Immunological studies of trophoblast antigens: no evidence for human leucocyte antigen (HLA) linkage

Parental disparity for trophoblast-lymphocyte crossreactive (TLX) antigens may promote successful pregnancy. A TLX antigen system has been defined on peripheral blood lymphocytes by heteroantisera. More recently, we have reported additional activity against antigens on B lymphocytes alone termed trophoblast-B lymphocyte crossreactive (TBX) antigens. In the present study we have investigated ten TLX sera in order to determine if their target antigens are linked to the human leucocyte antigen (HLA) gene complex. The sera showed no selective activity when tested against target B lymphocytes from ten normal donors. Cytotoxic activity of TLX antisera against peripheral blood lymphocytes from six normal donors was not reduced when the class I HLA antigens of the target cells were blocked with a monoclonal antibody (PA 2.6). Similarly the cytotoxic activity of both TBX antisera against B lymphocytes from six normal donors was not decreased when class II HLA antigens were blocked by a monoclonal antibody (FMC 4). Within a family the cytotoxic activity of the TLX antisera was absorbed equally by lymphocytes from siblings who shared neither HLA haplotype. Antibody content in TLX and TBX antisera is not directed toward the classically defined HLA class I or class II antigens and is not linked to the HLA gene complex.     

Stimulation of rat autologous mixed lymphocyte reaction with xenogeneic sera and their protein preparations.

Autologous mixed lymphocyte cultures were set up from nylon non-adherent T-enriched lymphocytes and mitomycin C-treated spleen cells of individual ACI/N rats and the effect on the reaction (AMLR) of sera in the culture medium was studied with regard to the xenogeneic nature of the sera. Not only foetal calf serum, but also sera of adult human, horse and swine stimulated the AMLR response, but autologous or rat serum did not. Albumin fractions of these sera were also effective in inducing the AMLR response. Furthermore, the presence of xenogeneic serum in the culture medium was also required for the blastogenic response of AMLR-primed lymphocytes against the secondary stimulation with syngeneic spleen cells. Both autologous and xenogeneic sera, however, supported the Con-A response of the same responder cell population as used in the AMLR. These results substantiated our previous finding (Endho & Hashimoto, 1981) and suggest that two signals, one from autologous non-T cells and another from the xenogeneic factor in culture medium, are required to cause AMLR, at least in the rat system.     

PPD-induced viral antibody production in human blood lymphocytes.

The ability of purified protein derivative of tuberculin (PPD) to induce polyclonal antibody production in cultures of human blood lymphocytes was studied. IgG and IgM were determined by the enzyme-linked immunosorbent assay (Elisa). PPD induced both IgM and IgG production, with a predominance of IgM. PPD usually stimulated a stronger IgM response but a weaker IgG response than did pokeweed mitogen (PWM). Supernatants of PPD- or PWM-stimulated lymphocyte cultures were tested for antibodies to morbilli, rubella and herpes simplex by Elisa. PPD as well as PWM induced viral antibody production in lymphocytes of donors who had serum antibodies to the corresponding viral antigens. Production of viral antibodies of IgG class but not of IgM class was demonstrated. The PPD-induced antibody response was T cell-dependent.     

In vitro proliferation of macrophage depleted human peripheral blood lymphocytes.

The incorporation of thymidine by normal human peripheral blood lymphocytes was tested in vitro following various culture conditions. A significant increase of thymidine uptake was observed in cultures depleted of plastic adherent, nylon wook adherent, or phagocytic cells. This proliferative activity occurred in the presence of various sera but was not due to a blastogenic response to a foreign protein, since it was also observed when autologous plasma was the only source of protein in the culture medium. The similarities and differences between this 'spontaneous' proliferative phenomenon and other blastogenic responses which are regulated by macrophages are discussed.     

Cell-mediated and humoral immune responses of renal transplant recipients with cytomegalovirus infections

Cell-mediated and humoral immune responses were determined in immunosuppressed renal transplant recipients. A micro-phytohaemagglutinin (PHA) stimulation test utilizing whole heparinized blood and a macro-PHA test utilizing separated, washed lymphocytes were used to study cell-mediated immunity. Humoral immune status was estimated by determining quantitative immunoglobulins and complement fixing (CF) antibody titres to cytomegalovirus (CMV) infection. Micro-PHA responses were found to be markedly depressed in patients undergoing immunosuppressive therapy and in patients with chronic uraemia. Macro-PHA responses were normal, indicating that serum factors were responsible for the depressed micro-PHA responses. Antibody responses to CMV infections were found to be ten to a hundred times higher than in normal persons. An inverse relationship was demonstrated between micro-PHA responses and peak CF antibody titres to CMV infections. Humoral immune responses appeared to compensate for depressed cell-mediated immunity as measured by the micro-PHA test. Four patients had very low micro-PHA responses, did not respond to their CMV infections with CF antibody, and died of mixed bacterial and viral infections. Serum immunoglobulins of two were studied and were shown to be greater than two standard deviations below the normal mean. These patients appeared more suppressed than other patients receiving similar therapy and thus probably retained higher concentrations of suppressive drugs.     

Anti-complement immunofluorescence test for antibodies to human cytomegalovirus.

An anti-complement immunofluorescence (ACIF) test that detects human cytomegalovirus (CMV) antigen in the nuclei of infected cells was used for assay of CMV antibodies in human sera. Various factors influencing the sensitivity and specificity of the ACIF test system were investigated, and results were applied to the development of a procedure which could be completed in a relatively short length of time and gave reproducible results. Results obtained in the ACIF test were compared with those obtained in complement fixation, indirect hemagglutination, and neutralization tests, and the ACIF test was shown to be suitable for detection of significant antibody titer rises and stationary levels of CMV antibody. Heterotypic antibody responses were not seen with sera from other human herpesvirus infections. The nonspecific cytoplasmic staining that occurs in indirect immunofluorescence tests for CMV did not occur in the ACIF system, and sera that were anti-complementary in complement fixation tests could be examined satisfactorily by ACIF. Thus, the test is a valuable supplemental or back-up procedure for the serodiagnosis of CMV infection.     

The maintenance of B-cell and T-cell function in frozen and stored human lymphocytes

The objective of this study was to identify and test a convenient means for long-term storage of lymphocytes taken from clinically characterized patients without losing B- or T-cell function. Accordingly, peripheral blood lymphocytes were frozen and stored, and portions of each sample were subsequently assayed for T-cell blastogenic response and B-cell Jerne plaquing at various time intervals after freezing. A comparison of the cell counts of fresh and frozen cultures indicated that all cells were recovered after freezing. Furthermore, these cells showed no significant differences in (i) cell viability; (ii) blastogenic response to antigens ofActinomyces naeslundii, Bacteroides melaninogenicus, Fusobacterium nucleatum, and tetanus toxoid; (iii) blastogenic response to phytohemagglutinin and pokeweed mitogen; and (iv) polyclonal B-cell response to pokeweed mitogen as measured by the direct Jerne plaque assay. The retained blastogenic and plaquing responses seen in frozen cultures indicated the maintenance of both T-cell and B-cell function, respectively. This is the first reported demonstration of Jerne plaquing of normal human lymphocytes after freezing. It appears that freezing techniques provide a means for repeating and extending both T- and B-cell assays using frozen stored portions of the same cell sample.     

Stimulation of human lymphocytes by cytomegalovirions and dense bodies

Lymphocytes from healthy adult individuals were examined for their ability to incorporate thymidine in the presence of cytomegalovirus (CMV) and dense bodies. It was found that lymphocytes from donors with antibodies to CMV were stimulated to incorporate thymidine-¹⁴C both by preparation of CMV and CMV dense bodies. Lymphocytes from CMV seronegative donors did not respond. Dilution experiments and the dose-response curve indicate that the stimulation induced by the dense body preparation was not caused by the small amounts of contaminating CMV particles. These results indicate that in healthy human adults there is a correlation between CMV seropositivity and in vitro lymphocyte transformation, induced by either CMV or by dense bodies.     

Blastogenic response of purified human T-lymphocyte populations to Epstein-Barr virus (EBV).

The blastogenic response of T-cell populations to infectious EBV (B95-8 strain) was studied. The addition of virus increased the [3H]thymidine uptake of unfractionated lymphocytes from seropositive and seronegative subjects, and from cord blood, but not from patients with infectious mononucleosis. The same amount of virus evoked a small response in purified T cells from seropositive donors, but not in T cells from the other sources. The addition of autologous B lymphocytes with EBV adsorbed on their surfaces caused a more significant blastogenic response, which was even more pronounced in the presence of mercaptoethanol.     

Biological response modifier enhances the activity of natural killer cell against human cytomegalovirus-infected cells

Peripheral blood lymphocytes (PBL) from two human cytomegalovirus (CMV)-seronegative donors and eight CMV-seropositive donors were cultured for 3 days with or without the biological response modifier OK-432 and examined for lysis of K562 cells and CMV-infected MRC-5 cells. OK-432-stimulated PBL exhibited significantly greater natural killer (NK) activity than did unstimulated PBL. There was no difference in activity of NK cells in PBL prepared from CMV-seronegative and -seropositive donors. Antibody-complement depletion studies suggested that OK-432-stimulated NK activity was associated with Leu-7-positive cells. The ability of OK-432 to sustain the NK activity in PBL was decreased when the CD4-positive population of lymphocytes was eliminated by antibody-complement depletion prior to OK-432 stimulation. The ability of OK-432 to sustain the NK activity of PBL was also significantly decreased in the presence of monoclonal antibody against recombinant human interleukin-2. The results suggest that the activity of human NK cells against K562 and CMV-infected MRC-5 target cells can be sustained in vitro by OK-432-stimulated T-helper cells and that the effect of the T-helper cells is mediated, at least in part, by interleukin-2.     

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.     

Lymphocyte transformation and interferon production in human mononuclear cell microcultures for assay of cellular immunity to herpes simplex virus.

Interferon production and transformation in response to herpes simplex virus antigen were studied in microcultures of human mononuclear cells. Mononuclear cells consisting of monocytes and both T and B lymphocytes were purified by Ficoll-Hypaque gradients. Lymphocytes, predominantly T with 5% B, were obtained by passage of buffy-coat cells through nylon fiber columns. For some experiments, autochthonous macrophages and column-purified lymphocytes were stimulated with herpesvirus antigen. The effect of specific antibody and cell concentration on reactivity is described. Crude and purified antigens were compared as cell culture stimulants. Significant differences in transformation and interferon were observed between donors with a history of herpes labialis and donors with no detectable antibody, both in cultures prepared by Ficoll-Hypaque gradients and by column purification of lymphocytes. Cultures from seronegative donors prepared by Ficoll-Hypaque gradients produced interferon but did not transform when stimulated by herpes simplex antigen. "Immune" interferon production, that is, type II as opposed to type I, occurred only with autochthonous macrophage and column-purified lymphocyte cultures. Interferon produced by Ficoll-Hypaque-purified mononuclear cultures was type I, and its production was unrelated to immune status. Similarly, column-purified lymphocytes responded to herpes simplex virus antigen with type I interferon if obtained from a seropositive donor.     

Human thymus cells: blastogenic response to mitogens, antigens and allogeneic cells.

Over 90 per cent of the thymus cells from each of twenty-six donors were T lymphocytes, identified by E-rosetting and less than 3 per cent of the cells were B lymphocytes identified by EAC-rosetting. With advancing age, the proportion of T lymphocytes decreased while that of B lymphocytes increased. The degree of (3H)thymidine incorporation of thymus cells was inversely proportional to the age of the thymus-cell donor. The PHA or PWM- induced blastogenic response of thymus cells gradually increased with advancing age when the response was expressed as the stimulation index. However, the actual rate of (3H)thymidine incorporation in all three groups was rather similar when cells were cultured with mitogens. The difference in stimulation index was due to the variation in incorporation rate in cultures without stimulants. The PHA response was approximately four-fold higher than that of PWM response. Thymus cell response to allogeneic lyphocytes, on the other hand, had no correlation with the age of thymus donor. The most surprising result in the present study was that the thymus cells from each of ten donors, aged 1-14 years, were incapable of responding to all four different recall antigens. Peripheral blood lymphocytes from nine to ten randomly selected age-matched children responded very well to one or more antigens.     

Failure of hydrocortisone to inhibit blastogenesis by pokeweed mitogen in human leucocyte cultures

Hydrocortisone was added to cultures of human peripheral blood leucocytes 30 min before the addition of phytohaemagglutinin (PHA) or pokeweed mitogen (PWM). A concentration of 10 μg/ml of hydrocortisone inhibited a major part of the blastogenic response to PHA at 3 days of incubation and prevented a decline in the number of macrophages. A portion of corticosteroid-resistant PHA-stimulated blasts was observed in all experiments. Hydrocortisone (10 μg/ml) caused a slight decrease in the mitogenic response to PWM at 2 and 3 days of incubation, but at 5 days the proportion of blasts equalled or exceeded that seen in cultures with PWM alone. Hydrocortisone did not prevent the virtual disappearance of macrophages from cultures incubated with PWM or with a combination of PHA and PWM. The effect of hormone on both lymphocytes and macrophages indicated that the principal action of PWM was on a subpopulation of lymphocytes not responsive to PHA. In addition a small number of corticosteroid-sensitive cells was transformed by PWM early in incubation. Results indicated that the prior addition of corticosteroids to leucocyte cultures was useful in determining the response of segments of the circulating lymphocyte population to different mitogenic stimulants.     

Rapid herpes simplex virus detection in clinical samples submitted to a state virology laboratory.

Cord sera and antepartum maternal sera from three congenitally cytomegalovirus (CMV)-infected infants and their mothers were CMV seronegative (titer, less than 8) in a complement fixation (CF) assay with a glycine-extracted CMV AD169 antigen; sera from two of the infants and mothers were also seronegative in a commercial indirect hemagglutination (IHA) assay with AD169 antigen. In tests with their own CMV isolates propagated and made into glycine-extracted CF antigen, all were seropositive. When 108 random cord sera were assayed for CF antibody with AD169, Davis, and A antigens (A is a locally derived antigen from one of the above infants), 44 were seropositive and 54 were seronegative for all three antigens. Of the remaining 10 sera, 4 were positive for A only, 3 were positive for A and Davis only, 2 were positive for Davis and AD169 only, and 1 was positive for AD169 only. All 10 were positive when a mixture of all three antigens was used. The IHA assay with AD169 antigen was positive with only 4 of these 10 sera. These results suggest that up to 6% of sera may be misclassified as seronegative in the CF and IHA assays if only a single antigen is used.     

An enzyme labelled nuclear antigen immunoassay for detection of cytomegalovirus IgM antibodies in human serum: specific and non-specific reactions

A mu-capture enzyme linked immunosorbent assay was developed for detection of IgM antibody to cytomegalovirus (CMV). Virus-specific IgM was detected using horseradish peroxidase labelled nuclear CMV antigen (CMV-ELA). False-positive reactions caused by Paul-Bunnell-Davidsohn (PBD) positive sera and antinuclear antibody (ANA) positive sera were identified in a combination assay employing enzyme labelled nuclear control antigen (CO-ELA) in parallel to the CMV-ELA. Four of five PBD positive and 30 of 31 ANA positive sera reactive with the CMV-ELA were identified as false positive reactions in the combined ELA-assay. The reactivity in PBD-positive sera could not be explained by antigenic cross reactivity between CMV and Epstein-Barr virus, and the results further suggested that different cell specified components of the CMV-ELA were responsible for the reactivity of PBD-positive as compared to ANA-positive sera. One of 314 healthy blood donors, 12 of 12 patients with primary CMV infection, and 11 of 15 patients with secondary CMV infection had detectable CMV IgM antibodies. Comparison of different CMV-ELAs revealed that pronounced differences in specificity as well as sensitivity may exist.     

Humoral immune response to virions and dense bodies of human cytomegalovirus determined by enzyme immunofluorescence assay

An enzyme immunoassay utilizing a fluorogenic substrate (EIFA) was employed to compare humoral antibody responses to purified virion and dense body antigens in human cytomegalovirus (CMV) infections. Results of these antibody assays were also compared to those obtained by plaque reduction neutralization and complement fixation (CF). In adults with CMV infections diagnosed by a significant rise in CF antibody titer, there was also a vigorous antibody response to the virion and dense body antigens. There was better correlation between antibody levels to the virion and dense body antigens than between antibody levels detected by other combinations of tests. Antibody responses to the virion and dense body antigens were similar, but not identical, suggesting that although the two structures share major antigens, they may also possess unique antigens to which the host can mount independent antibody responses. All individuals with CMV neutralizing antibody also had virion and dense body antibodies, but there was poor correlation between neutralization titers and those detected by EIFA, perhaps reflecting greater strain specificity of the neutralization test. Some individuals with serological diagnoses of Mycoplasma pneumoniae or influenza A infection showed fourfold or greater antibody titer rises to CMV in two or more assays, suggesting reactivation of latent CMV in these infections.     

A whole-blood lymphoproliferation assay for measuring cellular immunity against herpes viruses

A whole blood test system was established to study cell-mediated immunity to cytomegalovirus (CMV) and herpes simplex virus (HSV) in a large number of healthy blood donors. Cellular immunity was measured by the in vitro proliferative response (LP) of peripheral lymphocytes. These responded vigorously to several mitogens. Lymphocytes of most individuals responded to HSV, but only a limited number were reactive towards CMV. In parallel, antibodies against CMV and HSV were measured by an ELISA technique. For HSV, good correlation was observed between serological and lymphocyte proliferation results. For CMV, no clear correlation was obtained, only 21 of 40 donors positive in the antibody test being positive in the LP test. The majority of seronegatives were negative in the LP test. Use of virions purified by sucrose gradient centrifugation, or an additional strain of CMV (strain Davis) did not increase the number of donors positive in the LP test. One explanation might be that individuals possessing antibodies against CMV as measured by ELISA but no capacity to react in the LP test had suffered from a CMV infection a long time before, and now showed waning cellular immunity, but antibody still detectable. Use of the whole blood technique on 108 individuals showed that this very simple test works well with various mitogens and at least some antigens.     

Human antibody response to a pneumococcal vaccine in SCID-PBL-hu mice and simultaneously vaccinated human cell donors

Severe combined immunodeficient (SCID) mice were transplanted intraperitoneally with human peripheral blood lymphocytes (PBL) from nine healthy human donors (SCID-PBL-hu mice). None of the donors had ever received pneumococcal vaccine. Ten days after transplantation, 62 out of 111 transplanted mice and six of the nine donors were vaccinated with a 23-valent pneumococcal polysaccharide vaccine. For each donor, human IgG was detected in 91.7–100% of the SCID-PBL-hu mice, whereas specific human IgG antipneumococcal antibodies were demonstrated in 16.7–100% of the vaccinated SCID-PBL-hu mice. Most of the mice transplanted with cells from the same donor showed similar antibody response patterns in terms of kinetics and antibody levels. A significant antibody response was only obtained in mice that received cells from donors with relatively high antipneumococcal antibody levels at the time of transplantation, or donors that showed a substantial increase in antibody levels after vaccination. The immune response in the SCID-PBL-hu mice did not always reflect the ability of the respective donor to produce antipneumococcal antibodies. The donor dependency of the antipneumococcal antibody response has great practical importance for the use of the SCID-PBL-hu model. Donors should not be chosen randomly. By selecting donors whose cells have been found to result in successful engraftment, functional SCID-PBL-hu mice can be obtained for the study of human immune responses and function in an in vivo experimental model.     

Neutralizing Antibodies to Cytomegaloviruses in Normal Simian and Human Sera

Simian and human sera were examined for neutralizing antibodies to simian and human cytomegaloviruses (CMV). Neutralizing antibody to simian CMV was found in sera from 12 of 12 African green monkeys, 8 of 10 rhesus monkeys, and 7 of 7 baboons captured in the wild. The antibody did not cross-react with human CMV strain AD169 but cross-reacted with human strain C87, particularly in the presence of complement. Thirty-six baboons and 10 rhesus monkeys born and hand-reared in captivity remained free of neutralizing antibody both to simian and human CMV for as long as 4 years. Fifteen of 24 human sera (63%) revealed only species-specific neutralizing antibody.