CTLA4‐Ig inhibits allergic airway inflammation by a novel CD28‐independent,nitric oxide synthase‐dependent mechanism

The T‐cell response to antigen depends on coordinate signaling between costimulatory and inhibitory receptors. The altered function of either may underlie the pathophysiology of autoimmune and/or chronic inflammatory diseases and manipulation of these pathways is an important emerging area of therapeutics. We report here that the immunosuppressant drug CTLA4‐Ig inhibits the effector phase of allergic airway inflammation through a CD28‐independent, nitric oxide synthase (NOS)‐dependent mechanism. Using mice deficient in both B‐ and T‐lymphocyte attenuator (BTLA) and CD28, we demonstrate that simultaneous deficiency of an inhibitory receptor can rescue the in vivo but not the in vitro CD28‐deficient phenotype. Furthermore, we demonstrate that inflammation in CD28/BTLA‐double‐deficient mice is suppressed by CTLA4‐Ig. This suppression is reversed by treatment with the NOS inhibitor, N⁶‐methyl‐L ‐arginine acetate (L‐NMMA). In addition, CTLA4‐Ig is ineffective at inhibiting inflammation in NOS2‐deficient mice when given at the effector phase. Thus, CD28 and BTLA coordinately regulate the in vivo response to inhaled allergen, and CTLA4‐Ig binding to B7‐proteins inhibits the effector phase of inflammation by a CD28‐independent, NOS‐dependent mechanism.     

Soluble HLA‐I‐mediated secretion of TGF‐β1 by human NK cells and consequent down‐regulation of anti‐tumor cytolytic activity

Soluble HLA class I (sHLA‐I) molecules can regulate survival of NK cells and their anti‐tumor killing activity. Herein, we have analysed whether interaction of sHLA‐I with CD8 and/or different isoforms of killer Ig‐like receptors (KIR) induced secretion of transforming growth factor (TGF)‐β1. CD8⁺KIR^? NK cell clones secreted TGF‐β1 upon the interaction of sHLA‐I with CD8 molecule. sHLA‐Cw4 or sHLA‐Cw3 alleles engaging inhibitory isoforms of KIR, namely KIR2DL1 or KIR2DL2, strongly downregulated TGF‐β1 production elicited through CD8. On the other hand, sHLA‐Cw4 or sHLA‐Cw3 alleles induced secretion of TGF‐β1 by ligation of stimulatory KIR2DS1 or KIR2DS2 isoforms. TGF‐β1 strongly reduced NK cell‐mediated tumor cell lysis and production of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Also, TGF‐β1 inhibited NK cell cytolysis induced by the engagement of stimulatory receptors including NKG2D, DNAM1, 2B4, CD69, NKp30, NKp44 and NKp46. The IL‐2‐dependent surface upregulation of some of these receptors was prevented by TGF‐β1. Furthermore, TGF‐β1 hampered IL‐2‐induced NK cell proliferation but not IL‐2‐mediated rescue from apoptosis of NK cells. Depletion of TGF‐β1 restored all the NK cell‐mediated functional activities analysed. Taken together these findings suggest that sHLA‐I antigens may downregulate the NK cell‐mediated innate response by inducing TGF‐β1 release.     

Roles of the third Ig‐like domain of Necl‐5/PVR and the fifth Ig‐like domain of the PDGF receptor in its signaling

The immunoglobulin (Ig)‐like cell adhesion molecule nectin‐like molecule (Necl)‐5/poliovirus receptor is up‐regulated in many types of cancer cells and implicated in their abnormally enhanced cell proliferation and movement. We previously showed that Necl‐5 cis‐interacts with the platelet‐derived growth factor (PDGF) receptor β through the extracellular region and enhances its signaling. Although this cis‐interaction does not affect the PDGF‐induced tyrosine phosphorylation of the receptor, the interaction of the cytoplasmic region of Necl‐5 with sprouty2 and the regulation of its activity are required for the enhancement of the PDGF receptor β signaling by Necl‐5. We investigated here the more detailed mechanism for this cis‐interaction of Necl‐5 with the PDGF receptor β. Necl‐5 contains three Ig‐like domains and the PDGF receptor β contains five Ig‐like domains at their extracellular regions. We showed here that the third Ig‐like domain of Necl‐5 cis‐interacted with the fifth Ig‐like domain of the PDGF receptor β. The recombinant protein of the third Ig‐like domain of Necl‐5 inhibited the cis‐interaction of full‐length Necl‐5 with the PDGF receptor β and the PDGF‐induced activation of the ERK signaling pathway that was enhanced by Necl‐5. These results revealed the novel roles of the third Ig‐like domain of Necl‐5 and the fifth Ig‐like domain of the PDGF receptor β in its signaling.     

XLP1 inhibitory effect by 2B4 does not affect DNAM‐1 and NKG2D activating pathways in NK cells

X‐linked lymphoproliferative disease 1 (XLP1) is a rare congenital immunodeficiency caused by SH2D1A (Xq25) mutations resulting in lack or dysfunction of SLAM‐associated protein adaptor molecule. In XLP1 patients, upon ligand (CD48) engagement, 2B4 delivers inhibitory signals that impair the cytolytic activity of NK (and T) cells. This causes the selective inability to control EBV infections and the occurrence of B‐cell lymphomas. Here, we show that in the absence of SLAM‐associated protein, co‐engagement of 2B4 with different activating receptors, either by antibodies or specific ligands on target cells, inhibits different ITAM‐dependent signaling pathways including activating killer Ig‐like receptors. In XLP1 NK cells, 2B4 affected both the cytolytic and IFN‐γ production capabilities, functions that were restored upon disruption of the 2B4/CD48 interactions. Notably, we provide evidence that 2B4 dysfunction does not affect the activity of DNAM‐1 and NKG2D triggering receptors. Thus, while CD48⁺ B‐EBV and lymphoma B cells devoid of NKG2D and DNAM‐1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands. The study sheds new light on the XLP1 immunological defect and on the cross‐talk of inhibitory 2B4 with triggering NK (and T) receptors.     

What goes up must come down: A tripartite Dok‐3/Grb2/SHIP1 inhibitory module limits BCR signaling

Properly regulated immunity requires precise integration of activating and inhibitory signals. As for other lymphocytes, B cells express an antigen‐specific activating receptor, the B‐cell antigen receptor (BCR), and inhibitory receptors (e.g. FcγRIIb) that exercise checkpoint control on B‐cell activation. Moreover, following BCR engagement, CD19 recruits proteins that amplify BCR signaling, while CD22 initiates a negative feedback loop by recruiting proteins that inhibit BCR signaling. Initial BCR signaling is mediated by protein tyrosine kinases and lipid kinases; inhibitory receptors directly antagonize the actions of these enzymes by recruiting protein tyrosine phosphatases and lipid phosphatases and positioning them close to actively signaling BCRs. Previously it was thought that inhibitory receptors such as FcγRIIb and CD22 were essential for bringing these phosphatases near the BCR. In this issue of the European Journal of Immunology, Manno et al. show that a tripartite inhibitory module consisting of the adaptor proteins Dok‐3 and Grb2 and the lipid phosphatase SHIP1 binds directly to activated BCRs and limits the Ca²⁺ mobilization that is required for B lymphocyte activation. This reveals that the BCR can be both an activating and inhibitory receptor, one that activates signaling enzymes while initiating a negative feedback loop that prevents excessive signaling.     

Generation of precursor,immature, and mature murine B1‐cell lines from c‐myc/bcl‐xL‐overexpressing pre‐BI cells

Deregulated expression of c‐myc and bcl‐xL is long known to generate transformed B cells in humans and mice. We overexpressed these genes to induce in vitro and in vivo differentiation of fetal liver‐derived mouse pre‐BI cells to B1‐lineage pre‐BII‐like, immature and mature B‐cell lines, and to Ig‐secreting cells. In vitro, doxycycline‐controlled c‐myc/bcl‐xL‐overexpressing CD19⁺CD93⁺c‐kikt⁺IgM^? pre‐BI cells differentiate to and survive as CD19⁺CD93⁺c‐kit^?IgM⁺ immature B1 cells. Timed CpG stimulation of these oncogene‐overexpressing pre‐B or immature B1 cells generates either CD19⁺CD93^lowc‐kit^?IgM^?SLC^? pre‐BII‐like or IgM⁺MHCII⁺CD73⁺CD80⁺CD40⁺ mature B1‐cell lines and IgM‐secreting B1 cells in vitro and fixes their state of differentiation. All cell lines are clonable, but a majority of immature and mature B1‐cell clones eventually reach a nonproliferating, surviving G₀‐state. Transplanted in vivo, c‐myc/bcl‐xL‐overexpressing pre‐B cells expand to mature B1 cells, and to IgM‐ and IgA‐secreting plasmablasts and plasma cells. Within 2 months, plasmablasts have expanded most prominently in BM and spleen, indicating that the host selectively expanded development of these transformed plasma cells. The sIgM⁺ B1‐cell lines and clones offer the possibility to study their roles in the development of B1‐Ab repertoires, of B1‐cell‐mediated autoimmune diseases and of B1‐cell malignancies.     

KLRG1 impairs regulatory T‐cell competitive fitness in the gut

Immune homeostasis requires the tight, tissue‐specific control of the different CD4⁺ Foxp3⁺ regulatory T (Treg) cell populations. The cadherin‐binding inhibitory receptor killer cell lectin‐like receptor G1 (KLRG1) is expressed by a subpopulation of Treg cells with GATA3⁺ effector phenotype. Although such Treg cells are important for the immune balance, especially in the gut, the role of KLRG1 in Treg cells has not been assessed. Using KLRG1 knockout mice, we found that KLRG1 deficiency does not affect Treg cell frequencies in spleen, mesenteric lymph nodes or intestine, or frequencies of GATA3⁺ Treg cells in the gut. KLRG1‐deficient Treg cells were also protective in a T‐cell transfer model of colitis. Hence, KLRG1 is not essential for the development or activity of the general Treg cell population. We then checked the effects of KLRG1 on Treg cell activation. In line with KLRG1's reported inhibitory activity, in vitro KLRG1 cross‐linking dampened the Treg cell T‐cell receptor response. Consistently, lack of KLRG1 on Treg cells conferred on them a competitive advantage in the gut, but not in lymphoid organs. Hence, although absence of KLRG1 is not enough to increase intestinal Treg cells in KLRG1 knockout mice, KLRG1 ligation reduces T‐cell receptor signals and the competitive fitness of individual Treg cells in the intestine.     

Composite monoclonal B‐cell lymphocytosis and MYD88 L265P‐positive lymphoplasmacytic lymphoma in a patient with IgM light chain amyloidosis: Case report

Monoclonal B‐cell lymphocytosis (MBL) is an early or precursor asymptomatic proliferation of chronic lymphocytic lymphoma (CLL)‐like B‐cells. Lymphoplasmacytic lymphoma (LPL), often clinically associated with Waldenström macroglobulinemia, is a B‐cell neoplasm characterized by frequent MYD88 L265P mutation. Here, we report a rare composite MBL and LPL in a patient with IgM light chain (AL) amyloidosis. A 74‐year‐old male with a known IgM monoclonal protein developed proteinuria. No lymphocytosis was detected. Renal biopsy showed deposition of AL λ amyloid in the glomeruli and vessels. Subsequent bone marrow biopsy revealed nodular atypical CLL‐like small B‐cell proliferation and scattered peripheral LPL. Immunohistochemistry and/or flow cytometry revealed that the atypical CLL‐like population expressed CD19, CD20, CD5, weak CD23, LEF‐1 and diminished surface Igκ. The LPL was positive for CD19, CD20 and surface Igλ. Using laser‐capture microdissection and allele‐specific polymerase chain reaction, we confirmed that MYD88 L265P was detectable in the LPL but not in the atypical CLL‐like population. Thus, we demonstrated that these two populations were clonally independent, and made the diagnosis of composite MBL and LPL. An integrated clinical, pathological, immunophenotypic and genetic assessment is essential in such complicated cases, and especially ‘clone‐specific’ MYD88 genotyping may facilitate the differential diagnoses of low‐grade B‐cell lymphomas.     

Cis association of leukocyte Ig‐like receptor 1 with MHC class I modulates accessibility to antibodies and HCMV UL18

Leukocyte Ig‐like receptor (LIR) 1 (CD85j/ILT2/LILRB1) is an inhibitory receptor with broad specificity for MHC class I (MHC‐I) and the human CMV MHC‐I homologue UL18. LIR‐1 can inhibit NK cells through the conventional interaction with MHC‐I expressed on a target cell (in trans) but the nature and the effects of LIR‐1 interactions with MHC‐I in cis are not well understood. Here we show that MHC‐I expressed in cis has an impact on the detection of LIR‐1 with various antibodies. We found the cis interaction alters recognition by only one of two antibodies known to block functional trans recognition by LIR‐1 on NK cells. Specifically, we observed an enhancement of recognition with GHI/75 in the presence of various MHC‐I alleles on 721.221 cells. We found that blocking the LIR‐1 contact site with anti‐MHC‐I antibodies decreased detection of LIR‐1 with GHI/75. We also observed a decrease in GHI/75 following acid denaturation of MHC‐I. Finally, disruption of LIR‐1 cis interactions with MHC‐I significantly enhanced UL18‐Fc binding to NK92 cells and enhanced the relative inhibition of NK92 cells by HLA‐G. These results have implications for LIR‐1 function in scenarios such as infection when MHC‐I levels on effector cells may be increased by IFNs.     

Balance between activating NKG2D,DNAM‐1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast‐like synoviocytes (FLS) are central components of the aggressive, tumour‐like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA‐FLS and NK cells. We used cultured RA‐FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA‐FLS/NK cell cross‐talk. We show that RA‐FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM‐1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA‐FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA‐E expressed on RA‐FLS further enhanced Nishi cell degranulation in co‐culture with RA‐FLS. Using cultured RA‐FLS and the human NK cell line Nishi as an in vitro model system of RA‐FLS/NK cell cross‐talk, our results suggest that cell‐mediated cytotoxicity of RA‐FLS may be one mechanism by which NK cells influence local joint inflammation in RA.     

Inhibitory 2B4 contributes to NK cell education and immunological derangements in XLP1 patients

X‐linked lymphoproliferative disease 1 (XLP1) is an inherited immunodeficiency, caused by mutations in SH2D1A encoding Signaling Lymphocyte Activation Molecule (SLAM)‐associated protein (SAP). In XLP1, 2B4, upon engagement with CD48, has inhibitory instead of activating function. This causes a selective inability of cytotoxic effectors to kill EBV‐infected cells, with dramatic clinical sequelae. Here, we investigated the NK cell education in XLP1, upon characterization of killer Ig‐like receptor (KIR)/KIR‐L genotype and phenotypic repertoire of self‐HLA class I specific inhibitory NK receptors (self‐iNKRs). We also analyzed NK‐cell cytotoxicity against CD48⁺ or CD48^? KIR‐ligand matched or autologous hematopoietic cells in XLP1 patients and healthy controls. XLP1 NK cells may show a defective phenotypic repertoire with substantial proportion of cells lacking self‐iNKR. These NK cells are cytotoxic and the inhibitory 2B4/CD48 pathway plays a major role to prevent killing of CD48⁺ EBV‐transformed B cells and M1 macrophages. Importantly, self‐iNKR defective NK cells kill CD48^? targets, such as mature DCs. Self‐iNKR^? NK cells in XLP1 patients are functional even in resting conditions, suggesting a role of the inhibitory 2B4/CD48 pathway in the education process during NK‐cell maturation. Killing of autologous mature DC by self‐iNKR defective XLP1 NK cells may impair adaptive responses, further exacerbating the patients’ immune defect.     

Novel function of histamine signaling in hyperlipidemia‐induced atherosclerosis: Histamine H1 receptors protect and H2 receptors accelerate atherosclerosis

Histamine is not only essential for acute inflammatory reactions, but it also participates in a chronic inflammatory disorder. We generated apolipoprotein E (apoE) and histamine receptors (HHRs), including the major H1 and H2 receptors (HH1R, HH2R) double knockout mice (DKO) to clarify the role of HHRs in hyperlipidemia‐induced atherosclerosis, in which apoE‐KO and DKO mice were fed a high cholesterol diet. We found that pronounced hyperlipidemia‐induced atherosclerotic progression occurred in HH1R/apoE‐DKO mice, but in HH2R/apoE‐DKO mice less atherosclerosis, despite pro‐atherogenic serum cholesterol levels compared with apoE‐KO mice. Furthermore, the increased expressions of scavenger receptors (SRs), such as SR‐A, CD36 and lectin‐like oxidized low‐density lipoprotein receptor 1 (LOX‐1), nuclear factor‐kappa B (NFκB), monocyte chemoattractant protein (MCP‐1), matrix metalloproteinases (MMPs) or liver X receptor (LXR)‐related inflammatory signaling factors, were consistent with the pro‐atherogenic phenotype of HH2R/apoE‐DKO mice. We hypothesize that histamine/HH1R and HH2R signaling has conflicting innate functions, inflammatory/atherogenic and anti‐inflammatory/anti‐atherogenic actions, and that there are innate links between histamine signaling and hyperlipidemia‐induced atherosclerosis, independently of serum cholesterol metabolism. Specific histamine signaling blockers, in particular, HH2R blockers, are a possible novel therapeutic target for hyperlipidemia‐induced atherosclerosis.     

Interaction of KLRG1 with E‐cadherin: New functional and structural insights

The killer cell lectin‐like receptor G1 (KLRG1) is an inhibitory receptor expressed by memory T cells and NK cells in man and mice. It is frequently used as a cell differentiation marker and members of the cadherin family are ligands for KLRG1. The present study provides new insights into the interaction of mouse KLRG1 with E‐cadherin. Firstly, we demonstrate that co‐engagement of KLRG1 and CD3/TCR in a spatially linked manner was required for inhibition arguing against the notion that KLRG1‐ligation per se transmits inhibitory signals. Secondly, experiments with T cells carrying Y₇F‐mutant KLRG1 molecules with a replacement of the tyrosine residue to phenylalanine in the single ITIM indicated that the inhibitory activity of KLRG1 is counteracted to some degree by increased interaction of KLRG1⁺ T cells with E‐cadherin expressing target cells. Thirdly, we demonstrate that deletion of the first or the second external domain of E‐cadherin abolished reactivity in KLRG1‐reporter cell assays. Finally, we made the intriguing observation that KLRG1 formed multimeric protein complexes in T cells in addition to the previously described mono‐ and dimeric molecules.     

LAIR‐1 shedding from human fibroblast‐like synoviocytes in rheumatoid arthritis following TNF‐α stimulation

This study examined the expression of the inhibitory receptor, leucocyte‐associated immunoglobulin (Ig)‐like receptor‐1 (LAIR‐1) in fibroblast‐like synoviocytes (FLS) in rheumatoid arthritis (RA) patients to investigate its potential role in the modulation of inflammatory cytokines, matrix metalloproteinases (MMPs) and invasiveness of synoviocytes. LAIR‐1 expression in synovial tissues from RA patients, osteoarthritis patients and healthy donors was analysed by immunohistochemistry. The membrane‐bound form (mLAIR‐1) was detected by flow cytometry. Factors involved in inflammation and MMP activity in FLS were analysed by quantitative polymerase chain reaction (qPCR). LAIR‐1 expression was higher in the synovia of the RA patients than those of the osteoarthritis patients. Co‐immunostaining of vimentin/LAIR‐1 demonstrated that LAIR‐1 was localized mainly in FLS in the RA patients. Surprisingly, primary FLS isolated from the RA patients had low levels of mLAIR‐1 expression, with cytoplasmic distribution. The extracellular domain of LAIR‐1 was shed from the cell surface in response to tumour necrosis factor (TNF)‐α, and this process could be blocked by serine protease inhibitors. Additional experiments indicated that LAIR‐1 over‐expression reduced FLS invasion considerably, which reduced simultaneously the mRNA levels of interleukin (IL)‐6, IL‐8 and MMP‐13 in the presence of TNF‐α. Our study demonstrated that LAIR‐1 is an anti‐inflammatory molecule, and was up‐regulated in FLS in the RA patients; however, cell‐surface LAIR‐1 could be shed from cells in the inflammatory microenvironment in RA. This may weaken the interaction of LAIR‐1 with its ligand, thus reducing the anti‐inflammatory effects of LAIR‐1. These findings suggested that LAIR‐1 may be an important factor involved in the mediation of the progressive joint destruction in RA.     

A cytokine‐controlled mechanism for integrated regulation of T‐lymphocyte motility,adhesion and activation

The co‐ordination of T‐cell motility, adhesion and activation remains poorly understood. It is also unclear how these functions are co‐ordinated with external stimuli. Here we unveil a series of molecular interactions in cis at the surface of T lymphocytes with potent effects on motility and adhesion in these cells, and communicating with proliferative responses. These interactions were controlled by the signature cytokines of T helper subsets interleukin‐2 (IL‐2) and IL‐4. Low‐density lipoprotein receptor‐related protein 1 (LRP1) was found to play a key role for T‐cell motility by promoting development of polarized cell shape and cell movement. Endogenous thrombospondin‐1 (TSP‐1) enhanced cell surface expression of LRP1 through CD47. Cell surface expressed LRP1 induced motility and processing of TSP‐1 while inhibiting adhesion to intercellular adhesion molecule 1 and fibronectin. Interleukin‐2, but not IL‐4, stimulated synthesis of TSP‐1 and motility through TSP‐1 and LRP1. Stimulation of the T‐cell receptor (TCR)/CD3 complex inhibited TSP‐1 expression. Inhibitor studies indicated that LRP1 regulated TSP‐1 expression and promoted motility through JAK signalling. This LRP1‐mediated motogenic signalling was connected to CD47/Gi protein signalling and IL‐2‐induced signalling through TSP‐1. The motogenic TSP‐1/LRP1 mechanism antagonized TCR/CD3‐induced T‐cell proliferation. These results indicate that LRP1 in collaboration with TSP‐1 directs a counter‐adhesive and counter‐proliferative motogenic cascade. T cells seem programmed to prioritize movement before adhesion through this cascade. In conclusion, vital decision‐making in T lymphocytes regulating motility, adhesive interactions and proliferation, are integrated through a molecular mechanism connecting different cell surface receptors and their signalling pathways.     

TGF‐β downregulates KLRG1 expression in mouse and human CD8+ T cells

The inhibitory receptor killer cell lectin‐like receptor G1 (KLRG1) and the integrin α_E (CD103) are expressed by CD8⁺ T cells and both are specific for E‐cadherin. However, KLRG1 ligation by E‐cadherin inhibits effector T‐cell function, whereas binding of CD103 to E‐cadherin enhances cell–cell interaction and promotes target cell lysis. Here, we demonstrate that KLRG1 and CD103 expression in CD8⁺ T cells from untreated and virus‐infected mice are mutually exclusive. Inverse correlation of KLRG1 and CD103 expression was also found in human CD8⁺ T cells‐infiltrating hepatocellular carcinomas. As TGF‐β is known to induce CD103 expression in CD8⁺ T cells, we examined whether this cytokine also regulates KLRG1 expression. Indeed, our data further reveal that TGF‐β signaling in mouse as well as in human CD8⁺ T cells downregulates KLRG1 expression. This finding provides a rationale for the reciprocal expression of KLRG1 and CD103 in different CD8⁺ T‐cell subsets. In addition, it points to the limitation of KLRG1 as a marker for terminally differentiated CD8⁺ T cells if lymphocytes from tissues expressing high levels of TGF‐β are analyzed.     

Programmed death (PD)‐1 molecule and its ligand PD‐L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus‐1‐infected individuals

Human immunodeficiency virus (HIV)‐1 causes T cell anergy and affects T cell maturation. Various mechanisms are responsible for impaired anti‐HIV‐1‐specific responses: programmed death (PD)‐1 molecule and its ligand PD‐L1 are negative regulators of T cell activity and their expression is increased during HIV‐1 infection. This study examines correlations between T cell maturation, expression of PD‐1 and PD‐L1, and the effects of their blockade. Peripheral blood mononuclear cells (PBMC) from 24 HIV‐1⁺ and 17 uninfected individuals were phenotyped for PD‐1 and PD‐L1 expression on CD4⁺ and CD8⁺ T cell subsets. The effect of PD‐1 and PD‐L1 blockade on proliferation and interferon (IFN)‐γ production was tested on eight HIV‐1⁺ patients. Naive (CCR7⁺CD45RA⁺) CD8⁺ T cells were reduced in HIV‐1 aviraemic (P = 0·0065) and viraemic patients (P = 0·0130); CD8 T effector memory subsets [CCR7^‐CD45RA^–(T_EM)] were increased in HIV‐1⁺ aviraemic (P = 0·0122) and viraemic (P = 0·0023) individuals versus controls. PD‐1 expression was increased in CD4 naive (P = 0·0496), central memory [CCR7⁺CD45RA^– (T_CM); P = 0·0116], T_EM (P = 0·0037) and CD8 naive T cells (P = 0·0133) of aviraemic HIV‐1⁺versus controls. PD‐L1 was increased in CD4 T_EMRA (CCR7^‐CD45RA⁺, P = 0·0119), CD8 T_EM (P = 0·0494) and CD8 T_EMRA (P = 0·0282) of aviraemic HIV‐1⁺versus controls. PD‐1 blockade increased HIV‐1‐specific proliferative responses in one of eight patients, whereas PD‐L1 blockade restored responses in four of eight patients, but did not increase IFN‐γ‐production. Alteration of T cell subsets, accompanied by increased PD‐1 and PD‐L1 expression in HIV‐1 infection contributes to anergy and impaired anti‐HIV‐1‐specific responses which are not rescued when PD‐1 is blocked, in contrast to when PD‐L1 is blocked, due possibly to an ability to bind to receptors other than PD‐1.     

Targeting of CD22‐positive B‐cell lymphoma cells by synthetic divalent sialic acid analogues

CD22 is an inhibitory co‐receptor of the B‐cell receptor (BCR) on B cells. Since CD22 is ubiquitously expressed in the B‐cell lineage and CD22 endocytosis can be triggered efficiently, antibodies and antibody‐based immunotoxins against CD22 are used to target B cells both in B‐cell lymphomas and leukemias, as well as in autoimmune diseases. CD22 recognizes α2,6‐linked sialic acids as endogenous ligands. We have developed new synthetic sialosides as ligands for human CD22. These sialosides bind CD22 on human B cells with high affinity and can efficiently enhance IgM‐triggered Ca²⁺ signaling. We coupled these sialosides to Pseudomonas exotoxin A to generate a novel CD22 ligand‐based immunotoxin. This sialoside‐exotoxin‐A construct can specifically kill CD22‐positive B‐cell lymphoma cells. It binds specifically to CD22‐positive B‐cell lymphoma cells and is dominant over endogenous cis‐ligands on the B‐cell surface. The sialoside‐exotoxin‐A construct is efficiently internalized by endocytosis into B‐cell lymphoma cell lines. Thus we show the development of a new therapeutic compound for targeting CD22 on human B cells, both for B‐cell lymphoma, as well as for B‐cell‐mediated autoimmune diseases.     

Influence of PD‐L1 cross‐linking on cell death in PD‐L1‐expressing cell lines and bovine lymphocytes

Programmed death‐ligand 1 (PD‐L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death‐1 (PD‐1). However, the mechanism of PD‐L1‐mediated inhibitory signalling after PD‐L1 cross‐linking by anti‐PD‐L1 monoclonal antibody (mAb) or PD‐1–immunogloblin fusion protein (PD‐1‐Ig) is still unknown, although it may induce cell death of PD‐L1⁺ cells required for regular immune reactions. In this study, PD‐1‐Ig or anti‐PD‐L1 mAb treatment was tested in cell lines that expressed PD‐L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD‐L1‐mediated cell death. PD‐L1 cross‐linking by PD‐1‐Ig or anti‐PD‐L1 mAb primarily increased the number of dead cells in PD‐L1^high cells, but not in PD‐L1^low cells; these cells were prepared from Cos‐7 cells in which bovine PD‐L1 expression was induced by transfection. The PD‐L1‐mediated cell death also occurred in Cos‐7 and HeLa cells transfected with vectors only encoding the extracellular region of PD‐L1. In bovine lymphocytes, the anti‐PD‐L1 mAb treatment up‐regulated interferon‐γ (IFN‐γ) production, whereas PD‐1‐Ig treatment decreased this cytokine production and cell proliferation. The IFN‐γ production in B‐cell‐depleted peripheral blood mononuclear cells was not reduced by PD‐1‐Ig treatment and the percentages of dead cells in PD‐L1⁺ B cells were increased by PD‐1‐Ig treatment, indicating that PD‐1‐Ig‐induced immunosuppression in bovine lymphocytes could be caused by PD‐L1‐mediated B‐cell death. This study provides novel information for the understanding of signalling through PD‐L1.