Elevation of a gamma delta T cell subset in peripheral blood and synovial fluid of patients with rheumatoid arthritis.

We examined the levels of TcR delta 1+ T cells (total gamma delta T cell) and delta TCS1+ (gamma delta T cell subset) T cells in the peripheral blood (PB) and synovial fluid (SF) of 16 patients with rheumatoid arthritis (RA) and compared them to the levels in PB of patients with Felty's syndrome (FS) and 21 healthy control subjects (NML). Synovial fluid from eight patients with seronegative spondyloarthropathies (SSA) was also examined. The results demonstrated elevated levels of the delta TCS1+ subset in the PB of RA and FS patients relative to NML (P less than 0.05). No such differences were observed in the levels of PB TcR delta 1+ T cells. The results did not appear to reflect a non-specific inflammatory response since delta TCS1 T cells were elevated in the SF of RA patients relative to SSA SF and NML PB. delta TCS1 T cells in SSA PB and SSA SF were comparable to NML PB. TcR delta 1+ T cells levels in RA SF were higher than SSA SF levels but were comparable to those of NML PB. Taken together, the results support a pathogenic role for delta TCS1+ T cells in RA.

A critical analysis of serum and urine interleukin-2 receptor assays in renal allograft recipients.

A component of the interleukin 2 receptor (IL-2R) is released in soluble form during T cell activation and can be detected in the blood during acute renal allograft rejection. This study evaluates the diagnostic utility of a sandwich enzyme immunoassay test for serum and urine IL-2R in renal allograft recipients. A rise in serum IL-2R during the week prior to the clinical diagnosis of rejection correlated better with rejection than did isolated serum IL-2R levels or urine values. For the diagnosis of acute rejection, a rise in serum IL-2R (sensitivity 73%, specificity 87%) was comparable in overall test performance to a rise in serum creatinine (sensitivity 70%, specificity 84%). Overall, the two tests had equivalent receiver operating characteristic curves. Because the etiology of false positives in creatinine and IL-2R assays differed (primarily cyclosporine toxicity and infection, respectively), the predictive value of the combined tests was superior to either alone.

Soluble interleukin 2 receptors in sera of Japanese patients with adult T cell leukemia mark activity of disease.

Serum concentrations of soluble interleukin 2 receptors (sIL 2R) were measured by an enzyme-linked immunosorbent assay (ELISA) in 30 patients with adult T cell leukemia (ATL), in 9 patients with other hematopoietic malignancies, and in 17 asymptomatic individuals seropositive for human T cell leukemia virus type I (HTLV-I). Sixty HTLV-I seronegative, age-matched controls showed a normal range of form 63.2 to 480.8 U/mL. All asymptomatic carriers of HTLV-I had sIL 2R in their sera within the normal range. sIL 2R in sera was not related to the anti-HTLV-I antibody titer. Eleven patients with acute ATL, a clinical phenotype with median survival rate of 4.4 months, had markedly elevated sIL 2R (11,100 to 99,000 U/mL), but eight patients with smoldering ATL had low sIL 2R values (less than 480.8 U/mL) comparable to controls. Eleven patients with chronic ATL had intermediate elevated levels of sIL 2R (480.8 to 37,300.0 U/mL). Serum levels of sIL 2R correlated with the number of ATL cells (r = 0.812) and CD25-positive cells (r = 0.725) circulating in the peripheral blood. Longitudinal studies performed in four patients with ATL showed significant correlation between serum concentration of sIL 2R and activity of the malignancy. These findings suggest that the level of sIL 2R in serum indicated tumor load and, possibly, prognosis.

Plasma interleukin 2 receptor levels in renal allograft recipients.

Shed/soluble interleukin 2 receptor (IL2R) was measured by an enzyme-linked immunosorbent assay (ELISA) in serial samples of plasma from 32 patients with renal allografts. Patients on chronic dialysis (pretransplant) had elevated IL2R levels which fell toward normal after transplantation. Patients with acute rejection and viral infection had significantly higher levels of plasma IL2R than did patients with stable renal function or with cyclosporine nephrotoxicity (all P less than 0.005). Acute renal failure from other causes (renal artery stenosis, hemolytic-uremic syndrome) did not have a comparable rise in IL2R. The assay of shed/soluble IL2R may have diagnostic value in the clinical management of allograft recipients, a possibility that deserves further clinical evaluation.

Isolation and characterization of murine monoclonal antibodies specific for gram-negative bacterial lipopolysaccharide: association of cross-genus reactivity with lipid A specificity.

Somatic cell hybrids secreting monoclonal antibodies against the core-glycolipid portion of enterobacterial endotoxin were derived from mice immunized with Escherichia coli J5 or Salmonella minnesota R595 heat-killed organisms or lipopolysaccharide (LPS). Eight antibodies were selected for their ability to cross-react with several members of a panel of gram-negative bacterial antigens in a radioimmunoassay. This panel represented five genera and two families of organisms: E. coli O111:B4, E. coli O55:B5, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella minnesota, and Serratia marcescens. The binding sites for six of the antibodies were unequivocally localized within the lipid A moiety of the endotoxin molecule by using the radioimmunoassay on LPS and free lipid A. The anti-lipid A antibodies were further characterized for their ability to interact with LPS variants by using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining procedure. The monoclonal antibodies bound almost exclusively to the low-molecular-weight species of LPS on the polyacrylamide gel. These components corresponded to LPS isolated from rough strains of organisms (strains which lack O-specific carbohydrate). These results suggested that the cross-reactive component of antisera raised against rough mutants of gram-negative bacteria contain antibodies of lipid A specificity. Moreover, the determinant within the lipid A moiety of LPS may have been accessible to the monoclonal antibodies only in those endotoxin molecules on the outer membrane surface which lack the O-specific carbohydrate.

In vitro response to HBsAg of peripheral blood lymphocytes from recipients of hepatitis B vaccine.

Lymphocytes isolated from recipients of hepatitis B vaccine were studied for their immune response to HBsAg in vitro. Peripheral blood mononuclear cells (PBMs) from 70 to 80% of 40 vaccinees yielded proliferative indices larger than 2 after 5 to 7 days incubation with HBsAg. This in vitro proliferative response could be augmented by incubating the cells with HBsAg and supernatants of activated T cells for 2 weeks or longer. After 7 to 10 days, in vitro stimulation with antigen, PBMs (1 X 10(6] could yield 5 to 15 HBsAg-specific antibody-secreting plaque-forming cells. The antibody to HBsAg produced in vitro was greatly increased in cultures that contained antigen-specific B cells enriched by panning with HBsAg-coated plates and a T cell growth factor-dependent, HBsAg-specific autologous T cell line. The results indicate that HBsAg-specific B and T cells are present, although at low frequencies, in the circulation of hepatitis B vaccinees.

Use of monoclonal antibodies as sensitive and specific probes for biologically active human gamma-interferon.

Mouse monoclonal antibodies B1 and B3 are specific for natural and Escherichia coli-derived recombinant human gamma-interferon (IFN-gamma). The two antibodies recognize different epitopes of the IFN-gamma molecule and do not compete with each other's binding. We have used these two antibodies to construct a solid-phase, sandwich immunoradiometric assay for human IFN-gamma. Purified antibody B1 was coated on polystyrene beads (0.64 cm in diameter) and used as the solid-phase immunoadsorbent and antibody B3 was labeled with 125I and used as tracer. This assay can be completed in about 4 hr and is capable of detecting IFN-gamma levels in human serum or tissue culture fluids as low as 0.1 NIH reference unit/ml. Recombinant human IFN-gamma derived from E. coli was detectable at a concentration of 0.02 ng/ml. The assay appears to be specific for the biologically active forms of IFN-gamma, since after exposure to pH 2, 37 degrees C, or 56 degrees C, biological activity and reactivity in the immunoradiometric assay decreased in parallel. The immunoradiometric assay can be employed for the analysis of the structural characteristics of the human IFN-gamma molecule.

A cDNA clone encoding a product of activated human T lymphocytes.

A cDNA cloning approach was used to study the regulation of gene expression in human T lymphocytes upon mitogen stimulation. Poly(A)+ mRNA was prepared from phytohemagglutinin A (PHA) and 12-O-tetradecanoyl phorbol 13-acetate (TPA) activated human T cells and a cDNA clone library was constructed. After screening by colony hybridization with [32P]cDNA probes made from resting and activated T cell mRNA, several clones whose mRNA increased at least 10 to 20-fold upon stimulation were isolated. Northern blot analysis of the mRNA from various cell types using these cDNA clones as probes revealed that one of the cDNA clones, pNC5A, encoded a gene expressed only in PHA and TPA-stimulated human T lymphocytes and in a human neoplastic T cell line HUT102-SH9. Less than 20 copies of this mRNA species per cell was detected in resting human T lymphocytes, B lymphocytes and monocytes and in two other T cell lymphoma lines (CEM and MOLT4), two B lymphoblastoid cell lines (WIL2-729-HF2 and HFB-1), a myeloid cell line (HL60) and a human embryonic lung fibroblast cell line (MRC-5). Hybrid selection translation and sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis of the translated product indicated that a polypeptide of 30,000 to 32,000 Mr is encoded by this particular cDNA clone. Thus, this cDNA clone may define a novel gene that is expressed only in activated human T cells.

Hepatitis B virus-reactive human T lymphocyte clones: antigen specificity and helper function for antibody synthesis.

To study immunity to hepatitis B surface antigen (HBsAg) at the cellular level, lymphocytes were obtained from the peripheral blood of hepatitis B vaccine recipients and were examined for various immune responses to HBsAg in vitro. The peripheral blood mononuclear cells (PBM) from most of the vaccinees did not proliferate to a great extent to HBsAg in vitro. However, HBsAg-reactive lymphocyte lines and clones were obtained from some of these individuals if the PBM were stimulated in vitro with HBsAg and were maintained in the presence of T cell growth supplement. Most of the HBsAg-reactive T cell clones obtained were found to be antigen-specific and some of them provided help in the production of anti-HBsAg antibodies by a cell population enriched for HBsAg-binding cells. These results indicate that HBsAg-specific T and B cells exist in the circulation of hepatitis B vaccine recipients, although they are at limiting concentrations for the in vitro cell proliferation and antibody production assays.

Antigenic polymorphism of the T4 differentiation antigen expressed on human T helper/inducer lymphocytes.

The human TH lymphocyte population has been established to express a differentiation antigen (T4) which appears to function in cellular collaboration and T cell recognition of Class II MHC alloantigens. Because we observed altered immunofluorescence staining of the TH cells of some individuals using the OKT4 mAb, a systematic investigation on both the epitopic structure of the T4 glycoprotein molecule and possible polymorphism of these epitopes was undertaken. From competitive blocking assays using eight murine anti-T4 mAbs coupled with quantitative flow cytometry, at least five and possibly seven different epitopes can be recognized on the T4 molecule. Population studies showed some individuals had a reduced phenotypic expression of the OKT4 reactive determinant to one-half that of normal and others completely lacked this epitope. The OKT4 reactive epitope variations are common but have so far been racially restricted to American Blacks and do not appear related to the stage of TH cell differentiation, any identifiable immune abnormality in vitro, or a definable disease process. The OKT4 epitope cannot be unmasked by neuraminidase treatment or T cell stimulation with lectins, soluble antigens, or allogeneic lymphocytes. Coupled with a family study, the alterations in OKT4 phenotype are best explained by autosomal, codominant expression of the T4 gene product. The significance of this polymorphism on TH cell function remains unclear.

Five epitopes of a differentiation antigen on human inducer T cells distinguished by monoclonal antibodies.

A series of mouse monoclonal antibodies reacting with human T cells of the helper/inducer subclass, OKT4, 4A, 4B, 4C, and 4D, have been reported. Using double-fluorescent staining and complement-mediated depletion, it was shown that the antigen(s) recognized by OKT4, 4A, 4B, 4C, and 4D antibodies are present on the same cell. Using FITC-labeled OKT4, 4A, 4B, 4C and 4D, a lack of competition between the antibodies for their epitopes was shown. Immunoprecipitation of the antigen recognized by each antibody yielded a molecule of approximately 60,000-62,000 Da. Sequential precipitation with several antibodies resulted in a minimum of additional precipitated antigen following removal of the first antigen. Capping of cell surface antigen with OKT4, followed by staining with OKT4, 4A, 4B, 4C, or 4D, indicated that the epitopes for all five antibodies co-cap. A sandwich ELISA assay using OKT4 and the other antibodies showed that molecules binding to OKT4A, 4B, 4C, and 4D also bound OKT4. It can therefore be concluded that monoclonal antibodies OKT4, 4A, 4B, 4C, and 4D recognize distinct epitopes present on a molecule of approximately 60-62,000 Da on human helper/inducer T cells.

T-lymphocyte subsets in smoking and lung cancer: Analysis of monoclonal antibodies and flow cytometry.

In order to determine whether abnormalities of immunoregulatory T-cells occur in patients with lung cancer, we characterized peripheral T-lymphocytes in 26 patients with untreated lung cancer. The results in patients with primary squamous cancer (SC) (n = 10), primary adenocarcinoma (AC) (n = 7), and secondary lung metastases (M) (n = 9) were compared with each other and to subjects without cancer (n = 48), including nonsmokers (n = 29) and smokers (n = 19). We found that OKT3+ (mature, peripheral (T-lymphocytes, including both OkT4+ (inducer/helper) and OKT8+ (cytotoxic/suppressor) lymphocytes, were increased in light-to-moderate smokers, but that OKT4+ cells were decreased n heavy smokers (p less than 0.05). The ratio of OKT4+ to OKT8+ (4/8) lymphocytes, reflecting the balance of immunoregulatory cells, was normal in light-to-moderate smokers, but was decreased in heavy smokers (p less than 0.05). The profile of circulating T-cells in patients with SC was similar to the smokers. In contrast, in patients with AC, we found a decreased percentage of OKT8+ cells (p less than 0.05). The 4/8 ratio was elevated in patients with AC (p less than 0.05). In patients with M, there was a decreased percentage of OKT3+ cells reflected in both OKT4+ and OKT8+ subsets. The 4/8 ratio in patients with M was low. Thus, a number of abnormalities in circulating T-cells was found both in smokers and in patients with lung cancer. These results suggest that immunoregulatory abnormalities contribute to the pathogenesis of lung cancer.

Inducer and suppressor T-cells in hepatitis B virus-induced liver disease.

During acute type B hepatitis, the proportion of inducer to cytotoxic/suppressor T-cells is decreased due to an increase in the concentration of suppressor cells. Similar changes are seen in chronically infected subjects with evidence of active viral replication (HBeAg positive) and chronic hepatitis of varying severity. This imbalance of the regulatory cells returns to normal when viral replication decreases during the recovery phase of acute hepatitis and in patients who become chronic carriers with minimal liver disease (HBeAb positive patient). Patients in whom viral replication has subsided (HBeAb positive) but who continue to exhibit chronic active liver disease have increased inducer to cytotoxic/suppressor cell ratios due to a decrease in the concentration of the cytotoxic/suppressor cell population. Further studies are needed to determine whether these alterations of the regulatory cells of the immune system are a causal factor influencing the duration of active hepatitis B virus replication and the degree of inflammatory liver damage, or merely changes secondary to the presence of a replicating virus.

Cellular origin and interactions involved in gamma-interferon production induced by OKt3 monoclonal antibody.

OKT3 monoclonal antibody, a human T cell mitogen, induced interferon production by cultured mononuclear cells at 10(-11) M concentrations. Interferon was secreted only under conditions wherein OKT3 was mitogenic, and production was correlated with cell proliferation. Thus, like mitogenesis, interferon secretion reached a peak 3 days after OKT3 stimulation, was inhibited by a factor(s) in human serum, and required 1000 times higher concentrations of Fab and F(ab')2 fragments of OKT3 for induction. The interferon was most likely of "gamma" (immune) type, because pH 2 and 56 degrees C treatments denatured it, whereas anti-alpha or -beta interferon antibodies did not. Mononuclear cells were fractionated into subpopulations that contained OKT4+ cells (helper/inducer T cells), OKT8+ cells (cytotoxic/suppressor T cells), and OKM1+ cells (monocytes) by combining sheep red blood cell rosetting and complement-mediated lysis using monoclonal antibodies against specific cell types. Both OKT4+ and OKT8+ cells proliferated upon OKT3 stimulation with the absolute requirement of OKM1+ cells. However, OKT4+ cells plus OKM1+ cells were necessary for the secretion of interferon. Studies with selective pretreatments with mitomycin C suggested that gamma-interferon was secreted by the OKT4+ cells and that the OKM1+ population subserved an accessory function.

The relationship between T lymphocyte subsets and Ia-like antigen positive nonlymphoid cells in early stages of cutaneous T cell lymphoma.

Immunofluorescence studies were carried out in cutaneous T cell lymphoma (mycosis fungoides) in order to analyse the microanatomical relationship of the different T lymphocyte subsets (inducer and suppressor/cytotoxic cell populations) to large nonlymphoid Langerhans-type and so-called "indeterminate" or interdigitating cells. The conventional and mouse (monoclonal) antibodies were used in various combinations using fluorescein and rhodamine labeled second layers. In 5 of the 7 cases studied the dermal infiltrate consisted of numerous T (HuTLA+) lymphocytes, 80-90% of which expressed the inducer phenotype (HuTLA+,OKT4+). Most of these cells formed close contact with large cells exhibiting large amounts of Ia-like antigens. These cells corresponded to the interdigitating and indeterminate cells in the sections. By contrast, only small numbers (10-20%) of T cells of suppressor/cytotoxic type (HuTLA+,OKT8+) were seen. These did not show a close affinity to the Ia-like antigen positive nonlymphoid component but appeared to have a predilection for the epidermis. Epidermal Langerhans cells, also strongly Ia-like antigen positive, were further defined by 2 monoclonal antibodies reacting with a cortical thymocyte antigen HTA-1. Although Langerhans cells are probably related to the Ia-like antigen positive dermal cells only a few of the abundant latter population were HTA-1+. In the remaining 2 cases, larger populations of OKT8+ (suppressor/cytotoxic) cells were seen and could be heralding a particularly benign course. These observations indicate a close functional relationship between the lymphoid and Ia-like antigen positive dermal cells during the pre-malignant phase of cutaneous T cell lymphoma.

Immunoelectron microscopic identification of Langerhans cells using a new antigenic marker.

The specificity of a monoclonal antibody (OKT6) for epidermal Langerhans cells was examined by immunoelectron microscopy. Peroxidase-labeled OKT6 bound to 1-5% of suspended human epidermal cells, as determined by light microscopy. Electron microscopic examination of peroxidase-labeled cells revealed that all Birbeck granule-containing Langerhans cells bound OKT6. In addition, a small population of indeterminate cells, lacking the Birbeck granule, was also labeled with OKT6. The ultrastructural studies confirm the specificity of OKT6 for Langerhans cells and suggest that the indeterminate cell represents a related cell population.