Simultaneous determination of T,B, and mixed rosette-forming lymphocytes in human peripheral blood

Preliminary treatment of erythrocytes in suspension with trypan blue did not affect their ability to take part in the spontaneous rosette formation test, but enabled them to be distinguished when mixed with other erythrocytes after staining with methyl green-pryronine in fixed preparations. The presence of a small subpopulation of lymphocytes binding both types of indicator particles was demonstrated by the use of a mixture of these, erythrocytes with erythrocytes sensitized by antibodies and complement.Department of Immunology and Laboratory of Immunomorphology, Research Center, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. M. Lopukhin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 6, pp. 714–716, June, 1977.     

Immunological competence of T and B lymphocytes in clophosphamide-induced tolerance

Immunological competence of T and B lymphocytes of mice was studied 7 days after induction of tolerance by injection of a massive dose of sheep's red cells and cyclophosphamide. The ability of lymphocytes of the tolerant mice to influence cooperation between normal T and B lymphocytes also was investigated. This particular form of tolerance was shown to be due not to suppressor T cells, but to a true deficiency of helper T cells (in both the thymus and spleen) and, to some extent also, of B cells (in the spleen). The very small deficiency of B cells in the bone marrow was connected with the nonspecific action of cyclophosphamide. It is postulated that cyclophosphamide selectively eliminates cells proliferating under the influence of the antigen.Laboratory of Immunological Tolerance, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. V. Baroyan.) Translated from Byullenten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 4, pp. 445–447, April, 1976.     

The peripheral blood compartment in patients with Graves' disease: activated T lymphocytes and increased transitional and pre‐naive mature B lymphocytes

Graves'' disease (GD) is an autoimmune disease that involves aberrant B and T lymphocyte responses. Detailed knowledge about lymphocyte subpopulation composition will therefore enhance our understanding of the pathogenesis of GD and might support the development of new immunomodulatory treatment approaches. The aim of this study was to gain detailed insight into the composition of the peripheral blood lymphocyte compartment in GD before and during anti-thyroid drug therapy. Major B and T lymphocyte subpopulations were investigated by flow cytometry in peripheral blood from newly diagnosed GD patients (n = 5), GD patients treated with anti-thyroid drugs (n = 4), patients with recurrent GD (n = 7) and healthy controls (HC; n = 10). In GD patients, numbers of activated T lymphocytes [human leucocyte antigen D-related (HLA-DR)⁺ and CD25⁺] were increased. The B lymphocyte compartment in GD was characterized by significantly higher numbers of transitional (CD38^highCD27⁻, P < 0·03) and pre-naive mature (CD38^lowCD27⁻IgD⁺CD5⁺, P < 0·04) B lymphocytes, while memory populations were slightly decreased. The increased numbers of CD5⁺, transitional and pre-naive mature B lymphocytes correlated positively with fT4 plasma levels. GD is associated with increased numbers of activated T lymphocytes and transitional and pre-naive mature CD5⁺ B lymphocytes within the peripheral blood. The increase in CD5⁺ B lymphocytes was due mainly to an increase in transitional and pre-naive mature B lymphocytes. Increased fT4 plasma levels might be associated with this increase in transitional and pre-naive mature CD5⁺ B lymphocytes.     

Activated T and B lymphocytes in peripheral blood of patients with Chagas' disease

Whole blood preparations from patients with either the indeterminate(asymptomatic) or cardiac clinical forms of chronic Trypanosomacruzl infection were analyzed by flow cytometry using double-labelingto identify subsets of circulating lymphocytes. Several significantdifferences were demonstrated between the blood lymphocyte profilesof chagaslc patients and non-chagaslc controls. Clear increasein the percentages and actual numbers of double-positive cellsof the phenotype CD3⁺/HLA-DR⁺, as well as decrease in the percentageof CD45RA⁺/CD4⁺ and CD45RA⁺/CD8⁺ T cells, Indicate greater numbersof activated T cells circulating in the blood of infected patients.Consistent parallel increases were seen also in the B lymphocytesubset which stained double-positive for CD19/CD5. There wereno significant differences in the circulation of these chronicchagaslc patients in the CD4:CD8 ratios. Also, no substantivephenotyplc differences were observed in the lymphocyte populationsbetween the two ends of the clinical spectrum (Indeterminateversus cardiac) in chronic human Chagas' disease. These observationsdemonstrate that increased levels of activated T cells and CD5⁺B cells are present in the circulation of people with chronicChagas' disease. These are cell phenotypes that have been associatedin other conditions with autoimmune, polyclonal, and hyperlmmuneresponses. The specificities of these activated cells and theroles they may play in resistance or pathogenesis during chronicChagas' disease need now to be determined.     

A fluorescent probe to indicate differences between blood T and B lymphocytes

A new physicochemical marker for human peripheral blood lymphocytes is suggested. The lyphocytes were stained intravitally with the fluorescent probe 3-methoxybenzathrone and examined microfluorometrically. The blood lymphocyte population was shown to be heterogeneous. Two main groups of cells, differing in their intensity of fluorescence, are distinguished. Immunologic fractionation of the lymphocytes showed that one group of cells consists of T lymphocytes and the other of B lymphocytes.Research Center, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. M. Lopukhin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 1, pp. 51–53, January, 1979.     

Human natural killer (NK) cells present staphylococcal enterotoxin B (SEB) to T lymphocytes

Superantigen-mediated T cell activation requires the participation of antigen-presenting cells (APC). Once superantigen has bound class II MHC molecules on the surface of APC, it then can interact with the T cell receptor to induce T cell activation. Superantigen-mediated T lymphocyte activation, along with its consequent cytokine production is thought to be the basis for the pathophysiology of conditions such as toxic shock syndrome, Kawasaki''s disease and possibly rheumatoid arthritis. We examined the role of CD56⁺ NK lymphocytes in the interaction between superantigens and T lymphocytes. First, we found that a subpopulation of CD56⁺ cells freshly isolated from human peripheral blood expressed class II MHC molecules. The amount of HLA-DR expression varied between individuals, ranging from 9.3% to 37.7%. CD56⁺ (NK) cells were purified from the peripheral blood by cell sorting and were tested for their ability to support SEB-mediated T cell activation as assessed by surface expression of IL-2 receptor α-chain (CD25) on CD3⁺ lymphocytes. We observed that when enriched T cells were incubated with SEB in the presence of NK cells, there was a significant up-regulation of CD25 expression on the T cells. When HLA-DR⁺ cells were removed from sorted CD56⁺ populations, the remaining HLA-DR⁻ NK cells were unable to support SEB-mediated T cell activation. Also, SEB up-regulated the expression of HLA-DR on CD56⁺ cells in peripheral blood mononuclear cell (PBMC) populations after 24 h of incubation, implying that the ability of NK cells to function as superantigen-presenting cells is up-regulated by superantigens themselves. Together, these data demonstrate for the first time that human CD56⁺HLA-DR⁺ NK cells can function as superantigen-presenting cells, and imply that NK cells may be involved in the activation of non-specific T cell reactivity during early host defences against superantigen-elaborating microorganisms in vivo. Furthermore, the physical linkage of NK cells and T cells by the interaction of superantigen with HLA class II molecules and T cell receptors, respectively, may lead to NK cell activation and augmented lytic potential, helping to clear the body of superantigen-elaborating microorganisms.     

Antigen and helper T lymphocytes activate B lymphocytes by distinct signaling pathways

Resting murine B lymphocytes can be induced to proliferate by cross-linking membrane immunoglobulin, the antigen receptor, or by contact with activated helper T lymphocytes in the absence of a signal through membrane immunoglobulin. Little is known about the molecular nature of contact-dependent T cell help. To determine whether helper T cells activate B cells through different signal transduction and second messenger pathways from those used by membrane immunoglobulin, the effects of drugs which block activation of B cells through membrane immunoglobulin were measured on B cell activation by contact with anti-CD3-activated and fixed T helper cells. Cyclosporin A, phorbol esters added at the time of activation, and cAMP agonists all block activation of B cells through membrane immunoglobulin at concentrations at least 100-fold lower than those necessary to block B cell activation by contact with activated T_h1 or T_h2 helper T cells. Depletion of protein kinase C by pretreatment of B cells with phorbol ester inhibits the proliferative response to anti-immunoglobulin but not the response to contact with activated T cells. The B cell response to lipopolysaccharide is intermediate in sensitivity to cyclosporin A and cAMP agonists, and resembles the response to activated T cells in resistance to phorbol esters and protein kinase C depletion. Various protein kinase inhibitors did not distinguish among these B cell activation pathways, except for the tyrosine kinase inhibitor, herbimycin A, which inhibited anti-immunoglobulin responses at 3- to 5-fold lower concentrations.     

Participation of mouse T and B lymphocytes in the graft versus host reaction

Transplantation of spleen or lymph node cells from CBA mice into sublethally irradiated (CBAxC57BL/6)F₁ mice induced the development of a graft versus host reaction (GVHR). The lymphocytes lost their ability to give this reaction after treatmentin vitro with specific sera against both mouse T lymphocytes and B lymphocytes. The development of the GVHR in mice is evidently connected with cooperative interaction between T and B lymphocytes.Department of Immunology, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. M. Lopukhin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 6, pp. 713–715, June, 1976.     

B/T cell ratio of rabbit peripheral blood lymphocytes. Influence of separation technique on results

Spontaneous in vitro T cell rosette formation at room temperature leading to enrichment of B cells has been reported. We tested 10 individual New Zealand White rabbits sequentially, separating the lymphocytes either at room temperature or at 37 degrees C. T cells are lost constantly at room temperature but to a lesser extent at 37 degrees C. The determination of the yield of lymphocytes after Ficoll separation gives the best control for the accuracy of the results. If lymphocytes are separated at 37 degrees C and if the yield of lymphocytes is greater than 45%, the variation in T cells is small and their number is constant between 62 and 74%. These data show that the reported wide range of T cells in rabbit peripheral blood is due to methodological errors and not inherent in the rabbit.     

Increased expression of CTLA-4 (CD152) by T and B lymphocytes in Wegener's granulomatosis.

CTLA-4 (CD152) is a surface molecule of activated T cells with sequence homology to CD28. Both molecules bind to the same ligands, B7.1 (CD80) and B7.2 (CD86) but have antagonistic functions. While CD28 is an important costimulator, CTLA-4 has an essential inhibitory function in maintaining the homeostasis of the immune system. Furthermore, CTLA-4 has a role in inducing a Th1 response and suppressing Th2 cytokines, an effect which is antagonized by CD28. Many autoimmune diseases are characterized by an overwhelming production of Th1 cytokines. Recently, the predominance of the Th1 cytokine pattern has been directly observed in the granulomatous inflammation of patients with Wegener's granulomatosis. The balance between CD28 and CTLA-4 expression by T lymphocytes could be a factor in the pathogenesis of autoimmune diseases. Down regulation of CD28 predominantly on CD8+ T cells has been described in Wegner's granulomatosis; however, analysis of CTLA-4 is complicated by its low expression levels. Here we have used potent signal enhancement to study CTLA-4 on PBMC in patients with Wegener's granulomatosis (n = 25) in comparison with healthy controls (n = 19). Expression levels of CTLA-4 were significantly increased selectively on CD4+ and possibly also on CD4-/CD8- T cells in Wegener's granulomatosis. High CTLA-4 expression by T lymphocytes was associated with more severe disease. In contrast, after stimulation with the mitogen PHA, CTLA-4 levels were strongly increased on T cells from controls but in T cells from Wegener's granulomatosis patients this response was severely impaired. Interestingly, while CTLA-4 was seen exclusively on T cells in control individuals, about half of the Wegener's patients showed CTLA-4 expression by a fraction of peripheral B lymphocytes. CTLA-4 positive B cells in the periphery were associated with less acute disease.     

Content of T and B lymphocytes in healthy human peripheral blood

It was shown by the rosette-formation method that the number of T and B lymphocytes in human blood varies with the time of day and season of the year. The number of T cells reaches a maximum in the morning and the number of B cells in the evening. The relative percentage of B cells is higher in the fall than in winter.Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 7, pp. 872–874, July, 1976.     

Decreased perforin and granzyme B expression in senescent HIV-1-specific cytotoxic T lymphocytes

Cytotoxic T lymphocyte (CTL) senescence may be an important mechanism of immune failure in HIV-1 infection. We find that senescence of HIV-1-specific CTL clones causes loss of killing activity, preventable by transduction with telomerase. Furthermore, senescence is associated with reduced expression of the effector molecules granzyme and perforin, suggesting CTL "exhaustion" can result in hypofunction. These results agree with other studies showing that HIV-1-specific CTL exhibit abnormal phenotypes in vivo, and suggest the possibility that chronic turnover is an important mechanism of antiviral failure in HIV-1 infection.     

A B220(-), CD19(-) population of B cells in the peripheral blood of quasimonoclonal mice

We describe a new population of non-naive B cells in the peripheral blood of quasimonoclonal (QM) mice. Surface Ig of switched isotypes is expressed, but not B220 nor CD19. These cells are larger and denser than naive B cells but smaller than blasts or plasma cells; they do not stain with syndecan, a marker for plasma cells. Telomerase, which is usually expressed in B cell blasts, was not present in this population. We sorted the switched, idiotype-positive, B220(-) B cells from the peripheral blood of QM mice and sequenced Ig H chain and lambda L chain cDNA. There were many point mutations but no V gene replacements, gene conversions or other type of diversifications. As they express switched isotypes and have mutated their Ig genes, cells in the B220(-), CD19(-) population must have been in an immune response and we suggest that it includes the memory B cell subset.     

In vitro production of B cell growth factor and B cell differentiation factor by peripheral blood mononuclear cells and bronchoalveolar lavage T lymphocytes from patients with idiopathic pulmonary fibrosis.

The activation of B lymphocytes and formation of immune complexes have been suggested to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). To investigate the mechanisms of activation of B lymphocytes, we studied the production of B cell growth factor (BCGF) and B cell differentiation factor (BCDF) in patients with IPF and those with interstitial pneumonia associated with collagen vascular diseases (IP-CVD), in comparison with healthy controls. Culture supernatants of peripheral blood mononuclear cells from patients with IPF induced more IgM and IgA production by B lymphocytes than those from healthy controls, indicating a higher production of BCDF in the patients. Culture supernatants of T lymphocytes obtained from bronchoalveolar lavage fluids (BALF) of patients with IPF induced higher proliferation of B lymphocytes than those from healthy controls, indicating a higher production of BCGF. An increase in production of BCGF and BCDF was not observed in patients with IP-CVD. In the light of these results, it was suggested that there may be an imbalance in T lymphocyte subsets that release lymphokines like BCGF and BCDF in patients with IPF, and that the subsets may differ between blood and BALF. It remains to be elucidated whether the activation of B lymphocytes depending on T lymphocytes determines the development of disease in IPF.     

Plaque-forming cell response of frozen human lymphocytes stimulated with pokeweed mitogen. II. Results obtained in cultures of irradiated T and untreated B lymphocytes

The PFC capability of cryopreserved lymphocytes was tested after T/B cell separation and irradiation of the T cells. It is concluded that there is no selective loss of function of the involved subpopulations. Thus nitrogen-stored lymphocytes can safely be used in PFC assays after conventional separation and irradiation procedures.