Killer cells: a functional comparison between natural, immune T-cell and antibody-dependent in vitro systems.

Previous reports have shown that spleen cells from nonimmune adult mice of certain strains do regularly kill Moloney leukemia virus-induced lymphomas in short-term 51Cr release assays. This naturally occuring killer (NK) cell had low adherent properties and had the morphological appearance of a lymphocyte. Still it lacked surface characteristics of mature T or B lymphocytes. In the present report a functional study was carried out, comparing in parallel the NK system, the T-cell killing across an H-2 barrier (anti-P815), and the antibody-dependent cell-mediated chicken red blood cell (CRBC) system. In contrast to the effector cells in the CRBC system, the NK cells were insensitive to erythrocyte antibody complement (EAC) rosette depletion and would pass through nylon wool columns. NK activity was not inhibited by the presence of heat-aggregated human or mouse gamma globulin, in contrast to the strong inhibition noted in the CRBC system. Sensitivity to trypsin pretreatment was noted in the NK system as well as in the immune P815 system, whereas the CRBC system was relatively trypsin resistant. Antitheta plus complement eliminated the anti-P815 activity, but did not touch the NK activity. The present results thus further distinguish the NK cell from cytotoxic T lymphocytes or from antibody-dependent killer cells.

Allylamine derivatives: new class of synthetic antifungal agents inhibiting fungal squalene epoxidase.

A new class of synthetic antifungal agents, the allylamines , has been developed by modification of naftifine , a topical antimycotic. SF 86-327, the most effective of these compounds so far, is highly active in vitro against a wide range of fungi and exceeds clinical standards in the oral and topical treatment of guinea pig dermatophytoses. SF 86-327 is a powerful specific inhibitor of fungal squalene epoxidase, a key enzyme in sterol biosynthesis.

TCR gamma/delta bearing lymphocytes in peripheral blood of allogenic bone marrow transplanted patients.

The presence of two distinct T-cell receptors (TCR) alpha/beta and gamma/delta dimers as well as of the activated T cells was analysed in peripheral blood mononuclear cells from seventeen recipients of allogeneic bone marrow transplants for leukemia and for severe aplastic anemia. Nine of seventeen recipients expressed an elevated percentage of T cells bearing TCR gamma/delta receptors in their peripheral blood. Seven out of nine cases having elevated gamma/delta positive cells showed chronic graft-versus-host (GVH) disease; one patient was treated with Cyclosporin A, and one patient was asymptomatic. In the twelve patients with GVH or other clinical symptoms, activated T cells (CD3+/HLA-DR+) were elevated indicating an autoreactive or alloreactive cell population. Our results confirmed earlier in vitro data showing that TCR-gamma/delta-bearing lymphocytes may be an activated T-cell population, and this T cell subset might be involved in mediating GVH disease, or in prolonging immunodeficiency after transplantation.

Comparison of the lymphoid toxicities of mitobronitol and busulphan in mice: reduced B cell toxicity and improved thymic recovery as possible contributors to the reduced risk for complications following BMT with mitobronitol preconditioning.

It has previously been reported that the use of mitobronitol (dibromomannitol, DBM) instead of busulphan (BU) for myelosuppression is associated with significantly decreased risk for several complications of allogeneic bone marrow transplantation in accelerated chronic granulocytic leukemia. In exploring the pharmacologic basis for this observation, we have compared the acute and subacute cytotoxicities of DBM and BU on the spleen and thymus of mice. While there was comparable early (day 3) weight loss in both organs following these treatments, splenic B cells exhibited significantly less damage, and thymic regeneration (over weeks) was significantly faster following DBM treatment than with BU. These observations raise the possibility that improved post-BMT immune recovery could contribute to the clinical benefits observed with DBM-preconditioning.

Regulatory function of cell surface molecules CD2-, LFA- and beta 2-microglobulin in natural killer cell activity.

The functional importance of various cell membrane bound molecules was studied and compared in the NK cytotoxicity and CTL activity. LFA-1 and CD2 participate in both killing functions, while CD3 and CD8/CD4 as well as MHC class I molecules are involved only in CTL activity. Nevertheless CD2- and beta 2-microglobulin are representatives of the NK function. It was demonstrated that CD2-, LFA-1 and beta 2-microglobulin molecules have an additive and complementary function in the killing mechanism. The upregulation of alpha- and gamma-interferons on NK function seems not to be a consequence of the enhanced expression of these molecules on the cell surface induced by IF at the same time.

Ligand inhibition studies on the role of Fc receptors in antibody-dependent cell-mediated cytotoxicity.

Subjection of human peripheral blood lymphocytes to a temp shift from 4 to 37 degrees C resulted in a shedding of Fc receptors (termed FcRI) from 40-50% of FcR-positive cells followed by their re-expression within 4 hr; a phenomenon which had no effect on the cells' antibody-dependent killing capacity. Removal of lymphocytes having an immobile form of the Fc receptor resistant to the effects of the temp shift (termed FcRII), or removal of lymphocytes bearing both FcRI and FcRII, resulted in a similar amount of reduction in ADCC activity. This was attributed, therefore, to the loss of FcRII-positive cells. The influence of isolated (shedded) FcRI and Clq on ADCC activity was investigated. Soluble FcRI was shown to inhibit ADCC mediated through the immobile Fc receptors (FcRII), despite its lack of an ability to block EA rosette formation through these receptors. Clq also had a dose-dependent inhibitory effect on ADCC. These observations are consistent with earlier findings that FcRII possesses two active binding sites; and suggest that a prerequisite for killing in ADCC is the interaction of these with the C gamma 2 and C gamma 3 domains. The ability of synthetic peptides representative of human gamma 1-chain sequences to inhibit ADCC was determined, in an attempt to locate those sites within the IgG antibody Fc region involved in interaction with two FcR binding sites. Preliminary evidence was obtained to suggest that one of these is situated within the C gamma 2 domain, in the region of residues 274 (Lys)-294 (Glu).

Recognition of autologous and allogeneic lymphocytes and tumor cells by human natural killer cells.

The shedding of the mobile Fc receptor (FcR1) and the depletion of the immobile Fc receptor (FcR11) bearing human lymphocytes revealed that human natural killer cells belong to the FcR11-bearing population. Anti-beta-2-microglobulin treatment of the effector cells decreased natural cytotoxicity against some target cells and the detectability of HLA antigens, indicating that histocompatibility antigens or related structures may be involved in natural cytotoxicity. Using a panel of 29 autologous and allogenic PHA-stimulated target cells and peripheral lymphocytes from the same donors as the effector cells, distinct cytotoxic responses against allogeneic and autologous target cells were observed. A computer analysis of selective natural cytotoxicity distinguished seven different groups of target cells that may represent common structures for NK recognition.

Morphological changes induced by Naftifine, a new antifungal agent, in Trichophyton mentagrophytes.

The morphological changes in the hyphae of Trichophyton mentagrophytes treated with Naftifine (0.01-0.5 micrograms/ml) were studied by light and electron microscopy. The most striking changes observed following treatment with this new antimycotic agent were bulb-shaped thickenings at the hyphal tips and dose-dependent, spherical, or drop-shaped depositions of varying size within the cells. The abnormal formations were not only visible in the cytoplasm (discrete or aggregated in vacuoles), but also in the region of the cell membrane, in all layers of the cell wall and on the cell surface. Their lipid nature can be deduced from several significant characteristics including osmiophily, the conchoidal fracture surface observed in freeze-fracture replicas and their extractability with acetone. This evidence suggests that the antimycotic action of Naftifine results from interaction of the agent with fungal lipid metabolism and possibly from alterations of the cell wall structure. The latter is also suggested by the changes observed in the hyphal tips.

Analysis of the age-related refractoriness of T-lymphocyte reactivity in humans.

Aged individuals could be divided into two groups according to their T-lymphocyte transformation values. The relationship between the PHA (phytohemagglutinin) stimulation indices and spontaneous thymidine incorporation; the PHA dose-response type distribution and the relative number of resting T lymphocytes was similar to the control group in aged subjects of seemingly intact T lymphocyte transformation values. However, their B cell compartment was found to be reduced. On the other hand, the ratio between the stimulation indices and spontaneous thymidine incorporation values of aged subjects of impaired T lymphocyte reactivity deviated from that of the control group. This group had an increased frequency of subjects giving maximal transformation values at relatively high PHA doses (hyposensitives) at the expense of normosensitives and showed reduced numbers of resting T cells, but normal B cell compartment. These results suggest that immunodeficiencies developing with age can possibly be of individually different types.

Synthesis and antifungal activity of (E)-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphtha lenemethanamine (SF 86-327) and related allylamine derivatives with enhanced oral activity.

The allylamine derivatives are a new class of synthetic antifungal agents inhibiting fungal squalene epoxidase. A new subclass, which features an acetylene group conjugated with the allylamine double bond, is characterized by enhanced antifungal activity, especially on oral treatment of guinea pig dermatophytoses. Increased branching of the alkyl group next to the triple bond led to the tert-butylacetylene derivative SF 86-327, a compound with strikingly increased activity in vitro and in vivo, which is now under clinical evaluation. Versatile synthetic routes, comparative biological data, and structure--activity relationships are presented.

Synthesis and structure-activity relationships of benzo[b]thienylallylamine antimycotics.

Benzo[b]thiophene analogues of the allylamine antimycotic terbinafine (2) bearing the side chain at various positions and optionally substituted by halogen have been prepared and their antifungal activity studied. Derivatives bearing the side chain at positions 3, 4, or 7 are bioequivalents of 2. Compounds containing the allylamine side chain at position 7, with a further substituent at position 3, showed significantly enhanced activity against Candida albicans, an effect which appears to be specifically linked only to this particular substitution pattern. 3-Chloro-7-benzo[b]thienyl derivative 7m was found to be the most potent allylamine antimycotic identified so far. In general, substituted benzo[b]thiophenes can be used not only as potential equivalents of naphthalene in bioactive compounds but also as a tool to selectively modify biological activities.

Synthesis and structure-activity relationships of naftifine-related allylamine antimycotics.

Naftifine (1) is the first representative of the new antifungal allylamine derivatives. Its biological activity is strictly bound to specific structural requirements that are unrelated to those of known antifungals. A tertiary allylamine function seems to be a prerequisite for activity against fungi. By systematic variation of the individual structural elements in 1, detailed structure-activity relationships are defined in which the phenyl ring is the structural feature permitting the widest variations. Versatile synthetic routes to allylamine derivatives and comparative biological data are presented.

Selective effect of noncytotoxic blocking alloantibodies produced after platelet transfusions on MLC, and mitogen and soluble antigen-induced responses of human lymphocytes.

The transfusion of blood components (buffy coat and platelets) may induce characteristic alloimmune response or suppressive regulation that have, in certain cases, a beneficial effect on allograft survival. The blocking effect of the sera of donors immunized with platelets on mixed lymphocyte culture and on the response of lymphocytes to mitogen as well as soluble antigen (PPD, tetanus toxoid) stimulation was studied. Six sera from 7 volunteers displayed a strong and significant nonspecific MLC blocking effect that was detectable on the 10th day following the second platelet transfusion (PT). Incubation of isolated stimulator and effector cell population with this "blocking sera" showed that the latter are involved in the mediation of suppression in the MLC test. This inhibitory effect is associated with the serum IgG fraction lacking any correlation with either class I or class II specific cytotoxic antibodies. Selective blocking behavior was found on transformation activity induced by mitogens or soluble antigens. Thus, sera of platelet-transfused volunteers decreases the responsiveness of lymphocytes to phytohemagglutinin, while the response to concanavalin A, tetanus toxoid, and PPD was not suppressed. Separate treatment of T and B lymphocyte populations and monocytes with the blocking sera showed that only T and B lymphocytes are targets, and not the monocytes for the inhibition in the case of PHA-induced proliferation. Indirect evidence may support the notion that MLC-inhibiting and FcR-blocking antibodies may be analog products of a regulatory alloimmune response induced by leukocytes that are partially responsible for the beneficial transfusion effect in organ transplantation.

Decreased antibody-dependent and spontaneous cell-mediated cytotoxicity in patients with chronic renal failure.

Antibody-dependent and spontaneous lymphocytotoxicity of blood mononuclear cells from 15 patients with chronic renal failure was studied in allogeneic K-562 and xenogeneic chicken erythrocyte test systems. Both antibody-dependent and spontaneous cytotoxic activities were significantly decreased in uremic patients as compared to the corresponding mean values of healthy control persons and non-uremic patients with the same underlying diseases. The addition of autologous serum further reduced the impaired cytotoxic capacity of uremic lymphocytes. Uremic sera also inhibited the antibody-dependent and spontaneous lymphocytotoxicity of healthy individuals.

Effect of haemodialysis on the antibody-dependent and spontaneous cell-mediated cytotoxicity of patients with chronic renal failure.

Antibody-dependent and spontaneous lymphocytotoxicity of blood mononuclear cells from 10 patients with chronic renal failure was studied before and after haemodialysis in xenogeneic chicken erythrocyte and allogeneic K-562 test systems. The originally impaired antibody-dependent and spontaneous lymphocytoxicity of uraemic patients significantly improved following haemodialysis. Both pre- and post-haemodialysis uraemic sera strongly inhibited the antibody-dependent and spontaneous cytotoxicity of autologous and of healthy control lymphocytes.

Lectin-dependent cellular cytotoxicity by human lymphocytes is blocked by monoclonal antibodies to the sheep red cell receptor.

Investigating the functional effects of anti-T cell monoclonal antibodies (obtained through the Second International Workshop on Human Leucocyte Differentiation Antigens), antibodies to the sheep red cell receptor (CD2) were found to block lectin-dependent cytotoxicity. Blocking was seen with all anti-CD2 reagents tested regardless of epitope specificity, immunoglobulin subclass or other factors. Antibodies to other T cell differentiation antigens had no effect.

Non-cytotoxic blocking antibodies and suppressor cells induced by donor-specific transfusions in healthy volunteers and potential kidney transplant recipients.

Donor-specific transfusion (DST)-induced immunosuppression plays a significant role in clinical and experimental transplantation. To clarify the mechanism of suppression on alloreactivity the suppressor cell induction and the non-cytotoxic blocking antibody production and importance was studied in 15 healthy volunteers and 3 kidney transplant recipients (KTR) after DST on mixed lymphocyte culture (MLC). Significant decrease of anti-donor MLC response was found in all of KTR and in 12 cases of the 15 transfused volunteers. Of 18 cases, 15 had blocking factors in their post-DST serum which strongly (52-91%) inhibited the MLC response. The IgG fractions isolated from the "blocking sera" were responsible for this inhibition. Eighty-one percent of non-cytotoxic blocking antibodies affected the responder cells in MLC and reacted with third party responder cells as well. Both buffy coat and platelet transfusions evoked production of the non-specific blocking antibodies. Our data strongly suggest that DST induces not only the differentiation of suppressor cells and production of anti-idiotypic antibodies, but also the appearance of non-specific, non-cytotoxic antibodies, which may participate in the cell-mediated immune suppression of the alloimmune reactivity.

Functional and immunogenetic characterization of FcR-blocking antibody.

The characteristics and functional importance of FcR-blocking antibodies and their production were investigated after immunization with whole blood, "buffy coat" and purified platelets. We studied the presence of FcR-blocking antibody in haemodialyzed, transfused patients waiting for kidney transplantation, and we found strong correlation between the blocking effect and better graft survival. We suggest that this blocking antibody does not attack FcR as primary target. Investigation of blocking activity of ten different immune sera on 50 healthy panel cells showed that target antigen has some polymorphic varieties. On basis of family studies it seems that the target antigen is not linked to HLA haplotype. The blocking effect of sera could be removed by absorption of CD8+ cells, B lymphocytes, platelets and granulocytes, but not with erythrocytes, monocytes, CD8- cells and NK cells.

The different effect of alpha and gamma interferons and interleukin 2 on the expression of CD2, CD3, CD4 and CD8 antigens in comparison to histocompatibility antigens of human lymphocytes.

The effects of alpha- and gamma-interferons (IFN-alpha, -gamma) and of interleukin 2 (IL-2) on the expression of certain differentiation antigens were compared with those of major histocompatibility antigens on human lymphocytes. IFN-gamma and IFN-alpha in high doses significantly increased the expression of T11 (CD2) differentiation antigen, but did not affect the expression of T4 (CD4), T8 (CD8), T3 (CD3) and Leu-7 antigens (HNK-1). Both natural and recombinant IFN-alpha and -beta apparently increased the expression of HLA-ABC antigens and of beta-2 microglobulin (beta 2m) after 16 h incubation. The amount of HLA-DR antigen, however, doubled in a few hours following IFN-gamma treatment. IL-2 affected the expression of CD2 and CD8 antigens only marginally, but did not affect that of CD3 and Leu-7; however, it strongly enhanced the expression of HLA-ABC, HLA-DR, and beta 2m antigens.

Immunoglobulin variable regions usage by B-lymphocytes infiltrating a human breast medullary carcinoma.

Breast medullary carcinoma are heavily infiltrated by B-lymphocytes and associated with a good prognosis despite their high histological grade. We investigated the Ig repertoire of B-lymphocytes infiltrating one such tumour. A single cell suspension was obtained from a tumor specimen by enzymatic digestion. VH, Vkappa, and Vlambda regions were amplified by RT-PCR using mixtures of primers optimized to maximize the diversity of the PCR products. They were then cloned and sequenced. Analysis of 9 VH, 5 Vkappa, and 10 Vlambda sequences using the Kabat database indicated that several VH and VL region subgroups (I, II and III) are expressed by B-lymphocytes infiltrating this tumor. The analysis of CDR3 regions also showed a variability, although some VH and VL clones exhibited identical or nearly identical sequences. Thus, the B-cell infiltration observed in this breast medullary carcinoma does not reflect a monoclonal proliferation and represents an oligoclonal or a polyclonal B-cell proliferation.