Expression of human B-cell specific receptor FCRL1 in normal individuals and in patients with autoimmune diseases

The comparative analysis of expression level of FCRL1 gene encoding human B-cell surface receptor in healthy individuals and patients with autoimmune diseases was carried out. For the expression estimation we used results of DNA dot-hybridization on the membranes, containing cDNA samples from subpopulations of blood cells of patients with autoimmune diseases. The quantitative estimation of hybridization signals showed that expression level of FCRL1 gene in peripheral blood B-lymphocytes was significantly higher in patients with a multiple sclerosis, lupus anticoagulans, Takayasu's arteritis and also in von Willebrand disease than in healthy individuals. FCRL1-specific monoclonal and polyclonal antibodies were raised. They were proven to detect FCRL1 in Western blotting, immunohistochemistry and flow cytometry. It was found that FCRL1 is expressed on the surface of CD19+ mature B-cells. In tonsil FCRL1-positive cells were located in crypt area: in mantle zone of secondary lymphoid follicles and among cells of lymphoepithelium. FCRL1-positive cells were also found in B-cell follicles of the spleen.     

The distribution of cells expressing a natural killer cell marker (HNK-1) in normal human lymphoid organs and malignant lymphomas

A murine monoclonal antibody, HNK-1, is known to react with some human leukocytes including all natural killer (NK) cells in peripheral blood. The distribution of cells reacting with this antibody (HNK-1+ cell) was studied in human peripheral lymphoid organs, consisting of five lymph nodes, two specimens of gastric mucosa with lymphoid tissue, two tonsils, one appendix, and two thymuses. Fourteen cases of malignant lymphoma (ML) were also examined. For the demonstration of HNK-1+ cells, the peroxidase-antiperoxidase (PAP) bridge method was applied to cryostat sections of these specimens. It was found that in normal lymphoid organs most HNK-1+ cells were located in lymph follicles, especially in germinal centers, and some were found in 'mixed' regions which indicate outsides of both the follicles and T-zones. Amongst the ML, large clusters of HNK-1+ cells were observed only in two cases of follicular lymphoma, although a few scattered HNK-1+ cells were noted in other ML, including five diffuse B-cell lymphomas, six T-cell lymphomas and one null cell lymphoma. The possible significance of these findings is discussed.     

CD1a and CD1c activate intrathyroidal T cells during Graves' disease and Hashimoto's thyroiditis

Molecular studies have shown that CD1 proteins present self and foreign lipid Ags to T cells, but the possible roles of CD1 in human autoimmune diseases in vivo are not known, especially for the group 1 CD1 isoforms (CD1a, CD1b, and CD1c). To investigate the hypothesis that CD1-restricted T cells might be activated and home to target tissues involved in Hashimoto's thyroiditis and Graves' disease, we performed ex vivo analysis of lymphocytes from peripheral blood and autoinflammatory lesions of thyroid tissue. Immunofluorescence analysis identified two types of CD1-expressing APCs in inflamed thyroid tissues. CD1a, CD1b, and CD1c were expressed on CD83+ dendritic cells, and CD1c was expressed on an abundant population of CD20+ IgD+ CD23- CD38- B cells that selectively localized to the mantle zone of lymphoid follicles within the thyroid gland. CD1c-restricted, glycolipid-specific T cells could not be detected in the peripheral blood, but were present in polyclonal lymphocyte populations isolated from affected thyroid glands. In addition, polyclonal thyroid-derived lymphocytes and short-term T cell lines were found to recognize and lyse targets in a CD1a- or CD1c-dependent manner. The targeting of CD1-restricted T cells and large numbers of CD1-expressing APCs to the thyroid gland during the early stages of autoimmune thyroiditis suggests a possible effector function of CD1-restricted T cells in tissue destruction and point to a new model of organ-specific autoimmune disease involving lipid Ag presentation.     

Expression of human B-Cell specific receptor FCRL1 in healthy individuals and in patients with autoimmune diseases

The expression levels of the FCRL1 gene, which encodes a human B-cell surface receptor, were compared in healthy individuals and patients with autoimmune diseases. The expression levels were evaluated using DNA dot hybridization on membranes with spotted cDNA samples derived from blood-cell sub-populations of patients with autoimmune diseases. Quantification of the hybridization signals showed that FCRL1 expression in peripheral blood B-lymphocytes of patients with multiple sclerosis, lupus anticoagulants, Takayasu’s arteritis, and von Willebrand disease was significantly higher than in healthy individuals. Monoclonal and polyclonal FCRL1-specific antibodies that enable FCRL1 detection in Western blotting, immunohistochemistry, and flow-cytometry assays were generated. It was found that FCRL1 is expressed on the surfaces of mature CD19+ B-cells. In the tonsils, FCRL1-positive cells were located in the crypt area, i.e., in the mantle zone of secondary lymphoid follicles and among the cells of lymphoid epithelium. FCRL1-positive cells were also found in B-cell follicles of the spleen.     

Tolerance mechanism in experimental ovarian and gastric autoimmune diseases.

Neonatal splenocytes, neonatal thymocytes, or phenotypically mature adult thymocytes, transferred from normal BALB/c mice to syngeneic athymic nu/nu (or SCID) mice, led to autoimmune oophoritis and autoimmune gastritis, with corresponding serum autoantibodies, in the recipients. The overall disease incidence was 73%; the pathology ranged from mild to severe, with complete loss of ovarian follicles and gastric parietal cells. CD4+ neonatal spleen cells and CD4+ CD8- adult thymocytes were required for autoimmune disease induction. Adult spleen cells did not elicit disease, but they prevented disease when co-transferred with neonatal spleen cells. However, in confirmation of an earlier report by Sakaguchi et al., (J. Exp. Med. 161:72, 1985), a subset of adult splenic T cells expressing a low level of CD5 molecules elicited similar autoimmune diseases. Thus, self-reactive T cells responsible for autoimmune disease of the stomach and ovary are not effectively deleted in the thymus, and they exist in the peripheral lymphoid organs of normal mice. We conclude that the functional expression of the self-reactive T cells is ontogenetically regulated; whereas T cells in the neonatal mice readily elicited autoimmune diseases in nu/nu recipients, regulatory cells may render self-reactive T cells in the normal adults unresponsive.     

Molecular interactions mediating T-B lymphocyte collaboration in human lymphoid follicles. Roles of T cell-B-cell-activating molecule (5c8 antigen) and CD40 in contact-dependent help.

In lymphoid follicles, CD4+ T lymphocytes provide contact-dependent stimuli to B cells that are critical for the generation of specific antibody responses in a process termed Th function. The CD4+ T cell-restricted surface activation protein, 5c8 Ag (T-BAM), has recently been shown to be a component of the contact-dependent helper signal to B cells. To further dissect this process, we utilized a Jurkat T cell lymphoma clone, termed D1.1, that constitutively expresses T-BAM and activates peripheral B cells to express surface CD23 in a contact-dependent mechanism that is inhibited by mAb anti-T-BAM (5c8). Similar to its effect on peripheral B cells, Jurkat D1.1 activates B cells from lymphoid organs, as well as a B cell lymphoma clone, RAMOS 266,4CN 3F10 (RAMOS 266), to up-regulate surface CD23. Interestingly, mAb to the B cell surface molecule, CD40 (mAb G28-5 and B-B20), inhibit D1.1 induced activation of RAMOS 266 and peripheral and lymphoid B cells. In contrast, mAb to CR2 or the adhesion molecules, LFA1, LFA3, or ICAM-1, have little effect. The inhibitory effect of anti-CD40 mAb on B cell activation induced by D1.1 is specific because anti-CD40 potentiates, rather than inhibits, the up-regulation of CD23 on B cells induced by rIL-4. Moreover, cross-linking CD40 molecules by anti-CD40 mAb bound to Fc gamma RII+ (CD32) L cells induces B cell CD23 expression. In vivo, T-BAM-expressing cells are CD4+ T cells that are restricted to lymphoid organs and are localized in the mantle and centrocytic zones of lymphoid follicles and the spleen periarteriolar lymphoid sheath in association with CD40+ B cells. Taken together, these data demonstrate that T-BAM on T cells and CD40 on B cells are involved in contact-dependent T-B help interactions that occur in lymphoid follicles.     

DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells

Dendritic cells (DC) capture microorganisms that enter peripheral mucosal tissues and then migrate to secondary lymphoid organs, where they present these in antigenic form to resting T cells and thus initiate adaptive immune responses. Here, we describe the properties of a DC-specific C-type lectin, DC-SIGN, that is highly expressed on DC present in mucosal tissues and binds to the HIV-1 envelope glycoprotein gp120. DC-SIGN does not function as a receptor for viral entry into DC but instead promotes efficient infection in trans of cells that express CD4 and chemokine receptors. We propose that DC-SIGN efficiently captures HIV-1 in the periphery and facilitates its transport to secondary lymphoid organs rich in T cells, to enhance infection in trans of these target cells.     

Role of inducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity

Bronchus-associated lymphoid tissue (BALT) is occasionally found in the lungs of mice and humans; however, its role in respiratory immunity is unknown. Here we show that mice lacking spleen, lymph nodes and Peyer's patches generate unexpectedly robust primary B- and T-cell responses to influenza, which seem to be initiated at sites of induced BALT (iBALT). Areas of iBALT have distinct B-cell follicles and T-cell areas, and support T and B-cell proliferation. The homeostatic chemokines CXCL13 and CCL21 are expressed independently of TNFalpha and lymphotoxin at sites of iBALT formation. In addition, mice with iBALT, but lacking peripheral lymphoid organs, clear influenza infection and survive higher doses of virus than do normal mice, indicating that immune responses generated in iBALT are not only protective, but potentially less pathologic, than systemic immune responses. Thus, iBALT functions as an inducible secondary lymphoid tissue for respiratory immune responses.     

CD2 is expressed by a subpopulation of normal B cells and is frequently present in mature B-cell neoplasms

BACKGROUND: CD2 is expressed by T and natural killer (NK) cells and has been reported in T/NK cell lineage neoplasms as well as in immature B-lymphoblastic and myeloid leukemias. Although CD2+ B-cells have been identified in normal fetal and postnatal thymus, they have not been reported in adults. METHODS: We retrospectively reviewed flow cytometric immunophenotypic data on consecutive low-grade B-cell leukemias and lymphomas to investigate the frequency of CD2 expression. We also reviewed samples from normal healthy donors to determine whether there is a normal CD2+ B-cell population. RESULTS: CD2 expression (partial or complete) was observed in 13 of 83 (16%) chronic lymphocytic leukemias (CLL), 16 of 29 (55%) follicle center lymphomas (FCL), 3 of 12 (25%) hairy cell leukemias (HCL), 0 of 6 mantle cell lymphomas (MCL), 8 of 28 (29%) large cell lymphomas (LCL), and in 0 of 5 marginal zone/mucosa-associated lymphoid tissue lymphomas (MZL/MALT). We determined that 5.74 +/- 2.46% (mean +/- SD) of normal peripheral blood B cells and 6.48 +/- 1.62 % (mean +/- SD) of normal bone marrow B cells coexpress CD2. CONCLUSIONS: CD2 expression in B-cell neoplasia is a more prevalent phenomenon than previously appreciated. Normal CD2+ B-cell populations are observed in adults and may represent the nonmalignant counterpart of CD2+ B-cell neoplasms.     

Activation-induced cytidine deaminase deficiency causes organ-specific autoimmune disease

Activation-induced cytidine deaminase (AID) expressed by germinal center B cells is a central regulator of somatic hypermutation (SHM) and class switch recombination (CSR). Humans with AID mutations develop not only the autosomal recessive form of hyper-IgM syndrome (HIGM2) associated with B cell hyperplasia, but also autoimmune disorders by unknown mechanisms. We report here that AID-/- mice spontaneously develop tertiary lymphoid organs (TLOs) in non-lymphoid tissues including the stomach at around 6 months of age. At a later stage, AID-/- mice develop a severe gastritis characterized by loss of gastric glands and epithelial hyperplasia. The disease development was not attenuated even under germ-free (GF) conditions. Gastric autoantigen -specific serum IgM was elevated in AID-/- mice, and the serum levels correlated with the gastritis pathological score. Adoptive transfer experiments suggest that autoimmune CD4+ T cells mediate gastritis development as terminal effector cells. These results suggest that abnormal B-cell expansion due to AID deficiency can drive B-cell autoimmunity, and in turn promote TLO formation, which ultimately leads to the propagation of organ-specific autoimmune effector CD4+ T cells. Thus, AID plays an important role in the containment of autoimmune diseases by negative regulation of autoreactive B cells.     

Detailed analysis of intrahepatic CD8 T cells in the normal and hepatitis C-infected liver reveals differences in specific populations of memory cells with distinct homing phenotypes

In hepatitis C virus (HCV) infection the immune response is ineffective, leading to chronic hepatitis and liver damage. Primed CD8 T cells are critical for antiviral immunity and subsets of circulating CD8 T cells have been defined in blood but these do not necessarily reflect the clonality or differentiation of cells within tissue. Current models divide primed CD8 T cells into effector and memory cells, further subdivided into central memory (CCR7+, L-selectin+), recirculating through lymphoid tissues and effector memory (CCR7-, L-selectin-) mediating immune response in peripheral organs. We characterized CD8 T cells derived from organ donors and patients with end-stage HCV infection to show that: 1) all liver-infiltrating CD8 T cells express high levels of CD11a, indicating the effective absence of naive CD8 T cells in the liver. 2) The liver contains distinct subsets of primed CD8+ T cells including a population of CCR7+ L-selectin- cells, which does not reflect current paradigms. The expression of CCR7 by these cells may be induced by the hepatic microenvironment to facilitate recirculation. 3) The CCR7 ligands CCL19 and CCL21 are present on lymphatic, vascular, and sinusoidal endothelium in normal liver and in patients with HCV infection. We suggest that the recirculation of CCR7+/L-selectin- intrahepatic CD8 T cells to regional lymphoid tissue will be facilitated by CCL19 and CCL21 on hepatic sinusoids and lymphatics. This centripetal pathway of migration would allow restimulation in lymph nodes, thereby promoting immune surveillance in normal liver and renewal of effector responses in chronic viral infection.     

Germinal centers and the B-cell system

Summary The migration pattern of germinal center cells of the rabbit appendix was studied and compared with that of appendix dome cells, spleen cells, thymus cells and thoracic duct lymphocytes. To discriminate T-and B-cell migration pathways, normal or T-cell-depleted rabbits were used as donors. Cell suspensions were labeled in vitro with ³H-leucine followed by intravenous transfer. The migration of labeled cells in lymphoid organs was studied using autoradiography, particular attention being paid to the spleen of the recipient. B-cells from the appendix dome, spleen and thoracic-duct lymph migrate to primary follicles or the corona of secondary follicles via thymus-dependent areas of peripheral lymphoid organs. In contrast, a B-cell subpopulation from the germinal centers of the appendix migrates to the center of splenic primary follicles and into germinal centers. The migration of germinal center cells to splenic follicle centers is not enhanced by specific antigens. The migration properties of B-cells, possibly changing during differentiation, may be instrumental in the two types of immune reactions, i.e., plasma-cell reaction and germinal-center reaction.     

Cutting edge: nonproliferating mature immune cells form a novel type of organized lymphoid structure in idiopathic pulmonary fibrosis

Ectopic formation of secondary lymphoid tissue is initiated by the local attraction of naive T and B cells. In this study, we describe a novel type of organized lymphoid structure in the lung of human idiopathic pulmonary fibrosis, with key features of lymphoid neogenesis, including: 1) recently activated CD40 ligand (CD40L)+ T cells; 2) variable numbers of activated CD40+/CD40L+ B cells, sometimes organized in follicles; 3) fully mature dendritic cells (DC) expressing CD40, CD83, CD86, and DC-lysosome-associated membrane protein; 4) the expression of the chemokine CCL21; 5) the presence of vessels with characteristics of high endothelial venules; and 6) a dense network of follicular DC. Surprisingly, these structures are devoid of CCR7+ naive T cells, proliferating lymphocytes, and germinal centers, suggesting that newly recruited activated DC and Ag-experienced lymphocytes can drive lymphoid neogenesis and that factors present within the lymphoid aggregates, such as CD40L, are essential to induce DC maturation.     

CD57, a marker for B-cell activation and splenic ellipsoid-associated reticular cells of the chicken

We have demonstrated that the ellipsoid-associated reticular cells of chicken spleen express CD57, a marker for B-cell activation. These cells are characterised by their spindle-shaped morphology, tissue distribution and the absence of certain leucocyte-specific markers. They are phagocytotic and possess high endogenous non-specific esterase activity. Previous reports failed to detect CD57 expression on ellipsoid-associated reticular cells, probably because the tissue sections were differently treated before immunohistochemistry. CD57 is also expressed by a small number of T-cells in the spleen and the caecal tonsils. This number is highly variable between individual chickens depending on the activation state of the immune system. Moreover, CD57 is expressed by bursal lymphocytes (90% or more) but not by B-cells of the peripheral blood. More interestingly, we have been able to discriminate and quantify three B-cell populations of the secondary lymphoid organs, i.e. resting B-cells, germinal centre B-cells and plasma cells, based on their expression levels of CD57 and Bu-1 (a pan B-cell marker). Thus, CD57 should be considered as a B-cell activation marker, rather than as a marker for bursal B-cells; it is also a valuable marker for the immunohistochemical study of ellipsoid-associated reticular cells of chicken spleen.     

The lymphoid past of mouse plasmacytoid cells and thymic dendritic cells

There has been controversy over the possible lymphoid origin of certain dendritic cell (DC) subtypes. To resolve this issue, DC and plasmacytoid pre-DC isolated from normal mouse tissues were analyzed for transient (mRNA) and permanent (DNA rearrangement) markers of early stages of lymphoid development. About 27% of the DNA of CD8(+) DC from thymus, and 22-35% of the DNA of plasmacytoid pre-DC from spleen and thymus, was found to contain IgH gene D-J rearrangements, compared with 40% for T cells. However, the DC DNA did not contain IgH gene V-D-J rearrangements nor T cell Ag receptor beta gene D-J rearrangements. The same DC lineage populations containing IgH D-J rearrangements expressed mRNA for CD3 chains, and for pre-T alpha. In contrast, little of the DNA of the conventional DC derived from spleen, lymph nodes, or skin, whether CD8(+) or CD8(-), contained IgH D-J rearrangements and splenic conventional DC expressed very little CD3 epsilon or pre-T alpha mRNA. Therefore, many plasmacytoid pre-DC and thymic CD8(+) DC have shared early steps of development with the lymphoid lineages, and differ in origin from conventional peripheral DC.     

Anti-TCR antibody treatment activates a novel population of nonintestinal CD8 alpha alpha+ TCR alpha beta+ regulatory T cells and prevents experimental autoimmune encephalomyelitis

CD8alphaalpha+CD4-TCRalphabeta+ T cells are a special lineage of T cells found predominantly within the intestine as intraepithelial lymphocytes and have been shown to be involved in the maintenance of immune homeostasis. Although these cells are independent of classical MHC class I (class Ia) molecules, their origin and function in peripheral lymphoid tissues are unknown. We have recently identified a novel subset of nonintestinal CD8alphaalpha+CD4-TCRalphabeta+ regulatory T cells (CD8alphaalpha Tregs) that recognize a TCR peptide from the conserved CDR2 region of the TCR Vbeta8.2-chain in the context of a class Ib molecule, Qa-1a, and control- activated Vbeta8.2+ T cells mediating experimental autoimmune encephalomyelitis. Using flow cytometry, spectratyping, and real-time PCR analysis of T cell clones and short-term lines, we have determined the TCR repertoire of the CD8alphaalpha regulatory T cells (Tregs) and found that they predominantly use the TCR Vbeta6 gene segment. In vivo injection of anti-TCR Vbeta6 mAb results in activation of the CD8alphaalpha Tregs, inhibition of the Th1-like pathogenic response to the immunizing Ag, and protection from experimental autoimmune encephalomyelitis. These data suggest that activation of the CD8alphaalpha Tregs present in peripheral lymphoid organs other than the gut can be exploited for the control of T cell-mediated autoimmune diseases.     

CCR7 expression and memory T cell diversity in humans

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag alpha(4)beta(7) integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.     

Altered expression of self-reactive T cell receptor V beta regions in autoimmune mice.

Intrathymic tolerance results in elimination of T cells bearing self-reactive TCR V beta regions in mice expressing certain combinations of I-E and minor lymphocyte stimulatory (Mls) phenotypes. To determine if autoimmune strains of mice have a defect in intrathymic deletion of self-reactive TCR V beta regions, expression of V beta 3, V beta 6, V beta 8.1, and V beta 11 were examined in lpr/lpr and +/+ strains of mice; MRL/MpJ(H-2K, I-E+, Mlsb,), C57BL/6J(H-2b, I-E-, Mlsb,), C3H/HeJ(H-2k, I-E+, Mlsc), AKR/J(H-2k, I-E+, Mlsa); and in autoimmune NZB/N(H-2d, I-E+, Mlsa) and BXSB(H-2b, I-E-, Mlsb) mice. The results suggest that, during intrathymic development, self-reactive T cells are deleted in autoimmune strains of mice as found in normal control strains of mice. However, the TCR V beta repertoire is skewed in autoimmune strains compared to normal strains of mice. For example, MRL-lpr/lpr mice, but not other lpr/lpr strains, had increased expression of V beta 6 relative to expression in control MRL(-)+/+ mice, which is associated with collagen-induced arthritis. These data are consistent with a model of normal affinity for negative selection of self-reactive T cells in the thymus of autoimmune strains of mice followed by expansion of autoreactive T cell clones in the peripheral lymphoid organs. The peripheral lymphoid organs of lpr/lpr mice contain an expanded population of abnormal CD4-, CD8-, 6B2+ T cells. Elimination of self-reactive peripheral T cells suggests that these abnormal cells are derived from a CD4+ subpopulation in the thymus. Flow cytometry analysis of peripheral lymph node T cells from MRL-lpr/lpr mice reveal three populations of CD4+ T cells expressing low, intermediate and high intensity of B220 (6B2). This supports the hypothesis that in lpr/lpr mice, self-reactive CD4+ T cells are eliminated in the thymus, and that these cells lose expression of CD4 and acquire expression of 6B2 in the periphery.