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Peripheral blood lymphocytes from eighteen untreated patients with chronic lymphocytic leukaemia (CLL) were analysed for the proportions of T and B lymphocytes with receptors for IgM, IgG or IgA. T lymphocytes with Fc receptors for IgM (Tμ cells) or IgA (Tα) cells were found in proportions comparable to those found in the controls. However, the proportion of T lymphocytes with receptors for IgG (Tγ small gamma cells) was significantly increased (P < 0.001) resulting in an abnormally low ratio of Tμ/Tγ small gamma (P < 0.001), when compared with normal controls. The proportion of B cells bearing Fc receptors for IgM, IgG or IgA was determined simultaneously. No significant differences were found between the normal controls and the patients with CLL. In vitro treatment of the purified T and B lymphocyte preparations with human leucocyte interferon, did not alter the proportions of the lymphocytes expressing Fc receptors for various immunoglobulin isotypes. The significance of these findings is discussed. [ABSTRACT FROM AUTHOR]

E-rosette negative largely leukaemic B cells from patients with chronic lymphocytic leukaemia (CLL) were separated by density gradient electrophoresis, on the basis of their surface charge. The separated cells were pooled in seven fractions, according lo their relative position in the electrophoretic distribution, and analysed by functional tests and cell-surface phenotypes. The ability of electrophorelically separated B cells from patients with CLL to differentiate into plasma cells in the presence of T cells from normal donors and in the pokewced mitogen induced differentiation system was investigated. Lymphocytes able to differentiate into plasma cells under these conditions were highly enriched in the low-mobility fractions V, VI and VII These plasma cells were of leukaemic origin, because they expressed only the light chain present on the cell surface of the leukaemic B cells before stimulation. Lymphocytes with Fe (IgG) receptors were relatively enriched in the high and intermediate mobility fractions I IV, but they were present in the remaining of the fractions in smaller proportions. Lymphocytes with Fc (IgM) receptors were present in all fractions, but only in very small proportions in the very high mobility fraction I. Cells with complement receptors I and II were present in all fractions. Analysis of the density of cell surface immunoglobulin expression using the fluorescence-activated cell sorter, revealed that fractions of high and intermediate elcetrophoretic mobility (I-V) contained cells with both low and intermediate density of surface immunoglobulin. whereas the low mobility fractions VI and VII contained predominantly cells with low density of surface immunoglobulin. These results revealed significant phenotypic and functional heterogeneity of B cells from patients with CLL, suggesting the presence of subpopulations of leukaemic B cells in different differentiation or maturation stages. [ABSTRACT FROM AUTHOR]

We observed that highly purified E-rosette-negative largely leukaemic B cells from 9 out of 15 patients with chronic lymphocytic leukaemia (CLL) significantly suppressed immunoglobulin production by mixtures of T4 and B cells from normal donors in the presence of pokeweed mitogen (PWM). This suppression by leukaemic B cells was concentration-dependent. Addition of equal numbers of B cells from normal donors to the mixtures of normal T4 and B cells increased, or had no effect on the production of IgM, IgA, and IgG. Treatment of purified largely leukaemic B cells from patients with CLL with either the anti-B1 or anti-Leu 1 monoclonal antibody plus complement abolished their ability to suppress immunoglobulin production. In contrast, treatment with either the anti-Leu 5 or the OKM1 monoclonal antibody plus complement had no effect on the suppression. These results suggest that leukaemic B cells from certain patients with CLL may exhibit, or can be induced to exhibit, immunosuppressive properties. [ABSTRACT FROM AUTHOR]

We investigated the growth and differentiation of leukaemic B cells from patients with B-cell chronic lymphocytic leukaemia (CLL) in response to proliferation and differentiation factors present in conditioned medium and to anti-immunoglobulin antibodies. Highly purified E-rosette negative (E) B cells from 5 out of 15 patients with CLL exhibited moderate proliferative responses to I0 or 50 μg/ml of F(ab)′2 fragments of rabbit anti-human μ-chain specific antibody. Conditioned medium (CM), derived by stimulating human peripheral blood mononuclear leucocytes with PHA, induced significant proliferative responses of purified E cells in 13 out of 14 patients examined. The extent of these proliferative responses varied substantially, and was in the range of 2.6- to 91-fold. Stimulation of purified E cells from patients with CLL with both anti-μ and CM resulted in significant proliferation in all 15 patients examined. These responses were significantly higher than those induced by CM alone (P<0.02). Synergism between CM and anti-μ in inducing proliferative responses was observed in 11 out of 15 patients. Largely leukaemic B cell populations expressing on the cell surface more than one immunoglobulin heavy-chain isotype, exhibited significantly higher (P<0.009) proliferative response to CM and anti-μ than those expressing IgM only. Highly purified E- peripheral blood or tonsil lymphocytes from all normal donors examined responded by proliferation to anti-μ alone or to CM alone. Synergism in inducing proliferative responses was also observed when the cells were stimulated with both CM and anti-μ. In addition to inducing proliferative responses, culture with CM of purified E-rosette negative, largely leukaemic, B cells from patients with CLL for 6 days at 37°C resulted in differentiation into immunoglobulin synthesizing and secreting cells. Synthesis and secretion of IgM were observed in 7 out of 10 patients examined. A switch to IgG production was observed in three patients. Morphological examination of E- cells from patients with CLL after treatment with CM demonstrated that these cells were differentiated into plasma-like cells. These results suggest that leukaemic B cells from patients with CLL can be induced to proliferate and differentiate in response to growth and differentiation factors derived by mononuclear leucocytes, in a manner similar to that of normal B cells. [ABSTRACT FROM AUTHOR]

The abilities of Escherichia coli-derived human interferon gamma (IFN-γ) and E. coli-derived human interferon-αA (IFN-αA) or -α2 (IFN-α2) to augment natural killer (NK) cytotoxicity were compared. When low concentrations (less than 10 antiviral units/ml) of interferons were used, and equal numbers of antiviral units of E. coli-derived IFN-γ and E. coli-derived IFN-αA or IFN-α2 were compared for their ability to augment NK, E. coli-derived IFN-γ was found to be more active in augmenting NK against the K562 targets, than E. coli-derived IFN-αA or IFN-α2. Antiviral units in these experiments were determined by the standard cytopathic effect assay using vesicular stomatitis virus (VSV)-challenged human fibroblasts, trisomic for chromosome 21. However, when these interferons were compared on a weight basis (ng/ml) or on a molar basis, their ability to augment NK against the K562 targets was comparable. These differences in the relative abilities of these interferons (when their concentrations were expressed in antiviral units/ml) to augment NK, were due to an approximately 100-fold difference in their specific activities (antiviral units per mg of interferon). These were 1.8× 106 units/mg for E. coli-derived IFN-γ, 2.0×106 units/mg for E. coli-derived IFN-αA, and 1.8×106 units/mg for E. coli-derived IFN-α2. At concentrations higher than 10 units/ml, all these interferons showed a similar ability to augment NK. Studies on the kinetics of the augmentation revealed that in vitro treatment with E. coli-derived IFN-γ for several hours was necessary for augmentation of NK against targets from haemopoietic human tumour cell lines (K562, Daudi). In contrast, alpha interferons were able m augment NK after treatment in vitro for significantly shorter periods (30 min or less with certain donors). Augmentation of NK cytotoxicity of human peripheral blood mononuclear leucocytes by E. coli-derived IFN-γ was not accompanied by the induction of interleukin 2 (IL-2) production, suggesting that IL-2 is not involved in the augmentation of NK by IFN-γ. A monoclonal antibody specific for human IFN-γ blocked augmentation of NK by E. coli-derived IFN-γ and natural IFN-γ, but not by E. coli-derived IFN-αA or staphyloccocal enterotoxin A (SEA). Pretreatment (14 h at 37°C) of effector cells with this anti-IFN-γ monoclonal antibody or addition of this antibody directly into the 51chromium release assay had no effect on NK cytotoxicity, suggesting that constitutively produced IFN-γ is not required for maintaining NK cytotoxicity. [ABSTRACT FROM AUTHOR]

We have recently described the presence of perivascular CD3+ CD45RO+ T cells infiltrating the brains of children with AIDS. To determine whether these infiltrates contain oligoclonal populations of T cells, we amplified by PCR beta-chain T-cell receptor (TCR) transcripts from autopsy brains of four paediatric patients with AIDS. The amplified transcripts were cloned and sequenced. Sequence analysis of the beta-chain TCR transcripts from all four patients revealed multiple identical copies of TCR beta-chain transcripts, suggesting the presence of oligoclonal populations of T-cells. These TCR transcripts were novel. The presence of oligoclonal populations of T cells in the brains of these four paediatric patients with AIDS suggests that these T cells have undergone antigen-driven proliferation and clonal expansion very likely in situ, in the brains of these AIDS patients, in response to viral or self-antigens. Although the specificity of the clonally expanded beta-chain TCR transcripts remains to be elucidated, none of the beta-chain TCR transcripts identified in this study were identical to those specific for HIV-1 antigens that are currently reported in the GENBANK/EMBL databases. Certain common CDR3 motifs were observed in brain-infiltrating T cells within and between certain patients. Large proportions (24 of 61; 39%) of beta-chain TCR clones from one patient (NP95-73) and 2 of 27 (7%) of another patient (NP95-184-O) exhibited substantial CDR3 homology to myelin basic protein (MBP)-specific TCR derived from normal donors or TCR expressed in the brain of patients with multiple sclerosis (MS) or with viral encephalitis. These two patients (NP95-73 and NP95-184-O) also shared HLA class II with the normal donors and the MS patients who expressed these homologous TCR. Pathologic examination at autopsy of the brains revealed the presence of myelin pallor only in patient NP95-73. T-cell clones identified in the brain of patients NP95-73 and NP95-184-O may recognize MBP or another CNS self antigen and this recognition may be restricted by either DRB1*15 or DQB1*0602 specificities.

Background: We investigated the role of apoptosis (programed cell death) in the pathogenesis of chronic rejection., Methods: Epicardial coronary arteries from cardiac allografts with chronic rejection were examined for apoptosis by the TUNEL assay. Double labeling was carried out using anti-CD3, anti-CD68, and anti-von Willenbrand factor (vWF) monoclonal antibodies. Additional immunostaining was carried using anti-Fas, anti-Fas-L, and anti-Bcl-2 monoclonal antibodies. Apoptosis-associated oligonucleosomal DNA degradation was assessed by DNA agarose gel electrophoresis. The transcription level of apoptosis-related caspase genes were determined using microarrays., Results: Apoptotic cells (TUNEL+) were detected within the arterial wall and in perivascular areas. Double labeling demonstrated that apoptotic cells included T cells (CD3+), monocyte/macrophages (CD68+), and vascular endothelial cells (VWF+). Numbers and densities of TUNEL+ cells did not correlate with the degree of arterial stenosis. Apoptosis-associated oligonucleosomal DNA degradation was assessed by agarose gel electrophoresis of DNA, which showed DNA fragments of approximately 180 bp and multimers thereof (DNA laddering gel), which are characteristic for DNA fragmentation in apoptotic cells. Microarray analysis demonstrated that the apoptosis related caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, were all transcribed (caspases 8, 9, and 10 were highly up-regulated). These results are consistent with the involvement of apoptosis in chronic rejection. Immunoreactivity for Fas/Fas-L was present at the sites of apoptotic cells. Immunoreactivity for Bcl-2 was present in areas with very few apoptotic cells., Conclusions: Apoptotic cells include T cells, monocyte/macrophages, and endothelial cells. Apoptosis, likely through the Fas/Fas-L system, is involved in the pathogenesis of chronic rejection in cardiac allografts.

Intracranial inoculation of susceptible SJL mice with Theiler's murine encephalomyelitis virus (TMEV) results in biphasic disease consisting of early acute disease, followed by late chronic demyelinating disease, associated with mononuclear infiltrates and demyelinating lesions. In contrast, resistant C57BL/6 (B6) mice develop only early acute disease. We employed cytokine-specific RT-PCR to determine the expression of cytokine transcripts in the CNS of TMEV-infected SJL and B6 mice. During early acute disease, we have found a strong proinflammatory (Th1) cytokine response in the CNS of both TMEV-infected SJL and B6 mice, demonstrated by the expression of transcripts for IFN-gamma, IL-1, IL-6, IL-12p40, and TNF-alpha. At 8 days postinfection (p.i.), TGF-beta1 and TNF-alpha transcripts were present at significantly higher levels (P < 0.01) in the CNS of SJL susceptible mice in comparison to those found in the CNS of B6 mice. Immunohistochemical staining revealed that TGF-beta protein was expressed in leptomeningeal mononuclear inflammatory cell infiltrates in the brain of SJL mice but not in B6 mice, at 8 days p.i. TGF-beta may be responsible for the failure of SJL mice to develop an effective anti-TMEV CTL response. During late chronic demyelinating disease, high levels of proinflammatory Th1 cytokines were found in the CNS of SJL mice, but not B6 mice. Significantly higher levels (P < 0.01) of anti-inflammatory cytokine transcripts (IL-4, IL-5, and IL-10 (Th2 cytokines) and TGF-beta) were found in the spinal cord of TMEV-infected SJL mice with chronic demyelinating disease than in the spinal cord of B6 mice during the same time period (39 or 60 days p.i.). These anti-inflammatory cytokines may contribute to the downregulation of the proinflammatory response in SJL mice. High levels of IL-2 transcripts and protein appeared transiently in the spinal cord of TMEV-infected SJL mice before the onset of demyelinating disease and coincided with an influx of new T cells into the CNS and/or expansion of remaining T cells that have not been eliminated after viral clearance., (Copyright 2000 Academic Press.)

To identify strategies that enhance tumor-specific immunity in patients with ovarian carcinoma, 22 patients received four to six doses of i.p. recombinant IFN-gamma (rIFN-gamma), 200 microg/m2 on days 1, 3, 5, 8, 10, and 12, and i.p. recombinant interleukin 2 (rIL-2), either 6.0 x 10(5) IU/m2 (group A) or 1.0 x 10(5) IU/m2 (group B), on days 9, 10, and 11. Two patients in group A also received T-cell lines expanded from peritoneal tumor-infiltrating lymphocytes (TILs) obtained after i.p. rIFN-gamma/rIL-2 administration. Toxicity was manageable and included five nonhematological grade 3 or 4 events in 22 patients (23%). A patient had normalization of CA-125 values and a progression-free interval of 18 months, after receiving i.p. rIFN-gamma/rIL-2 without TILs. Another patient who received i.p. rIFN-gamma/rIL-2 plus TILs had stabilization of ascites and intra-abdominal tumors and >50% reduction in serum CA-125 values over 6 months. A third patient who received i.p. rIFN-gamma/rIL-2 had stabilization of intra-abdominal tumors and ascites accompanied by CA-125 values of 50 to 100 units over 6 months. T-cell lines for adoptive immunotherapy were developed for only 3 of 20 patients who were treated with rIFN-gamma/rIL-2. Large numbers of CD3- CD56+ adherent cells were expanded in rIL-2 in the remaining patients, precluding the development of T-cell lines. i.p. rIFN-gamma, either alone or followed by rIL-2, increased proportions of human leukocyte antigen (HLA) class I+ and class II+ tumor cells and increased HLA class I staining intensity on peritoneal carcinoma cells. i.p. rIFN-gamma plus rIL-2 also enhanced cytotoxic activity against Daudi and K562 cells and against allogeneic ovarian tumor cells. Increased cytotoxic activity was associated with an increase in the proportion of CD56+ cells. IFN-gamma and IL-2 transcripts were expressed more frequently after rIFN-gamma and rIL-2 treatment. In addition, the proportions of CD45RA+ (naive lymphocytes) were increased, and CD8+ DR+ lymphocytes were increased relative to CD8+ CD69+ cells, which were decreased. IL-10 concentrations in peritoneal fluids were increased after treatment with rIFN-gamma and the higher rIL-2 dosing (group A) but not in those treated with rIFN-gamma and the lower rIL-2 dosing (group B). These results demonstrated that patients with ovarian carcinoma can tolerate treatment with rIFN-gamma and rIL-2 and that rIFN-gamma alone or rIFN-gamma combined with rIL-2 enhances the expression of HLA class I and class II antigens on ovarian tumor cells, although immunosuppressive cytokines, such as transforming growth factor-beta and IL-10, may persist. Treatment with rIFN-gamma/rIL-2 i.p. did not facilitate the production of TIL-derived T-cell lines ex vivo.

A newly described subset of monocytes has been identified in peritoneal exudate cells (PEC) from the malignant ascites from patients with ovarian cancer. These cells were characterized by the production of IL-10 and TGF-beta2, but not IL-12, IL-1alpha, or TNF-alpha, and they expressed CD14, CD16, and CD54, but not HLA-DR, CD80, CD86, CD11a, CD11b, or CD25 cell surface Ags. Since this subset of monocytes could affect the modulation of tumor immune responses in vivo, studies were undertaken to determine their effect on the activation and proliferation of autologous T cells from the peritoneal cavity of patients with ovarian carcinoma. Expression of cytokine-specific transcripts in T cells was determined by RT-PCR. Transcripts for the following cytokines were detected in patient specimens that also contained the IL-10-producing monocytes IL-2 (12 of 17 specimens), GM-CSF (9 of 17 specimens), IFN-gamma (6 of 17 specimens), and TNF-alpha (4 of 17 specimens). Cytokine production by T cells was determined by intracellular flow cytometry and by ELISA. IL-2 and IFN-gamma proteins, unlike their transcripts, were detected only in specimens that lacked IL-10-producing monocytes. IL-10-producing monocytes cocultured with autologous T cells inhibited the proliferation of the T cells in response to PHA. However, T cells cocultured with PEC from which the IL-10-producing monocytes had been removed did not inhibit T cell proliferation. Moreover, the inhibition of T cell proliferation by IL-10-producing monocytes could be reversed by adding neutralizing Abs to both IL-10R and TGF-beta2. These results suggest that this subset of monocytes may modulate immune responses by inhibiting T cell proliferation and cytokine protein production.

Transforming growth factor beta (TGF-beta) is an important family of cytokines that may promote tumor growth in vivo through several mechanisms including interference with antitumor T-cell immune responses, alteration of factors in the stroma and matrix, and the promotion of angiogenesis. TGF-beta isotypes have been detected in malignant and normal ovarian tissues. We have determined by quantitative immunohistochemistry the density of TGF-beta1, TGF-beta2, and human leukocyte antigen (HLA) Class I and Class II antigens on malignant cells in paired primary and metastatic specimens from 10 patients with ovarian carcinoma. Cryostat sections of specimens from the carcinomas and from normal ovaries of three women of similar age without ovarian cancer were stained respectively with specific antibodies to TGF-beta1, TGF-beta2, and HLA Class I and II antigens, and with isotype-matched control antibodies. Antigen density was quantitated blindly as mean absorbance on a SAMBA 4000 image analyzer. TGF-beta1 and TGF-beta2 were overexpressed in both primary and metastatic tumor specimens in comparison with normal ovarian tissue. No statistical correlation was found between the expression of TGF-beta1 or TGF-beta2 and HLA class I or HLA class II, which suggests that TGF-beta isotypes could have effects on the immune system other than down-modulation of these HLA molecules. Furthermore, the lack of association between levels of TGF-beta expression and the reduced expression of HLA molecules could suggest that tumor cells expressing both HLA and TGF-beta may be suitable targets for adaptive immunotherapy. Additional studies are necessary to determine whether TGF-beta expressed by ovarian cancer cells merits evaluation as a therapeutic target.

Recent in vitro studies have shown that interleukin 4 (IL-4) induces and gamma interferon (IFN-gamma) inhibits collagen production. To define the TH1(IFN-gamma) and TH2(IL-4) cytokine profiles in systemic sclerosis (Sscl), a disease characterized by widespread fibrosis, we investigated IL-4 and IFN-gamma transcripts in peripheral blood mononuclear cells and plasma protein levels in 13 patients with Sscl. Two previously identified IL-4 transcripts, a full-length transcript and an alternatively spliced (truncated) transcript (designated IL-4delta2), were identified in patients and normal controls. Significantly increased levels of total IL-4 transcripts (full-length plus IL-4delta2 transcripts) were found in patients with Sscl in comparison to those found in healthy controls (P = 0.003), and this increase was primarily due to an increase in the level of the alternatively spliced IL-4delta2 form. The IL-4delta2/full-length-IL-4 transcript ratio was significantly increased in Sscl patients (P < 0.0001, versus healthy controls). Sequencing analysis revealed that the frequency of IL-4 clones carrying the IL-4delta2 transcript was also substantially increased in patients with Sscl. Plasma IL-4 protein levels were increased in Sscl patients compared to those in healthy controls (P = 0.001) and correlated with total IL-4 transcript levels. The up-regulation of the fibrogenic IL-4 (a TH2 cytokine) in Sscl suggests a pathogenic role for IL-4 in this disease.

A significant proportion of brain tissue specimens from children with AIDS show evidence of vascular inflammation in the form of transmural and/or perivascular mononuclear-cell infiltrates at autopsy. Previous studies have shown that in contrast to inflammatory lesions observed in human immunodeficiency virus type 1 (HIV-1) encephalitis, in which monocytes/macrophages are the prevailing mononuclear cells, these infiltrates consist mostly of lymphocytes. Perivascular mononuclear-cell infiltrates were found in brain tissue specimens collected at autopsy from five of six children with AIDS and consisted of CD3(+) T cells and equal or greater proportions of CD68(+) monocytes/macrophages. Transmural (including endothelial) mononuclear-cell infiltrates were evident in one patient and comprised predominantly CD3(+) T cells and small or, in certain vessels, approximately equal proportions of CD68(+) monocytes/macrophages. There was a clear preponderance of CD3(+) CD8(+) T cells on the endothelial side of transmural infiltrates. In active lesions of transmural vasculitis, CD3(+) T-cell infiltrates exhibited a distinctive zonal distribution. The majority of CD3(+) cells were also CD8(+) and CD45RO+. Scattered perivascular monocytes/macrophages in foci of florid vasculitis were immunoreactive for the p24 core protein. In contrast to the perivascular space, the intervening brain neuropil was dominated by monocytes/macrophages, microglia, and reactive astrocytes, containing only scant CD3(+) CD8(+) cells. Five of six patients showed evidence of calcific vasculopathy, but only two exhibited HIV-1 encephalitis. One patient had multiple subacute cerebral and brainstem infarcts associated with a widespread, fulminant mononuclear-cell vasculitis. A second patient had an old brain infarct associated with fibrointimal thickening of large leptomeningeal vessels. These infiltrating CD3(+) T cells may be responsible for HIV-1-associated CNS vasculitis and vasculopathy and for endothelial-cell injury and the opening of the blood-brain barrier in children with AIDS.