Preserved specific anti-viral T-cell response but associated with decreased lupus activity in SLE patients with cytomegalovirus infection.

OBJECTIVES: SLE is an autoimmune disease characterized by aberrant autoantibody production and immune dysfunctions. Whether the anti-CMV immunity is impaired in SLE patients is poorly understood. We investigated the specific anti-viral T-cell response in SLE patients with CMV infection and its possible impacts on clinical manifestations in lupus. METHODS: CD28 null T-cell percentages were measured by flow cytometry in 89 SLE patients and 58 healthy controls. A specific anti-CMV CD8 T-cell response was assessed ex vivo by the production of intracellular cytokines in response to CMV phosphoprotein 65 (pp65) by flow cytometry. Clinical manifestations and immune parameters were analysed in SLE patients according to their CMV serostatus. RESULTS: CD28 null T cells were significantly expanded in SLE patients. When the anti-CMV pp65 CD8 polyfunctional T cell response was analysed, as defined by production of at least three of four functional cytokines or effectors (intracellular IFN-γ, IL-2, TNF-α and surface CD107a), the results demonstrated that it was not impaired in SLE patients. In contrast, when comparing clinical manifestations, there were lower anti-ds-DNA levels and decreased SLEDAI in SLE patients with CMV infection. Furthermore, the expansion of CD4+CD28 null T cells was negatively associated with anti-ds-DNA levels and SLEDAI in these lupus patients. CONCLUSION: In SLE patients with CMV infection, the specific anti-CMV CD8 T-cell response is preserved but is associated with decreased disease activity and lower anti-DNA levels among these patients, suggesting CMV infection may mitigate lupus activity.

Role of increased CD8/CD28(null) T cells and alternative co-stimulatory molecules in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease; it is a leading cause of death and existing treatments have no proven disease-modifying effect. The mechanisms underlying this resistance are largely unknown, but suggest the presence of some self-maintaining pathogenic process, possibly initiated by cigarette smoking, that prevents the normal resolution of inflammation. We have previously reported increased production of proinflammatory cytokines and granzyme b by CD8(+) T cells in COPD; costimulatory receptor/ligand interactions required include CD80:86/CD28, B7-1/CTLA4, 4-1BB/1BBL and OX40/OX40L. We hypothesized that a dysregulated expression/function of these molecules may play a role in inflammatory/autoimmune components of COPD. We analysed T cell co-stimulatory molecules in blood from 34 controls, 15 smokers and 48 COPD subjects. We assessed the potential functional relevance of CD8/CD28(null) cells in COPD by measuring their production of proinflammatory cytokines, co-stimulatory molecules, granzyme and perforin. A smoke-exposed murine model was applied to investigate the relative expression of CD8/CD28(null) T cells in blood, lung tissue and airway. CD8/CD28(null) cells were increased in both current- and ex-smoker COPD groups; these cells expressed significantly more interferon (IFN)-γ, OX40, 4-1BB, CTLA4, granzyme and perforin when stimulated than CD8/CD28(+) T cells. There were no changes in CD4/CD28(null) T cells. In mice exposed to cigarette smoke for 12 weeks, CD8/CD28(null) T cells were significantly increased in the airway with a trend for an increase in lung tissue and blood. Increased production of proinflammatory cytokines and expression of alternative co-stimulatory molecules by CD8/CD28(null) T cells may play a role in inflammatory or autoimmune responses in COPD and identify therapeutic targets.

VEGF-mediated cross-talk within the neonatal murine thymus.

Although the mechanisms of cross-talk that regulate the hematopoietic and epithelial compartments of the thymus are well established, the interactions of these compartments with the thymic endothelium have been largely ignored. Current understanding of the thymic vasculature is based on studies of adult thymus. We show that the neonatal period represents a unique phase of thymic growth and differentiation, marked by endothelium that is organized as primitive, dense networks of capillaries dependent on vascular endothelial growth factor (VEGF). VEGF dependence in neonates is mediated by significantly higher levels of both VEGF production and endothelial VEGF receptor 2 (VEGF-R2) expression than in the adult thymus. VEGF is expressed locally in the neonatal thymus by immature, CD4(-)CD8(-) "double negative" (DN) thymocytes and thymic epithelium. Relative to adult thymus, the neonatal thymus has greater thymocyte proliferation, and a predominance of immature thymocytes and cortical thymic epithelial cells (cTECs). Inhibition of VEGF signaling during the neonatal period results in rapid loss of the dense capillaries in the thymus and a marked reduction in the number of thymocytes. These data demonstrate that, during the early postnatal period, VEGF mediates cross-talk between the thymocyte and endothelial compartments of the thymus.

Surface CD152 (CTLA-4) expression and signaling dictates longevity of CD28null T cells.

CD28(null) T cells are a highly enriched subset of proinflammatory T cells in patients with autoimmune diseases that are oligoclonal and autoreactive. In this study, we analyzed the role of CD152 signaling on the longevity of human CD28(null) T cells. Using a sensitive staining method for CD152, we show that human CD4(+)CD28(null) and CD8(+)CD28(null) T cells rapidly express surface CD152. Serological inactivation of CD152 using specific Fab or blockade of CD152 ligands using CTLA-4Ig in CD4(+)CD28(null) and CD8(+)CD28(null) T cells enhances apoptosis in a Fas/FasL-dependent manner. CD152 cross-linking on activated CD28(null) cells prevents activation-induced cell death as a result of reduced caspase activity. Apoptosis protection conferred by CD152 is mediated by phosphatidylinositol 3'-kinase-dependent activation of the kinase Akt, resulting in enhanced phosphorylation and thereby inhibition of the proapoptotic molecule Bad. We show that signals triggered by CD152 act directly on activated CD28(null) T lymphocytes and, due to its exclusive expression as a receptor for CD80/CD86 on CD28(null) T cells, prevention of CD152-mediated signaling is likely a target mechanism taking place during therapy with CTLA-4Ig. Our data imply strongly that antagonistic approaches using CD152 signals for chronic immune responses might be beneficial.

Infection with Nippostrongylus brasiliensis or injection of anti-IgD antibodies markedly enhances Fc-receptor-mediated interleukin 4 production by non-B, non-T cells.

Non-B, non-T cells from spleen and bone marrow of naive mice produce IL-4 upon stimulation by plate-bound IgE or IgG2a in the presence of IL-3. Infection of mice with Nippostrongylus brasiliensis (Nb) or injection of anti-IgD antibodies, treatments known to cause striking polyclonal IgE responses, increase the number of splenic non-B, non-T cells and cause 10-30-fold increase in IL-4 production by a standard number of these cells. In Nb-infected mice, IL-4 producing non-B, non-T cells can be found in the lungs, a site through which Nb larvae migrate. Non-B, non-T cells from anti-IgD-injected mice produce IL-4 in response to anti-IgE antibodies, indicating that these cells have been sensitized in vivo with IgE and that crosslinkage of such IgE can lead to stimulation of lymphokine production. Similarly, non-B, non-T cells from Nb-infected mice produce IL-4 upon stimulation with Nb-antigen, indicating that antigen can also crosslink receptors on in vivo sensitized non-B, non-T cells and stimulate lymphokine production. The striking increases in the IL-4-producing capacity of the splenic non-B, non-T cell population in anti-IgD-injected and Nb-infected mice and the in vivo sensitization of these cells strongly suggests that they may have an important role in lymphokine production in helminthic infections and other situations marked by striking elevations of serum IgE levels.

Differentiation antigens identify subpopulations of rabbit T and B lymphocytes. Definition by flow cytometry.

A panel of six monoclonal antibodies produced against cell surface glycoproteins of a rabbit T lymphocyte line was used with flow cytometry to define rabbit lymphocyte subpopulations. Four thymocyte populations were characterized by size and expression of cell surface antigens and appear to represent stages in thymocyte differentiation. Rabbit spleen contained five subpopulations: two of T lineage, two of B, and a null cell subset. Bimodal distribution of staining of thymocytes and peripheral T cells was observed using an antibody (9AE10) directed against a Thy-1 analogue in the rabbit, suggesting two separate T cell lineages. One of the monoclonal reagents, L11/135, reacted strongly with peripheral rabbit T cells as shown by two-color immunofluorescence. In functional studies, only the L11/135-bearing cells responded to the T cell mitogens concanavalin A and phytohemagglutinin and to allogeneic splenocytes. The thymocyte subpopulations and the peripheral T and B cell subsets differ from those described in mouse and man.

Null cell senescence and its potential significance to the immunobiology of aging.

The null cell compartments of human bone marrow and mouse spleen were arbitrarily divided into three subpopulations based upon the ability of cells to acquire T or B cell membrane markers when incubated with poly A:U or ubiquitin. There was an accumulation of T cell precursors with congenital absence of the thymus. In contrast, T cell precursors were reduced and there was an accumulation of uninduced null cells with old age. These observations suggest that there is an intrinsic defect of null cell differentiation with a drift towards more differentiated precursors in T cell differentiation with aging. This could result in a diminution in the range of responses by their progeny, mature T lymphocytes.

Phenotypic expression of non-Hodgkin's lymphomas in China.

Monoclonal antibodies were used to label malignant lymphomas obtained from 57 patients. On the basis of morphologic criteria, 18 lymphomas were the B-cell type, 10 were the T-cell type, and 6 were histiocytic; for 23 the type could not be determined. After monoclonal antibody labeling, 18 lymphomas of B-cell lineage were confirmed, 16 of the T-cell type were demonstrated, 6 were true histiocytic, and 17 were the null cell (non-T, non-B) type. Of the 16 lymphomas of T-cell lineage, 6 were lymphoblastic and 10 were the peripheral type. The percentages of cell types in the non-Hodgkin's lymphomas were as follows: B-cell, 31.5%; T-cell, 28%; null cell, 29%; and histiocytic, 10%. Of the 16 lymphomas of T-cell origin, 15 belonged to helper T-cell subsets (Leu1+, Leu4+, and Leu3a+), and the la marker was positive in all 16. Of the 18 B-cell lymphomas, 14 were kappa-positive and 4 were lambda-positive. Eleven were both B1- and kappa-positive, and 1 was kappa-positive but B1-negative. In the 4 cases that were lambda-positive, 2 were both lambda- and B1-positive. The results indicate that Leu4, Leu2a, Leu3a, and B1 are the most important markers to differentiate T-cell and B-cell lymphomas for pathologic classification. The findings also show a higher percentage of T-cell neoplasm in China as compared to that in Western countries.

Heterogeneity of acute lymphocytic leukemia cell surface markers as detected by monoclonal antibodies.

Monoclonal antibodies 3A1, 4F2, 5E9, OKT3, OKT6, and OKT8 were tested by flow microfluorometry for reactivity with cells from patients with T-cell or null cell acute lymphocytic leukemia (ALL). The 3A1 antibody reacted with a greater percentage of cells from 1 group of patients that could be classified as T-cell leukemia patients as compared to its reaction in the group classifiable as null cell leukemia patients. Reactivity patterns of other monoclonal antibodies with lymphocytes from patients with ALL did not clearly define groups of ALL patients. All cells from individual leukemia patients reacted differently with the seven monoclonal reagents. On the basis of patterns of reactivity, T-cell leukemias could not be classified as malignant clones of cells from the previously described stages of thymus cell maturation. The results demonstrated that the cells undergoing malignant change express a variety of combinations of antigens not found on normal lymphocytes.

Proliferation of infected lymphoid precursors before Moloney murine leukemia virus-induced T-cell lymphoma.

NFS/N mice inoculated with Moloney murine leukemia virus (M-MuLV) developed T-cell lymphoma after a 10-week latent period. Expression of lymphoid differentiation antigens, appearance of M-MuLV-encoded cell surface antigens, and rates of cellular proliferation were measured in splenic and bone marrow subpopulations during this latent period. At 2 weeks of age, Thy-1-and surface immunoglobulin-negative null cells of spleen and bone marrow expressed M-MuLV antigens whereas T- and B-lymphocytes did not. During the 3d and 4th weeks, the number of splenic null cells increased to six times the number found in uninfected controls. These null cells included the precursors of lymphocytes and hematopoietic cells. For the remainder of the latent period, the percentage of null cells undergoing proliferation was three times greater in the infected mice, while the total number of null cells remained constant. This proliferation was not accompanied by terminal differentiation or emigration of mature cell types from the spleen. Proliferation was substantially delayed in CBA mice, which are resistant to lymphoma induction.

Bone marrow differentiation in vitro.

In vitro techniques have proven particularly fruitful for the study of the differentiation of the hemopoietic cells in bone marrow. Progenitor cells can be readily obtained in suspension and in many cases can be induced to grow and differentiate as isolated clones in vitro. At least in vitro, growth and differentiation appear to be regulated by a series of soluble molecules. Recent advances in immunology (the production of inducible T-cell hybridomas and specific T-cell clones) have defined the T-cell as a source of a number of these soluble regulators. Study of the generation of lymphocytes from bone-marrow precursors in vitro, however, remains a challenge.

Immunohistochemical staining of human brain with monoclonal antibodies that identify lymphocytes, monocytes, and the Ia antigen.

Using immunoperoxidase histochemistry, human brain sections obtained at biopsy were labeled with monoclonal antibodies which identify human lymphocyte subsets, monocytes, and the Ia antigen. Staining of a population of cells in white matter was present with the anti-Ia and the anti-M1 (monocyte-associated) antibodies but not with any of the 8 monoclonal antibodies which react with human T-cell subsets (anti-T1, 3, 4, 5, 6, 8, 10 and 12). The Ia antigen was present on 1-2% of cells in white matter, and approximately 5% of cells in white matter were M1-positive. Ia-positive cells demonstrated a pattern of diffuse surface membrane staining, whereas the M1 antigen appeared to cluster at proximal cell processes. Definitive identification of these cells as microglial cells, astrocytes or oligodendrocytes was not possible. These findings demonstrate that: (1) cells which bear the Ia and M1 determinants can be found in histologically normal human white matter, and (2) human oligodendrocytes do not react with monoclonal antibodies (anti-T5 and anti-T8) that identify human suppressor/cytotoxic cells.

Autologous mixed lymphocyte reaction in man: further characterization of responding cells.

Experiments were performed to determine the type of cells undergoing thymidine incorporation in 7-day autologous mixed lymphocyte reactions (AMLR). Peripheral blood mononuclear cells (PBM) were separated into B cells, T cells, B + Null cells and T + Null cell-enriched populations. Cells were cultured in various combinations. Monocytes were removed to determine their influence on AMLR. The main thymidine-incorporating cells in cultures were shown to be Null cells and to a lesser extent T cells. Monocytes were found to have a more pronounced suppressor effect on stimulation of T cells by non-T cell populations in younger individuals than in the elderly. Whether Null cells undergo a spontaneous blastogenesis in culture or are stimulated by T or B cells, could not be answered by the present investigation.

Expression of HLA-DR alloantigens on acute lymphoblastic leukemia lymphoblasts.

We have described the expression of HLA-DR alloantigens on the surface of lymphoblasts from patients with acute lymphoblastic leukemia (ALL). The patient groups included 49 acute lymphoblastic leukemia (ALL) patients (15 common ALL [CALLA +]; 11 "Null" ALL [CALLA-]; 19 T-Cell ALL; 4 Pre-B ALL, and one patient with hairy cell leukemia (HCL). Thirty one of these patients, who exhibited Ia-like antigens demonstrable by monoclonal antibodies, expressed HLA-DR utilizing alloantisera to Class II histocompatibility alloantigens (25/26 non-T-ALL; 2/4 Pre-B ALL; 3/19 T-ALL, and one HCL). The expression of HLA-DR on lymphoblasts was confirmed by family studies of five patients, indicating that ALL lymphoblasts can be used to perform HLA-DR phenotyping or genotyping of such patients. Another important finding was the coexpression on T-cell ALL lymphoblasts of markers for T-helper, T-supressor/cytotoxic, and thymic differentiation marker T6, together with Ia-like and HLA-DR, in one patient, and markers for T-helper, T6, and CALLA in another patient.

Human cell lines express multiple populations of Ia-like molecules.

Three monoclonal antibodies have been used to isolate Ia-like antigens from three human cell lines; two of which are thought to be homozygous at the HLA-D/DR locus. Complete extraction of the Ia antigens identified by one antibody leaves those recognized by the two remaining antibodies in three parallel sets of experiments, indicating that the antigenic determinants recognized by these antibodies are present on three different populations of Ia molecules from cells of single individuals. These three populations of Ia-like molecules may reflect serologic variants of the product of a single genetic locus or may represent the products of as many as three nonallelic genetic regions. Demonstration of the existence of these multiple populations of Ia-like molecules on presumed homozygous typing cells indicates that this antigenic system is much more complex than has been generally realized. Further study may clarify the relationship between HLA-D/DR type and susceptibility to a variety of diseases and ultimately lead to better matches and improved survival for allogenic transplants. Since the HLA-D region is intimately involved in regulation of the immune response and susceptibility to a variety of diseases, use of monoclonal antibodies specific for discrete Ia antigens, the only identified products of the HLA-D region, may facilitate dissection of its many biological functions.

Specificity of monoclonal antibodies directed against human and murine class II histocompatibility antigens as analyzed by binding to HLA-deletion mutant cell lines.

The specificity of 70 monoclonal anti-Ia monoclonal antibodies (MoAbs) (18 mouse allo-induced and 52 rodent anti-human) was studied with a panel of 17 HLA-deletion mutants that were derived from a single parent line and vary in expression of Ia antigens due to deletion of different subregions of HLA. MoAb binding was analyzed both by ELISA and flow microfluorometry. Characterization of the MoAbs with respect to specificity for products of subregions of a DR1-DC1-SB2 haplotype revealed great complexity. Many antibodies were quite specific for DR-linked determinants (26 MoAbs), DC-linked determinants (5 MoAbs), or determinants indistinguishable from SB (4 MoAbs). However, many MoAbs bound to products of more than one subregion: DR + SB (+/- weak DC) (22 MoAbs); DR + DC (3 MoAbs); or DR + DC + SB (1 MoAb). Furthermore, a number of the MoAbs bound unequally to products of the two HLA haplotypes analyzed, particularly among those recognizing DC1-linked determinants and the murine alloinduced MoAbs. Finally, despite strong structural homologies of murine I-A to human DC and murine I-E to human DR, the intraspecies cross-reactions of MoAbs do not closely follow that pattern. These data: (1) illustrate the usefulness of HLA-deletion mutant cell lines for analysis of the specificity of MoAbs and for delineation of HLA subregions; (2) demonstrate the great diversity of MoAbs specific for class II molecules and the high frequency of MoAbs that bind to products of more than one Ia subregion, particularly DR and SB. In view of such complexity, many (perhaps most) MoAbs cannot be relied on to unambiguously identify products of a particular Ia subregion, without extensive characterization.

Heterogeneity of non-T, non-B acute lymphocytic leukemia defined by the quantitative expression of Ia and common all antigens.

The Ia antigens and the common acute lymphoblastic leukemia antigens (CALLA) accessible on the cell surface were quantitated in newly diagnosed patients with non-T, non-B ALL using monoclonal antibodies and a cellular radioimmunoassay. The levels of both antigens expressed in molecules of RAM-Fc bound per cell exhibited a wide range of variation amongst patients. Ia levels, measured with monoclonal antibody 21w4 which recognizes a monomorphic epitope of human Ia molecules, were between 5.0 X 10(4) and 87 X 10(4) molecules per cell in 37 patients. CALLA levels measured with BA-3 or J-5 antibody varied from 3.4 X 10(4) to 22 X 10(4) molecules per cell in 13 patients. In 12/13 cases for which Ia and CALLA were quantitated simultaneously, the amount of Ia was found to be higher than that of CALLA. A positive correlation (p less than 0.02) between the levels of these two antigens was observed suggesting that ALL cells with the highest levels of Ia also had the highest levels of CALLA. In addition, our results suggest a possible correlation (0.05 less than p less than 0.1) between the amount of Ia expressed on the leukemic cells and the white blood cell count of the patient at the time of diagnosis. The data indicate that non-T, non-B ALL are heterogeneous with respect to their expression of Ia and CALLA antigens. A longitudinal study of non-T, non-B ALL patients will allow us to assess if the levels of Ia and CALLA at diagnosis are correlated with prognosis of the disease in these patients.

Immunophenotypic classification of lymphoblastic leukaemia and lymphocytic lymphoma--an experience in the south-western area of the Cape Province of South Africa.

Adequate tumour material was obtained for phenotypic classification using a standard library of monoclonal antibodies from 81 previously untreated patients with acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL), or lymphocytic lymphoma (LL). Sixty-one individuals were adults and 20 were children of 14 yr or younger. Fifty-eight of the patients (72%) had acute lymphoblastic leukaemia and the remaining 23 (28%) had chronic lymphocytic leukaemia or lymphocytic lymphoma. Considering only the patients with acute lymphoblastic leukaemia (n = 58) the median age was 19 yr (range 3-69 yr): 9% were black, 43% were coloured, 48% were white, and the distribution between adult (n = 38) and paediatric patients (n = 20) was comparable. Complete remission rate in the adults was 58% and in the paediatric group 85%. For the total group (n = 58) median duration of survival was 59 weeks for common, 39 weeks for null, 63 weeks for T-ALL, and 13 weeks for B-ALL subtypes. In both the common and the null groups overall and disease-free survival was superior in the children. In contrast, no difference was evident in the T-ALL group, which was also notable for its high incidence in young coloured males. The 15 patients with CLL and eight with LL were adults and all the cells were phenotypically of B lineage: in view of the small numbers no comments are possible about ethnic differences. A multi-centre collaborative study is needed to define the epidemiology of haematologic malignancy in South Africa, with emphasis on differences among ethnically distinct subpopulations.

Concentration-dependent augmentation of anti-K562 activity following short-term culture of null cell-enriched human blood lymphocyte populations.

The increase in natural cell-mediated cytotoxicity against K562 cells following short-term culture (e.g. 12 hours) of null cells was dependent upon the concentration of cells during incubation. The increased lytic activity by cells cultured at higher concentrations was associated with increased numbers of target-binding cells. It occurred with a variety of serum supplements in the medium. The augmentation of natural killer activity required close cellular proximity or contact and was not associated with detectable increased release of interferon or interleukin 2, although the participation of these molecules cannot be excluded. The lower recovery of lytic activity from cells incubated at lower concentrations was not associated with increased PGE synthesis.

Characterization of hematogenous cellular constituents of the murine decidua: a surface marker study.

Decidual tissue, which includes typical (stromal type) decidual cells as well as infiltrating leukocytes, appears to play a local immunoregulatory role in the maintenance of pregnancy in nature. The present study evaluated the contribution of numerous leukocyte subsets characterized on the basis of morphology combined with cell surface markers to the development of murine decidua during syngeneic (CBA female X CBA male) and allogeneic (CBA female X C57BL/6 male) pregnancy. Collagenase dispersed decidua were subjected to total and differential counts and cell surface labeling for a radioautographic identification of various markers: S-IgM on B cells, Thy-1 on T cells, neither marker on null lymphocytes, Lyt- (1 or 2 or 1,2) antigens on T cell subsets, Mac-1 and I-A on macrophages, using 125I-labeled monoclonal antibodies or a sandwich labeling with 125I-protein A. The total cellularity of decidua basalis showed a biphasic rise in both pregnancies, with peaks on day 11 and days 15 and 16, but the allopregnant decidua showed a higher accumulation of all cell types indicating that an allogeneic conceptus causes an augmented deciduogenesis. The number of decidual cells, the most frequent cell class, rose to a peak on day 11 followed by a decline possibly due to cell death. The number of lymphocytes, the next frequent cell class, showed a parallel pattern initially, followed by a sharp secondary rise on day 16. This rise may be due to a withdrawal of progesterone, an antiinflammatory hormone. Null cells predominated amongst decidual lymphocytes (45-80%), as well as in the progestational endometrium (53%), indicating a hormonal control of their accumulation. The frequency of B cells was low (10-13%) and T cells (25-45%) comparable to that in the blood, with Lyt-1 only class being the most common T cell subset. Allopregnant decidua also showed a late rise in the total number of Lyt-2 only cells which may have a suppressor function. Macrophages, the next common leukocyte class, all expressed Mac-1. Their number rose to a plateau by day 12, but at a higher level in allopregnancy. I-A (needed for antigen presentation) was expressed by an increasing proportion (5-60%) of macrophages with advancing gestation. These findings provide a basis for further functional studies.