+, CD4+ or CD8+ blood cells were depleted with selective antibodies. Expression of CCR5 and IL-16 in lymphocytes was studied by flow cytometry and IL-16 blood levels were measured by ELISA. IL-16 and CD8 were detected by immunofluorescence. Key findingsIL-16 and CCR5 expression were demonstrated in CD4+ and CD8+ T-lymphocytes by flow cytometry. Furthermore, CCL4-induced hyperalgesia was abolished by reducing circulating T-lymphocyte levels or by selectively depleting CD4+ lymphocytes. In contrast, when the anti-CD4 antibody was acutely administered, CCL4 induced analgesia instead of hyperalgesia. A similar response was obtained when administering A-770041, that prevents CD4-mediated CCR5 desensitization by inhibiting p56lck kinase. As occurred with the analgesic effect evoked by low doses of CCL4, analgesia evoked by combining CCL4 and A-770041 was reverted by naloxone, naltrindole or an anti-met-enk antibody. Interestingly, flow cytometry assays showed that the number of CD8+, but not CD4+, T-cells expressing IL-16 is reduced after the acute administration of CCL4, a result compatible with the description that CD8+-lymphocytes can rapidly release preformed IL-16. Accordingly, the rise in IL-16 blood concentration evoked by CCL4 was prevented after CD8+ lymphocyte depletion. SignificanceCCL4-evoked hyperalgesia is related to the desensitization of CCR5 in CD4+ T-cells and to the release of IL-16 from CD8+ lymphocytes.

The Inhibitory Signaling Receptor Protocadherin-18 Regulates Tumor-Infiltrating CD8+ T-cell Function.

Cancers are infiltrated with antitumor CD8+ T cells that arise during tumor growth, but are defective in effector phase functions because of the suppressive microenvironment. The reactivation of TILs can result in tumor destruction, showing that lytic dysfunction in CD8+ tumor-infiltrating lymphocytes (TIL) permits tumor growth. Like all memory T cells, TILs express inhibitory signaling receptors (aka checkpoint inhibitor molecules) that downregulate TCR-mediated signal transduction upon TIL interaction with cells expressing cognate ligands, thereby restricting cell activation and preventing the effector phase. Previously, we identified a novel murine CD8+ TIL inhibitory signaling receptor, protocadherin-18, and showed that it interacts with p56lck kinase to abrogate proximal TCR signaling. Here, we show that TILs from mice deleted in protocadherin-18 had enhanced antitumor activity and that coblockade of PD-1 and protocadherin-18 in wild-type mice significantly enhanced TIL effector phase function. These results define an important role for protocadherin-18 in antitumor T-cell activity. Cancer Immunol Res; 5(10); 920-8. ©2017 AACR.

P-194 Use of CEACAM5 Synthetic Peptides to Induce Activation of CD8+ Suppressor Regulatory T Cells

CD8+ regulatory T (TrE) cells can be activated by intestinal epithelial cells (IECs) through the complex formed by the non-classical I molecule, CD1d and gp180 both expressed on IECs. mAb B9, an anti-gp180 Ab, can block this induction. We have previously demonstrated sequence homology between gp180 and CEACAM5. Furthermore, CEACAM5 has properties attributed to gp180, such as CD1d and CD8α binding and activation of CD8-associated LcK. Furthermore, the N70, 81A CEACAM5 mutant, which is devoid of the sugar bridge, appeared to lose the capacity to induce the phosphorylation of Lck when compared to CEACAM5. We suggest that unique set of interactions between CEACAM5, CD1d and CD8α can render CD1d more class I-like, facilitating antigen presentation by CD1d to T cells and the activation of CD8+ Tregs. Moreover, we showed that CEACAM5-activated CD8+ T cells acquire suppressive function. The aim of this study is to determine which amino acids of CEACAM5 are functional in the activation of CD8+ T cells in order to design synthetic peptides with immunoregulatory function. We generated an overlapping peptide library of CEACAM5 N domain to identify the amino acids that contribute to its functionality (19 peptides with offset of 5). We tested the ability of the entire pool of peptides, 3 combinations of peptides and each single peptide to phosphorylate CD8 associated p56Lck kinase both in human isolated CD8+ T cells and in a murine T cell line transfected with human CD8α, 3G8, using In-cell Western Blot assay. OKT8, an anti-CD8 antibody, was used as positive control. The entire collection of peptides induced phosphorylation of Lck compared to OKT8 and to the full length CEACAM5 in CD8+ T cells but not in CD4+ T cells. When pooled peptides were tested, pool 3>>2 (pool 2: G30-T55; pool 3: P66-Y107) resulted in the activation of CD8+ T cells. The peptides 12 (T56-N70) and P14 (I66-Q80) within pool 3 induces phosphorylation of LcK to the greatest extent. Our data suggest the crucial role of the N domain sugar bridge site in CEACAM5 binding to CD8α and in the activation of CD8+ Tregs. Furthermore, we identified the regions of the N domain where the functional amino acid residues are located. These finding are critical in designing peptides that can be used to induce CD8+ TrE cells as in Crohn’s disease in which a defect of these cells have been described.

P60src and p72Syk Tyrosine Kinases Activate Both Lw and CD44 to Mediate Sickle Red Blood Cell Adhesion to Endothelium

Abstract 3223The vaso-occlusive process in patients with sickle cell disease is complex and involves interactions between hemoglobin S red blood cells (SSRBCs) and vascular endothelial cells (ECs). However, the pathophysiologic triggers inducing SSRBC adhesion and vaso-occlusion are poorly understood. Elucidation of these mechanisms at the molecular level would allow development of new preventive and treatment strategies to abrogate vaso-occlusive events. Because non-receptor tyrosine kinases in non-erythroid cells are known to mediate cell-cell interactions, we have now investigated the role of non-receptor tyrosine kinases in modulating SSRBC adherence to cultured ECs, identified the kinases involved, and defined both the receptors on activated SSRBCs and the ligands on ECs involved in these interactions. Less than 20% of non-treated SSRBCs were able to adhere to non-stimulated ECs in vitro in intermittent flow conditions at a shear stress of 2 dynes/cm2. However, treatment of SSRBCs with sodium orthovanadate (Na3VO4), a potent broad spectrum inhibitor of protein tyrosine phosphatases, was able to significantly up-regulate RBC adhesion to cultured ECs by 4.6±1-fold over baseline adhesion in vitro. Na3VO4 in contrast, completely failed to increase adhesion of normal RBCs to cultured ECs. The increased SSRBC adhesion induced by Na3VO4 was significantly inhibited with piceatannol (p<0.001), which inhibits non-receptor tyrosine kinases p72Syk and p56Lck, and PP1 (p<0.001), an src-selective tyrosine p56Lck, p59Fyn and p60src kinase inhibitor. However, genistein, a broad range inhibitor of tyrosine kinases, and damnacanthal, a highly potent and selective inhibitor of p56Lck, completely failed to inhibit the effect of Na3VO4 on SSRBC adhesion. In addition, phenylarsine oxide, which specifically activates p56Lck tyrosine kinase, did not affect SSRBC adhesion to ECs. Together, these data suggest that SSRBC adhesion to ECs can be upregulated via activation of at least p72Syk and p60src tyrosine kinases, but not via the src-tyrosine kinase p56Lck. We further confirmed that increased SSRBC adhesion by Na3VO4 treatment is indeed a result of the activation of p72Syk and p60src tyrosine kinases. Slight basal phosphorylation of p72Syk and p60src was detected in all SSRBC samples tested. However, Na3VO4-treatment of SSRBCs significantly enhanced phosphorylation of both p60src and p72Syk kinases over basal phosphorylation (p<0.05), and this effect induced by Na3VO4 was completely blocked with the src-selective inhibitor PP1 or PP2, and piceatannol, respectively, suggesting that p72Syk and p60src undergo enhanced activation and are involved in up-regulation of SSRBC adhesion to endothelium. Moreover, at a shear stress of 2 dynes/cm2, both anti-LW (ICAM-4) and anti-CD44 antibodies individually inhibited adhesion of activated SSRBCs to ECs, by up to 83% and 78%, respectively. Recombinant soluble LW (srLW) and CD44 (srCD44) proteins also completely abolished adhesion of activated SSRBCs to ECs, identifying LW and CD44 as the RBC receptors involved in this interaction. The EC ligands for activated SSRBCs were also identified using antibody inhibition studies, as the αvβ3 integrin, a ligand previously shown to be important to SSRBC adhesion to activated ECs in vivo, and the endothelial CD44. These data demonstrate that activation of p72Syk and p60src-dependent pathways can act to activate LW- and CD44-mediated SSRBC adhesion to endothelial αvβ3 integrin and CD44, respectively, suggesting that this mechanism may initiate or exacerbate vaso-occlusion by increasing SSRBC adhesion to the endothelium. RBC CD44 is also the first adhesion molecule shown to be involved in SSRBC adhesion to endothelium. Disclosures:No relevant conflicts of interest to declare.

Protocadherin-18 Is a Novel Differentiation Marker and an Inhibitory Signaling Receptor for CD8+ Effector Memory T Cells

CD8+ tumor infiltrating T cells (TIL) lack effector-phase functions due to defective proximal TCR-mediated signaling previously shown to result from inactivation of p56lck kinase. We identify a novel interacting partner for p56lck in nonlytic TIL, Protocadherin-18 (‘pcdh18’), and show that pcdh18 is transcribed upon in vitro or in vivo activation of all CD8+ central memory T cells (CD44+CD62LhiCD127+) coincident with conversion into effector memory cells (CD44+CD62LloCD127+). Expression of pcdh18 in primary CD8+ effector cells induces the phenotype of nonlytic TIL: defective proximal TCR signaling, cytokine secretion, and cytolysis, and enhanced AICD. pcdh18 contains a motif (centered at Y842) shared with src kinases (QGQYQP) that is required for the inhibitory phenotype. Thus, pcdh18 is a novel activation marker of CD8+ memory T cells that can function as an inhibitory signaling receptor and restrict the effector phase.

In vivo study of tyrosine protein kinase Lck inhibited by siRNA in T cells of asthmatic mice

Objective Using the technology of siRNA to inhibit the gene expression of no-receptor tyrosine protein kinase Lck in T cells of asthmatic mice,and to study the therapeutic effect of Lck specific siRNA in asthmatic mice.Methods Receptor tyrosine protein kinase Lck specific siRNA fragments were taken from chemosynthesis.In vivo-jetPEITM was used to transfect the siRNA into mice body through tail vein injection.The mice were killed 48 hours later,and the levels of IL-4,IL-17 in bronchoalveolar lavage fluid (BALF) were detected with respondent ELISA kits.The change of inflammatory histopathology in lung was observed with H.E.staining.The expression of Lck in lung was detected with immunohistochemistry (IHC),and the level of Lck in lung tissue homogenate was detected with Western Blot.Results Compared with asthmatic group[ (234.68 ± 11.15 ) pg/ml,( 96.76 ± 8.28 ) pg/ml],the levels of IL-4,IL-17 [ (234.68 ± 11.15)pg/ml,(96.76 ±8.28) pg/ml] in the BALF of siRNA interference group decreased, and the inflammation in the lung relieved.IHC indicated that the expression of Lck in lung decreased and the level of Lck in lung tissue homogenate decreased ( P ＜ 0.05 ).Conclusions Lck specific siRNA could reduce the level of IL-4,IL-17 in the lung tissues of asthmatic mice,and relieve the inflammatory reaction in lung. Key words: RNA, small interfering ; Asthma/TH/ME ; Lymphocyte specific protein tyrosine kinase p56(lck)/ME

Update on lymphocyte specific kinase inhibitors: a patent survey

Importance of the field: Lck (p56lck or lymphocyte specific kinase) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (NK) cells. Genetic evidence from knockout mice and human mutations demonstrates that Lck kinase activity is critical for T cell receptor (TCR)-mediated signaling, leading to normal T-cell development and activation. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection.Areas covered in this review: This review covers the patents, patent applications and associated publications for small molecule kinase inhibitors of Lck since 2005 and attempts to place them in context from a structural point of view.What the reader will gain: Readers will gain an overview of the structural classes and binding modes of Lck inhibitors, the major players in this area and an insight into the current state of the field.Take home message: The search for a potent and orally active inhibitor of Lck has been an intense area of research for a number of years. Despite tremendous efforts, the identification of a highly selective and potent Lck inhibitor suitable for use as an immunosuppressive agent remains elusive.

A generalized numerical approach to steady-state enzyme kinetics: Applications to protein kinase inhibition

A generalized numerical treatment of steady-state enzyme kinetics is presented. This new approach relies on automatic computer derivation of the underlying mathematical model (a system of simultaneous nonlinear algebraic equations) from a symbolic representation of the reaction mechanism (a system of biochemical equations) provided by the researcher. The method allows experimental biochemists to analyze initial-rate enzyme kinetic data, under the steady-state approximation, without having to use any mathematical equations. An illustrative example is based on the inhibition kinetics of p56lck kinase by an ATP competitive inhibitor. A computer implementation of the new method, in the modified software package DYNAFIT [Kuzmič, P. (1996) Anal. Biochem. 237, 260–273], is freely available to all academic researchers.

Spatial distribution, kinetics, signaling and cytokine production during homeostasis driven proliferation of CD4+ T cells

During recovery from lymphopenia, the naïve T-cells undergo homeostasis driven proliferation (HDP) and acquire a memory phenotype. The HDP of T-cells requires signals derived from T-cell-receptor, p56lck kinase, IL-7R and IL-15R. However, the role of other signaling molecules during HDP of CD4+ T-cells remains speculative. The differentiation of naïve T-cells into Th1/Th2/Th17 or Treg populations during HDP is not well understood. Present report describes the spatial and signaling characteristics of HDP of CD4+ T-cells and their cytokine profiles. The HDP of CD4+ T-cells was found to occur only in specific areas (T-cell zones) of secondary lymphoid organs of lymphopenic mice. The inhibitors of MEK and PKC and their combination with inhibitors of PI3kinase and mTOR suppressed mitogen induced T-cell proliferation without affecting their HDP. The CD4+ T-cells taken from reconstituted lymphopenic mice showed activation of proteins involved in NF-κB pathway, significantly higher production of pro-inflammatory cytokine IL-6, and lower production of IL-4 as compared to T-cells from normal mice. Plumbagin, a known NF-κB blocker inhibited survival as well as HDP of CD4+ T-cells and IL-6 production in activated T-cells. Our results demonstrate the essential role of NF-kappaB during HDP of T-cells.

Protein tyrosine kinase p56 lck-deficiency confers hypersusceptibility to ρ-fluorophenylalanine (pFPhe)-induced apoptosis by augmenting mitochondrial apoptotic pathway in human Jurkat T cells

Phenylalanine analog, ρ-fluorophenylalanine (pFPhe)-mediated cytotoxicity and several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, Bid cleavage, degradation of PARP and PLCγ-1, and DNA fragmentation were more significant in p56 lck-deficient Jurkat T cells (JCaM1.6) than in wild-type Jurkat T cells (E6.1). The susceptibility of JCaM1.6 toward apoptogenic activity of pFPhe decreased after acquisition of p56 lck by transfection. The p56 lck kinase activity increased 1.6-fold at 15–30 min after pFPhe treatment. The pan-caspase inhibitor (z-VAD-fmk) completely blocked the pFPhe-mediated apoptotic changes except caspase-9 activation. The caspase-8 inhibitor (z-IETD-fmk), which failed to influence pFPhe-induced caspase-9 activation, completely blocked caspase-8 activation and PLCγ-1 degradation with a marked reduction in caspase-3 activation and PARP degradation, indicating pFPhe-induced caspase-8 activation as a downstream event of mitochondria-dependent activation of caspase-9. These results indicate that the deficiency of p56 lck augments pFPhe-induced mitochondrial cytochrome c release and resultant apoptotic cell death in Jurkat T cells.

Chronic Disseminated Mycobacterium xenopi Infection in a Cat with Idiopathic CD4+ T Lymphocytopenia

A 3-year-old castrated male domestic shorthair cat was referred for examination in August 2000 with a 3-week history of poor appetite, vomiting, and weight loss. One week before referral, evaluation by the referring veterinarian identified anemia (PCV 21% [reference range 29–48%]) and moderate renal insufficiency (BUN 55mg/dL [reference range 14–36mg/dL], serum creatinine 4.2mg/dL [reference range 0.6–2.4mg/dL], urine specific gravity 1.015) with an inflammatory urine sediment (proteinuria, white blood cells, bacteria) on a voided sample. The results of enzyme-linked immunosorbent assays for feline leukemia virus (FeLV) antigen and feline immunodeficiency virus (FIV) antibodies were negative. Radiographs disclosed bilateral renomegaly and microcardia. Abdominal ultrasound examination identified a thin cortex in the left kidney and both kidneys were hyperechoic. The cat was treated with lactated Ringer’s solution IV and enrofloxacin 6mg/kg/d IV for 3 days. Upon examination at the referral hospital, the cat was quiet and responsive. Physical examination abnormalities were limited to bilaterally enlarged kidneys that appeared to be painful on palpation and a body condition score of 3/9. Body weight was 3.6 kg. On CBC there was a severe nonregenerative macrocytic normochromic anemia (PCV 17% [reference range 30–47%], MCV 55 fL [reference range 41–51 fL], MCHC 31 g/dL [reference range 31–35 g/dL], aggregate reticulocytes 20,880/mL) in the presence of normal serum erythropoietin concentration (17mU/mL [reference range 10–30mU/mL]). The WBC count (10,200/mL [reference range 5,500–19,500/ mL]), differential cell count (segmented neutrophils 7,040/mL [reference range 2,500–12,500/mL], lymphocytes 2,860/mL [reference range 1,500–7,000/mL], monocytes 260/mL [reference range 0–800/mL], eosinophils 50/mL [reference range 0–1,500/mL]), and platelet count (485,000/mL [reference range 300,000–800,000/ mL]) were normal. Serum chemistry results indicated moderate azotemia (BUN 36mg/dL [reference range 16–34mg/dL], serum creatinine 3.5mg/dL [reference range 0.7–2.3mg/dL]), increased AST activity (173U/L [reference range 5–30U/L]), and hypoalbuminemia (1.8 g/dL [reference range 2.0–3.1 g/dL]). Results of urinalysis collected by cystocentesis indicated isosthenuria (USG 1.011), proteinuria, and bacteruria. Aerobic urine culture was negative, and plasma prothrombin and partial thromboplastin times were within normal limits. Ultrasonographic examination of the abdomen identified moderate bilateral renomegaly with pyelectasia, mesenteric lymphadenopathy, and an enlarged hypoechoic pancreas. By means of ultrasound guidance, fineneedle tissue aspirates were collected from kidney, mesenteric lymph node, and pancreas, and slides were prepared for cytologic evaluation using Wright-Giemsa stain. Preparations obtained from the kidney were very cellular, consisting primarily of large, epitheliod macrophages arranged in sheets and individually. The cytoplasm of many of the macrophages contained long, thin, negatively staining, rod-shaped inclusions consistent withMycobacteria spp. (Fig 1). Renal epithelial cells were also observed in the preparation.Macrophages containing similar negatively staining rods were observed in tissue aspirate preparations from the pancreas and a mesenteric lymph node, along with pancreatic epithelium and lym-

An octapeptide analogue of HIV gp120 modulates protein tyrosine kinase activity in activated peripheral blood T lymphocytes.

Following infection with HIV, patients exhibit lymphocyte dysfunction before the loss of CD4+ T cells. The major HIV surface glycoprotein, gp120, can modulate lymphocyte function in vitro; however, the mechanism by which gp120 affects T lymphocyte signal transduction is controversial. We have used Peptide T, a synthetic octapeptide derived from a conserved, CD4 binding region of gp120, to examine gp120-related modulation of lymphocyte signal transduction. Activation of lymphocytes through the T cell receptor (TCR) in collaboration with cell surface accessory molecules results in rapid increases in tyrosine phosphorylation, probably through the recruitment and activation of src-family protein tyrosine kinases (PTK) such as lck and fyn which have been implicated in mediating the proximal signalling events mediated through the TCR. To identify potential mechanisms by which gp120 could modulate the function of T lymphocytes, we determined the effect of Peptide T on normal, activated peripheral blood lymphoblasts. Treatment of normal, activated peripheral blood lymphoblasts with Peptide T (10(-9) M) for 60 min transiently reduced levels of protein tyrosine phosphorylation (ptyr). Reduction in levels of cellular ptyr was associated with transient inhibition of the activity of total cellular and CD4-associated p56lck kinase activity (80%). Peptide T also induced a small delayed reduction in the p59fyn activity (up to 42%). Despite the decrease in total cellular ptyr levels, pp60c-src kinase activity was increased 11-fold following treatment with Peptide T. Peptide T pretreatment also induced tyrosine phosphorylation of a 48-kD CD4-associated protein, indicating that Peptide T may have multiple effects. Peptide T did not alter the levels of total cellular p56lck enzyme, nor did it directly inhibit the activity of purified p56lck. These results are consistent with a Peptide T-dependent modulation of PTK regulation, and support the potential of gp120 to interfere with T lymphocyte signal transduction in activated T lymphocytes.

The Siva protein is a novel intracellular ligand of the CD4 receptor that promotes HIV-1 envelope-induced apoptosis in T-lymphoid cells

In addition to its positive signaling function in the antigen presentation process, CD4 acts as the primary receptor for HIV-1. Contact between CD4 and the viral envelope leads to virus entry, but can also trigger apoptosis of uninfected CD4+ T-cells through a mechanism that is poorly understood. We show that Siva-1, a death domain-containing proapoptotic protein, associates with the cytoplasmic domain of CD4. This interaction is mediated by the cysteine-rich region found in the C-terminal part of the Siva-1 protein. Expression of Siva-1 specifically increases the susceptibility of both T-cell lines and unstimulated human primary CD4+ T-lymphocytes to CD4-mediated apoptosis triggered by the HIV-1 envelope, and results in activation of a caspase-dependent mitochondrial pathway. The same susceptibility is observed in T-cells expressing a truncated form of CD4 that is able to recruit Siva-1 but fails to associate with p56Lck, indicating that Siva-1 participates in a pathway independent of the p56Lck kinase activity. Altogether, these results suggest that Siva-1 might participate in the CD4-initiated signaling apoptotic pathway induced by the HIV-1 envelope in T-lymphoid cells.

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

The biological effects of rIgG(1) 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-kappaB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG(1) 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG(1) 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37 degrees C, together with recruitment of TCR, CD3zeta, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Ctheta, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cgamma1, and p95(vav). Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr(292) and Tyr(319) phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10-30 s following rIgG(1) 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-kappaB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG(1) 13B8.2 acts to induce its biological effects.

Lcking ERK Activation

Tapinos and Rambukkana uncovered an unexpected pathway to activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) while investigating the response of Schwann cells to long-term infection with the bacillus that causes leprosy. Damage to peripheral nerves, which involves bacterial invasion of nonmyelinating Schwann cells and leads to sensory loss, is one of the key features of leprosy. Tapinos and Rambukkana grew human Schwann cells in primary cultures and found that infection with Mycobacterium leprae stimulated their proliferation and accelerated progression through the G 1 phase of the cell cycle. Gene array analysis, reverse transcription polymerase chain reaction (RT-PCR), and Western analysis indicated that expression and abundance of the G 1 regulator cyclin D1 was increased 30 days after infection. Moreover, overexpression of a cyclin D1 dominant-negative mutant blocked the effects of M. leprae on G 1 progression. Infected cells showed increased phosphorylation of ERK1/2 compared with controls, and transfection with a dominant-negative ERK2 protein implicated that kinase in the stimulation of cyclin D1 abundance and cell cycle progression. However, pharmacological and peptide inhibitor analysis indicated that neither the SOS-Ras-Raf-mitogen-activated protein kinase kinase (MAPK) signaling pathway, which is critical to growth factor activation of ERK1/2, nor phosphatidylinositol 3-kinase (PI3K) signaling were involved in M. leprae activation of ERK1/2. Rather, M. leprae activated ERK1/2 and thereby stimulated cell proliferation through a pathway involving protein kinase Cε (PKCε)-dependent activation of the Src family tyrosine kinase lymphoid cell kinase (p56Lck). Thus, M. leprae appears to activate a previously unknown pathway to cell proliferation and thereby to provide itself with a niche for long-term survival. N. Tapinos, A. Rambukkana, Insights into regulation of human Schwann cell proliferation by Erk1/2 via a MEK-independent and p56Lck-dependent pathway from leprosy bacilli. Proc. Natl. Acad. Sci. U.S.A. 102 , 9188-9193 (2005). [Abstract] [Full Text]

Epitope‐specific crosslinking of CD45 down‐regulates membrane‐associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

CD45 engagement by monoclonal antibodies on human activated T cells triggers tumour necrosis factor-alpha (TNF-alpha) gene transcription in an epitope-specific manner. To dissect the early signalling events leading to TNF-alpha gene expression, we established that CD45 crosslinking resulted in tyrosine phosphorylation of p56lck, ZAP-70, CD3-zeta, LAT and Vav. This was accompanied by down-regulation of membrane-associated protein tyrosine phosphatase activity in the absence of demonstration of enhanced p56lck, p72syk and ZAP-70 kinase activity, which remained constitutive. These early events eventually triggered an intracellular Ca(2+) rise and phosphoinositide turnover. We conclude that down-regulation of membrane-associated tyrosine phosphatase activity by CD45 extracytoplasmic domain multimerization led, in an epitope-specific fashion, to unopposed tyrosine kinase activity and to the activation of the T-cell receptor/CD3 complex signalling cascade, resulting in TNF-alpha gene expression. This model strongly suggests that CD45 extracytoplasmic tail multimerization may contribute to the modulation T-cell functions.

T-Cell Function in Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL) Following Alemtuzumab (Campath®) or Fludarabine Treatment.

Fludarabine and alemtuzumab (humanized anti-CD52 antibody, Campath®) are both used as routine therapy for patients with B-CLL. Fludarabine and in particular alemtuzumab, in addition to their antitumor effects, induce long-lasting suppression of T-cell numbers in blood. T-cell suppression, in combination with advanced disease, may result in an increased risk of opportunistic and other infections. In addition to a decrease in circulating T cells, functional defects in T cells have been described following alemtuzumab therapy for non-malignant disorders; however, only a limited amount of information exists about the effect of alemtuzumab on T-cell function in patients with B-CLL. The aim of the present study was to characterize in detail various aspects of T-cell function in patients with B-CLL who were in stable unmaintained PR or CR following fludarabine (n = 9) and alemtuzumab (n = 10) treatment as well as in age-matched healthy control donors (n = 10). CD4 and CD8 T-cell subsets of freshly obtained peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry to measure the expression of TCR-CD3 ζ chain, p56Lck, p59Fyn, ZAP-70, and PI3 Kinase as well as intracellular production of IFN-γ and IL-4. Additional analyses were performed to measure the proliferative response capability to a recall antigen (PPD) in alemtuzumab-treated patients and (as a control) indolent, untreated B-CLL patients (n = 11). The expression of IFN-γ and IL-4 (measured as mean fluorescence intensity [MFI]), but not the total number of positive cells, was significantly higher in CD4 and CD8 T cells for both CLL groups compared to healthy donors with the highest expression observed in alemtuzumab-treated patients. The total number of T cells that expressed the five signaling molecules was significantly lower in treated patients versus control donors; however, there were no differences between fludarabine- and alemtuzumab-treated patients. MFI analysis showed a significant increase in the TCR-CD3 ζ chain in fludarabine-treated patients compared to control donors; however, MFI analysis revealed no other significant differences between control group patients and fludarabine- and alemtuzumab-treated patients. Moreover, the proliferative response to PPD was not significantly different in alemtuzumab-treated patients and indolent CLL patients. In conclusion, these results suggest that the intrinsic signal transduction machinery in T cells is relatively well-preserved, such that T cells remain functionally intact following fludarabine or alemtuzumab therapy despite a marked reduction in their numbers. These results provide a better understanding of the immune suppression induced by alemtuzumab and fludarabine therapy in B-CLL patients.

CD28 Costimulation Controls Histone Hyperacetylation of the Interleukin 5 Gene Locus in Developing Th2 Cells

Interleukin 5 (IL-5) plays a unique role in allergic inflammatory responses, and the understanding of molecular mechanisms underlying the generation of IL-5-producing cells is crucial for the regulation of allergic disorders. Differentiation of naive CD4 T cells into type-2 helper (Th2) cells is accompanied by chromatin remodeling including hyperacetylation of histones H3 and H4 in the nucleosomes associated with the IL-4, IL-13, and IL-5 genes. Histone hyperacetylation of the IL-5 gene displayed a delayed kinetics compared with that of the IL-4 and IL-13 genes, suggesting a distinct remodeling mechanism for the IL-5-gene locus. Here we studied the role of CD28 costimulation in the generation of IL-5-producing cells and the histone hyperacetylation of the IL-5 gene locus. CD28-costimulation selectively enhanced histone hyperacetylation of the IL-5 gene locus that appeared to be mediated through NF-kappaB activation and subsequent up-regulation of GATA3. The CD28 costimulation-sensitive histone hyperacetylation spanned almost the entire intergenic region between the IL-5 and RAD50 accompanied with intergenic transcript. Thus, this is the first demonstration that CD28 costimulation controls a chromatin-remodeling process during Th2 cell differentiation.

A regulatory role for CD37 in T cell proliferation.

CD37 is a leukocyte-specific protein belonging to the tetraspanin superfamily. Previously thought to be predominantly a B cell molecule, CD37 is shown in this study to regulate T cell proliferation. CD37-deficient (CD37(-/-)) T cells were notably hyperproliferative in MLR, in response to Con A, or CD3-TCR engagement particularly in the absence of CD28 costimulation. Hyperproliferation was not due to differences in memory to naive T cell ratios in CD37(-/-) mice, apoptosis, or TCR down-modulation. Division cycle analyses revealed CD37(-/-) T cells to enter first division earlier than wild-type T cells. Importantly, proliferation of CD37(-/-) T cells was preceded by enhanced early IL-2 production. We hypothesized CD37 to be involved in TCR signaling and this was supported by the observation that CD4/CD8-associated p56(Lck) kinase activity was increased in CD37(-/-) T cells. Remarkably, CD37 cross-linking on human T cells transduced signals that led to complete inhibition of CD3-induced proliferation. In the presence of CD28 costimulation, CD37 engagement still significantly reduced proliferation. Taken together, these results demonstrate a regulatory role for CD37 in T cell proliferation by influencing early events of TCR signaling.

An oncogenic tyrosine kinase inhibits DNA repair and DNA-damage-induced Bcl-x L deamidation in T cell transformation

A transgenic mouse model of T cell lymphoma was used to investigate the transforming events mediated by an oncogenic tyrosine kinase in pretumorigenic CD4 −CD8 − (DN) thymocytes. Parental CD45 −/− and p56 lck-F505Y mice do not develop tumors, whereas their CD45 −/−p56 lck-F505Y progeny develop T lymphomas. Increased but nononcogenic p56 lck kinase activity in p56 lck-F505Y mice DN thymocytes causes cell-cycle progression, survival, and Bcl-X L upregulation. Additional unique oncogenic signals occur in pretumorigenic CD45 −/−p56 lck-F505Y thymocytes in which p56 lck kinase activity is 2- to 3-fold higher relative to p56 lck-F505Y: inhibition of DNA repair, inhibition of DNA-damage-induced Bcl-X L deamidation, Bax conformational change and mitochondrial translocation, cytochrome c release, and the apoptotic caspase execution cascade. Inhibition of Bcl-X L deamidation may be a critical switch in oncogenic kinase-induced T cell transformation.