Immunosuppressive properties of methotrexate: apoptosis and clonal deletion of activated peripheral T cells.

The folate antagonist methotrexate (MTX) is extensively used in graft-versus-host disease, rheumatoid arthritis, and other chronic inflammatory disorders. In addition to its antiinflammatory activity associated with increased release of adenosine, MTX exerts antiproliferative properties by inhibition of dihydrofolate reductase and other folate-dependent enzymes. However, the mechanisms of immunosuppressive properties associated with low-dose MTX treatments are still elusive. We report here that MTX (0.1-10 microM) induces apoptosis of in vitro activated T cells from human peripheral blood. PBL exposed to MTX for 8 h, then activated in drug-free medium, underwent apoptosis, which was completely abrogated by addition of folinic acid or thymidine. Apoptosis of activated T cells did not require interaction between CD95 (Fas, APO-1) and its ligand, and adenosine release accounted for only a small part of this MTX activity. Apoptosis required progression of activated T cells to the S phase of the cell cycle, as it was prevented by drugs or antibodies that interfere with IL-2 synthesis or signaling pathways. MTX achieved clonal deletion of activated T cells in mixed lymphocyte reactions. Finally, in vitro activation of PBL taken from rheumatoid arthritis patients after MTX injection resulted in apoptosis. Altogether, the data demonstrate that MTX can selectively delete activated peripheral blood T cells by a CD95-independent pathway. This property could be used as a new pharmacological end point to optimize dosage and timing of MTX administration. It may account for the immunosuppressive effects of low-dose MTX treatments.

B-cell maturation stages of Burkitt's lymphoma cell lines according to Epstein-Barr virus status and type of chromosome translocation.

This study addressed the possible relationship between B-cell maturation stage of Burkitt's lymphoma (BL) cell lines and Epstein-Barr virus (EBV) status, ethnic group, or type of chromosome translocation. Fifty-seven cell lines obtained at the International Agency for Research on Cancer from 51 patients were studied. Cytogenetic analyses of 54 cells lines were available. Cell size, surface immunoglobulins (sIgs), cytoplasmic immunoglobulins (cIgs), mouse red blood cell receptors, and reactivity with various monoclonal antibodies were assessed. Immunoglobulin (Ig) class secretions were measured in the supernatant of 2- and 5-day cultures from 33 cell lines, with the use of a sensitive enzyme-linked immunosorbent assay technique. From this study, BL appears to cover a broad range of the B-cell differentiation sequence, since the following Ig phenotypes were observed: null cells (sIg-, cIg-), large pre-B-cells (intracytoplasmic mu-chains), small B-cells (sIg+, cIg-), and various types of secreting B-cells (sIg+, cIg+). Among the latter, various patterns of cIg could be defined (perinuclear, paranuclear, and vesicular). B-cell maturation stages were correlated with the amount of secreted Ig. In sIg+ cell lines, different classes of Ig were found: 35 IgM, 10 IgM plus IgD, 4 IgG, and 1 IgA. None of the different monoclonal antibodies used was specific to a precise stage of maturation. The stages of maturation were correlated with neither the type of chromosome translocations of BL nor the presence of EBV genome, but the most immature cell lines were all EBV positive and most of them originated from African patients. In contrast with acute lymphoblastic leukemia, the common acute lymphocytic leukemia antigen (CD10) was expressed on nearly all BL cell lines of intermediate maturation stages but only on half of the pre-B ones. In addition, none of the cell lines tested was found to react with CD5 antibodies, which recognize most of the chronic lymphocytic leukemia of the same stage of maturation as that in the B-lymphocyte lineage.

Presence of estrogen binding sites and growth-stimulating effect of estradiol in the human myelogenous cell line HL60.

In the present study, we investigated the effects of estrogens on the growth of the HL60 line in vitro and the presence of estrogen binding sites in the same cells. Cell proliferation was estimated by cell counts, [3H]thymidine incorporation, and determination of the percentage of cells in the S phase by flow cytometry. Cells maintained in a medium containing physiological concentrations of estradiol (10(-9) M, 10(-8) M, 10(-7) M) exhibited a growth stimulation, shown by an increase in the percentage of cells in the S phase, whereas a pharmacological concentration (10(-6) M) produced a growth inhibition. Furthermore, the addition of the specific antihormone, tamoxifen, inhibited the stimulating effect of the estrogens. Receptor analysis showed the presence of specific estrogen-binding sites with an apparent dissociation constant of about 5.3 X 10(-10) M. The effect of estrogen was therefore associated with the presence of estrogen receptors in the human leukemic cell line HL60.

Anthracyclines trigger apoptosis of both G0-G1 and cycling peripheral blood lymphocytes and induce massive deletion of mature T and B cells.

The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both nonactivated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and typical nuclear alterations reached a maximum after 48 h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and camptothecin) and antimetabolites (methotrexate and 5-fluorouracil) that induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the G0-G1 phases of the cell cycle. In agreement with in vitro data, a single i.p. injection of daunorubicin or doxorubicin in BALB/c mice induced T- and B-cell depletion in spleen, lymph nodes, and to a lesser extent in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies, as well as the p55 tumor necrosis factor receptor-immunoglobulin fusion protein, did not inhibit drug-induced apoptosis. The level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in nonactivated lymphocytes. However, antioxidants such as N-acetyl-L-cysteine or glutathione did not prevent apoptosis. Activation of caspase-3 after daunorubicin or doxorubicin treatment of either nonactivated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Finally, daunorubicin and doxorubicin induced a rapid production of ceramides. These data indicate that anthracyclines may induce major peripheral T-cell deletion, a property not shared by many cytotoxic agents.

Rapid binding of beta 2-microglobulin to renal brush-border membranes.

125I-labelled human beta 2-microglobulin binding to rat renal brush-border membranes was assessed by an in vitro assay under near physiological incubation conditions (i.e. low content of albumin). Binding rate was 55 pmol/min per mg protein in the presence of 200 nM of beta 2-microglobulin and degradation rate was negligible versus binding rate. The binding rate was in reasonable agreement with the in vivo reabsorption rate, supporting the hypothesis of proteins binding to the luminal membrane during the process of reabsorption. Mild solubilizing treatment (Triton 0.1%) of brush border after beta 2-microglobulin binding yielded the labelled molecule associated with a high-molecular-weight component. Aminopeptidase activity and binding ability were to a certain extent co-purified during the course of the brush-border preparation, suggesting that most of the beta 2-microglobulin binding sites were localized in the brush-border membranes.

The binding of beta-2-microglobulin to renal brush-border membrane: affinity measurement, inhibition by serum albumin.

In the kidney, filtered proteins are rapidly reabsorbed so that the final excretion is less than 0.1% of the filtered amount for low molecular weight proteins such as beta 2-microglobulin and a few percent for albumin. In order to investigate the affinity of proteins for luminal membranes, rat renal brush-border membranes were incubated with 125I-labelled human beta 2-microglobulin and the initial binding rate determined by the filtration method. Scatchard plot analysis of binding rate revealed two types of binding sites: one with Km = 0.25.10(-6) M and Vmax = 0.1 nmol/min per mg protein and another with Km = 1.10(-5) M and Vmax = 1.3 nmol/min per mg protein. The lower affinity type is likely to represent non-specific binding the physiological role of which is to be discussed. The higher affinity sites seem to play the major role in binding rate. beta 2-Microglobulin initial binding is reversible, and inhibited by bovine serum albumin. Comparison of the time course of bound beta 2-microglobulin removal by unlabelled beta 2-microglobulin and by albumin suggests that these two proteins have a different internalization mechanism.

The topoisomerase inhibitors camptothecin and etoposide induce a CD95-independent apoptosis of activated peripheral lymphocytes.

The effect of etoposide and camptothecin, two topoisomerase inhibitors directed against topoisomerases II and I, respectively, was evaluated on human peripheral blood lymphocytes. Etoposide and camptothecin induced apoptosis of mitogen-activated but not resting CD4+ and CD8+ T lymphocytes. Cell sensitivity to these agents required G1 to S-phase transition of the cell cycle. Conversely, daunorubicin, an intercalating agent and topoisomerase II inhibitor, induced apoptosis of both resting and activated lymphocytes. Although etoposide and camptothecin induced CD95-ligand mRNA expression, drug-induced apoptosis of activated human lymphocytes was not inhibited by CD95 antagonists. Drug-induced cell death was also not inhibited by p55 TNFR-Ig fusion protein. Activation of the caspases cascade was suggested by the partial inhibitory effect of the tripeptide zVAD-fmk and documented by activation of caspase 3. Finally etoposide and camptothecin induced a rapid production of ceramide in activated but not resting peripheral blood lymphocytes, suggesting that ceramide might initiate the signaling apoptotic cascade in sensitive cells.

A1/Bfl-1 expression is restricted to TCR engagement in T lymphocytes.

We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.

Alteration of dealcylated lipopolysaccharide antagonistic properties by interaction with plasma factors.

The acyl poly(1,3)galactoside (APG) from Klebsiella pneumoniae is a bis-acylated lipopolysaccharide (LPS) devoid of ester-linked fatty acids. APG interacts with CD14 and CD11b/CD18 on monocytes. This study addressed the role of serum proteins in the binding and functional properties of APG as a candidate LPS antagonist. In the absence of serum, APG did not induce tumor necrosis factor alpha (TNF-alpha) synthesis by human mononuclear cells and dose-dependently inhibited their activation induced by different LPS. Conversely, in the presence of 5% autologous plasma, APG activated cells and did not antagonize LPS. Serum decreased APG but not LPS binding to monocytes. Binding competition experiments indicated that APG and LPS competed for the same receptors in serum-free conditions but bound to different receptors in the presence of plasma. The data indicate that serum-dependent LPS receptors do contribute to LPS activation of monocytes but do not recognize deacylated LPS analogues.

Activation of human monocyte chemiluminescence response by acylpoly(1,3)galactosides derived from Klebsiella pneumoniae.

The stimulating activity of several preparations isolated from a membrane proteoglycan of a nonencapsulated smooth strain of Klebsiella pneumoniae (Kp-MPG) on the oxidative burst of human blood monocytes was assessed by luminol-enhanced chemiluminescence (CL). Five Kp derivatives were studied: a 34-kd acylpoly(1,3)galactoside (APG), obtained by drastic alkaline hydrolysis and purified by chromatography; an APG preparation subjected to acid hydrolysis that removed the core part and all fatty acids, leaving intact the galactose chain of APG (GC-APG); an APG preparation subjected to mild oxidation (ox APG); a preparation obtained by mild alkaline hydrolysis of Kp-MPG, containing additional ester-linked C14 and C16 fatty acids bound to the APG molecule (EFA-APG); and a polymer of the latter compound, APG pol. EFA-APG directly stimulated monocyte CL, whereas Kp-MPG, APG pol, and the whole bacterial cells had little or no activity. APG itself and ox APG induced a weaker response than EFA-APG. Polymyxin B sulfate completely inhibited the CL response to bacterial lipopolysaccharide (LPS) but not to EFA-APG. The stimulating action of EFA-APG on blood monocytes was dependent on the extracellular levels of both calcium and magnesium. Preincubation of monocytes with monoclonal antibody anti-Mac-1 directed against CD11b, the alpha chain of complement receptor type 3 (CR3; CD11b/CD18), strongly inhibited CL activation by EFA-APG and to a lesser extent CL activation by unopsonized zymosan and rough LPS. Altogether, these findings provide indirect evidence for the contribution of the CD11b/CD18 integrin in the functional interaction of EFA-APG with monocyte membranes. They demonstrate the role of fatty acids in the triggering of monocyte oxidative burst, while the polysaccharide chain itself does not contribute to induction of the CL response in this model. In keeping with the effects of EFA-APG and APG, we show that the monocyte CL response was triggered by bacterial LPS from the rough strain of Salmonella minnesota Re 595 and its lipid A, but not by LPS from smooth strains, again suggesting a critical role for the lipid moiety.

Auto-antibodies specific for beta 2 microglobulin in normal human serum.

Serum components with binding activity towards free human beta 2 microglobulin (beta 2m) were investigated in healthy adults. The binding activity increased after treatment of the serum components by dissociating buffers (acid pH, 2-8 M urea, 3 M NaSCN, 6 M guanidine hydrochloride). This activity resided in serum IgG as shown by the following evidence: (1) recovery in the 160 K region after AcA 44 filtration, (2) association with the IgG fraction after purification by DEAE chromatography and AcA 34 filtration, (3) after immunopurification on beta 2m-Sepharose immunosorbent, the labeled eluted fraction was shown to bind to beta 2m-Sepharose and to protein A or anti-IgG-Sepharose. Pepsin-digested F(ab')2 fragments from serum IgG were treated by 3 M urea, then passed on beta 2m-Sepharose immunosorbent in order to prepare specific anti-beta 2m F(ab')2. Those fragments retained all the beta 2m binding capacity of the IgG fraction. Saturation analysis studies showed estimated K values between 1.5 and 9.5 X 10(9) L/M, depending on the preparation it was concluded that normal human serum contains minute amounts of auto-antibodies of relatively high affinities, specific for beta 2m.

Internalization and degradation of anti-CD4 monoclonal antibodies bound to human peripheral blood lymphocytes.

Treatment of patients with anti-CD4 mAbs induces both functional alterations of CD4+ cells and depletion of circulating CD4+ lymphocytes. Some of these effects depend on the amount of mAb molecules bound per CD4+ cell and on the properties of the Fc part of the mAb (isotype specificity). We have investigated the fate of anti-CD4 monoclonal antibodies (mAbs) after their interaction with CD4 protein on the surface of peripheral blood lymphocytes (PBL). We used seven anti-CD4 mAbs whose epitope specificity, equilibrium constant and kinetics of binding are reported. Lymphocytes were saturated with anti-CD4 mAbs either at +4 degrees C or 37 degrees C then washed and incubated in antibody-free medium. At different time intervals cells were processed for analysis. By indirect immunofluorescence, it was shown that the amount of surface-bound mAb decreased rapidly when cells were incubated at 37 degrees C, but not at 4 degrees C. With 125I-mAbs, we demonstrate that there was a rapid internalization of the molecules followed by the re-expression on the cell surface of a part of initially bound mAbs and by the release of partially degraded antibody in the cell supernatant. In the presence of sodium azide (10 mM) only a slow dissociation of intact antibody occurred, without internalization. The radioactive material eluted in the 100-200 kDa zone from supernatants was only partly adsorbed on protein A and hardly on CD4+ cells, indicating that alterations of the Fc region and loss of antigen binding activity, possibly by formation of CD4-anti-CD4 complexes, had occurred during the process of internalization and release into the extracellular medium. These data may be important to consider for adjusting the dosage of anti-CD4 mAbs to be administered.

Charge heterogeneity of beta 2-microglobulin in lymphoid cells.

Extracts of blood lymphocytes, polymorphonuclear neutrophils and B, T or monocytic cell lines were analyzed by two-dimensional gel electrophoresis and immunoradiometric assay after electro-transfer to nitrocellulose sheets with radiolabelled polyclonal or monoclonal antibodies specific for beta 2-microglobulin. Four different forms of the molecule were identified with an apparent Mr of 12,000 and pI values of 5.7, 5.3 and lower. Lymphocyte activation by phytohemagglutinin and concanavalin A, or incubation with recombinant alpha 2b interferon, resulted in an increased beta 2-microglobulin cell content and release of the protein in supernatants with a predominant elevation of the more acidic minor forms. Recombinant interleukin-2 and recombinant gamma interferon increased the expression of the molecule without significant shift in the relative proportion of beta 2-microglobulin forms. Tumor necrosis factor alpha did not increase cell beta 2-microglobulin (beta 2-m) content and release and did not alter the relative distribution of the different forms of the molecule. Several mechanisms may be considered for the generation of beta 2-m microheterogeneity, including intracytoplasmic post-translational modifications such as proteolysis or modification of the amide groups of internal amino acids.

Complexes of alpha 1-microglobulin and monomeric IgA in multiple myeloma and normal human sera.

alpha 1-Microglobulin (alpha lm), a glycoprotein heterogeneous in charge, was reported to occur both as a 31-kilodalton (kd) monomer [low mol. wt alpha lm (LMW-alpha lm)] and as polymers or complexes formed with other plasma proteins including IgA [high mol. wt alpha lm (HMW-alpha lm)]. The present study was designed to characterize HMW-alpha lm in normal human serum and in myeloma sera. The following sera were selected: five IgG, 16 IgA and four Bence-Jones protein myelomas. alpha lm was identified by specific monoclonal antibodies in competitive radioimmunoassay and solid-phase ELISA. HMW-alpha lm was found to be associated almost exclusively with monomeric IgA and possibly in very small proportion with dimeric IgA. Ever in cases of predominantly dimeric IgA myelomas, alpha lm was associated with the monomeric form of the monoclonal IgA. The molar ratio of HMW-alpha lm to monomeric IgA never exceeded 3.5% and it was estimated to range from 0.5 to 1.4% in normal serum. No association with other proteins than IgA and no alpha lm polymers were found in IgA myeloma. Two types of HMW-alpha lm-IgA complexes were found: (a) those that were dissociable into IgA and LMW-alpha lm after mild reduction, and (b) those which were dissociated only after complete reduction of the complexes into IgA and an 88-90-kd component bearing alpha lm but no IgA epitopes. It was concluded that either of the two molecular species of alpha lm bearing common epitopes, with apparent mol. wts of 31,000 and 88,000-90,000, respectively, could form stable complexes with monomeric IgA. The association is likely to be performed through disulfide bridges. Nearly all the 88-90-kd but only a small proportion of the 31-kd component is associated with IgA.

Acylation of the lipid A region of a Klebsiella pneumoniae LPS controls the alternative pathway activation of human complement.

Two mechanisms of direct activation of the complement system by LPS have been extensively documented: (i) activation of the alternative pathway (AP) by the polysaccharide region, and (ii) activation of the classical pathway (CP) by the lipid A region. Here we demonstrate that LPS from the Klebsiella pneumoniae I-145 strain activates the AP by a mechanism dependent on the acylation of the lipid A region. Cleavage of C3 by K. pneumoniae LPS in EGTA was blocked by polymyxin B. Two 34 kDa derivatives were prepared from a membrane extract of this K. pneumoniae strain: (i) an acyl-poly (1,3) galactoside containing two galactosamine-bound ester-linked and two amide-linked fatty acids (EFA-APG), and (ii) an acyl-poly (1,3) galactoside devoid of ester-linked fatty acids (APG). APG and EFA-APG share the structure of LPS molecules, with a long polysaccharidic chain, a core, and a lipid A region. The AP was activated by EFA-APG but not by APG nor by the isolated polygalactose chain GC-APG, indicating a critical role for ester-linked fatty acids in AP activation. Polymyxin B which binds to the lipid A region of LPS completely inhibited AP activation by EFA-APG. A small part of EFA-APG was shown to form aggregates in saline, but aggregation was not decreased by polymyxin B. Furthermore, APG formed aggregates of similar size although it was not able to activate AP. Therefore the role of lipid A acylation in triggering AP activation is not exclusively mediated by aggregation of the molecule. LPS from the rough strain of Salmonella minnesota (Sm Re LPS) directly activates the CP but not the AP. However, when mixed with the polygalactose chain GC-APG, Sm Re LPS activated the AP. The data demonstrate a cooperation between the lipid A region and the polysaccharidic chain in activation of the AP. Similar cooperation may occur with other LPS molecules.

Apoptosis without decrease of cell DNA content.

Apoptosis of human B cells and murine T and B cells was analyzed by DNA agarose gel electrophoresis, clamped homogeneous electric field, measurement of cell DNA content by flow cytometry, transmission electron microscopy and by UV microscopy. Apoptosis was induced by etoposide (an inhibitor of topoisomerase II), by the calcium ionophore ionomycin or by cross-linking of membrane immunoglobulins (Ig) with anti-Ig-antibodies. Two types of apoptosis could be defined. Apoptosis resulting in small DNA fragments (180-200 base pairs and multiples thereof) was associated with a typical 'ladder' in agarose gel electrophoresis and a decrease in cell DNA content assessed by flow cytometry. Conversely apoptosis with large DNA fragments (100-150 kilobase pairs) was only demonstrated by clamped homogeneous electric field but was not associated with decreased cell DNA content or the observation of DNA ladders. Nuclear condensation without fragmentation was more frequent when apoptosis generated large DNA fragments. The type of apoptosis appears to be an intrinsic property of each cell type.

Role for protein kinase C activation in IgA B cell terminal differentiation.

In the present study, we demonstrate that the PKC-activating phorbol ester PMA selectively induced IgA synthesis by PP B cells. PKC activation triggered neither B cell proliferation nor the switching rate of IgA- to IgA+ cells. Together with the fact that the rate of IgA secretion by the myeloma cell line MOPC 315 was not altered by PMA, the data demonstrate that activation of PKC enhances IgA secretion by promoting terminal differentiation of IgA-committed B cells into IgA-secreting plasma cells.

Polymeric Ig receptor expression in hepatocellular carcinoma.

The cellular localisation of the polymeric Ig receptor (pIg-R) and carcinoembryonic antigen (CEA), hepatic and biliary cell markers, were investigated in patients with hepatocellular carcinoma (HCC) and high serum levels of secretory component. Serum SC were increased 6-20-fold in 8 HCC patients compared with normal subjects. Serum free SC was positively correlated bilirubin (r = 0.95, P less than 0.04). In normal liver tissue, cytokeratin (CK) 8 and 18 were localised in hepatocytes and biliary cells while pIg-R and CK 19 expression was restricted to biliary cells. In tumoral liver tissue, malignant cells expressed CK 8 and 18 weakly; pIg-R and CK 19 were not detected in tumoral cells. CEA was expressed by biliary cells in normal and proliferating ducts. In peritumoral fibrosis, proliferating biliary cells were strongly stained by anti-cytokeratins and anti-pIg-R antibodies. In one case, pIg-R was localised in isolated cells close to fibrosis without co-staining of anti-CK 19. Thus increased serum SC is not associated with pIg-R expression by tumoral cells, and pIg-R may be considered an additional marker of biliary cells. High SC might be explained either by reflux from bile to serum and/or release of unbound SC from the vascular pole of non-functional, proliferating biliary structures.

Sandwich-type ELISA for free and bound secretory component in human biological fluids.

Three sandwich-type enzyme-linked immunosorbent assays (ELISA) are described for the measurement of free secretory component (SC) and SC bound to IgA (S-IgA) or IgM (S-IgM). These assays do not require preliminary fractionation of the biological fluids to be tested. The specificity of the assays is achieved with monoclonal antibodies specific for free SC (855 SC) and for SC bound to IgA or IgM (8545 SA). The amount of the three SC molecules in various biological fluids is reported. We demonstrate the presence of low levels of free SC in most of these fluids, including normal serum. Moreover our results suggest that S-IgM in serum may result from a non-covalent association between serum IgM and free SC.

Comparison of radio-immunoassay and lymphocytotoxicity inhibition techniques for the determination of beta2 microglobulin.

Rabbit anti-human beta2 microglobulin antisera can lyse human lymphocytes in the presence of rabbit complement. Inhibition of the lymphocytotoxic reaction by highly purified beta2m was applied to the measurement of beta2m concentration in biological fluids. Parallel determinations were also performed using a radioimmunoassay. Lymphocytotoxicity inhibition is simple and more sensitive than radial immunodiffusion but less sensitive than the radioimmunoassay. beta2m was measured by these two techniques in serum and urine from normal individuals, uremic or transplanted patients.