Chronic lymphocytic leukemia cells diversify and differentiate in vivo via a nonclassical Th1-dependent, Bcl-6-deficient process.

Xenografting primary tumor cells allows modeling of the heterogeneous natures of malignant diseases and the influences of the tissue microenvironment. Here, we demonstrate that xenografting primary chronic lymphocytic leukemia (CLL) B lymphocytes with activated autologous T cells into alymphoid mice results in considerable CLL B cell division and sizable T cell expansion. Nevertheless, most/all CD5+CD19+ cells are eventually lost, due in part to differentiation into antibody-secreting plasmablasts/plasma cells. CLL B cell differentiation is associated with isotype class switching and development of new IGHV-D-J mutations and occurs via an activation-induced deaminase-dependent pathway that upregulates IRF4 and Blimp-1 without appreciable levels of the expected Bcl-6. These processes were induced in IGHV-unmutated and IGHV-mutated clones by Th1-polarized T-bet+ T cells, not classical T follicular helper (Tfh) cells. Thus, the block in B cell maturation, defects in T cell action, and absence of antigen-receptor diversification, which are often cardinal characteristics of CLL, are not inherent but imposed by external signals and the microenvironment. Although these activities are not dominant features in human CLL, each occurs in tissue proliferation centers where the mechanisms responsible for clonal evolution operate. Thus, in this setting, CLL B cell diversification and differentiation develop by a nonclassical germinal center-like reaction that might reflect the cell of origin of this leukemia.

Observational study on the occurrence of muscle spindles in human digastric and mylohyoideus muscles.

Although the occurrence of muscle spindles (MS) is quite high in most skeletal muscles of humans, few MS, or even absence, have been reported in digastric and mylohyoideus muscles. Even if this condition is generally accepted and quoted in many papers and books, observational studies are scarce and based on histological sections of a low number of specimens. The aim of the present study is to confirm previous data, assessing MS number in a sample of digastric and mylohyoideus muscles. We investigated 11 digastric and 6 mylohyoideus muscles from 13 donors. Muscle samples were embedded in paraffin wax, cross-sectioned in a rostrocaudal direction, and stained using haematoxylin-eosin. A mean of 5.1 ± 1.1 (range 3-7) MS was found in digastric muscles and mean of 0.5 ± 0.8 (range 0-2) in mylohyoideus muscles. A significant difference (P < 0.001) was found with the control sample, confirming the correctness of the histological procedure. Our results support general belief that the absolute number of spindles is sparse in digastric and mylohyoideus muscles. External forces, such as food resistance during chewing or gravity, do not counteract jaw-opening muscles. It is conceivable that this condition gives them a limited proprioceptive importance and a reduced need for having specific receptors as MS.

The soluble form of CTLA-4 from serum of patients with autoimmune diseases regulates T-cell responses.

Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) is a costimulatory receptor transducing a potent inhibitory signal. Increasing evidence showed that CTLA-4 gene is an important susceptibility locus for autoimmune disorders. Alternatively spliced mRNA generates a soluble form, called sCTLA-4. Whereas low levels of sCTLA-4 are detected in normal human serum, increased/high serum levels are observed in several autoimmune diseases. The biological significance of increased sCTLA-4 serum level is not fully clarified yet. It can be envisaged that sCTLA-4 specifically inhibits the early T-cell activation by blocking the interaction of CD80/CD86 with the costimulatory receptor CD28. On the other hand, higher levels of sCTLA-4 could contend the binding of the membrane form of CTLA-4 with CD80/CD86, in later activation phase, causing a reduction of inhibitory signalling. We showed that sCTLA-4 from sera of patients with different autoimmune diseases is able to display functional activities on an in vitro system acting on the proliferation capability and modulating the secretion of cytokines. We observed a dual effect of sCTLA-4: inhibiting the secretion of IFN-γ , IL-2, IL-7, and IL-13 and activating the secretion of TGF-ß and IL-10. This study underlines the role of sCTLA-4 in modulating the immune response and its relevance in autoimmune disease pathogenesis.

Serum LAIR-2 is increased in autoimmune thyroid diseases.

Leukocyte-associated Ig-like receptor (LAIR) is a small family-receptor able to inhibit immune cell function via collagen binding. It exists as both membrane-bound and soluble forms. LAIR-1 functions as an inhibitory receptor on natural killer cells, T lymphocytes and monocytes. In addition to LAIR-1, the human genome encodes LAIR-2, a soluble homolog. Several studies have focused on LAIR-1, whereas few investigations concentrate on the expression and function of LAIR-2. We demonstrate the presence of high LAIR-2 levels in 74/80 sera from patients with autoimmune thyroid diseases (both Graves' disease and autoimmune thyroiditis). LAIR-2 levels seemed not to be related to specific clinical manifestations, such as thyroid functions (hypo- or hyperthyroidism), or specific clinical features (such as ophtalmopathy). In addition, serum LAIR-2 is able, in vitro, to bind its natural ligand, collagen. Since LAIR-2 has been found to have higher affinity for collagens than LAIR-1 did, we hypothesize a potential regulating capability of serum LAIR-2 in finally regulating the inhibitory capability of LAIR-1.

A soluble form of CTLA-4 is present in paediatric patients with acute lymphoblastic leukaemia and correlates with CD1d+ expression.

CTLA-4 is a key factor in regulating and maintaining self tolerance, providing a negative signal to the T cell and thus limiting immune responses. Several polymorphisms within the CTLA-4 gene have been associated with an increased risk of developing autoimmune diseases and, very recently, with susceptibility to human cancer. Acute lymphoblastic leukemia is a clonal disorder of lymphoid progenitors representing the most frequent malignancy of childhood. Here, we show the presence at significantly elevated levels of a circulating soluble form of CTLA-4 in 70% of B-ALL pediatric patients with active disease, the positive correlation between the percentage of leukemic B lymphocytes and the amount of serum sCTLA-4, and the expression of sCTLA-4 transcript by B cells in patients. Finally, a correlation between CD1d expression (a negative prognostic marker) and the sCTLA-4 in B-ALL patients was observed. This suggests a possible role of this soluble molecule as a marker of progression or severity of the neoplastic disease.

A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease.

Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology. Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has been impeded by lack of a reproducible adoptive transfer model. We report a simple, reproducible system in which primary CLL cells proliferate in nonobese diabetes/severe combined immunodeficiency/γc(null) mice under the influence of activated CLL-derived T lymphocytes. By co-transferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth of primary CFSE-labeled CLL cells in vivo is achieved and quantified. Using this approach, we have identified key roles for CD4(+) T cells in CLL expansion, a direct link between CD38 expression by leukemic B cells and their activation, and support for CLL cells preferentially proliferating in secondary lymphoid tissues. The model should simplify analyzing kinetics of CLL cells in vivo, deciphering involvement of nonleukemic elements and nongenetic factors promoting CLL cell growth, identifying and characterizing potential leukemic stem cells, and permitting preclinical studies of novel therapeutics. Because autologous activated T lymphocytes are 2-edged swords, generating unwanted graph-versus-host and possibly autologous antitumor reactions, the model may also facilitate analyses of T-cell populations involved in immune surveillance relevant to hematopoietic transplantation and tumor cytoxicity.

Ligation of the BT3 molecules, members of the B7 family, enhance the proinflammatory responses of human monocytes and monocyte-derived dendritic cells.

BT3 is a new family of immunoreceptors belonging to the extended B7 family. BT3 molecules are expressed on the surface of resting and activated monocytes and monocyte-derived dendritic cells (iDC). We show that BT3 cross-linking, in the absence of other survival factors, provides a survival signal for monocytes and iDC and induces up-regulation of costimulatory molecules, such as CD80 and CD86, and HLA-DR. We further analyzed the effects of BT3 cross-linking on various proinflammatory responses on monocytes and iDC. The results obtained showed that BT3 engagement is able to modulate the production of IL8/CXCL8, IL-1ß and IL-12/p70. Moreover, we demonstrated a synergistic effect between BT3 and Toll-like receptors ligands on both monocytes and iDC in up-regulating the production of proinflammatory cytokines. Thus, BT3 could be involved in the regulation of the balance between immune activation and suppression. A better understanding of its physiological role of these families of receptors awaits the precise identification of the nature, origin, expression, and distribution of their ligands.

CTLA-4 is expressed by human monocyte-derived dendritic cells and regulates their functions.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is the major negative regulator of T-cell responses, although growing evidence supports its wider role as an immune attenuator that may also act in other cell lineages. Here, we have analyzed the expression of CTLA-4 in human monocytes and monocyte-derived dendritic cells (DCs), and the effect of its engagement on cytokine production and T-cell stimulatory activity by mature DCs. CTLA-4 was highly expressed on freshly isolated monocytes, then down-modulated upon differentiation toward immature DCs (iDCs) and it was markedly upregulated on mature DCs obtained with different stimulations (lipopolysaccharides [LPS], Poly:IC, cytokines). In line with the functional role of CTLA-4 in T cells, treatment of mDCs with an agonistic anti-CTLA-4 mAb significantly enhanced secretion of regulatory interleukin (IL)-10 but reduced secretion of IL-8/IL-12 pro-inflammatory cytokines, as well as autologous CD4+ T-cell proliferation in response to stimulation with recall antigen purified protein derivative (PPD) loaded-DCs. Neutralization of IL-10 with an anti-IL-10 antibody during the mDCs-CD4+ T-cell co-culture partially restored the ability of anti-CTLA-4-treated mDCs to stimulate T-cell proliferation in response to PPD. Taken together, our data provide the first evidence that CTLA-4 receptor is expressed by human monocyte-derived mDCs upon their full activation and that it exerts immune modulatory effects.

The soluble CTLA-4 receptor and its role in autoimmune diseases: an update.

CTLA-4, initially described as a membranebound molecule, is a costimulatory receptor transducing a potent inhibitory signal. Increasing evidence shows the CTLA-4 gene to be an important susceptibility locus for autoimmune endocrinopathies and other autoimmune disorders. A soluble form of cytotoxic T-lymphocyte-associated antigen-4 (sCTLA-4) has been established and shown to possess CD80/CD86 binding activity and in vitro immunoregulatory functions. sCTLA-4 is generated by alternatively spliced mRNA. Whereas low levels of sCTLA-4 are detected in normal human serum, increased serum levels are observed in several autoimmune diseases (e.g. Graves' disease, myasthenia gravis, systemic lupus erythematosus, type 1 diabetes, systemic sclerosis, coeliac disease, autoimmune pancreatitis and primary biliary cirrhosis). The biological significance of increased sCTLA-4 serum levels is not fully clarified yet. On the one hand, it can be envisaged that sCTLA-4 specifically inhibits early T-cell activation by blocking the interaction of CD80/CD86 with the costimulatory receptor CD28. On the other hand, higher levels of sCTLA-4 could compete for the binding of the membrane form of CTLA-4 with CD80/CD86 in the later phases of T-lymphocyte activation, causing a reduction in inhibitory signalling. This double-edged nature of sCTLA-4 to block the binding of CD28 to CD80/CD86 may result in different outcomes during the clinical course of an autoimmune disease.

Identification of a novel antibody associated with autoimmune pancreatitis.

BACKGROUND: Autoimmune pancreatitis is characterized by an inflammatory process that leads to organ dysfunction. The cause of the disease is unknown. Its autoimmune origin has been suggested but never proved, and little is known about the pathogenesis of this condition. METHODS: To identify pathogenetically relevant autoantigen targets, we screened a random peptide library with pooled IgG obtained from 20 patients with autoimmune pancreatitis. Peptide-specific antibodies were detected in serum specimens obtained from the patients. RESULTS: Among the detected peptides, peptide AIP(1-7) was recognized by the serum specimens from 18 of 20 patients with autoimmune pancreatitis and by serum specimens from 4 of 40 patients with pancreatic cancer, but not by serum specimens from healthy controls. The peptide showed homology with an amino acid sequence of plasminogen-binding protein (PBP) of Helicobacter pylori and with ubiquitin-protein ligase E3 component n-recognin 2 (UBR2), an enzyme highly expressed in acinar cells of the pancreas. Antibodies against the PBP peptide were detected in 19 of 20 patients with autoimmune pancreatitis (95%) and in 4 of 40 patients with pancreatic cancer (10%). Such reactivity was not detected in patients with alcohol-induced chronic pancreatitis or intraductal papillary mucinous neoplasm. The results were validated in another series of patients with autoimmune pancreatitis or pancreatic cancer: 14 of 15 patients with autoimmune pancreatitis (93%) and 1 of 70 patients with pancreatic cancer (1%) had a positive test for anti-PBP peptide antibodies. When the training and validation groups were combined, the test was positive in 33 of 35 patients with autoimmune pancreatitis (94%) and in 5 of 110 patients with pancreatic cancer (5%). CONCLUSIONS: The antibody that we identified was detected in most patients with autoimmune pancreatitis but also in some patients with pancreatic cancer, making it an imperfect test to distinguish between these two conditions.

A functional soluble form of CTLA-4 is present in the serum of celiac patients and correlates with mucosal injury.

Celiac disease (CD) is a multifactorial disorder influenced by environmental, genetic and immunological factors. Increasing evidence showed CTLA-4 gene as an important susceptibility locus for autoimmune disorders. A native soluble cytotoxic T-lymphocyte-associated protein-4 (sCTLA-4), lacking of transmembrane sequence, has been described in several autoimmune diseases. We aimed to evaluate the presence of increased sCTLA-4 concentration in the serum of patients with CD and the possible immunoregulatory function. Blood samples were collected from 160 CD patients; sCTLA-4 levels were evaluated by ELISA, western blot and reverse transcription-PCR. The capability of serum sCTLA-4 to modulate T-lymphocyte proliferation in vitro was evaluated by two-way mixed leukocyte reaction assay. We demonstrated high levels of sCTLA-4 in serum of untreated celiac patients. Additionally, we observed that sCTLA-4 concentrations are related to gluten intake and that a correlation between autoantibodies to tissue transglutaminase and sCTLA-4 concentration exists. Moreover, sCTLA-4 levels correlate with the degree of mucosal damage. Conversely, no correlation between sCTLA4 levels and the HLA-related risk was observed. Finally, we show that sCTLA-4 from sera of CD patients displays functional activities. These results strongly suggest a regulation of sCTLA-4 synthesis depending on the presence or absence of dietary gluten and imply a possible immunomodulatory effect on cytotoxic T lymphocyte functions. In gluten-exposed patients, serum sCTLA-4 levels might provide insight about mucosal injury.

The inhibitory co-receptors: a way to save from anergy the HIV-specific T cells.

The functional impairment of HIV-specific CD4(+) T cells during chronic HIV infection is thought to be closely linked to viral replication and to T cell exhaustion. T cell exhaustion in the presence of ongoing antigen exposure is a common feature of chronic viral infection, in which dysfunctional T cells fail to eliminate the virus. Otherwise, antiviral T cell function impairment is a poorly understood mechanism. Increasing evidences show that HIV-specific T lymphocytes up-regulated inducible co-receptors, such as the Cytoxic T Lymphocyte Antigen-4, (CTLA-4, or CD152) and Programmed Death-1 (PD-1) and that blockade of the CD152 or PD-1 pathway restores HIV-specific CD4(+) T cell function in HIV infection. This review will focus on finding a possible role for inhibitory receptors on virus-specific CD4(+) T cells. The analysis of the role of CD152 and PD-1 in HIV-1 infection could provide important insight into the mechanism of viral induced immune dysfunction and lead to immunotherapeutic strategies to reverse immune suppression in this pathology.

Stimulation of human CD4⁺ T lymphocytes via TLR3, TLR5 and TLR7/8 up-regulates expression of costimulatory and modulates proliferation.

The cells of innate and adaptive immunity, although activated by different ligands, engage in cross talk to ensure a successful immune outcome. Toll-like receptors (TLRs) are key components of the innate immune system and have the ability to detect microbial infection and trigger host defence responses. Otherwise, human T lymphocytes are able to produce most TLRs. Thus, we analyze the capability of some TLR ligands to modulate the function of highly-purified CD4⁺ T cells. We found that agents acting via TLRs (poly I:C, a TLR3 ligand; flagellin, a TLR5 ligand; and R848, a TLR7/8 ligand) are able to regulate the expression of costimulatory molecules both on purified antigen presenting cells and on purified T lymphocytes. Moreover, the activation mediated by TLRs determines a kinetic expression of B7-family members such as through an inhibition of T lymphocytes delayed proliferation. These findings suggest a functional role of some invading microorganisms in regulating acquired immunity.

The frequency of regulatory CD3+CD8+CD28- CD25+ T lymphocytes in human peripheral blood increases with age.

Aging is commonly associated with immune deficiency and dysregulation. The aging of the immune system involves a progressive reduction in naïve T cell output associated with thymic involution and peripheral expansion of oligoclonal memory T cells. We have investigated frequency, phenotype, and function of CD3+CD8+CD28(-)CD25+ T cells in healthy volunteers over a wide age range. We demonstrate that the frequency of CD3+CD8+CD28(-)CD25+ T cells in healthy volunteers increases with age. Peripheral CD3+CD8+CD28(-)CD25+ T cells share phenotypic and functional features with CD3+CD4+CD25+ regulatory T cells (Tregs): In particular, they strongly express CTLA-4 and forkhead box P3. We observed that in vitro, functional titration assays of CD3+CD8+CD28(-)CD25+ T cells show equivalent regulatory function in young and elderly donors, with suppression of proliferation and cytokine production in response to polyclonal T cell stimulation. Finally, CD3+CD8+CD28(-)CD25+ T cells seem to specifically express the CD122 receptor. Altogether, these observations demonstrate an increase in peripheral blood CD8+ Tregs associated with aging.

Soluble CTLA-4 in autoimmune thyroid diseases: relationship with clinical status and possible role in the immune response dysregulation.

CTLA-4 molecule, expressed by activated T and B lymphocytes, transduces an inhibitory signal. Increasing evidence showed CTLA-4 gene as an important susceptibility locus for autoimmune endocrinopathies and other autoimmune disorders. The aim is to evaluate the augmented sCTLA-4 serum levels in different autoimmune thyroid diseases when compared with normal donors or with non-autoimmune hyperthyroidism and to investigate the functional activities and suggest the possible pathogenetic role of sCTLA-4. We demonstrate the presence of a soluble form of CTLA-4 in 59/90 sera from patients with autoimmune thyroid diseases (both Graves' disease and autoimmune thyroiditis). sCTLA-4 levels were not related to specific clinical manifestations, such as clinical thyroid status (hypo- or hyperthyroidism), circulating thyroid hormones, or other clinical features (ophthalmopathy). sCTLA-4 production does not seem to be affected by disease evolution during time. We showed that sCTLA-4 from sera of patients with thyroid autoimmunity is able to bind its physiological ligands CD80/CD86 and displays functional activities on different in vitro systems (T-cell proliferation induced by specific soluble antigens, bi-directional mixed lymphocyte reaction). In conclusion, we demonstrate an increment of sCTLA-4 in serum of patients with autoimmune thyroid diseases. Its possible pathogenetic role during autoimmune processes can be speculated: sCTLA-4 can specifically inhibit the early T-cell activation by blocking the interaction of CD80/CD86 with the co-stimulatory receptor CD28. Conversely, higher levels of sCTLA-4 could compete with membrane-bound CTLA-4 for CD80/CD86, in later T lymphocytes activation phase, causing a reduction of inhibitory signaling.

CD85j (leukocyte Ig-like receptor-1/Ig-like transcript 2) inhibits human osteoclast-associated receptor-mediated activation of human dendritic cells.

Immature dendritic cells (DCs) derived from freshly isolated human monocytes were used to evaluate the effect of the inhibiting receptor CD85j (leukocyte Ig-like receptor-1/ILT2) on activation induced by cross-linking of the human osteoclast-associated receptor (hOSCAR). CD85j and hOSCAR were expressed consistently at the same density on monocytes and on monocyte-derived DCs (both immature and mature). Cross-linking of hOSCAR, which activates via the FcR-associated gamma-chain, induced Ca(2+) flux in DCs. Concomitant cross-linking of anti-CD85j mAb abolished this early activation event. Likewise, CD85j stimulation strongly reduced IL-8 and IL-12 production by hOSCAR-activated DCs. Inhibition of DCs via CD85j also impaired their ability to enhance Ag-specific T cell proliferation induced by hOSCAR. Finally, because hOSCAR prevents apoptosis of DCs in the absence of growth/survival factors, CD85j cross-linking was able to counteract completely this antiapoptotic effect and to reduce Bcl-2 expression enhanced by hOSCAR stimulation. Thus, CD85j is an inhibiting receptor that is functional in human DCs.