Chemokine receptor expression and modulation by Mycobacterium tuberculosis antigens on mononuclear cells from human lymphoid tissues

Chemokine receptor switching on lymphoid cells is an important factor regulating migration and homing, but little is known about the expression of such molecules during Mycobacterium tuberculosis infection in humans. We describe CCR2, CCR5 and CCR7 expression on human cells from blood, spleen and pulmonary hilar lymph nodes (PHLN) stimulated by M. tuberculosis antigens. CCR2 was not expressed by CD3+ cells regardless of the presence of antigen, but was highly expressed on CD14+ CD63+ monocytes/macrophages. CCR2 decreased on splenic monocytes/macrophages by nearly 50% in culture, independent of antigen, but remained high in blood and PHLN. CCR5 was low in CD3+ cells and was down-regulated by M. tuberculosis antigens on blood and splenic cells but not in PHLN. CCR5 was highly expressed on monocytes/macrophages and was down-regulated by M. tuberculosis antigens at 48 hr only in blood. Less than 15% of CD3+ cells from spleen and PHLN were CCR7+, whereas nearly 40% from blood expressed this receptor on primary isolation. However, CCR7 in PHLN increased in culture, independent of antigen. Monocytes/macrophages did not express CCR7. Thus, we characterize, for the first time, chemokine receptor expression and differential modulation by M. tuberculosis antigens on human mononuclear cells from spleen, blood and PHLN. Knowledge of chemokine receptor switching in human lymphoid tissue provides novel insight into mechanisms of the immune response to M. tuberculosis with potential effects on directing cell trafficking.     

Electrophoresis of lymphoid cells. Characterization of human B and T cells in peripheral lymphoid tissues

Small lymphocytes from human blood, tonsils, spleen and lymph nodes showed a bimodal distribution on electrophoresis, the electrophoretic mobility (EPM) of one group being slow, the other fast. The difference in mean EPM between the slow and fast groups was about 30%. The percentage of slow cells in each organ was similar to that of cells with surface-bound immunoglobulin as judged by immunofluorescence studies. It was therefore concluded that a major proportion of B cells has a relatively low net surface charge while that of the T cells is higher.     

B and T cells in lymphoid tissues of human appendix.

Human appendix lymphoid cells (HAL) react very strongly to stimulation with concanavalin A, strongly to stimulation with phytohaemagglutinin, and weakly but definitely to stimulation with lipopolysaccharide. From the results of rosette formation assay, cytotoxicity tests with anti-T cell antiserum or anti-B cell antiserum, cell surface or intracellular immunoglobulin staining with fluorescein-conjugated rabbit anti-Fab of human immunoglobulin serum, and plaque-forming cell (PFC) assay, it was concluded that human appendix lymphoid tissue is a B cell pool but includes T cells. However, both direct and indirect PFC could not be significantly demonstrated against sheep red blood cells in a 5-day HAL culture.     

Phenotypes of infiltrating cells in trehalose dimycolate-induced interstitial pneumonitis.

Trehalose dimycolate is a glycolipid component of the cell walls of mycobacteria, nocardia, and corynebacteria. When trehalose dimycolate is injected into certain strains of mice, they develop interstitial pneumonitis that is characterized by mononuclear cell infiltration of the alveolar walls, intra-alveolar hemorrhages, and in some animals, granuloma formation. The disorder is seldom fatal, and in approximately 4 weeks, the lungs are normal. There is strong evidence that T lymphocytes are essential for production of interstitial pneumonitis by trehalose dimycolate, but little is known about the mechanisms of lung injury in this model. The experiments described in this report were conducted to identify the roles of the various cells that accumulate in the lungs of mice with this form of interstitial pneumonitis. We found that Mac3+ macrophages were the first cells to appear in the alveolar walls. Increases in the number of L3T4+ T lymphocytes, Lyt2+ T lymphocytes, and surface-immunoglobulin-positive lymphocytes followed, but significant increases in the number of lymphoid cells were not observed until day 7, when the pulmonary lesions were well developed. Treatment of the mice with cyclophosphamide or anti-T-cell sera significantly reduced the number of lymphoid cells in the alveolar walls but did not affect the number of Mac3+ cells and did not affect development of intra-alveolar hemorrhages. Treatment with poly(I.C) significantly decreased the number of Mac3+ cells in the lungs, and these mice did not develop pulmonary hemorrhages. We conclude that although development of pulmonary lesions in trehalose dimycolate-treated mice is a T-cell-dependent process, macrophages are also essential and are more directly involved in production of the lung injury. We postulate that the lung lesions are the direct effect of macrophage-produced cytokines, such as tumor necrosis factor.     

Biased immunoglobulin variable region gene expression by Ly-1 B cells due to clonal selection

Most, if not all, autoantibodies specific for bromelain-treated mouse erythrocytes recognize the common membrane phospholipid, phosphatidyl choline (PtC). Anti-PtC antibodies are produced by 5%-15% of CD5+ Ly-1 B cells of normal unimmunized mice, but not by detectable numbers of conventional CD5- B cells. At 1 week of age PtC-specific B cells are undetectable but then increase dramatically over the next 3 to 4 weeks to reach adult numbers. We report here that PtC-specific Ly-1 B cells in B10.H-2aH-4bp/Wts mice predominantly express either of two heavy and kappa chain variable (V) region gene combinations. In addition, the sequence and length of DH genes are conserved among cells expressing the same V gene combination, and the V kappa-J kappa junctions of one group involve unusual splice sites. Preferential V gene rearrangement models are insufficient to explain the DH and V kappa-J kappa junctional sequences or the delayed appearance of this specificity, and so they cannot solely account for the high frequency of PtC-specific cells. These characteristics are more consistent with antigen selection. We therefore attribute the frequent use of the two V region gene combinations to selection for cells that express them and conclude that the expressed V gene repertoire of Ly-1 B cells in adult mice is influenced by antigen selection. Apparently, there is no selection for mutant anti-PtC antibodies of higher affinity during the formation of the Ly-1 B repertoire because the V region genes expressed by PtC-specific cells are unmutated. Our findings are consistent with an important, germ line-encoded function for the immunoglobulin products of these gene combinations.     

Progressive decrease in VH3 gene family expression in plasma cells of HIV-infected patients

We have analysed the expression of V_H gene families In totalperipheral plasma cells of µ and isotypes from a groupof HIV-infected patients. CD19^– and CD20^– cellswere separated from B lymphocytes, and anchored RT-PCR productswere hybridized with specific V_H gene family probes and witha consensus J_H region probe. The V_H/J_H hybridization Intensityratios were taken as a parameter to measure V_H expression. Invivo V_H3 gene family expression is reduced In plasma cells ofall HIV-infected patients compared with adult healthy donors.This decrease is maximal in patients with AIDS: >90%. Thisis true for both studied Isotypes. Conversely, the two othermain V_H gene families, V_H1 and V_H4, show no significant variationIn expression. We and others have previously shown that V_H3gene family expression is first expanded and then decreasedIn peripheral B lymphocytes of HIV-infected patients. The presentresults extend these observations and show that V_H3 gene familyexpression is also affected In plasma cells. The existence ofa B cell superantlgen In HIV Infection may explain these data.This pronounced reduction In Vh3 family expression may participateIn the Impaired humoral responses against bacterial agents foundIn HIV-infected patients.     

Differential expression of HLA class-I antigens on B and T lymphocytes obtained from human lymphoid tissues

The amount of HLA class-I antigens was determined on the surface of enriched populations of B and T lymphocytes obtained from human peripheral blood, lymph nodes and spleens. The assays were performed using monoclonal antibodies that recognize different determinants on HLA class-I antigens and utilizing a simple and sensitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that B lymphocytes obtained from human peripheral blood, lymph nodes and spleens express at least twice as many HLA class-I antigens as T lymphocytes obtained from the same organs.     

Comparative analysis of light chain expression in germinal center cells and mantle cells of reactive lymphoid tissues. A four-color flow cytometric study

We studied skewing of light chain ratios (LCRs) in germinal center cells (GCCs) relative to mantle cells (MCs) by flow cytometry (FC) in 98 reactive lymphoid tissues. LCRs were assessed using a 4-color lambda/kappa/CD20/CD38 tube. GCCs and MCs were discriminated by CD20 and CD38 density. Of 98 cases, 65 contained distinct GCCs and MCs. Light chain expression usually was dimmer on GCCs than on MCs; in 22 cases, the kappa and lambda clusters converged and accurate LCRs could not be determined. Of the remaining 43 cases, the mean GCC LCR was 1.78 (range, 1.10-3.07) vs 1.56 (range, 1.00-2.24) in the MCs (P = .001). The overall kappa/lambda ratio in cases containing GCCs and MCs was 1.65 (range, 1.18-2.69) compared with 1.46 (range, 1.00-1.98) in cases containing MCs only. Of 43 cases, 19 (44%) showed differences of 20% or more between the LCRs of GCCs and MCs. LCRs of GCCs and MCs may differ substantially in reactive lymphoid tissues. These subsets may form distinct clusters and skews in their LCRs and should not be misinterpreted as evidence of occult lymphoma.     

Detection of clonal B cell populations in paraffin-embedded tissues by polymerase chain reaction.

A method was established to detect clonal B cell populations in frozen and paraffin-embedded tissues. The method is based on the polymerase chain reaction amplification of rearranged VH and V kappa genes using V gene family-specific primers. Monoclonal B cell populations could be detected in peripheral blood lymphocyte DNA in all 16 cases of B-CLL and immunocytoma investigated and in 8 of 10 cases of B cell non-Hodgkin's lymphoma using frozen and paraffin sections. The amplification of V kappa rearrangements in addition to the VH amplification is a useful tool to verify the results of the heavy chain rearrangement and to detect proliferation of a B cell clone in cases in which no VH product was obtained. In spite of the degradation of DNA in paraffin-embedded tissues, we were able to find amplified polymerase chain reaction products of about 350 bp length in 8 of 10 cases analyzed. The method presented here may be helpful in routine diagnosis of B cell non-Hodgkin's lymphoma using frozen or paraffin-embedded specimens.     

T cells in the lung of patients with hypersensitivity pneumonitis accumulate in a clonal manner

Hypersensitivity pneumonitis (HP) is characterized by an alveolitis sustained by CD8(+) T lymphocytes showing a limited expression of the T cell receptor (TCR). We previously demonstrated that a bias in T cell selection occurs in the lower respiratory tract of patients with HP, with a compartmentalization in the lung of CD8(+) T cells bearing (TCR)-beta variable (TCRBV) #2, 3, 5, 6, 8, and 13 gene segments. We herein characterized the clonal T cell populations present in the lung and in the blood of patients with HP. Heteroduplex analyses, cloning, and sequencing T cells bearing TCR indicate oligoclonal expansions of T cells expressing homologous or identical complementary-determining region 3. Furthermore, T cell clones isolated from the two compartments expressed similar, sometimes identical, junctional regions. Removal from antigenic exposure led to the disappearance of T cell clones. Our findings indicate that expansions of T lymphocytes bearing clonal TCRBV region gene segments take place in the lung of patients with HP during exposure. The evidence that identical T cell clones are present in the lung and the blood of the same patient suggests that the immune reaction occurring at lung level gives rise to a systemic reaction.     

Phenotype characteristics of human B cells studied by Epstein-Barr virus infection. I. Immunoglobulin expression on human lymphoblastoid cells established from various lymphoid cells

Forty-seven lymphoblastoid cell lines were established from human fetal lymphoid tissues, cord blood lymphocytes (CBL) and adult peripheral blood lymphocytes (PBL) by Epstein-Barr virus (EBV) infection. Their surface immunoglobulin (sIg), intracytoplasmic immunoglobulin (cIg) expression, and immunoglobulin (Ig) content in the culture supernatant were tested. Expression of sIgM, sIgG and sIgA were predominant on fetus-derived cell lines, while sIgD was the most prominent on CBL-derived cells. Though cIg expression did not vary between cell lines of different origin, Ig content in the culture supernatant differed greatly. Fetus- and CBL-derived cells secreted IgM exclusively, but PBL-derived cells secreted not only IgM, but also IgG and IgA abundantly. These results indicate that the lymphoblastoid cells established by EBV infection reflect the Ig phenotype of the cell from which they originated.     

Distinct role of surface lymphotoxin expressed by B cells in the organization of secondary lymphoid tissues

In order to definitively ascertain the functional contribution of lymphotoxin (LT) expressed by B cells, we produced mice with the LTbeta gene deleted from B cells (B-LTbeta KO mice). In contrast to systemic LTbeta deletion, in B-LTbeta KO mice only splenic microarchitecture was affected, while lymph nodes and Peyer's patches (PP) were normal, except for PP's reduced size. Even though B-LTbeta KO spleens retained a small number of follicular dendritic cells (FDC) which appeared to be dependent on LTbeta produced by T cells, IgG responses to sheep red blood cells were markedly reduced. Thus, the organogenic function of B-LTbeta is almost entirely restricted to spleen, where it supports the correct lymphoid architecture that is critical for an effective humoral immune response.     

Agreement analysis of variables involved in lipodystrophy syndrome definition in HIV-infected patients

BACKGROUND: Lipodystrophy studies in HIV-infected patients have usually defined abnormalities in body fat by clinical evaluation and patient questionnaires. Despite the risk for bias with these subjective approaches, agreement analysis among the large number of variables employed was seldom performed. OBJECTIVE: To analyze consistency between the usual approaches for definition of abnormalities in body fat distribution. DESIGN: We evaluated agreement between the clinical and questionnaire findings for abnormalities in body fat in an HIV patient population under antiretroviral treatment followed in our institution, using different criteria for definitions of body fat abnormalities within the same data set. METHODS: Kappa analysis for consistency and receiver-operator characteristic (ROC) curve analysis were performed. RESULTS: Low levels of agreement between clinical and patient perspectives were observed. Only one combination of criteria showed adequate agreement results. The waist/hip ratio showed low levels of agreement with all other variables, and no clear discriminative point was observed by ROC curve analysis. The ratio between the trunk fat content and the leg fat content assessed by dual energy x-ray absorptiometry (DEXA) scan demonstrated better agreement and more clear discriminative values for both male and female patients. CONCLUSION: Agreement analyses may help in the selection of the subjective variable methodology and in the inclusion of consistent and nonredundant objective measurements for diagnosis of abnormalities in body fat.     

L-selectin expression differentiates T cells isolated from different lymphoid tissues in cattle but does not correlate with memory.

L-selectin (LECAM-1, LAM-1) was expressed by a high proportion of CD4+ and CD8+ T cells, as well as almost all of the gamma delta T-cell receptor (TcR)+ (WC1+) T cells, isolated from blood, lymph nodes or tonsils. CD4+ T cells in the lamina propria of the gut villi and CD8+ T cells in the villous epithelium as well as the majority of WC1+ T cells in the gut mucosa were L-selectin-. The proportion of T cells from Peyer's patches that synthesized the molecule was intermediate between the value for blood and gut mucosa. Expression of L-selectin therefore marks T cells in cattle with a distinct tissue distribution that correlates with its function as the peripheral node homing receptor. The proportion of CD4+ and CD8+ T cells in the circulation that were L-selectin+ decreased with age. Unlike CD45R, expression of L-selectin was not related to CD4 T-cell memory as judged by proliferation in transformation assays to soluble antigen. Three-colour immunofluorescent staining demonstrated four subpopulations of CD4 and CD8 T lymphocytes in peripheral blood mononuclear cells (PBMC) that were CD45R+, L-selectin+; CD45R+, L-selectin-; CD45R-, L-selectin+; CD45R-, L-selectin-. CD4(4) memory cells were CD45R- and L-selectin+ or L-selectin-. Taken with earlier studies the reported observations demonstrated that only one of the four phenotypes of the CD4+ T cells in blood is present in the lamina propria of the gut villi and these are CD45R-, L-selectin-. Two of the four phenotypes of CD8+ T cells were present in the gut epithelium; these were CD45R+, L-selectin- or CD45R-, L-selectin-. Expression of the bovine molecule was not rapidly down-regulated on T cells following activation by exposure to phorbol myristate acetate.     

Phenotypic expression of T lymphocytes in thymus and peripheral lymphoid tissues.

The pattern of expression of cell surface antigens and their relationship to the sequence of T-cell differentiation were delineated by the application of a series of monoclonal antibodies against T cells on tissue sections. The morphologic features and location of T-cell differentiation can be categorized into four stages: 1) subcapsular thymocytes, 2) cortical thymocytes, 3) medullary thymocytes, and 4) peripheral T cells. Phenotypically, on the basis of reactivity with monoclonal antibodies, T cells could be separated into two groups: immature and mature T cells. All stages of T cells expressed T200, Lyt 3, Leu 1, OKT3, Leu 9, and TA1, although staining intensities varied between thymic cortex and medulla. Mature T cells (medullary thymocytes and peripheral T cells) stained more intensely than immature T cells for some antigens, such as Leu 1, OKT3, and Leu 9, and, therefore, probably have a greater antigen density. Immature T cells, exemplified by subcapsular and cortical thymocytes, were characterized by the expression of Tdt, Leu M3, OKT6, OKT9, J5, BA-2, OKT10, and usually coexpressed both helper (T4/Leu 3a) and suppressor (T8/Leu 2a) antigens. With further maturation, medullary thymocytes and peripheral T cells lost reactivity for the above-mentioned markers, acquired A1G3 and Leu 8, and segregated into either T4+/Leu 3a+ or T8+/Leu 2a+ cells. A subpopulation of T cells in germinal centers differs from the majority of peripheral T cells by virtue of an absence of expression of Leu 8/A1G3. This histologically localized subset of T cells may be responsible for the mediation of helper function within the germinal center.