Hereford cattle protected against Boophilus microplus with antigens purified by immunoaffinity chromatography from larval and adult ticks.

The subpopulations of lymphocytes in the pregnant and non-pregnant mammary glands of the sheep were delineated by a panel of monoclonal antibodies. The most striking feature observed was that in the mammary gland of both pregnant and non-pregnant sheep the great majority of the lymphocytes in the ductal and alveolar epithelium were agranulated CD8+ CD5- cells. A small subpopulation of granulated lymphocytes in the epithelium expressed the CD45R antigen but not the major histocompatibility complex (MHC) class II molecules. Other subpopulations, especially B lymphocytes, were present in much lower concentrations and were located mainly in the periductal and intralobular connective tissues. Patches of lymphocytes clustering around venules were observed and the majority of them were shown to be CD5+ CD4+, while some were CD5+ CD8+ but none were CD45R+ (B cell). It is suggested that selective traffic of T cells occurs at these sites.     

Morphological and immunocytochemical examinations on development of B-cells in human embryonic and fetal liver

Morphological characteristics of B cell in human embryonic and fetal livers during the first trimester of gestation were examined. Light microscopically, CD9+, CD10+, CD19+, and CD20+ cells of B cell lineage became detectable as small lymphoid cells from 8 weeks gestation. Tdt+ cells first appeared also as small lymphoid cells on the 43th day of gestation. Ia+ or CD34+ cells in embryonic livers between the 33th and 43th day of gestation were large blastic cells resembling myeloblasts while some of Ia+ or CD34+ cells after the 43th day of gestation as well as Tdt+ cells were similar to lymphocytes. Electron-microscopically, all Ia+, CD10+, and CD19+ cells existed solitarily in intercellular spaces of hepatocytes, but not in intravascular spaces. Ultrastructural aspects of these cells were distinguishable each other. These findings indicate that 1) B cells developed and differentiated in the fetal liver, but the fetal liver during the first trimester was not a lymphoid organ, 2) lymphohemopoietic progenitor cells were derived from Tdt+ cells in the livers between 43th and 56th day of gestation. Ia+ cells detected as a small lymphoid cell in the liver at the 50th day were considered to be progenitor cells of lymphocytic lineage.     

Rasmussen encephalitis: a clinicopathologic and immunohistochemical study of seven patients

We retrospectively reviewed the clinicopathologic features and immunohistochemical profiles of 7 patients with Rasmussen encephalitis (age range, 3.5-15 years at surgery). All had medically intractable seizures (6 months' to 7 years' duration); all but 1 developed unilateral hemiparesis. Histologically, all cases were characterized by leptomeningeal and parenchymal perivascular chronic inflammation consisting primarily of T lymphocytes (CD3+, CD5+, CD7+). In all but 1 case, a predominance of CD8+ T-cytotoxic/suppressor lymphoid cells over CD4+ cells was observed. All cases had rare B lymphocytes (CD79a+, CD20+). Rare CD10+ and no CD56+ cells were noted. All cases were marked by diffuse proliferation of microglial cells, highlighted on CD68 immunostaining. Focal microglial nodule formations were observed in 4 cases and focal cortical atrophy in 5 cases. Viral inclusions were not noted. There was no evidence of Epstein-Barr virus by LMP-1 antibody immunostaining. The histologic findings of Rasmussen encephalitis resemble those of viral meningoencephalitis. The pathologicfindings may be only focally present, and missed, if diagnosis is made or confirmed with biopsy alone. Most lymphoid cells have a T-cell immunophenotype, with a predominance of CD8+ cells in most cases.     

Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositol-anchored lymphocyte differentiation antigen ecto-5''-nucleotidase (CD73).

A panel of monoclonal antibodies to the 69 kDa glycosyl phosphatidylinositol anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (ecto-5'-NT, CD73) was produced using highly purified human placental 5'-NT as immunogen. Antibodies 1E9.28.1 and 7G2.2.11 inhibit soluble placental 5'-NT activity and recognize lymphocyte CD73 in indirect immunofluorescence and immunoprecipitation assays. In addition, 1E9.28.1 induces vigorous T cell proliferation in the presence of submitogenic doses of phorbol myristate and F(ab')2 goat anti-mouse Ig. Both antibodies can be used to purify the three major forms of placental 5'-NT by affinity chromatography. By two-color immunofluorescence, CD73 was found to be expressed on 19 +/- 5% of CD3+, 11 +/- 4% of CD4+, 51 +/- 14% of CD8+, 25 +/- 8% of CD28+, 15 +/- 5% of CD29+, 27 +/- 7% of CD45RA+, and 70 +/- 6% of CD19+ lymphocytes. Within T cells, CD73 expression is restricted to the CD28+ subset. Thus, CD73 is found on subsets of both T and B lymphocytes, with the highest expression on B cells and CD8+ T cells. In sections of hyperplastic tonsil, CD73 expression is restricted to the small lymphocytes of the follicular mantle zone, a small subset of extrafollicular lymphocytes situated within the epithelium of the tonsillar crypt, and to follicular dendritic cells within the lower part of the "light-zone." CD73 is also detected on subsets of endothelial cells of capillaries and venules and the basal layer of non-keratinizing squamous epithelium and transitional cell type mucosa of many tissues. Given the tissue distribution of CD73, along with its glycosyl phosphatidylinositol membrane anchoring and the observation that some CD73 antibodies are mitogenic, we propose that this interesting antigen may play a role in cell activation, lymphocyte homing, and/or cell adhesion.     

Detection of CD1 positive cells in the peripheral blood of patients suffering from cancer-associated malnutrition

The immunological phenotyping of peripheral blood mononuclear cells (PBMC) was assayed in a series of 22 patients suffering from severe cancer-associated malnutrition. A marked decrease of the T-lymphocyte subsets (CD3+, CD4+, CD8+) and of the CD20+ B lymphocytes occurred; there was however an increased percentage of monocytes but their absolute number was normal. Interestingly, 5% of the PBMC expressed "activated T-cell antigens". The specificity of two different monoclonal antibodies (MoAbs) towards CD1 epitopes (OKT6 and D47) was assessed by indirect immunofluorescence (IIF): about 5% of CD1+ thymocytes were detected with no antigen (Ag) cross reactivity with the small subset of activated T cells. It is hypothesized that some relationship may exist between such cells and malnutrition and/or cancer.     

Limit dilution analysis of peripheral blood T lymphocytes specific to periodontopathic bacteria.

A characteristic of active cytomegalovirus (CMV) infection is its suppressive effect on in vitro assays of immune function. The expression of CD11b by the Cd4+ and Cd8+ lymphocytes allows the identification of subsets with distinct regulatory functions of pokeweed mitogen (PWM) induced B cell differentiation. In order to relate that result with our previous observation that CMV carriers have significantly increased numbers of CD4+, HNK1+ and CD8+, HNK1+ lymphocytes in their peripheral blood compared with non-carriers, we performed a three-colour flow cytometric analysis of the co-expression of Cd11b and HNK1 by CD4+ and CD8+ lymphocytes obtained from 27 CMV carriers and 42 non-carriers. The differences between CMV carriers and non-carriers were significant for the CD4+, HNK1+ lymphocytes (median [5th and 95th percentiles], 59 [18 and 123 versus 24/7 and 73 per mm3, respectively; P less than 0.001) and CD8+, HNK1+ lymphocytes (59 [18 259] versus 52 [23 and 139] per mm3; P less than 0.001), but not for the CD4+, CD11b+ lymphocytes (59 [18 and 135] versus 52 [17 and 104] per mm3) and the CD8+, CD11b+ lymphocytes (85 [34 and 293] versus 82 [21 and 248] per mm3). The CD4+, HNK1+ and CD8+, HNK1+ lymphocytes that were increased in CMV carriers compared with non-carriers included mostly CD11b-, but also CD11b+ lymphocytes. After sorting CD4+ and CD8+ lymphocytes for four CMV carriers into HNK1+ and HNK1- fractions, we analyzed their regulatory functions on PWM-driven B cell Helper function to PWM-driven B cell differentiation was exclusively associated with the CD4+, HNK1- lymphocytes; the CD4+, HNK1+ generally did not show helper or suppressor activity in this assay. Both CD8+, HNK1+ and CD8+, HNK1- lymphocytes showed suppressor activity. Thus, the NHK1 marker does not constitute a phenotypical correlate for suppressor cells of PWM-driven B-cell differentiation.     

Follicular lymphoma of compartmentalized small cleaved center cells and mantle zone lymphocytes. Evidence for a common derivation.

The authors have observed a unique case of follicular lymphoma in which the central zones of neoplastic nodules were composed predominantly of small cleaved cells (SCC) that were surrounded by small lymphoid cells proliferating in wide mantles as in mantle zone (MZ) lymphoma. The central SCC component displayed a follicular SCC lymphoma-like phenotype (IgD-, CD10+, CD5-, CD68-), whereas the neoplastic cells of the peripheral zones had an MZ lymphoma-like phenotypic profile (IgD+, CD10-, CD5+, CD68+). In extranodal involved tissues, either follicular or diffuse (leukemic-like) patterns of lymphoma infiltration were noted. Flow cytometric analyses showed in the bone marrow or the peripheral blood two leukemic B cell populations, one mimicking the phenotypic profile (IgM+, IgD+, CD5+, CD10-, Leu8-) of small lymphoid cells with MZ-like features, and the other with phenotypic features (IgM+, IgD-, CD5+, CD10+, Leu8+) intermediate between those of MZ-like cells and those of the SCC component (follicular center-like) detected in the lymph node. Immunomagnetic sorting and gene rearrangement studies indicated that both CD10+ and CD10- B lymphocytes and lymph node neoplastic B cells shared the same clonal origin. This unusual follicular lymphoma can be viewed as the result of the proliferation of a single follicular progenitor capable of differentiating toward both a germinal center and an MZ phenotype. The simultaneous presence in the same patient of at least three neoplastic B-cell populations at different maturation stages, encompassing follicular center and MZ phenotypes, and showing the same clonal derivation, indicates a close lineage relationship between follicular SCC and MZ lymphomas.     

Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue.

Two alternative integrins involved in mucosal homing (alpha 4 beta 7) or epithelial retention (alpha E beta 7) of lymphocytes were examined in the human gut. The distribution of the beta 7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by alpha E beta 7 + cells but weakly by alpha 4 beta 7 + cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T-cell receptor (TCR) gamma/delta +, and CD3- cells, expressed alpha E beta 7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B-lineage cells (CD19+) and macrophages (CD68+) were negative. In gut-associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (< 5%) cells were positive for alpha E beta 7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30-50%) expressed alpha 4 beta 7 (mAb Act-1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of alpha 4 beta 7 as in the lamina propria whereas relatively few B lymphocytes (< 50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for alpha 4 and the former three subsets as well as appendix CD8+ cells also for beta 7 while only lamina propria CD8+ cells had mRNA for alpha E. Together, the results suggested that alpha E beta 7 and alpha 4 beta 7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly alpha 4 beta 7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because alpha E beta 7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of beta 7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle-associated epithelium of GALT expressed alpha E beta 7, perhaps reflecting epithelial interaction during primary immune responses.     

Extrathymic derivation of gut lymphocytes in parabiotic mice

In adult mice, c-kit+ stem cells have recently been found in their liver, intestine and appendix, where extrathymic T cells are generated. A major population of such thymus-independent subsets among intraepithelial lymphocytes is T-cell receptor (TCR)gamma delta+ CD4- CD8alpha alpha+(beta-) cells, but the origins of other lymphocyte subsets are still controversial. In this study, we examined what type of lymphocyte subsets were produced in situ by such stem cells in the small intestine, large intestine and appendix. To investigate this subject, we used parabiotic B6.Ly5.1 and B5.Ly5. 2 mice which shared the same circulation by day 3. The origin of lymphocytes was identified by anti-Ly5.1 and anti-Ly5.2 monoclonal antibodies in conjunction with immunofluorescence tests. Lymphocytes in Peyer's patches and lamina propria lymphocytes (especially B cells and CD4+ T cells) in the small intestine became a half-and-half mixture of Ly5.1+ and Ly5.2+ cells in each individual of parabiotic pairs of mice by day 14. However, the mixture was low in CD8alpha alpha+, CD8alpha beta+ and gamma delta T cells in the small and large intestines and in CD3+ CD8+ B220+ cells in the appendix. These cells might be of the in situ origin. When one individual of a pair was irradiated before parabiosis, the mixture of partner cells was accelerated. However, a low-mixture group always continued to show a lower mixture pattern than did a high-mixture group. The present results suggest that extrathymic T cells in the digestive tract may arise from their own pre-existing precursor cells and remain longer at the corresponding sites.     

Phenotyping of peripheral blood lymphocytes in adult coeliac disease.

In coeliac disease immunological abnormalities are not confined to the small bowel and it has been suggested that changes in peripheral blood lymphocytes may predispose to autoimmune or malignant complications. Using dual-colour immunofluorescence with labelled monoclonal antibodies, multiparameter flow cytometry was used to analyse peripheral blood lymphocytes in 32 untreated coeliacs, 29 treated coeliacs and 20 healthy volunteers. When the absolute numbers were considered, a decrease of CD3+, CD4+, CD8+ and CD19+ lymphocytes was found in untreated coeliacs compared with treated coeliacs and healthy volunteers. The proportion of CD3+ was significantly higher in untreated coeliacs (P<0.05) than in healthy volunteers. No differences were observed in CD4+, CD8+ and CD19+ subsets between the three groups studied. The proportion of CD3+ CD25+ and CD3+ HLA-DR+ cells were higher in untreated coeliacs (P<0. 001 and P>0.005) and in treated coeliacs (P<0.005 and P<0.05) than in healthy volunteers. On the contrary, natural killer cells and cytotoxic cells were lower in untreated and treated coeliacs than in healthy volunteers. As regards B-cell subsets, the only difference was the increase in FcepsilonR+ B cells in untreated coeliacs. The absolute reduction of peripheral lymphocytes in coeliac disease probably reflects their compartimentalization in intestinal mucosa. The decrease of natural killer cells and cytotoxic cells may be in keeping with the increased prevalence of malignancy in this condition. Finally, the phenotypic changes found in untreated coeliacs indicate T-cell activation.     

In rat B lymphocyte genesis sixty percent is lost from the bone marrow at the transition of nondividing pre-B cell to sIgM+ B lymphocyte, the stage of Ig light chain gene expression

The cycling B precursor cells in rat bone marrow (BM) that carry the B220 antigen and no surface Ig daily produce 780 million new cells. The pool of recirculating B lymphocytes in the rat, however, renew at a rate of only about 40 million cells/day. To analyze at which stages in B lymphocyte genesis the cell loss occurs, we identified post-mitotic cells in the rat BM B lineage, and determined their renewal rates. We used 5-bromo-deoxyuridine (BrdUrd) to label DNA-synthesizing cells, identifying incorporated BrdUrd with the mouse monoclonal antibody BU-1. B lineage cell subsets were identified by the markers HIS24 antigen (rat B220), terminal deoxynucleotidyl transferase (TdT), Ig mu heavy chain, and complete Ig. By use of double and triple immunocytology, we determined the extent of BrdUrd incorporation in the various B lineage compartments [HIS24+TdT-Ig-, TdT+, cytoplasmic mu chain (c mu)+ surface (s) IgM- pre-B, sIgM+ B]. Both sIgM+ B lymphocytes and all B precursors with cell diameters less than 11-12 microns were virtually devoid of DNA synthesis, as indicated by S-phase indices below 2%. In contrast, S-phase indices of large B precursors ranged between 43%-66%. We established the renewal rates of nondividing BM B lineage cells by placing osmotic minipumps containing BrdUrd subcutaneously in the flank of rats. The nondividing BM B lineage cells all renewed rapidly at rates between 2.4% and 5.6%/h, representing average half-lives of 29 to 12 h. In absolute numbers, the renewal/day/whole body BM was 165 X 10(6) for sIgM+ B lymphocytes, 422 X 10(6) for small c mu+ sIgM- pre-B cells, 89 X 10(6) for small TdT+ cells and 35 X 10(6) for small HIS24+TdT-Ig- cells. Assuming that recirculating B lymphocytes in the periphery are the descendants of BM sIgM+ B lymphocytes, which in their turn are the progeny of small pre-B cells, the renewal data indicate the following. Of the 165 million potentially available BM B lymphocytes, only 40 million cells become incorporated in the pool of recirculating B lymphocytes, representing a loss of 75%. BM B lymphocytes, in turn, use only (165/422 X 100% = ) 40% of the potential output from their immediate precursors. The 60% loss that occurs here may reflect the extent of aberrant Ig light chain gene rearrangement in normal B lymphocyte genesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical characterization of lymphocyte development in human embryonic and fetal livers

Lymphohemopoietic progenitor cells and the development of lymphocytes in human embryonic and fetal livers during the 4 to 11 weeks of gestation were examined immunocytochemically by using a panel of monoclonal antibodies. CD9+, CD10+, CD19+, and CD20+ cells of B cell lineage became detectable from the 8th gestational week. CD2+ and CD3+ cells of T cell lineage were observed from the 10th gestational week. Tdt+ cells first appeared on the 43rd day of gestation. Both CD34+ and Ia+ cells were observed in all examined livers, and these cells appeared morphologically as small lymphoid cells from the 43rd day of gestation. These seemed to suggest that B lymphocytes developed in fetal liver from 8 weeks of gestation and lymphohemopoietic progenitor cells were comprised in Tdt+ cells in liver during the 43rd to 56th day of gestation.     

Marked increase in L-selectin-negative T cells in neonatal pertussis. The lymphocytosis explained?

The detailed immunophenotype of peripheral blood lymphocytes from a neonate with pertussis was determined by flow cytometry and compared with results from cord blood from healthy newborns. Most (72%) of the lymphocytes were CD3+ T cells with a normal CD4/CD8 ratio (2.5). The T cells were largely HLA-DR negative and CD45RA+, consistent with unstimulated na?ve T cells. Almost all of the CD4+ T cells were Leu8 (L-selectin, CD62L) negative, while almost all of the CD8+ T cells were CD28+. There was no increase in CD7- CD4+ T cells (Th2-like). No relative increase in CD16/56+ NK cells (5%) or CD19/20+ B cells was seen. The most dramatic finding in this case was the remarkable lack of expression of L-selectin by the T cells. L-selectin expression is associated with homing of peripheral blood lymphocytes to lymph nodes. The dramatic reduction in L-selectin expression of the T lymphocytes in pertussis, perhaps induced by pertussis toxin, likely prevents homing of the T cells to peripheral lymphoid tissues and provides a likely explanation for the marked lymphocytosis noted in this disease.     

Non-random migration of CD4+, CD8+, gamma delta + T19+, and B cells between blood and lymph draining ileal and prescapular lymph nodes in the sheep fetus

We have examined the circulation of CD5+, CD4+, CD8+, gamma delta + T19+, and B cells through ileal and prescapular lymph nodes in the sheep fetus in an environment uninfluenced by foreign antigen and ongoing immune responses or circulating immunoglobulins, and have contrasted this circulation with that occurring through the same tissue in 1-year-old sheep. The vast majority of lymphocytes circulating through fetal prescapular lymph nodes and fetal ileal lymph were T cells; however, there was a significantly higher concentration of B cells in ileal lymph compared to prescapular lymph. Furthermore, in contrast to 1-year-old sheep, there was an imbalance in the distribution of CD4+ cells and CD8+ cells in fetal prescapular and ileal lymph, with CD4+ cells enriched in prescapular lymph relative to other T cell subsets and CD8+ cells enriched in ileal lymph. Our results suggest that in the fetus either there is preferential migration of CD4+ cells through peripheral lymph nodes and/or CD8+ lymphocytes through the ileal gut, or newly formed CD8+ lymphocytes are being released from the ileum or ileal lymph node directly into ileal lymph.     

小气道病变患者小气道黏膜层免疫病理的变化

探讨小气道病变患者小气道黏膜层的免疫病理学特点。86例因肺局限性病变需行肺叶切除患者,分为小气道病变组34例、COPD组22例及肺功能正常组30例。收集术后肺标本,应用免疫组化法检测小气道黏膜层内CD3^ 、CD8^ 、CD68^ 、CD45RA^ 、CD45RO^ 、CD20^ 细胞,并与FEV1．0、V50、V25做相关性分析。小气道病变组、COPD组CD3^ 、CD8^ 、CD68^ 细胞数明显高于正常组;而小气道病变组与COPD组相比无差异;小气道病变组CD8^ 细胞数与V50呈显著负相关;正常组内吸烟者CD45R0^ 细胞数明显高于非吸烟者;在小气道病变组或COPD组内,吸烟与非吸烟者之间CD3^ 、CD8^ 、CD45RO^ 细胞数均无差异;小气道病变组内非吸烟者CD68^ 细胞数高于吸烟者及正常非吸烟者。T淋巴细胞尤其CD8^ 细胞及CD68^ 细胞在小气道病变及COPD炎性过程中有重要意义,吸烟可导致CD45RO^ 细胞在小气道黏膜集聚,CD68^ 在非吸烟因素致小气道病变中意义更大。     

Cytotoxic T cells in AIDS colonic cryptosporidiosis

BACKGROUND/AIMS: It is not known how enteric cryptosporidiosis induces severe intestinal impairment despite minimal invasion by the parasite. The aim of this study was to analyse the histological features and locally implicated immune cells in colonic biopsies of AIDS related cryptosporidiosis. PATIENTS/METHODS: Colonic biopsies from patients with AIDS related cryptosporidiosis (n = 10, group I), patients with AIDS but without intestinal infection (n = 9, group II), and human seronegative controls (n = 9, group III) were studied. Using immunohistochemistry the infiltrating mononuclear cells were analysed in both the epithelium and lamina propria for the expression of CD3, CD8, TiA1, granzyme B, and CD68 and for glandular expression of human major histocompatibility complex DR antigen (HLA-DR). RESULTS: Severe histological changes, resulting in abundant crypt epithelial apoptosis and inflammatory infiltrate in the lamina propria, were seen in all biopsies from group I. A significant increase of CD8+, TiA1+, and granzyme B+ T cells in the lamina propria and HLA-DR glandular expression was noted in group I compared with groups II and III. However, the number of intraepithelial lymphocytes, lamina propria CD3+ T cells, and macrophages was not significantly increased in cryptosporidiosis specimens compared with controls. CONCLUSION: Epithelial apoptosis mediated by granzyme B+ cytotoxic host T cells might play a major role in the development of colonic lesions in AIDS related cryptosporidiosis.     

Characterization of early lymphoid precursor cells in the human fetus using monoclonal antibodies and anti-terminal deoxynucleotidyl transferase.

Monoclonal antibodies (MoAbs) directed primarily against immature lymphoid cells (VIL-A1, BA-2, OKT10) or recognizing antigens associated with the B cell lineage (VIB-C5, OKI1) were used for the identification of lymphoid cells in liver, bone marrow, spleen and thymus of human fetuses between 8 and 20 weeks of gestational age. Many lymphocytes in liver, bone marrow and spleen reacted with the MoAbs used. In the fetal thymus, however, cells did not bind to the VIL-A1 and VIB-C5 MoAbs and only a few cells were BA-2+ or OKI1+. In the liver and bone marrow the VIL-A1, VIB-C5 and BA-2 MoAbs reacted almost exclusively with terminal deoxynucleotidyl transferase (TdT) containing cells, pre-B and B cells. TdT+ cells were present in liver, bone marrow and thymus, but not in the spleen. In liver and bone marrow the relative numbers of TdT+ cells decreased during gestation, in the thymus they increased. The antigenic make-up of the TdT+ cells in liver and bone marrow was comparable to that of pre-B and B cells in these organs: most of them reacted with VIL-A1, VIB-C5 and OKT10 MoAbs and many were BA-2+ and OKI1+. TdT+ cells in liver and bone marrow did not bind to T-cell-markers, i.e. OKT6 and WT-1. A few lymphoid cells in these organs contained TdT and mu heavy chains. TdT+ cells in the thymus had a completely different phenotype: most of them were OKT6+ and they did not react with the VIL-A1 and VIB-C5 MoAbs. These findings suggest that TdT+ cells in fetal liver and bone marrow are precursors of the B cell lineage, whereas those in the thymus probably belong to the T cell lineage. In the fetal spleen almost all B cells displayed the VIB-C5 and OKI1 antigens. At 12 weeks of gestation greater than 80% of splenic B cells were also VIL-A1+ and BA-2+; with ongoing gestation far less B cells in spleen expressed these antigens, however, indicating that these B cells are more mature than those in fetal liver and bone marrow, but still less mature than the B cells in postnatal blood and bone marrow, which do not display the VIL-A1 and BA-2 markers. These findings suggest that some further maturation of B cell stages takes place in the spleen during human fetal life.     

A large quantity of CD3-/CD19-/CD16- lymphocytes in malignant pleural effusion from a patient with recurrent cholangio cell carcinoma

Tumor infiltrating lymphocytes (TILs) are candidates for adoptive cellular immunotherapy. Here we report on a patient whose TILs presented unusual lymphocyte antigens. Pleural effusions were collected from a 47-year-old man with recurrent cholangio cell carcinoma and malignant effusion. Effusion-associated lymphocytes (EALs) were separated by Ficoll-Hypaque gradient, and the EAL phenotype was determined by flow cytometry. The percentage of positive cells was determined for each lymphocyte-related differentiation antigen. The percentages of CD3+, CD19+, and CD16+ lymphocyte subpopulations among EALs were 20%, 7%, and 3%, respectively. Nearly 70% of EALs were CD3-/CD19-/CD56-/CD16- cells. The phenotypes of peripheral blood lymphocytes (PBLs) collected simultaneously from the patient's peripheral blood were CD3+ (52%), CD19+ (20%), and CD16+ (20%). When EALs were cultured in medium without pleural effusion, T cell-related antigens, but not B cell- or natural killer (NK) cell-related antigens, were newly expressed on EALs, and this expression reached a plateau after 48 h in culture. The proportions of CD3+, CD19+, and CD16+ cells were 69%, 7%, and 3%, respectively. However, when EALs were cultured in medium with pleural effusion, increased expression of T cell-related antigens was not observed; the proportions of CD3+, CD19+, and CD16+ cells were 16%, 6%, and 1%, respectively. Neither total cell numbers nor cellular viability of EALs changed significantly after in-vitro culture, suggesting that significant proliferation or death of EALs did not occur during the culture period. Co-culture of the patient's PBLs with autologous pleural effusion for 96 h did not alter the expression of lymphocyte-related antigens on the PBLs. These results indicate that expression of T cell-related antigens, but not B cell- or NK cell-related antigens, on EALs was blocked temporarily by the malignant pleural effusion. This is the first report concerning the existence of a large quantity of unclassified lymphocytes in which the T cell-related antigens were reversibly masked in the malignant pleural effusion.     

B cells expressing CD5 are increased in cerebrospinal fluid of patients with multiple sclerosis.

By two-colour flow cytometric analysis, we found increased numbers of B cells co-expressing the pan-T cell marker CD5 and the B cell marker CD19 in cerebrospinal fluid (CSF) of 21 patients with multiple sclerosis (MS), compared with 17 control subjects with muscular tension headache. Only one patient with MS, but nine controls lacked CD5+ B cells in CSF. This difference was not observed in peripheral blood. Numbers of CD5+19+ B cells were increased in CSF compared with blood in MS, but not in the controls. In both groups, CD5+19+ B cells were not restricted to small resting lymphocytes, but were also found among larger-sized lymphocytes. The relative density of CD5 molecules and of CD19 molecules was lower in CD5+19+ than in CD5-19+ B cells and CD5+19- T cells. CD5+ B cells are assumed to be responsible for autoantibody production, and our results suggest a pathogenetic role of such cells, predominantly within the central nervous system, in MS.     

Immunohistochemical study of necrotizing lymphadenitis: A possible mechanism for apoptosis involving perforin and granzyme-producing cytotoxic T cells

Twelve lymph node specimens with necrotizing lymphadenitis and which had florid necrotic lesions were studied immu-nohistochemically. The majority of viable lymphoid cells in the necrotic foci were CD8+ lymphocytes and KP1+ or PGM1+ phagocytizing macrophages. The CD8+ T cells were Leu1+, Leu2+, Leu3^--, Leu4+, Leu5b+, Leu7^--, Leu11b^- and Leu19^--, indicating a suppressor/cytotoxic T cell phenotype. In addition, the cytoplasm of these cells was immunoreactive for perforin and granzyme B in a granular pattern. With a nick end-labeling technique, fragmented nuclei and some lymphoid cell nuclei were positively stained. These results suggest that the necrosis in necrotizing lymphadenitis is apoptotic necrosis of T cells targeted by CD8+, perform and granzyme-producing, activated cytotoxic T cells, supporting a viral infection etiology.