Preparative electrophoresis of human lymphocytes. II. Use of non-immunoglobulin bearing lymphocytes obtained by electrophoretic levitation for the elicitation of a rabbit anti-human "T-cell" serum.

By intradermal injection (in Freund's complete adjuvant) of rabbits with only a few million human T-lymphocytes, obtained by preparative electrophoresis, an anti-human "T-cell" serum can be obtained. Although the antiserum is best used in a fairly concentrated form, its anti-human "T-cell" specificity (by immunofluorescence and E-rosetting) appears excellent, without the need to resort to absorption with various unwanted antigens. There may however, also be other specificities in the antiserum, as it also reacts with human granulocytes.     

Precipitin reactions of anti-human myoglobin serum with several human and animal muscle extracts

Myoglobin content of several human muscles was determined immunologically. The content of cardiac and skeletal muscle was similar. There was no antigenic difference noted between cardiac and skeletal muscle myoglobin. Uterine smooth muscle contained no myoglobin.

Details of the precipitin reaction between human myoglobin and its antiserum are presented. There may be approximately three regions on the human myoglobin molecule which acted as antigenic sites in a rabbit.

Several animal muscle extracts cross-reacted with the anti-human myoglobin. All primate skeletal muscle samples tested were equally potent, while beef, rodent and chicken thigh muscle extracts were poorer cross-reactors. It is postulated that these differences were due to differences in the myoglobin molecules of these species. Chicken breast muscle did not cross-react, and appeared to be deficient in myoglobin.

     

Reactivity of anti-HLA sera against mouse lymphocytes.

The reactivity of anti-HLA hyper-immune sera was tested by the microlymphocytotoxicity technique (LCT) against a panel of mouse lymphocytes. Three levels of reactivity were observed: negative (immune sera titre 1:20 or less), weak (titre 1:40 to 1:80 with a percentage of dead cells less than 50%) or strong (titre 1:60 or more with 100% killing). Thirteen normal human sera were non-reactive. Eleven out of 60 hyper-immune sera were strongly reactive. Tests using congenic lines showed that the reactivity was controlled at the H-2 complex. One serum (CODRON) was studied in detail. When tested against a panel of strains carrying 10 different H-2 haplotypes, it reacted strongly against lymphocytes H-2d, ja, k, p and r; and did not react, or at least only weakly, against lymphocytes H-2b, f, q, s and v. Tests using mouse strains carrying the recombinant H-2 haplotypes h4, i5, i, y2, g, g2 and t1, suggested that the observed reactivity was directed against structures controlled at the K end of the H-2 complex (H-2.47?). Absorption-elution experiments with human and murine lymphocytes and platelets confirmed that the structures recognised by serum CODRON were determined at the major histocompatibility complex.     

Reactivity of monoclonal antibodies against human leucocyte antigens with lymphocytes of non-human primate origin

The phylogenetic distribution of antigens present on human lymphocytes was investigated by incubating human or simian cells with murine anti-human monoclonal antibodies and then determining the level of reactivity with a radiolabelled anti-murine IgG reagent. The monoclonal antibodies used were specific for a T-cell antigen, lymphoid and lymphoid:myeloid antigens, Ia antigens, and beta 2 microglobulin. The cells examined included B- and T-lymphoblastoid cell lines and fresh peripheral blood lymphocytes separated by sheep erythrocyte rosetting into T-cell and non T-cell fractions. Results of these studies showed that the antibodies gave complete cross-reactivity with gorilla and chimpanzee cells while B-cell lines of orangutan origin had lost lymphoid and beta 2 microglobulin markers. Gibbon cells and cells of Old World and New World monkeys reacted strongly only with monoclonal antibodies against Ia antigenic determinants. These Ia antigens were found on the non T-cell fraction of fresh peripheral lymphocytes, on B-cell lines and on some virus induced T-cell tumour lines. Immunoprecipitation analysis using the anti-Ia antibodies showed a degree of molecular diversity on owl monkey and marmoset cells compared to the Ia antigens associated with human cells.     

Reactivity of antibodies in human serum with antigens of an enteropathogenic bovine coronavirus

Antibodies in human serum against an enteropathogenic bovine coronavirus were detected by double immunodiffusion (DID), neutralization of infectivity, indirect immunofluorescence, and immune electron microscopy. Human sera reacting in the DID test neutralized the infectivity of the bovine coronaviruses to indices of 2.5 to > 5. Nineteen of 40 DID-negative, heat-inactivated sera had neutralizing indices of 1 to 3.0. Human serum with neutralizing and DID antibodies produced juxtanuclear and cytoplasmic fluorescence identical to that of bovine immune serum in cells infected with the bovine coronavirus. Antibodies in human and bovine sera interacted with the peplomeres of the bovine coronavirus, matting and bridging them, when present in excess, and facilitated formation of large viral aggregates when present in equivalent concentrations. Complement added to the virus-antibody complexes did not alter specifically the morphology of single, antibody-laden viral particles or viral particles in aggregates. Evidence of the transmission of coronavirus from experimentally inoculated calves to man, with ensuing gastroenteritis, was found by electron microscopic tracing of the coronavirus and its virus-antibody complexes.     

Reactivity of anti-human C-reactive protein (CRP) and serum amyloid P component (SAP) monoclonal antibodies with limulin and pentraxins of other species.

Limulus polyphemus C-reactive protein (CRP) (limulin) has approximately 30% amino acid sequence homology and shares at least one idiotypic determinant associated with ligand-binding activity with human CRP (hCRP); limulin also shares amino acid sequence homology and lectin activity with human serum amyloid P component (hSAP). In the present study panels of 14 anti-hCRP monoclonal antibodies (mAb) directed to distinct hCRP epitopes and 11 anti-hSAP mAb directed to distinct epitopes of hSAP were tested for reactivity with limulin and pentraxins of other species including rabbit CRP (raCRP), rat CRP and hamster female protein (FP) by ELISA and Western blot analyses. None of the anti-human pentraxin mAb showed strong cross-reactivity with limulin; only five mAb reacted with limulin at all, and cross-reactivities of these mAb with the other pentraxins, when present, also were weak. Cross-reactivity of limulin with hCRP and hSAP was similar, and in light of comparable amino acid sequence homology, suggests this molecule can be considered the limulus SAP as well as the limulus CRP. Several anti-hCRP mAb cross-reacted strongly with rabbit CRP and rat CRP; a few anti-hSAP cross-reacted strongly with FP; and weak cross-reactions were observed between hCRP and hSAP, but cross-reactivities between the pentraxins generally were limited and weak. A rabbit polyclonal antibody raised to highly conserved limulin peptide 141-156 and strongly reactive with limulin reacted weakly with hCRP and raCRP but failed to react with rat CRP, hSAP or FP. These studies emphasize a limited but distinct antigenic similarity between limulin, hCRP and other pentraxins, and identify mAb reactive with potential regions of shared structure and/or function between pentraxins of different species.     

Reactivity of human monoclonal IgM with nerve glycosphingolipids.

We examined the reactivity of monoclonal IgM of sera from patients with neuropathy and monoclonal IgM, with or without antibody activity to myelin-associated glycoprotein (MAG), as well as sera from non-neurologic patients with Waldenström's macroglobulinaemia, with various nerve glycolipids extracts or with purified gangliosides. As expected from previous studies, all (five cases) anti-MAG IgM stained two glycolipids, the chemical characteristics of which corresponded to sulphated glucuronyl-paragloboside (SGPG) and sulphated glucuronyl-lactosaminyl-paragloboside (SGLPG). Five of 12 sera from patients with neuropathy whose IgM was devoid of anti-MAG reactivity stained nerve extracts greatly enriched (98%) with SGPG and SGLPG. Three of these five sera reacted with additional glycolipids and/or gangliosides. Two of 16 sera from patients with macroglobulinaemia without neuropathy reacted strongly with both SGPG and SGLPG. The latter finding as well as the detection of low titre of anti-sphingolipid antibodies in normal sera may cast a doubt on the pathogenetic significance of this antibody activity.     

Effect of complement inactivated rabbit antichick brain serum on thymic and splenic lymphocytes.

The effects on in vitro exposure of chicken thymic-dependent lymphocytes to complement inactivated rabbit antichick brain serum (RACB) previously absorbed with normal serum (ns) and kidney (k) [RACBns,k] was investigated. Spleen lymphocytes which normally initiate a 'graft vs. host' reaction give rise to much more intense reactions when treated with (RACB)ns,k. This may be due to increased antigenicity of the donor cell and/or stimulation of the donor T cell. Migration studies showed that chicken thymocytes are capable of in vitro migration which can be inhibited by exposure to (RACB)ns,k. Thymocytes pretreated with (RACB)ns,k were found to express factor(s) capable of inhibiting thymocyte migration. The correlation between lymphokines and increased splenomegaly was discussed.     

Reactivity of presumed anti-natural killer cell antibody Leu 7 with intrafollicular T lymphocytes.

This report describes the presence of a T lymphocyte subpopulation in germinal centres of lymph follicles. This subpopulation is defined by reactivity with Leu 7 antibody, in addition to OKT11, OKT1, OKT3 and OKT4 positivity. The functional activity of this T lymphocyte subpopulation is a matter of discussion and has to be clarified by functional studies of purified populations of these cells.     

Stimulation of pig lymphocytes with anti-immunoglobulin serum and mitogens.

Mitogenic stimulation of pig lymphocytes by anti-immunoglobulin serum and by Con A, PHA, PWM and LPS has been measured by 3H-thymidine incorporation. Lymphocytes from blood, spleen and lymph node were readily stimulated by antiglobulin. Specific anti-micron serum was mitogenic for blood lymphocytes, indicating that those B cells with surface IgM can be stimulated by complexing of this surface component with antibody. An initial 2-hour incubation with anti-Ig was as effective as continuous incubation. Differentiation of stimulated cells into high rate synthesising plasmablasts was not observed. Pig blood lymphocytes were reactive to Con A, PHA, PWM and LPS. LPS was also tested against spleen and lymph node cells, which were responsive to this mitogen.     

Cellular cooperation in lymphocyte activation. II. Cooperative response of human peripheral T and B lymphocytes to rabbit anti-human beta 2-microglobulin.

In the present study we attempted to clarify the effects of anti-beta 2-microglobulin (a-beta 2m) on lymphocyte activation. Neither a-beta 2m IgG fraction nor F(ab')2 had a mitogenic effect on either highly purified T or B lymphocytes alone, while their mitogenic effect was observed when T and B lymphocytes were appropriately reconstituted. When T lymphocytes were reconstituted with mitomycin C (MMC) treated B lymphocytes, a negligible decrease in the response to a-beta 2m was observed compared to the response of an untreated mixture to a-beta 2m. On the other hand, when B lymphocytes were reconstituted with MMC-treated T lymphocytes, the response was markedly diminished. It was found, moreover, that the response of T lymphocytes separated by a semipermeable membrane from MMC-treated B lymphocytes was not enhanced, while a mixture of T and MMC-treated B lymphocytes in the same chamber showed a marked response. These results lead to the conclusion that the cells responding to a-beta 2m are mainly T lymphocytes whose response is strongly enhanced by B lymphocytes, and that for the mitogenic effect of a-beta 2m direct cell-to-cell interaction between T and B lymphocytes is necessary.     

Effects of serum amyloid P component on human lymphocytes

The P component of amyloid is a normal serum protein designated SAP. SAP has substantial homology with C-reactive protein (CRP). Recent studies have established the structure, tissue distribution and binding reactivities of SAP; however, as yet, very minimal insight into its function has been achieved. Recent studies, though somewhat controversial, have suggested a regulatory role for CRP on the immune response. In view of these studies, we wanted to evaluate the in vitro effects of SAP on several immunological properties of human lymphocytes. We found that SAP had a marked inhibitory effect on the proliferative response of lymphocytes to a variety of T-dependent mitogens. In addition, SAP markedly enhanced the formation of active E rosettes, a marker of activated T cells.     

In vivo effects of rabbit anti-mouse brain serum on theta bearing lymphocytes of AKR mice.

The in vivo effect of rabbit anti-AKR mouse brain-associated serum (RAMB) was determined on theta bearing lymphocytes present in the spleens and thymuses of mature C3H mice and AKR mice staged into preleukemic, leukaemic and overtly leukaemic states.Following seven daily injections of RAMB serum, the splenic plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) and the percentage of theta-bearing lymphocytes in the spleen were significantly decreased in the C3H and the preleukaemic AKR mice. Decreases in thymic weight and thymocyte numbers were also apparent. Determinations of theta antigen density using in vitro cytoxicity tests indicated that splenic and thymic T lymphocytes (thymus-derived) remaining in the RAMB-treated C3H and preleukemic AKR mice consisted primarily of cells bearing less of the theta surface antigen. Histopathological studies of tissues from these two treated groups revealed cortical lymphocyte depletion in the thymus, and marginal and periarteriolar depletion in the spleen. Leukaemic AKR mice, administered seven injections of RAMB serum, demonstrated less dramatic changes in thymus weight, histopathology and theta-bearing cell percentages when compared with the data from the preleukaemic AKR or matureC3H mice. The results from testing overtly luekaemic AKR mice administered RAMB serum for the 7 or 9 days did not demonstrate differences from findings from groupsof overtly leukaemic control mice. These data indicate that the in vivo activity of RAMB serum in C3H mice and preleukaemic AKR mice is directed primarily toward the less mature T-lymphocyte population. This influence of RAMB serum is lesspronounced in leukaemic and the more overtly leukaemic mice, suggesting that a decreasedpopulation of RAMB-susceptible lymphocytes are present in these animals.     

Phospholipase C-labeled anti-human IgG: inhibition by human IgF.

We report here the inhibition of enzyme activity of phospholipase C-anti-human rabbit IgG conjugate complexed with human IgG. Phospholipase C activity was measured by assaying the release of hemoglobin from erythrocytes. The degree of inhibition varies depending on the relative amount of IgG utilized. A water soluble carbodiimide linking yields a conjugate which is inhibited by IgG, while conjugate prepared by glutaraldehyde is not. Immunodiagnostic assays requiring detection of low levels of antigen or antibody may be possible utilizing the phospholipase C-erythrocyte technique.     

Interaction of pertussis toxin with human T lymphocytes.

The binding of pertussis toxin (PT) to the human T-cell line Jurkat was examined by using flow cytometry. Fluorescein isothiocyanate (FITC)-labeled PT bound rapidly to the cells in a specific manner as determined by blocking experiments with unlabeled toxin, B oligomer, and the S2-S4 and S3-S4 dimers. Monoclonal antibodies against the S3 subunit of the toxin also significantly inhibited the binding of FITC-PT. Sialidase treatment of the cells resulted in decreased binding of FITC-PT, indicating that sialic acid residues are involved in the binding process. In addition, we studied the effect of PT binding on the expression of cell surface molecules. On binding of PT to the cell surface, a rapid down-regulation of the T-cell receptor (TCR)-CD3 complex was observed. The modulation of the TCR-CD3 complex was independent of the toxin's enzymatic activity, as the B oligomer and a nonenzymatic toxin mutant induced modulation comparable to that caused by the native holotoxin. Isolated dimers did not cause down-regulation. Stimulation of the TCR-CD3 complex, leading to reduced cell surface expression of this complex, provides a possible explanation for the second messenger production associated with the interaction of PT or B oligomer with T lymphocytes. We therefore conclude that PT activates T cells by divalent binding to the TCR-CD3 complex itself or by binding a structure closely associated with it.     

Identification of koala T lymphocytes using an anti-human CD3 antibody

Previous attempts to identify T lymphocytes in the koala using cross reacting monoclonal antibodies (mAbs) against other species T cell antigens (Ags) and classical T cell lectins have proved unsuccessful. Recently a polyclonal rabbit Ab preparation directed at the epsilon chain of the human CD3 complex (anti-CD3) has become commercially available and has been shown to have broad cross-species reactivity. We have demonstrated that this anti-CD3 is suitable for labelling T cells from peripheral blood of koalas if the purified peripheral mononuclear cells (PMC) are first made permeable with mild fixation in buffered formal acetone. We have also been able to identify koala T cells in both formalin-fixed and frozen lymphoid tissues. Immunoprecipitation and Western blotting studies demonstrated that this anti-CD3 bound to a single 23 kDa polypeptide, probably representing the koala homologue of the human epsilon chain. This is the first report of successful identification of koala T cells and the first reported use of this anti-CD3 for the identification of peripheral circulating T lymphocytes in any species.     

Effect of rabbit anti-human B-cell antigens on the response of lymphocytes stimulated by blastogenic factor.

It has been shown that specific antisera to B-cell determinants can block stimulation in the human mixed lymphocyte reaction. Therefore, it is of interest to study the effect of anti-human B-cell serum on blastogenic activities of the cell-free culture medium (CFM) derived from cultures of human blood lymphocytes. B-cell antigen was prepared from human B-cell line as a glycoprotein complex of mol. wt 27,000 and 33,000. Rabbit antisera to the B-cell antigen after absorption with human platelets or T-cell line (MOLT 4) was shown to react only against B cells but not T cells. The antisera suppressed human mixed lymphocyte reaction but did not affect the response of lymphocytes to PHA. The proliferative response of T-cell enriched population induced by blastogenic factor from lyphocyte cultures was markedly suppressed by the antisera. The inhibited reactivity irrespective of the source (autologous, allogeneic, mixed, T or B cells) of CFM. This is compatible with an effect caused by their interactions with the responding cell rather than blastogenic factor in the CFM. The results of the kinetic experiments suggest that addition of the antiserum at intervals after initiation of the culture only prevents the CFM stimulation of the responder cells that have not yet become committed to divide.