Graft vs graft reaction resulting in the elimination of maternal cells in a SCID patient with maternofetal GVHd after an HLA identical bone marrow transplantation

In a young girl with a severe combined immunodeficiency, the presence of circulating maternal T lymphocytes was proven by HLA typing. Manifestations of skin graft vs host disease were associated with the persistence of maternal cells. The patient received an HLA identical bone marrow transplantation from her brother without any conditioning. The bone marrow transplantation was quickly followed by a transient and dramatic increase in skin lesions associated with fever and the finding of a high number of circulating lymphocytes and eosinophils. Lymphocytes were shown to be of donor origin and exerted a spontaneous cytotoxic activity toward maternal cells. This activity progressively disappeared within 90 days, whereas maternal cells were no longer detected in patient's blood, and skin graft vs host disease was resolved within 8 wk. Cytotoxic activity was proven to be mediated by donor T lymphocytes specific for the mother's HLA antigens. The cytotoxic activity was demonstrated to be specific for the HLA class I molecules of the mother not shared with her daughter (HLA A1, B17) as shown by the use of a series of HLA typed cells as targets. In addition, cold K562 target cells did not block the cytotoxic activity, and the kinetics of the cytotoxic activity did not correlate with that of natural killer activity emergence after the bone marrow transplantation. Patient's serum did not contain antibodies toward maternal specific HLA class I antigen. Cytotoxic activity was totally blocked by anti-T3 monoclonal antibodies and partially by anti-T8 and anti-T4. It is thus likely that donor origin cytotoxic T lymphocytes were promptly activated after bone marrow transplantation and provoked the elimination of the maternal graft after a transient exacerbation of graft vs host disease manifestations. This observation represents one of the first examples of the possible role in vivo of allogeneic cytotoxic lymphocytes in humans.     

Monoclonal anti-human tumor antibodies of six isotypes in cytotoxic reactions with human and murine effector cells

Eighty-seven murine monoclonal antibodies (MAb) produced against human tumors of various origins and representing six different immunoglobulin classes were tested for antitumor reactivity in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. Mouse splenocytes, thioglycolate-elicited mouse peritoneal macrophages, freshly obtained nonadherent human peripheral blood lymphocytes, and human monocytes were used as effector cells, and human or rabbit serum as the source of complement. Of all four effector cell types tested, mouse macrophages showed the highest cytotoxic activity, based on net cytotoxicity, minimum requirement for Mab concentration, and effector cell number. Different immunoglobulin classes were associated with characteristic patterns of reactivity with the various effector cells or complement, independent of the target cell type used. MAb able to mediate ADCC were found among all IgG subclasses, with IgG2a and IgG3 MAb inducing lysis with all effector cell types. IgM and IgA MAb were nonreactive in the various ADCC assays, but IgM MAb were highly cytotoxic with complement.     

Lysis of leukemia cells with a monoclonal antibody-cocktail (e.g. VIP-pool) and human complement

During the lysis of leukemic cells with a monoclonal antibody cocktail (the so-called VIB pool) and complement the attempt was made to replace rabbit serum as a complement source by human serum. For identifying the lysis of leukemic cells the complement-dependent in vitro cytotoxicity test was used and for excluding stem cell toxicity the CFU-c test according to PIKE and ROBINSON. In combination with the applied monoclonal antibody pool against B and c-ALL the human complement could be shown to be suitable to produce a lysis in the same manner as rabbit complement. Similarly to the pretested rabbit serum the treatment with the human complement had no impact on stem cell recovery. An optimal cytotoxic activity (95% against ALL blasts of patients, 100% against NALM) could be identified up to an antibody dilution of 1:32 with a volume percentage of 50% of human complement, an incubation temperature of at least 37 degrees C and an incubation time of 30 mins. With proved high reactivity against leukemic cells and lacking impairment of the haemopoietic power of the bone-marrow, this method can be recommended for "purging" protocol with the possibility of using human serum as a source of complement having advantages as far as clinical application is concerned.     

The effect of heating and drugs on HL-A antigens and their reactions with cytotoxins]

In some instances the heating of lymphocytes for 2 to 3 minutes at 56 degrees C enhances HL-A antigens or makes it possible to detect these antigens by a twostage microlymphocytotoxic test on preheated lymphocytes only. Similar phenomenons are observed in some tannin (1: 40 000) or 0.1% phenol-treated lymphocytes and after the addition of these solutions during the first stage of the lymphocytotoxic test. Prolonged heating at 56 degrees C leads to nonspecific polyreactivity of lymphocytes, giving false results with all sera. For similar reasons also higher concentrations of tannin and phenol solutions were found dissatisfactory for the pretreatment of lymphocytes. The pretreatment of lymphocytes with 36-0.5% formaline induces full inhibition of specific cytotoxic reactivity of HL-A antigens and their absorption ability with regards to respective HL-A sera. The addition of formaline at 0.06-0.3% concentration during the first and second stages of the test and at the end of the second stage (or simultaneously with eosine) gives also negative results of the cytotoxic test owing to the inhibitory effect of formaline on rabbit complement and HL-A antigens.     

Cytological observations on the cytotoxic interaction between lymphocytes and antibody-coated monolayer cells

Chang cells in monolayers were killed when exposed to highly purified human peripheral blood lymphocytes in the absence of complement and in the presence of a concentration of heat inactivated rabbit anti-Chang cell serum (ACS) chosen for inability by itself to cause cytolysis. As evident by plaque formation, the cytotoxic reaction was confined to areas of the cultures covered with lymphocytes. In the presence of ACS, lymphocytes attached to and infiltrated the monolayers. Uropod formation indicated activation of the lymphocytes. Close contact was established between effector and target cells. The cytotoxic reaction was not accompanied by protracted cytopathological changes but led to detachment and rapid lysis of the target cells. Horse anti-thymocyte globulin, added to the cultures, completely inhibited plaque formation and lymphocyte infiltration.     

Human lymphoblastoid cell lines secreting antibodies with restricted HLA specificity

Peripheral B lymphocytes obtained from three healthy individuals who had been immunized against peripheral blood lymphocytes from appropriate HLA-incompatible donors were transformed by the use of Epstein-Barr virus. The transformed blastoid B cells were repeatedly subcultured by means of cluster picking, and the HLA antibody-producing cultures were identified by testing the culture supernatants by means of the cytotoxicity assay, using the corresponding donor cells. Thus far, four cell lines that secrete cytotoxic HLA antibodies (MP1, 3, 4, and 5) have been established. Specific immunoabsorption experiments revealed that the antibody activity is carried by lambda-type IgM for MP1, by kappa-type IgM for MP3 and MP5, and by both for MP4. Specificity analysis of a panel of HLA-pretyped cells indicated that MP1 detects DQw2, whereas MP5 recognizes B7. The specificity of MP3 was similar to a DQ specificity termed DC5 (probably equivalent to TA10) but not the same. In the case of MP4, both of the lambda-type and kappa-type antibodies appeared to be directed toward new HLA class 11 determinants.Abbreviations used in this paper HLA human major histocompatibility - EBV Epstein-Barr virus - B-LCL Blymphoblastoid cell line - NA not absorbed - PBS phosphate-buffered saline - SPA Sepharose protein A - NRS normal rabbit serum     

Two distinct cytotoxic T lymphocyte subpopulations in patients with Vogt-Koyanagi-Harada disease that recognize human melanoma cells

The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease ( VKH ) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1+ Leu-2a+ or Leu-1+ Leu-3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.     

Human lymphoid cell lines as targets for DRw.

Cultured human lymphoid cell lines (LCL) are useful as a source of target cells in several immunologic assays. More recently such cells have been used for the serological characterizations of the HLA-DR antigens. Typing of the same LCL in various laboratories during the VII Histocompatibility Workshop has given comparable results with a discordancy rate of less than 10%. This discordancy is likely to reflect the different sources of complement that can greatly alter the results of cytotoxic assays. The presence of naturally occurring antibody in rabbit complement to human cells can be avoided by: (a) absorbing with human cells at 0 degrees C; (b) dilution with human serum; (c) dilution with heat-inactivated rabbit serum; (d) repeated freeze-thawing of the complement; or (e) careful selection of complement by screening procedures. Comparison of the results of HLA-DR typing of LCL with peripheral B-cells of the same donor show good correlations. However, LCL will occasionally give extra reactions perhaps due to the expression of new antigens. LCL can be coated with F(ab')2 fragments from antihuman beta2-microglobulin antibodies that block reactions of HLA-A, -B and -C antibodies allowing for discrimination of anti-DRw activity.     

Effect of rabbit anti-asialo GM1 treatment in vivo or with anti-asialo GM1 plus complement in vitro on cytotoxic T cell activities

The susceptibility of cytotoxic effector lymphocytes and their induction to in vivo or in vitro treatment with rabbit anti-neutral glycolipid ganglio-N-tetraosylceramide (anti-ASGM1) antiserum was investigated. Intravenous injection of anti-ASGM1 antiserum eliminated measurable natural killer (NK) cell activity in spleen cells of mice infected for 5 days with Vaccinia virus, or for 8 days with lymphocytic choriomeningitis virus (LCMV) if injected 24 hr prior to testing. In addition, this treatment lowered measurable virus-specific cytotoxic T cell activity by 60 to 95%. Virus-specific cytotoxic T cell and NK cell activity generated during a primary infection in vivo was also sensitive to treatment in vitro with anti-ASGM1 antiserum (1/300 to 1/600 dilution) plus rabbit complement at a dilution of 1/15 (20 to 50% cell death, more than 30-fold decrease of cytotoxic activity); in vitro treatment with rabbit complement alone often enhanced NK and cytotoxic T cell activity slightly. In vivo treatment with anti-ASGM1 before primary immunization decreased generation of primary CTL only if high doses of anti-ASGM1 antiserum were injected twice. Antiviral T cells generated during secondary stimulation in vitro and alloreactive cytotoxic T cells from a mixed lymphocyte culture were resistant to treatment in vitro with anti-ASGM1 plus complement at the end of the culture period. Treatment in vitro of in vivo-primed responder spleen cells with anti-ASGM1 plus complement before their addition to a secondary restimulation culture resulted in complete inhibition of a secondary antiviral cytotoxic T cell response. In vivo treatment with anti-ASGM1 24 hr before their spleen cells were harvested and restimulated in vitro significantly reduced the virus-specific T cell activity of mice that had been immunized with virus several weeks previously. A cloned T cell line exclusively exerting NK-like activity was resistant, and two cloned virus-specific cytotoxic T cell lines were susceptible to treatment with anti-ASGM1 plus complement in vitro. These results caution the general use of rabbit anti-ASGM1 as a marker to distinguish NK from CTL cells; they indicate a possible relationship between NK and CTL cells and suggest that in vitro culture of lymphocytes may alter or select the cell surface expression or availability of the ASGM1 marker(s).     

Sera from lipopolysaccharide (LPS)-injected mice exhibit complement-dependent cytotoxicity against syngeneic and autologous spleen cells.

To determine if lymphocytes are able to discriminate between self and nonself, the polyclonal B-cell activator lipopolysaccharide (LPS) was injected into mice, and sera from those mice were tested at different times for their cytotoxic effect against autologous and syngeneic isotope-labeled spleen cells in the presence of complement. It was regularly found that LPS caused the appearance of cytotoxic activity in sera detectable against autologous and syngeneic spleen cells. This cytotoxicity was found to be complement dependent, and it was abolished by absorbing the sera with the target cells. LPS did not induce cytotoxic serum activity in the LPS nonresponder strain C3H/HeJ. When the serum was passed through an anti-mouse Ig column, the eluted sample completely lost its cytotoxicity. It is likely, therefore, that these cytotoxic factors are immunoglobulins with specificity for self, suggesting that tolerance to thymus-dependent autoantigens does not exist at the B-cell level. The implications of this possibility for the understanding of the triggering mechanism of B lymphocytes and for self-nonself discrimination are discussed.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. I. Lysis restricted by HLA class II MB and DR antigens

In contrast to general findings that mouse and human cytotoxic T lymphocytes (CTL) are restricted in cytotoxic activity by major histocompatibility complex (MHC) class I antigens, we previously found that some herpes simplex virus (HSV) type I-infected cells that shared no HLA class I antigens with the HSV-1-stimulated lymphocytes were lysed. In this study, we addressed the question of the role of HLA antigens in human T cell-mediated lysis of HSV-1-infected cells by generating clones of HSV-1-directed CTL from two HSV-1-seropositive individuals. CTL clones that lysed autologous HSV-1-infected lymphoblastoid cell lines (LCL), but not natural killer-sensitive K562 cells or uninfected or influenza virus-infected LCL, were tested for cytotoxicity against a panel of allogeneic HSV-1-infected LCL. Clone KL-35 from individual KL lysed only HSV-1-infected LCL sharing the HLA class II MB1 antigen with KL. With all four CTL clones isolated from individual PM, only HSV-1-infected LCL sharing DR1 with PM were lysed. Monoclonal antibody s3/4 (directed against MB1 ), but not TS1/16 or B33 .1 (directed against a DR framework determinant), blocked lysis of autologous HSV-1-infected cells by KL-35. In contrast, B33 .1, but not s3/4, blocked lysis of autologous HSV-1-infected cells by the PM CTL clones but not by KL-35. Together, these results indicate that our five human CTL clones which are directed against HSV-1-infected cells, and which are all OKT3+, OKT4+, OKT8-, are restricted in lytic activity by HLA class II MB and DR antigens. These results suggest that the HLA D region-encoded class II antigens may be important in the recognition and destruction of virus-infected cells by human CTL.     

Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen I (OFA-I)

Summary Oncofetal antigen I (OFA-I) has been identified by immunofluorescence and immune adherence (IA) as a membrane antigen on human tumor cells, which cross-reacts with fetal brain tissue. This antigen induces humoral antibody in patients with cancer. The present work was designed to evaluate the complement-dependent cytotoxic potential of anti-OFA-I antibody produced in melanoma patients against an OFA-I-positive melanoma cell line, UCLA SO M14 (M14). Patients' sera were chosen on the basis of anti-OFA-I activity by IA. Alloantibodies to M14 were removed by absorption of the sera with lymphoblastoid cells autologous to M14. Fourteen sera were tested, and all demonstrated cytotoxic activity in the presence of rabbit complement. Human complement was also shown to mediate cytotoxicity, although less effectively than rabbit complement. The specificity of the reaction was confirmed when the cytotoxic capability could be absorbed by fetal brain, but not by autologous fetal liver tissues. These results indicate that a patient's serum antibody to OFA-I can lyse tumor cells expressing this antigen.     

Cytotoxicity of anti-beta 2-microglobulin xenoantisera to human and murine myeloid progenitor cells and lack of cross-reactivity with colony-stimulating activity

A previous report has suggested an antigenic relationship between beta 2-microglobulin (beta 2 mu) and granulocyte colony-stimulating activity (CSA). Since human myeloid progenitor cells (CFU-C) express HLA antigens and beta 2 mu is a known molecular component of HLA antigens, we wondered whether the reported effect of anti-beta 2 mu heteroantisera on in vitro granulopoiesis might result from cytotoxicity to CFU-C rather than from cross-reactivity with CSA. To test this, we used rabbit antibody reactive with human and murine beta 2 mu (anti-beta 2 mu). Treatment of human and murine bone marrow cells with anti-beta 2 mu and complement resulted in 95+% inhibition of CFU-C colony formation compared to controls. To test for an effect on CSA, anti-beta 2 mu was incubated with human and murine sources of CSA. After addition of goat anti-rabbit Ig antiserum to precipitate immune complexes and unbound anti-beta 2 mu, the supernatant fluid retained CSA but was no longer cytotoxic to CFU-C. These results indicate that human and murine CFU-C express membrane beta 2 mu and that anti-beta 2 mu antibody does not cross-react with human or murine CSA.     

Human lymphoid cell lines as targets for DRw

Summary Cultured human lymphoid cell lines (LCL) are useful as a source of target cells in several immunologic assays. More recently such cells have been used for the serological characterizations of the HLA-DR antigens. Typing of the same LCL in various laboratories during the VII Histocompatibility Workshop has given comparable results with a discordancy rate of less than 10%. This discordancy is likely to reflect the different sources of complement that can greatly alter the results of cytotoxic assays. The presence of naturally occuring antibody in rabbit complement to human cells can be avoided by: (a) absorbing with human cells at 0°C; (b) dilution with human serum; (c) dilution with heat-inactivated rabbit serum; (d) repeated freeze-thawing of the complement; or (e) careful selection of complement by screening procedures. Comparison of the results of HLA-DR typing of LCL with peripheral B-cells of the same donor show good correlations. However, LCL will occasionally give extra reactions perhaps due to the expression of new antigens. LCL can be coated with F(ab′)₂ fragments from antihuman β₂-microglobulin antibodies that block reactions of HLA-A, −B and −C antibodies allowing for discrimination of anti-DRw activity. Recipient of an American Heart Established Investigatorship. This work was supported by the Naval Medical Research and Development Command, Work Unit No. ZF51.524.013.1025, and by Grant Nos. AI 13154 AND CA 16071 from the National Institutes of Health.     

Elimination of mycoplasma contaminants from cell cultures with animal serum

Repeated treatment with guinea pig or rabbit serum, but not with human serum, was found to eliminate mycoplasma contaminants from mammalian cell cultures as judged by staining with the fluorescent dye Hoechst 33258. Following treatment with rabbit serum and several passages, M. hyorhinis could not be detected by staining, isolation on agar, or specific immunofluorescence in a human prostate carcinoma cell line heavily contaminated with this organism. There was no evidence for the involvement of antimycoplasma antibodies in the bactericidal activity of rabbit serum. Mycoplasmacidal activity of rabbit serum was associated with a heat-labile component(s) which could be inactivated by incubation of the serum with goat antirabbit complement component C3.     

Antileptospiral activity of serum. I. Normal and immune serum

Johnson, Russell C. (University of Minnesota, Minneapolis), and Louis H. Muschel. Antileptospiral activity of serum. I. Normal and immune serum. J. Bacteriol. 91:1403-1409. 1966.-Normal serum was found to exert a leptospiricidal effect, mediated by the complement system, against the nonpathogenic leptospires. Although resistant to normal serum, the pathogenic serotypes were susceptible to antiserum plus complement. Several variables in these immune leptospiricidal reactions were investigated. A reaction period of 3 hr at 37 C between serum substances and 1-day-old cells provided a maximal leptospiricidal effect. The normal serum of the rabbit, guinea pig, bovine, and human were leptospiricidal against the nonpathogenic serotypes, and, in conjunction with rabbit antiserum, rabbit and bovine complement were leptospiricidal against the pathogenic serotypes. Studies with C(14)-labeled leptospires indicated that the immune leptospiricidal reaction was associated with a loss of permeability control. Thus, like the gram-negative bacteria, the treponemes, erythrocytes, and nucleated mammalian cells, the leptospires may be included as cell types susceptible to the antibody-complement system.     

Cell-mediated cytotoxicity of lymphocytes from chickens inoculated with herpesvirus of turkey against a Marek's disease lymphoma cell line (MSB-1).

Using a new device which increases the sensitivity of detection of specific immune lysis of target cells by labeling them with [35S]-methionine, the in vitro cell-mediated cytotoxic response of spleen lymphocytes and peripheral blood lymphocytes from chickens vaccinated with herpesvirus of turkey (HVT), O1 strain, against MSB-1 line cells was clearly demonstrated. The cytotoxic activity was clearly inhibited by pretreatment of effector lymphocytes with anti-T lymphocyte serum and complement. The activity was greater using T cells purified from spleen lymphocytes and peripheral blood lymphocytes than with the unfractionated cells, indicating that T lymphocytes play the main role in effector activity. Using sera from HVG-vaccinated chickens, no significant cytotoxic effects were detected in the complement-dependent antibody cytotoxicity test against MSB-1 cells. These results suggest that cellular immunity against the surface antigen of Marek's disease (MD) lymphoma cells is mainly related to the preventive mechanism against MD incidence by HVT vaccination.     

Identification and quantitation of human T-cell antigen by antisera purified from antibodies crossreacting with hemopoietic progenitors and other blood cells

Anti-T cell globulin (ATCG) prepared from antihuman thymocyte serum by absorption with kidney, cells from patients with chronic lymphatic leukemias, and several lymphoblastoid cell lines was shown to react specifically with human thymus-derived lymphocytes. While high activity against thymocytes and a T-lymphoblastoid cell line could be demonstrated, ATCG remained negative against several chronic lymphatic leukemias and B-lymphoblastoid cell lines. The ATCG was used in the cytotoxic test, electronmicroscopy, and immunoautoradiography for identification of T cells in thymus, tonsils, spleen, blood, bone marrow, lymphatic leukemias, and lymphoblastoid cell lines. A comparison of these results with the ability to form spontaneous SRBC-rosettes revealed remarkable deviations between both markers in leukemias. Absorption with human brain failed to remove specific activity of ATCG. Labeling experiments by immunoautoradiography and investigations by complement fixation permitted quantitation of relative T-cell antigen concentration on different cell populations. As further evidence for specificity it could be shown that ATCG was no longer toxic for hemopoietic progenitors, whereas unabsorbed globulin reduced the number of colonyforming cells considerably.Abbreviations ALL acute lymphatic leukemia - ATCG anti-human T cell globulin (absorbed) - ATG anti-human thymocyte globulin (not absorbed) - Bm-C bone marrow cells - CFA complete Freund's adjuvant - CLL chronic lymphatic leukemia - EBV Epstein-Barr virus - GPC' guinea pig complement - HBSS Hanks' balanced salt solution - Ig immunoglobulin - PBS phosphate buffered saline - Per-Ly peripheral blood lymphocytes (normal) - Spl-Ly spleen lymphocytes - SRBC sheep red blood cells - VBS veronal buffered saline - Thy-Ly thymus lymphocytes - Ton-Ly tonsil lymphocytes     

In vitro studies of the rabbit immune system. VI. Rabbit anti-mouse cytotoxic T effector cells are inhibited by anti-rabbit T cell serum in the absence of complement.

Xenogeneic rabbit anti-mouse cell-mediated cytotoxic activity could be generated by culturing lymphoid cells from mesenteric lymph nodes (MLN), spleen, or peripheral blood of rabbits primed 2 to 8 weeks earlier with mouse tumor or spleen cells. MLN cells, which provided the best source of activity after being cultured with 5 to 10 X 10(6) mitomycin C-treated mouse spleen cells for 4 to 6 days, produced 30 to 90% specific isotope release after 4 to 7 hr incubation with 15Cr-labeled tumor target cells. Xenogeneic cytotoxic activity was primarily H-2 specific and could not be blocked by immune complexes but was abrogated by treatment with goat anti-rabbit thymocyte serum plus complement (ATS + C) before or after culture. Therefore, the activity appeared to be mediated by cytotoxic T lymphocytes (CTL). Furthermore, ATS without C abrogated cytotoxic activity when included in the CTL assay at concentrations of 5 to 15 microliter/10(7) effector cells. The inhibitory activity of ATS was directed to the rabbit effector population and could be absorbed completely by rabbit thymocytes. Antisera to mouse T cells with comparable cytolytic activity in the presence of C did not inhibit murine allogeneic CTL.     

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.