Studies on the cytolytic attack mechanism of the cytotoxic T lymphocyte (CTL): preparation of antisera against cellfree cytosolic extracts of a CTL clone capable of blocking the lethal hit stage of CTL cytolysis and analysis of the cytolytic structure

Rat antiserum (as well as purified IgG and F(ab')2 fragments) raised against cellfree cytosolic extracts (CFE) of an alloimmune cytotoxic T lymphocyte (CTL) clone (B6.1.SF.1) is a potent inhibitor of CTL-mediated cytotoxicity. Inhibition by this antiserum (termed alpha CTLL) occurred during the postbinding lethal hit stages of cytolysis, because it did not inhibit target cell binding, nor did it prematurely dissociate CTL-target cell conjugates; inhibition was observed regardless of the H-2 haplotype of the target cell or CTL employed; inhibition was reversible when pretreated, and washed CTL were used as effectors; and in Ca++ pulse experiments alpha CTLL inhibited cytolysis beyond the Ca++-dependent (lethal hit) stage of cytolysis. This antiserum did not inhibit lysis of P815 cells by activated murine macrophages or by human cytotoxic cells, and extensive absorption of the antiserum on viable thymocytes, normal spleen cells, or CTL did not reduce its blocking activity. CFE prepared from several sources of CTL, including in vivo elicited peritoneal exudate lymphocytes (PEL), secondary MLC-generated CTL, alloimmune splenic T cells, and CTL clones, contained material(s) that inhibited the ability of alpha CTLL to block CTL-mediated cytolysis. The inhibitory activity was not detected in CFE from a variety of noncytotoxic cell sources, including thymocytes, normal C57BL/6 spleen cells, EL4 or P815 tumor cells, macrophages, and helper T cell clones. It was also absent in CFE prepared from human CTL cells. Furthermore, although alpha CTLL neutralizing activity was not detectable in CFE prepared from memory CTL, it rapidly appeared in CTL parallel to the development of cytolytic activity during secondary MLC cultures. The inhibitory material in CTL-CFE appeared to be specific for alpha CTLL antibody, as it did not affect the CTL blocking activity of anti-Lyt-2 or anti-target cell antisera. Finally, CTL-CFE did not contain proteases that degraded the alpha CTLL antibody. By the use of a soluble-phase immunoabsorbent assay, the biochemical properties of materials present CFE derived from CTL and reactive with alpha CTLL antibody were examined. CTL cytosolic material(s) reactive with alpha CTLL IgG was unstable to brief heating (50 degrees C) or acidic pH, but not to high ionic strength buffers. The material was inactivated by treatment with pronase but not by DNase, collagenase, or trypsin. Gel filtration chromatography of CTL-CFE revealed multiple peaks of alpha CTLL neutralizing activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of human lymphocyte-mediated mitogen-induced cytotoxicity of murine L-929 cells by heterologous anti-human lymphotoxin antisera in vitro.

Heterologous anti-human lymphotoxin (LT) antisera have been employed to investigate the role of LT in mitogen-(Con-A, PHA) induced destruction of murine L-929 cells by human lymphocytes in vitro. These various antisera will effectively neutralize human LT molecules associated with the stable (70 to 90,000 dalton) alpha-LT class of cytotoxin (anti-alpha-LT), the more unstable (35 to 50,000 dalton) beta-LT class of cytotoxins (anti-beta-LT), and antisera which will neutralize all classes of these cytotoxins in vitro, anti-whole supernatant (anti-W.S.). These anti-LT sera will greatly inhibit lysis of L-929 cells by using mitogen-activated human effector lymphocytes in vitro. This blocking was shown to be mediated by whole serum, purified IgG, or IgG-Fab fragments, which had been extensively absorbed with bovine serum, human serum, mitogens, and normal human lymphocytes. Inhibition of lysis was not apparently due to interference with either lymphocyte-target cell contact or lymphocyte activation step(s). The blocking effects of these sera were also shown to occur during the lymphocyte-independent phase of the lytic reaction. These data support the concept that the lymphocyte deposits an LT-like effector molecule on the target-L cell surface during the lymphocyte-dependent phase, which mediates cell lysis at a later time during the lymphocyte-independent phase.     

Specificities of human heterophile Hanganutziu and Deicher (HD) antibodies to glycosphingolipids and a glycoprotein

The specificities of five heterophile Hanganutziu and Deicher (HD) antibody-containing sera from four different cancer patients and one other diseased patients were compared. Three glycosphingolipids and one glycoprotein antigens and their chemically modified derivatives were used. The antibodies of all whole sera showed similar specificities. IgG and IgM antibody fractions of each serum were separated. Although antibodies of the same class showed similar specificities, differences were detected between the specificities of IgG and IgM. IgG antibody specificities were dependent on the hydrophobic (ceramide) group while IgM antibodies were directed more to the terminal sialic acid moiety of the glycosphingolipid antigens. The results suggested that a similar population of IgG-producing lymphocytes is stimulated in patients. Due to the similarities in specificities of HD antibodies, the results of this study will facilitate the future isolation of either IgG or IgM antibody-producing lymphocyte(s) from a patient with HD antibodies and the establishment of a monoclonal antibody through hybridization with a human myeloma cell line.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

The role of lymphotoxin in target cell destruction induced by mitogen-activated human lymphocytes in vitro. II. The correlation of temperature and trypsin-sensitive phases of lymphotoxin-induced and lymphocyte-mediated cytotoxicity.

The in vitro destruction of phytohemagglutinin (PHA) coated Beta L cells by non-immune human lymphocytes was resolved into two distinct phases--lymphocyte dependent and lymphocyte independent. The initial or lymphocyte-dependent phase occurred within the first 2 hr and proceeded equally well at 34 and 37 degrees C. The amount of lymphotoxin (LT) secreted by PHA-activated human lymphocytes in vitro to PHA stimulation was the same at 34 and 37 degrees C. Antiserum and complement inactivation of the aggressor lymphocytes at various intervals revealed that target cell lysis was lymphocyte independent. However, the latter phase was temperature dependent, i.e., proceeding at the permissive temperature of 37 degrees C, but inhibited at the restrictive temperature of 34 degrees C. Further experiments revealed that LT-induced destruction had the same temperature sensitivity as target cell cytolysis occurring during the lymphocyte-independent step. Trypsin treatment of target cells during an early period of the lymphocyte-independent phase protected the target cell from subsequent death, indicating the aggressor lymphocyte has deposited a cytotoxic effector material on its surface. These results suggest the lymphocyte-dependent stage involves the processes required for the induction of LT synthesis and secretion. The actual cytolysis occurring during the lymphocyte-independent stage may be caused by LT or LT-like material(s) deposited on the target cell surface by the mitogen-activated human lymphocyte.     

Sources of bovine lymphocyte antigen (BoLA) typing reagents*

Sera from about 1000 cows were tested for cytotoxicity against a panel of up to 100 lymphocyte samples. Cytotoxic antibodies presumably resulting from trans-placental immunization of the cow by her calf were found in about 45% of these sera. The antibody titers of sera from parous cows rarely exceed 4², some persisted for over one year, but decreased notably at calving. Thirty-five immune sera were also produced by alloimmunization with lymphocytes. They usually reached peak titers of up to 4⁴ at 2 or 3 weeks after the initial immunization. Subsequent immunizations produced sera with very high titers but they were much more polyspecific. High-titered antibodies were also produced by skin graft recipients. Useful cytotoxic antibodies were found in 19 of 111 colostrum whey samples. Studies on 13 dam-calf pairs showed that the newborn calf may acquire cytotoxic antibodies from its mother's colostrum, but the only cytotoxic antibodies detectable in this calf s serum are those not directed against its own lymphocyte antigens. It is concluded that efficient lymphocyte typing requires antibodies from a variety of sources.     

In vitro depression of cellular immunity by Friend virus leukemic spleen cells.

Four distinct sublines of mouse L 929 cells (termed alpha, beta, gamma, and delta) were derived and shown to differ markedly in their in vitro sensitivity to human lymphotoxin (LT). The alpha L cell is most sensitive and is rapidly destroyed by very low dilutions of LT. This cell is 100 times more sensitive to LT than the most resistant (delta) L cell. The highly lymphotoxin-sensitive alpha cell makes it possible to reproducibly detect LT activity in as little as 0.0005 ml of supernatant medium. Additional studies revealed a direct correlation between the sensitivities of the four L cell sublines to LT and to direct cytolysis mediated by mitogen-stimulated human lymphocytes. The alpha, beta, gamma, and delta L cells were shown to be equally sensitive to antibody-mediated complement-dependent lysis, indicating that the sequence of sensitivities of these L cell sublines to the direct lymphocyte and to LT does not merely reflect a general susceptibility to cell destruction. These results lend further support to the view that lymphotoxin is an important mediator of in vitro target cell destruction by human effector lymphocytes.     

A comparison of the role of protein synthesis in cell-mediated cytotoxic reactions.

Pactamycin, an irreversible inhibitor of protein synthesis, was employed to investigate the requirement for protein synthesis during in vitro cell-mediated cytotoxic reactions. The cellular reactions examined included direct cell-mediated cytolysis (DCMC) of EL-4 tumor cells by alloimmune lymphocytes, antibody-dependent cell-mediated cytolysis of HEp-2 tumor cells (ADCC-T), and antibody-dependent cell-mediated cytolysis of chicken erythrocytes (CRBC) (ADCC-E). Pretreatment of alloimmune lymphocytes with pactamycin (PAC) did not alter the DCMC reactivity osf the effector cells even though protein synthesis was inhibited by >90%. Similarly, inhibition of protein synthesis followed by 6 hr of in vitro incubation prior to the assay did not significantly reduce reactivity. Pretreatment of normal lymphocytes failed to inhibit cytotoxic reactivity when employed in an ADCC assay against HEp-2 cells, but produced partial inhibition of ADCC reactivity against CRBC. Incubation following PAC treatment had no effect on ADCC-T, but abrogated all ADCC-E activity within 3 hr. The data presented indicates that the effector cells mediating ADCC-E and those mediating both ADCC-T and DCMC differ markedly in their requirements for continued protein synthesis.     

The human LT system. II. Immunological relationships of LT molecules released by mitogen activated human lymphocytes in vitro.

Materials with LT activity present in supernatants from PHA stimulated human lymphocytes in vitro are very heterogeneous and can be separated into multiple molecular weight classes, termed complex, α, β, and γ. Several of these classes can be further resolved into subclasses by other physical and chemical methods. The immunologic relationships of these materials one to another were examined employing various rabbit anti-humn LT sera which will neutralize LT activity on L-929 cells in vitro. These studies reveal: (a) LT activities are due to a distinct group of substances which are immunologically related one to another and can exist in several molecular weight forms; (b) a high MW class of molecules, termed complex, appears to contain all currently known LT classes and subclasses; (c) LT classes and subclasses both have common (public) and discrete (private) antigenic specificities; (d) human LT classes and subclasses do not appear to share Ag determinants with materials with LT activity released by lectin stimulated lymphoid cells from rabbit, rat, hamster, guinea pig, or mouse; and (e) human LT molecules are not immunologically related to cell toxins released by glass adherent human peripheral blood monocytes or PMN cells. These data indicate human LT molecules form a “discrete system” of lymphocyte derived cell toxins, which can associate together into various related but different MW forms in the supernatant.     

Immunodiagnosis of angiostrongyliasis with monoclonal antibodies recognizing a circulating antigen of mol. wt 91,000 from Angiostrongylus cantonensis

In the analysis of excretory-secretory (ES) antigens from infective third-stage larvae (L3) of Angiostrongylus cantonensis, one major component of mol.wt 91,000 was not precipitated by pooled sera of patients with eosinophilic meningoencephalitis. Monoclonal antibodies (Mc Ab) secreted from two hybridoma cell lines, established against somatic antigens of L3, recognized this molecule but with different epitope specificities indicated by an additivity index (A.I.) of 83%. The 2 Mc Ab (TD2 and 3A5) belonged to IgG2a and IgM classes, respectively. Combinations of TD2 and 3A5 were used in a sensitive enzyme-linked fluorescent assay (ELFA) for the immunodiagnosis of human angiostrongyliasis. The double-antibody sandwich ELFA method was applied firstly to sera from experimentally infected rats using either TD2 or 3A5 to coat the assay plates. Two fluorescence unit (F.U.) peaks appeared in sera from infected rats collected 18 and 44 days after infection. Specimens from 35 patients were tested, all cerebrospinal fluids (CSF) and most sera (88%) showed positive reactions and the average F.U. of CSF was greater than that of serum.     

Characterization of warm-reactive IgG anti-lymphocyte antibodies in systemic lupus erythematosus. Relative specificity for mitogen-activated T cells and their soluble products

In addition to previously described cold-reactive IgM anti-lymphocyte antibodies maximally cytotoxic for resting cells at 15 degrees C, sera from patients with systemic lupus erythematosus (SLE) were found to contain a new type of antibody preferentially reactive at physiologic temperatures with mitogen-activated lymphocytes. This antibody lacked specificity for unstimulated lymphocytes, and was shown to be of the IgG class both by indirect immunofluorescence and in immunochemical experiments. Certain SLE sera also contained IgG antibodies with the capacity to develop plaques with mitogen-activated T lymphocyte preparations used in a reverse hemolytic plaque assay, indicating reactivity with products released by activated cells. The elimination of the ability of SLE sera to develop plaques after absorption with viable mitogen-stimulated lymphocytes, but not with resting cells, suggested that these antibodies were directed toward activation "neoantigen(s)" shed from the cell surface membrane. Surface membrane phenotype analyses performed by using a variety of monoclonal antibody reagents indicated that the plaque-forming cells (PFC) detected with SLE sera were activated T lymphocytes not restricted to single OKT4+, OKT8+, or Ia antigen+ subpopulations. Essentially all PFC expressed transferrin receptors. The present data raise the possibility that certain of the interesting effects of anti-lymphocyte antibodies on immunologic function in SLE may be mediated by interactions of these new type(s) of antibodies with activated lymphocytes or their products, rather than through blocking or depletion effects on resting precursor cells.     

The in vitro antibody response to cell surface antigens. I. The xenogeneic response to human leukemia cells.

An in vitro spleen fragment culture system has been developed for the production and analysis of xenogeneic antibody responses to cell surface antigens. Depending on the methods of immunization and in vitro stimulation employed, mouse spleen fragments can produce antibody of both IgG and IgM classes directed against human cell surface antigens for more than 30 days in culture. A saturation binding analysis of the antibody products indicates that their range of specificities was more restricted than that of serum antibody. Approximately 5% of the in vitro antibody products raised against a homogeneous population of human leukemia cells could distinguish between the antigens present on the leukemia cells and those present on normal human lymphocytes. Methods previously employed to influence the range of serum antibodies expressed against complex immunogens, such as suppression of certain responses by passive administration of antibody at the time of immunization, were tested in the in vitro spleen culture system and resulted in successful modulation of the antibody response patterns observed.     

Evaluating the A-Subunit of the Heat-Labile Toxin (LT) As an Immunogen and a Protective Antigen Against Enterotoxigenic Escherichia coli (ETEC)

Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit’s potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines against ETEC.     

A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus

Peripheral blood lymphocytes of rabbits immunized with live rubella vaccine respond to rubella virus antigens in tissue culture with increased DNA synthesis as measured by incorporation of ³H-thymidine. This reaction can be inhibited by rubella antibody. A dose dependent effect was observed when antibodies in whole serum were mixed with virus prior to addition to lymphocyte cultures. When antisera were fractionated and their individual immunoglobulins tested, a paradoxical effect was obtained. Immune IgG although it was highly effective in neutralizing the virus was incapable of inhibiting the lymphocyte response and at times caused an increased response. In contrast, immune IgM which was less efficient in neutralizing virus caused significant suppression of the blastogenic reaction. By themselves these results might have signified that IgG and IgM antibodies have different specificities or different binding properties with respect to viral surface antigens. However, immune complexes consisting of virus and IgM reduced response of both rubella immune and normal rabbit lymphocytes to PHA. This nonspecific inhibitory action required a specific step of antigen and IgM antibody interaction and normal IgM-virus mixtures or mixtures of anti-rubella IgM and poliovirus or influenza virus did not suppress lymphocyte response to PHA. Anti-rubella IgG complexed with rubella virus did not suppress the PHA response. The IgG function was apparently limited to neutralization of the infectivity of rubella virus whereas the major role of IgM was manifested through its suppressive effect on lymphocyte reactions.     

The human LT system. III. Characterization of a high molecular weight LT class (complex) composed of the various smaller MW LT classes and subclasses in association with Ig-like molecules.

Cytotoxic activity (lymphotoxin (LT)) present in supernatants from lectin stimulated human lymphocytes in vitro is composed of a heterogeneous system of biological macromolecules which can be separated into multiple classes and subclasses on the basis of their molecular weight and charge. These studies further characterize a large molecular weight human LT class, termed complex (MW >200,000 d), which elutes in the void volume off Sephadex G-150 or Ultrogel AcA 44. Immunological studies on the complex, employing various rabbit anti-LT class and subclass antisera, revealed this material is a macromolecular assemblage of the smaller MW α, β, γ LT classes and subclasses. Furthermore, the reactivity of this material with anti-human Fab′₂ (IgG) indicates these smaller molecular weight LT components can associate with immunoglobulin or Ig-like molecules. The materials present in the LT complex class appear to be noncovalently associated, since conditions of high ionic strength dissociate certain small MW LT components, while low ionic strength buffers may cause these components to reaggregate with the complex. When subjected to velocity sedimentation on sucrose gradients or gel filtration on Ultrogel AcA 22, LT complex activity elutes as several discrete peaks of activity in the 200,000 to 1,000,000 MW range. These findings suggest the concept that LT molecules can form discrete and specific macromolecular structures which contain the smaller MW LT classes. Moreover, these structures can also associate with immunoglobulin-like molecules to form secondary LT-Ig complexes. This may have important biological significance in explaining how nonspecific cell toxins could play a role in specific or nonspecific cell lytic reactions in vitro.     

Lymphocyte cytotoxicity to influenza virus-infected cells. II. Requirement for antibody and non-T lymphocytes.

Peripheral blood lymphocytes (PBL) obtained from humans were cytotoxic for influenza virus-infected target cells. The PBL were shown to have associated influenza virus anti-hemagglutinin antibody (AHAb) detectable only by radioimmunoassay. This antibody could be removed by incubating PBL at 37 degrees C for 30 min. The lymphocyte population that was effective in this system was nonadherent and nonphagocytic cells. PBL gave comparable levels of cytotoxicity when tested by using either a xenogeneic or allogeneic virus-infected target cell. These results indicate that lymphocyte cytotoxicity to influenza virus infected cells may be mediated by small quantities of antibody and by lymphocytes that possess characteristics of K cells. No evidence for T cell-mediated cytolysis was found with this xenogeneic system.     

The human LT serum. X. The initial form released by T-enriched lymphocytes is 150,000 m.w., associated with small nonlytic components, and can dissociate into the smaller alpha, beta, and gamma m.w. classes

The present studies examine the various lymphotoxin (LT) forms released in vitro by phytohemagglutinin- (PHA) activated T-enriched (Te) human peripheral blood lymphocytes. It is clear that Te cells rapidly released (24 to 48 hr) these molecules in vitro. The 1st cell-lytic form detected in these supernatants is a 140-160,000 m.w. molecule(s) termed precursor alpha heavy (P alpha H). This form does not express alpha-LT antigenic determinants but is neutralized by antisera from animals injected with serum-free PHA-activated unseparated lymphocyte supernatants (anti-WS). The P alpha H is converted into alpha H, which expresses alpha determinants, by passage through molecular sieving columns or by treatment with low levels of Nonidet P-40 or urea. These treatments dissociate a small nontoxic 10-20,000 m.w. molecule(s), termed precursor factor (Pf), which masks the alpha-LT determinant on the P alpha H molecule. The dissociation of Pf is reversible, since alpha H from the molecular sieving columns will reassociate with the Pf. The alpha H LT class can further dissociate into the smaller alpha, beta, and gamma LT forms upon chromatography on a molecular sieving column, and a certain small percentage of the alpha H forms appear capable of associating to form the high m.w. complex (Cx) LT class. These findings suggest P alpha H may represent an intermediate that requires additional processing in order to proceed down 1 of 2 pathways: a) formation of complexes that are highly cell-lytic, or b) degradation by dissociation into the smaller weakly cell-lytic molecules identified as LT forms.     

Characterization of a serum inhibitor of MLC reactions. III. Specificity: HL-A-related antibodies.

A mixed leukocyte culture (MLC)-inhibitory serum from a healthy multipara, JH, has been characterized with regard to the specificity of its inhibitory antibody. When added directly to MLC, JH serum will inhibit most combinations. However, when lymphocytes intended as responder or stimulator cells in MLC were preincubated with this serum, the specificity narrowed considerably. Four groups of lymphocyte donors were recognized, depending upon whether their lymphocytes were inhibited as responders, as stimulators, as both, or as neither. Absorptions of inhibitory activity, followed by assay of the absorbed sera in pretested MLC combinations, yielded reliable data for determining which donors' cells shared pertinent antigens. An association of MLC inhibition by JH serum with the HL-A types of the involved lymphocytes was observed and these relationships are summarized in Table 4. The three HL-A specificities identified, W19, W29, and 12, correspond with the HL-A typing of the husband of the serum donor. Various cell types absorbed relevant inhibitory activity (against responder and stimulator functions) in the following order of efficiency: LCL cells, B lymphocytes, T lymphocytes, fibroblasts, and erythrocytes. When the above three HL-A specificities were removed by absorption, the serum was no longer inhibitory in any combinations. Whether the inhibitory activity of JH serum is directly related to anti-HL-A antibodies or to antibodies against closely related MLR determinants will depend to a large extent upon the degree of linkage disequilibria found between W19, W29, and 12 antigens and the MLR locus.     

Natural Hidden Autoantibodies React with Negatively Charged Carbohydrates and Xenoantigen Bdi

Immunoglobulin preparations from sera of healthy donors contain polyspecific autoantibodies interacting with DNA and other charged antigens. These antibodies belong to the IgG class and can exist in the free or hidden state. The hidden antibody activity can be revealed after ion-exchange chromatography on QAE-Sephadex A-50. Immunoenzyme assay was used to assess the interactions of both free and hidden antibodies with different carbohydrates. The hidden antibodies were only able to interact with different polyanionic carbohydrates and neutral xenoantigen Bdi.     

Inhibition of human NK-induced cell lysis and soluble cell-lytic molecules with anti-human LT antisera and various saccharides

The present study examines and compares the cytolysis of K-562 and MOLT-4 cells mediated by human natural killer (NK) cells from fresh peripheral blood and lymphotoxins (LT) derived from human lymphoid cell populations after lectin stimulation in vitro. Lymphotoxins were obtained from 5-hr concanavalin A (Con A)-restimulated human peripheral blood lymphocytes (PBL) which were precultured for 5 days in medium and fetal calf serum or with allogeneic human B-lymphoid cell lines. Two classes of probes were employed in both direct (cell) and indirect (supernatant) induced target-cell lysis: (a) various saccharides and (b) antibodies reactive with human LT forms. Two sugars, N-acetylglucosamine and α-methylmannoside, were able to inhibit direct cell lysis of both MOLT-4 and K-562 target cells. However, saccharide inhibition was distinct for each type of target even when effector cells were obtained from the same donor. These same saccharides were also able to inhibit 20–30% of the total LT activity in a supernatant for L-929 cells and 50–90% of the lytic activity on MOLT-4 cells. Anti-human F(ab′)₂ (IgG) and rabbit anti-α₂ LT sera blocked direct cell lysis of MOLT-4 and K-562 targets in 50% of the experiments. The anti-α₂ LT serum only recognizes a portion of the LT forms in these supernatants. These results reveal that, while both direct and indirect cell lysis are complex phenomena, they may both occur in some cases by a common mechanism(s).