Rescue of haemopoiesis by a combination of growth factors including stem-cell factor.

We observed an unexpectedly rapid rise in platelet counts with complete haematological recovery after a BEAM regimen, in a patient who could not be rescued by autologous transplant but who received filgrastim, epoetin alfa, and ancestim. We feel that these results may be attributed to this specific growth factor combination, including ancestim, a cytokine known to act on primitive stem cells. If confirmed, this observation may open new possibilities in intensive chemotherapy for patients for whom haematopoietic progenitors are difficult to harvest. This may also represent an alternative to ex-vivo expansion and deserves further investigation.

Abrogation of post-myeloablative chemotherapy neutropenia by ex-vivo expanded autologous CD34-positive cells.

We show that absolute and severe neutropenia after high-dose therapy with melphalan with or without total body irradiation can be abrogated by cells generated ex vivo. This may change the clinical practice of haematopoietic cell transplantation and high-dose chemotherapy because the morbidity and hospitalisation associated with neutropenia could be avoided or reduced.

[Characterization of chronic lymphocytic proliferation in a female patient having rheumatoid polyarthritis with neutropenia]. / Caractérisation d'une prolifération lymphocytaire chronique chez une patiente atteinte d'une polyarthrite rhumatoïde avec neutropénie.

The authors have studied the case of a female patient with rheumatoid polyarthritis, who developed a lymphocytic proliferation in the blood, the marrow, and the liver, associated with a neutropenia. Several similar cases have been recently reported in the literature. The cellular proliferation is made of large granulous lymphocytes and the study of membrane markers enables to find the following homogeneous phenotype: E rosette+, CD8+, HNK-1+, FcR+, CD4-luminal diameter "divided by degrees - -, IgS-, HLA class II-. This lymphocytic sub-population produces little interleukin-2, responds weakly to mitogens (PHA, CON A, PWM), and inhibits the response of normal lymphocytes to the same mitogens. These lymphocytes have a weak natural killer activity but, on the contrary, develop a very strong cytotoxic activity which is antibody-dependent. Clinically, splenomegaly, anemia and infections are frequent and hepatomegaly or thrombopenia more rare. Adenopathies are never present. The evolution is chronic in nature and not very aggressive, although the lymphocytic proliferation is monoclonal in origin, as demonstrated in molecular biology studies. The neutropenia might be secondary to an inhibiting effect of lymphocytes on the granular precursors of the bone marrow. There is a definite association between this lympho-proliferative syndrome and rheumatoid polyarthritis, and this association appears to be different from the Felty's syndrome.

Enzyme immunoassay for human chorionic gonadotropin using monoclonal antibodies elicited with a synthetic peptide.

We recently described the generation of mouse monoclonal antibodies directed against hCG using a synthetic peptide immunogen: the carboxy-terminal peptide (CTP) of beta-hCG. The CTP, residues 109-145, is a portion unique to hCG and is not present in other glycoprotein hormones. These monoclonal antibodies react with beta-hCG-CTP and whole hCG but do not react with hLH. In the present study, a two-site sandwich ELISA specific for hCG has been developed using these CTP-induced monoclonal antibodies. Two monoclonal antibodies were paired: beta-hCG-CTP-a6 was immobilized to give a hCG-specific solid phase, and a biotinylated monoclonal antibody against beta-hCG was used as the indicator antibody. This second antibody was introduced during the hCG-capture step because of its agonist effect on hCG-capture by beta-hCG-CTP-a6 solid phase. This ELISA system can detect 2.2 IU/l hCG in a serum-free medium and 10.4 IU/l in a medium containing 20% human serum. We did not observe any cross-reactivity with hLH up to concentrations of 5000 IU/l. The hCG-ELISA correlated well (r = 0.99) with a control RIA in an assay of 78 human sera from healthy males, pregnant or non-pregnant females and from patients with choriocarcinoma, hydatidiform mole, or teratocarcinoma.

Characterization of an expanded subpopulation of large granular lymphocytes in a patient with rheumatoid arthritis.

We describe a rheumatoid arthritis patient who was found to have chronic T cell lymphocytosis and neutropenia. She had an increased number of lymphocytes in the peripheral blood, bone marrow, and liver, and the expanded lymphocyte subset consisted of large granular lymphocytes with a homogeneous phenotype. Of the previously described patients with these large granular lymphocytes, almost one-fourth have had rheumatoid arthritis.

Non-cross-reactive monoclonal antibodies to human chorionic gonadotropin generated after immunization with a synthetic peptide.

Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.

Human autologous rosettes. IV. Their relation with interleukin 2 activity production and natural killer cells in cancer patients.

Peripheral blood lymphocytes (PBL) of solid-tumor-bearing cancer patients produced a lower interleukin 2 (IL-2) activity after lectin stimulation than did those from normal subjects. Moreover natural killer (NK) cell activity and autologous rosette forming (ARF) cell rate are found significantly correlated with IL-2 production in these patients. No direct relation is observed between ARF cell ratio and NK cell activity in a given patient. A central role for IL-2 in cancer patient immune dysfunctions is suggested. Two lines of pathogenetic mechanisms are documented. First, PBL exhibited cellular function defects, namely, autologous receptor expression, IL-2 production, and NK activity. Second, these dysfunctions involved, at least partly, plasma factors. The possibility of specific deficiency, (e.g., thymic factors) is not documented. Conversely it is demonstrated that patient plasma contain immunosuppressive factor(s) that block(s) IL-2 production and ARF cell expression. Involvement of ARF cell receptor in T-cell activation is discussed.

Anti-idiotype antibody-dependent cell-mediated cytotoxicity (ADCC) against idiotype-bearing cells.

The present report demonstrates that cells expressing idiotypic determinants on their surfaces can be specifically destroyed by anti-idiotypic antibodies acting as inducers of antibody-dependent cell-mediated cytotoxicity. Lysis of surface-idiotype-positive hybridoma cells was effected by mouse spleen cells, mouse peritoneal cells, or human blood mononuclear cells, in the presence of anti-idiotypic antibodies. In order for lysis to occur, the simultaneous presence of Fc receptor-bearing cytotoxic cells and anti-idiotypic antibodies was required. Specificity of lysis was conferred by the anti-idiotypic antibody. Hybridoma cells expressing a different idiotype could not be lysed. Anti-idiotypic antibody-dependent cell-mediated cytotoxicity against 51Cr-labeled cells expressing a particular idiotype could be induced by the specific anti-idiotypic antibodies and inhibited by unlabeled idiotype-positive cells or by idiotype in a soluble form. Cytotoxicity could not be inhibited by the presence of bystander cells or monoclonal antibodies expressing a different idiotype. It is proposed that ADCC lysis of idiotype-positive cells has important implications for understanding the regulation of immune responses by an idiotype-anti-idiotype network. This cytolytic pathway provides an extremely specific and effective mode of control of idiotype-positive cells by anti-idiotype antibody. Previous observations that an intact Fc fragment was necessary for anti-idiotype antibodies to exert suppressive effects in vivo further support this hypothesis.

Human soluble Fc gamma-binding material. I. Immunochemical properties of the material produced by the T cell line KE37.

A soluble Fc gamma-binding material (S-Fc gamma BM) is produced by the Fc gamma receptor-bearing human T cell line KE37 but not by the two Fc receptor-negative cell lines Molt 4 and Reh 6. The S-Fc gamma BM has specific affinity for the Fc fragment of IgG and none for the Fab fragments. It can be obtained in radioactive form after internal labeling with radioactive amino acids. It can then be isolated from the supernatant of the KE37 cell line by affinity chromatography, gel filtration and isoelectric focusing. The subunit components of S-Fc gamma BM are 23,000-dalton polypeptide chains. The soluble form is a dimer (42,000 to 45,000 m.w., pl 6.2), which retains Fc gamma-binding activity after isolation. Higher m.w. polymers of the 23,000-dalton subunits are also present in the supernatant of the KE37 cell line. These polymers are relatively insoluble in aqueous solvents but possess Fc gamma-binding activity. The S-Fc gamma BM molecules might be derived from a membrane-inserted form by restricted proteolytic cleavage at connecting regions of a larger polypeptide chain folded into 23,000-dalton subunits.

Surface immunoglobulins as targets for anti-immunoglobulin-dependent cell-mediated lysis of B cells.

Human K cells are able to lyse human lymphoblastoid B cells in the presence of specific anti-human immunoglobulin isotype antibodies. The human lymphoblastoid B-cell line DAUDI (surface mu- and kappa-chains positive) was lysed in the presence of anti-mu and anti-kappa antisera, or IgG fraction of these antisera, and B-cell-depleted human lymphocytes. Lysis was not induced by anti-isotypic antisera alone or human lymphocytes alone. Lysis was not induced by antisera directed at isotypes which were not expressed on the DAUDI cells. The human lymphoblastoid B-cell line RAJI, which does not express surface membrane isotypes, could not be lysed by the anti-isotype-dependent cell-mediated mechanism. Lysis of DAUDI cells by this mechanism was mediated by Fc gamma receptor-bearing human non-B lymphocytes and required an intact Fc piece in the inducing anti-isotypic antibody. These observations are discussed as a possible role for K cells in the regulation of immune responses by interaction between anti-idiotypic antibodies and idiotype-bearing cells.

Amplification of the polyclonal activation of human T cells. I. Null-cell products promote the polyclonal proliferation of T cells.

Synergy can be observed in the proliferative response to mitogens of cultures containing human T and Null cells when compared with those containing only highly purified cells of those two types. This synergy was analysed (i) by evaluation of the proliferative response at each step of the purification process leading to separation of T and Null cells; (ii) by back-mixing T and Nul cells at different rations; and (iii) by evaluation of the proliferative response of free suspension cultures of T cells overlaying a semi-solid layer containing Null cells, or of free suspension cultures of Null cells over a semi-solid culture layer of T cells. The following conclusions were reached: (i) purified Null cells are unresponsive to mitogen when cultured alone or in the presence of diffusible T-cell products; (ii) the T cells are less responsive when cultured alone than in the presence of Null cells or diffusible Null cell products. Thus the synergistic effect observed between T and Null cells is not due to the promotion of Null-cell proliferation by T -cell products but can be accounted for by diffusible Null-cell products enhancing the process of T lymphocyte activation by mitogens.

Nature and mechanisms of action of co-operating cells controlling human T-colony formation.

The mechanisms of action and the nature of the co-operating cells (CC) controlling human T-lymphocyte-colony formation were investigated. Media conditioned by PHA-stimulated blood mononuclear cells (MC) were tested for their capacity to induce T-colony formation in the effluent cell population, obtained after anti (Fab')2 cell affinity chromatography of MC. This cell population has been previously shown to still contain T-colony-forming cells (TCFC), but to be devoid of a co-operating cell population essential for T-cell-colony growth, thus requiring a feeder layer containing media conditioned by PHA-stimulated MC in order to generate T colonies. Further evidence is also presented that: phytohaemagglutinin (PHA) was necessary to two steps of T-colony formation: (i) for the production of colony promoting activity (CPA) by PHA-stimulated MC; (ii) for the induction of the TCFC to generate a colony, in the presence of CPA. There were high producers and low producers of CPA. The low CPA production observed with some donors could be explained by a suppressive effect mediated by phagocytic cells, presumably monocytes, whereas the cells retained on the anti-(Fab')2 immunoadsorbent (mainly B cells) were able to produce very high CPA levels.

Human T-lymphocyte colonies: generation of colonies in different lymphocyte subpopulations.

The generation of human T-lymphocyte colonies from different lymphocyte subpopulations in the presence of PHA alone, or PHA plus media conditioned by PHA-stimulated lymphocytes (PHA-LCM) has been investigated. The separation technique consisted of phagocytic cell depletion by carbonyl iron treatment and fractionation of non-phagocytic cells (NP cells) into B cells and T + null cells by affinity chromatography on an anti-F(ab')2 column. The T cells were separated from the null cells by E-rosette sedimentation. Under these conditions, we showed that: no T-lymphocyte colonies were obtained from the null-cell subset in the presence of PHA of PHA + PHA-LCM; T-lymphocyte-colony formation potential was retained in the T-cell subset. Some variability was observed in the production of T colonies using peripheral blood lymphocytes (PBL) from different donors. Low producers and high producers of T-lymphocyte colonies were encountered. The low production of T-lymphocyte colonies observed in some donors was due to a suppressive effect mediated by the phagocytic cells, probably monocytes. The anti-F(ab')2 immunoadsorbent retained a cell population necessary for T-lymphocyte colony growth.

Human T lymphocyte colonies. I. Surface markers and cytotoxic potential of colony cells.

Colonies were obtained from peripheral blood lymphocytes (PBL) grown in soft agar in the presence of PHAM or PHAp mitogens. One out of 130 PBL was able to generate a colony. Colony cells were mass harvested and assayed for surface markers and cytotoxic potential. Most of the colony cells (83%) form spontaneous rosettes with sheep-red blood cells (RBC) and bear the human T lymphocyte antigens (HTLA) (92%). A significant amount of colony cells able to bind autologous RBC was detected (24%). The capacity of PBL and colony cells to bind Ox-RBC sensitized with rabbit anti-Ox-RBC IgM (EAM complexes) was measured: only 15% of colony cells compared to 49% of the PBL formed EAM rosettes. The capacity of cells to bind the Fc portion of antigen-complexed IgG was investigated by two rosette assays: using Chicken or Ox-RBC sensitized with a rabbit anti-Chicken-RBC or Ox-RBC IgG (Chicken EAG or Ox-EAG complexes). The percentage of colony cells forming Chicken EAG rosettes was low (3.6%) compared to PBL (12%). This percentage was significantly increased with PHAp, and not PHAM stimulation (11%). Using Ox-EAG complexes, we confirmed the low percentage of EAG rosettes in colony cells under PHAM stimulation (4.7%) compared to PBL (21%). A significant cytotoxic capacity (spontaneous or antibody dependent) was found in colony cells after PHAM stimulation. This method of culture is able to generate clones of T cells and conserve T cell subsets and cytotoxic potential usually found in a T purified population. In further studies, it will be interesting to investigate if each clone possesses specific markers and cytotoxic potential and is able to maintain this differentiation step in long term culture.

Human K cell activity. I. Relation to effector cells buoyant density.

In order to disclose a relation between human K cell activity and effector cells buoyant density, this activity was tested, against antibody sensitized 51Cr labelled L1210 cells, in lymphocytes subsets obtained by centrifugation to equilibrium in discontinuous bovine serum albumin gradients. Since K cell activity is mainly present in null and T cells, this study focused on the buoyant density subsets of human T and null cells. Our results show that, in these T and null cells, K cell activity is directly related to cell density, the less dense fraction having the highest cytotoxic capacity. From the recovery of Lytic Units, it is shown that the K cell pool can be associated with the population of activated or precursor cells in human peripheral blood.

Ability of thymosin to decrease in vivo and in vitro suppressor cell activity in tumor bearing mice and cancer patients.

We have demonstrated that splenic lymphocytes from normal syngeneic animals can stimulate tumor growth. This effect is T-cell dependent. Preincubation of these lymphocytes with thymosin not only blocks facilitated tumor growth but can induce a significant reduction of local tumor growth and number of pulmonary metastases. Furthermore, thymosin can also block the expression of suppressor activity of lymphocytes either stimulated by Con A or originating from various advanced solid tumor bearing patients. These results therefore suggest that thymosin is able to modulate, directly or indirectly, functional expression of suppressor cells.

Immune imbalance in cancer patients.

The immune status of the tumor-bearing patient remains poorly defined. In various solid-tumor-bearing patients, we demonstrated the absence of ADCC modifications in the patient in relapse or in evolution. These same patients presented a significant increase in immune complexes when compared with patients in remission. Furthermore, we noted a decreased NK activity, a decreased number of ARFC, corresponding to a helper T cell subpopulation, and a corollary increase in T-dependent suppressor activity. These results, on the whole, suggest an immune imbalance and that the helper cell-suppressor cell ratio should be investigated in greater depth within the context of the immune response in the cancer patient.