The Immune Reactivity Role of HCV-Induced Liver Infiltrating Lymphocytes in Hepatocellular Damage

Liver infiltrating lymphocytes (LIL) were isolated from HCV-positive (+) and HCV-negative (–) end-stage livers. Phenotypic analysis and functional studies using proliferative and lymphocytotoxic assays were performed with the isolated LIL. Two CD3+ lymphocyte populations were found in LIL using FITC anti-CD3 monoclonal antibodies (mAb). One was a bright fluorescence intensity population (as in PBL), and the other dim. We calculated the number of FITC-anti-CD3 mAbs bound per lymphocyte on PBL and LIL and found 80,040 ± 4628 and 39,615 ± 3932, respectively. Therefore, HCV+ and HCV– patient PBL contained approximately twice the number of CD3 molecules per cell than patient CD3+ LIL. LIL also contained approximately a threefold higher concentration of TCR+, CD4–CD8–, and CD56,16 (NK) cells than the patient PBL. Thus, a major subset of LIL is phenotypically similar to mouse NK1.1+ intermediate T cells. LIL freshly isolated from HCV+ livers exhibited weak CTL activity against EBV- or Con A-transformed lymphoblast targets infected with vaccinia–HCV recombinant virus (rHCV) or primary hepatocyte cultured cells. However, after in vitro coculture of LIL with rHCV, these cells developed a strong cytotoxicity for the above targets. In contrast, LIL from HCV– livers were not cytotoxic against the same targets. Histochemical studies (in situ) demonstrated that these hepatocytes express CD95, and stains demonstrated apoptosis. The HCV+ hepatocytes also express class I MHC molecules and ICAM-1. The addition of mAb specific for these adhesion molecules inhibited CML activity. Short-term cultured hepatocytes (targets) from HCV+ and HCV– patients produced low levels of cytokines IL-1, IL-2, IL-6, TNF, and IFN- but a high level of IL-8. It is speculated that LIL expressing reduced numbers of CD3 molecules may even function as immune regulators as proposed for intermediate T cells in mice.     

Use of liposomes to studycAMP transport by lymphocyte membranes

Institute of Immunology, Ministry of Health of the USSR. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 3, pp. 246–248, March, 1990.     

Fibronectin on the surface of human lymphocytes

Fibronectin was detected by immunofluorescence technique on the surface of one part of separated normal peripheral blood lymphocytes by using FITC-conjugated anti-human fibronectin antibodies. Approximately one-fifth of isolated B cells and 7% of O cells contained surface-bound fibronectin but T cells failed to stain. There were no detectable free receptors for fibronectin on the surface of lymphocytes of different subsets as it was studied with FITC-labelled purified fibronectin. The percent of B and O cells bearing surface bound fibronectin was markedly decreased in patients with acute and chronic lymphocytic leukemias.     

Age-dependent changes in the susceptibility to apoptosis of peripheral blood CD4+ and CD8+ T lymphocytes with virgin or memory phenotype

Susceptibility to apoptosis changes with age and most of the available data on lymphocytes refer to mitogen stimulated cells. We studied this susceptibility in quiescent, purified CD4+ or CD8+ T cells from a group of Italian old people compared with a group of young people. We found that an apoptotic agent such as 2-deoxy-D-ribose (dRib), which acts via glutathione depletion and oxidative stress, was more effective in CD4+ T cells from young donors, while no difference was found in CD8+ T cells. On the contrary, another agent such as TNF-alpha, which acts via receptor engagement, was more effective in CD8+ T cells from old subjects, and no difference was found in CD4+ T cells. When marker of activation-memory were investigated, no difference between young and old subjects was found when dRib was used. Differently, when TNF-alpha was used, memory and activated CD4+ T cells from old donors were less sensitive than younger counterparts, while memory CD8+ T cells from old donors were more sensitive than younger counterparts. This suggests that age-related changes in susceptibility to apoptosis of resting T cells largely depend on the type of the apoptotic stimulus which is used as well as on the memory phenotype of the cells. These results may also account, at least in part, for the deep remodelling of T cell repertoire that occurs during ageing.     

Mouse monoclonal antibodies to the human C3b receptor

Mouse monoclonal antibodies were raised against the human C3b receptor (CR1) molecule that had been purified from solubilized erythrocytes membranes. Four hybridomas were selected, cloned and expanded because their supernatants reacted strongly with insolubilized CR1 by ELISA and intensely stained B-dependent areas of the spleen and glomerular podocytes by indirect immunofluorescence. The four monoclonal antibodies, named J3D3, J8B10, J3B11 and J7C2, were IgG1 immunoglobulins. J3D3 immunoprecipitated two protein bands of apparent mol. wts 200,000 and 220,000 from 125I-surface-labeled human erythrocytes, which correspond to the two major allotypic forms of CR1. By indirect immunofluorescence, monoclonal antibodies stained polymorphonuclear leucocytes (PMN), most peripheral blood B-cells and a small subset of peripheral blood T-cells. J3D3 bound to CR1 on erythrocytes, PMN and lymphocytes with an affinity of 1-3 X 10(9) M-1 and recognized 170-1330 antigenic CR1 sites with an average of 740 sites/erythrocyte in 100 healthy individuals, approx. 50,000 sites/PMN and 15,000 sites/lymphocyte. There was a bimodal distribution of CR1 numbers on erythrocyte in the normal population. The four monoclonal antibodies similarly inhibited CR1-mediated decay of preformed cell-bound alternative- and classical-pathway C3 convertase sites. Two antibodies, J3D3 and J3B11, inhibited C3b-dependent rosette formation with lymphocytes, although much less efficiently than F(ab')2 polyclonal anti-CR1 antibody. Differences that were observed in the relative capacity of the antibodies to inhibit some of the functions of CR1 and in their ability to compete for binding of 125I-J3D3 to CR1 on erythrocytes, suggested that they are directed against different epitopes on CR1. Monoclonal antibodies provide useful means to assess and analyze the biological and immunoregulatory functions of the C3b receptor.     

Appearance of extrachromosomal circular DNAs during in vivo and in vitro ageing of mammalian cells

Appearance of extrachromosomal circular DNAs with in vivo and in vitro cellular ageing was examined by using a new technique of mica-press-adsorption for electron microscopy. The size distribution and the copy number of circular DNA complexes varied, depending on the cellular age. Extrachromosomal circular DNA complexes of variable length of more than 0.5 microns or 1.5 kilobase (kb) appeared during in vivo ageing of rat lymphocytes and in vitro ageing of cultured human lung fibroblasts. A restricted size class of circular forms of less than 0.5 microns in contour length was amplified in human skin fibroblasts from aged normal or Werner's syndrome subjects. These circular DNA molecules are suggested to be products of DNA rearrangements or gene amplification occurring in the chromosome.     

T suppressor lymphocytes reverse ongoing acute allograft rejection

(LEW X BN)F1 cardiac allografts are rejected within 8 days in untreated LEW recipients. At the critical time point of 5 days after transplantation, the obviously rejecting grafts are enlarged and maximally infiltrated by host cells as shown by 111In-labeled lymphocyte tracer studies. However, when such hearts were retransplanted back to naive (LEW X BN)F1 secondary hosts, they survive indefinitely, showing that even late rejection is reversible in the absence of sustained host immunological drive. Attempts were then made to abrogate this advanced immune responsiveness using Cyclosporine (CsA). CsA therapy (15 mg/kg/day for 7 days) starting from day 5 produced indefinite graft survival, similar as if initiated at the time of operation. Addition of exogenous IL-2, which drives the proliferation of Tc, could not reverse this effect. Serial changes in phenotype of lymphocyte subpopulations infiltrating both acutely rejecting and indefinitely functioning cardiac allografts in unmodified and CsA treated hosts, respectively, were then studied. Ratio of Th:Tc/s cells in acutely rejecting grafts was 1.6 by day 3; it inverted abruptly to 0.7 by day 5-6, suggesting predominance of Tc/s during the later stages of allograft rejection. Similarly, treatment with CsA produced a transient depression of Th, with recovery of original Th:Tc/s ratio during the next 2-3 weeks. Adoptive transfer experiments were then performed to investigate the functional significance of these findings.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-12 inhibits in vitro immunoglobulin production by human lupus peripheral blood mononuclear cells (PBMC)

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by polyclonal B cell activation and by the production of anti-double-stranded (ds) DNA antibodies. Given the inhibitory effects of IL-12 on humoral immune responses, we investigated whether IL-12 displayed such an activity on in vitro immunoglobulin production by SLE PBMC. Spontaneous IgG, IgG1, IgG2, IgG3 and IgM antibody production was dramatically reduced by addition of IL-12. These results were confirmed by Elispot assays detecting IgG- and anti-dsDNA-secreting cells. While IL-6 and TNF titres measured in PBMC supernatants were not modified by addition of IL-12, interferon-gamma (IFN-γ) titres were up-regulated and IL-10 production down-regulated. Since addition of IFN-γ did not down-regulate immunoglobulin production and since the inhibitory activity of IL-12 on immunoglobulin synthesis was not suppressed by anti-IFN-γ antibody, we concluded that the effect of IL-12 on immunoglobulin production was not mediated through IFN-γ. Our data also argue against the possibility that down-regulation of endogenous IL-10 production was responsible for the effect of IL-12. Thus, inhibition of IL-10 production by IFN-γ was not accompanied by inhibition of immunoglobulin production, and conversely, restoration of IL-10 production by anti-IFN-γ antibody did not suppress the inhibitory activity exerted by IL-12 on immunoglobulin production. Taken together, our data indicate that reduction of excessive immunoglobulin and anti-dsDNA antibody production by lupus PBMC can be achieved in vitro by IL-12, independently of IFN-γ and IL-10 modulation.     

Electrophoretic fractionation of murine lymphoid cells. I. Enrichment of peripheral T lymphocyte subsets with distinct surface phenotypes

The purpose of this work was to evaluate the efficiency of free-flow electrophoresis as a method for separating mouse lymphocyte subsets. The surface phenotype of the cells contained in the various fractions collected after electrophoresis of CBA/J lymph node cells was investigated by means of single- and 2-color flow cytofluorometry (FCF) analysis. In agreement with previous works, B cells (sIg+, Thy-1-) were found to segregate in the low mobility (LM) fractions and T cells (sIg-, Thy-1+) in the high-mobility (HM) fractions. While the mean fluorescence intensity of sIg staining did not significantly vary as a function of electrophoretic mobility (EPM) that of Thy-1 staining tended to decrease with increasing EPM. The distribution of Lyt-1+ cells was roughly parallel to that of Thy-1+ cells. However, 2-color FCF analysis suggested the existence, in addition to a major Thy-1+ Lyt-1+ subpopulation, of a minor subset of Thy-1- Lyt-1+ cells. Lyt-2+ cells made up a peak in the cathodic HM region where they were enriched by up to 3-fold, and a trail in the more anodic HM fractions. Two-color FCF analysis showed that all Lyt-2+ cells recovered in these various electrophoretic fractions expressed the Lyt-1 antigen. Taken together, these data demonstrate that free-flow electrophoresis provides a powerful tool for the delineation and substantial enrichment of phenotypically distinct mouse peripheral T cell subsets.     

The enhancement of antibody response by IgM antibodies is dependent on antigen-specific T helper cells

The enhancement of antibody responses by IgM antibodies administered with low doses of antigen has been studied in a T-dependent (SRBC) and an T-independent (alpha 1,6 dextran) system. It has been found that IgM anti-SRBC antibodies do not enhance a SRBC response in nude mice. The T-cell dependency was also directly demonstrated by showing the effect of IgM on T-cell priming in transfer experiments. The simultaneous injection of antigen and IgM antibody also induced a polyclonal increase of IgM, PFC, which was not due to a non-specific "adjuvant" effect of IgM, as we could not detect a similar effect on an ongoing response to HRBC in mice simultaneously given SRBC and IgM anti-SRBC antibodies. The specificity of the helper cell for either the antibody or the antigen was investigated in a response to alpha 1, 6 dextran, in which we could demonstrate antibody-specific helper T cells, but no antigen-specific help. We have found that IgM anti-dextran antibodies do not enhance and rather suppress the response of normal, high-responder mice, to dextran, suggesting that the T cells mediating the "19S enhancement" are antigen-specific. The magnitude of the enhancement response, as compared to the responses induced by either antigen or antibody alone, implies a synergistic mechanism, possibly involving antigen-specific and antibody(idiotype)-specific T helper cells.