IRI-514, a synthetic peptide analogue of thymopentin, reduces the behavioral response to social stress in rats.

Thymopentin, a synthetic pentapeptide (Arg-Lys-Asp-Val-Tyr) corresponding to amino acids 32-36 of the thymic polypeptide thymopoietin, has been reported to block adrenocorticotrope responses to stress. The purpose of the present study was to explore potential antistress properties of a synthetic analogue of thymopentin, IRI-514 (Ac-Arg-Pro-Asp-Phe-NH2) using a behavioral response to a stressor. The behavioral response to social conflict stress (resident-intruder paradigm) was evaluated by the elevated plus-maze test of anxiety in adult Wistar rats. A single subcutaneous (SC) administration of IRI-514, 48 h before stress, dose-dependently reversed the anxiety-like behavior induced by the social stress. The effect of IRI-514 was present over an extended period (24-72 h) following SC administration and was maximally effective at a dose of 1 mg/kg. These results indicate that IRI-514 has a long-lasting modulatory effect on behavioral responses to a stressor, and suggest that thymopoietin-derived peptides may have a role in modulating both behavioral and neuroendocrine responses to stress.

Expression of CZ-1: a CD45RB epitope on progenitors of natural killer and other haematopoietic cells.

CZ-1 is a novel sialic acid-dependent epitope of the murine CD45RB molecule which is expressed on cells that proliferate when cultured in IL-2. Because IL-2 appears to be important in the differentiation of NK cells, the authors examined the expression of CZ-1 on immature NK-lineage cells within the bone marrow. All mature NK1.1+ cells as well as their NK1.1- IL-2 responsive precursors were CZ-1+. Furthermore, IL-2 unresponsive transplantable NK progenitor cells expressed CZ-1 also. To examine expression of CZ-1 on other immature lymphoid progenitor cells, CZ-1+ and CZ-1- marrow cells were transplanted into lightly irradiated scid mice. Transfer of CZ-1+ cells resulted in rapid and sustained generation of thymocytes and splenic B cells, whereas CZ-1- cells caused delayed repopulation. This suggested that the slowly repopulating pluripotent stem cells lacked CZ-1. Therefore, expression of CZ-1 on Ly6+ Lin- c-kit+ cells, highly enriched for pluripotent stem cells, was examined. This population appeared to be homogeneously CZ-1dull. Thus, it appears that expression of CZ-1 is developmentally regulated, with differentiation associated with increased expression. Since CZ-1 is expressed on a protein tyrosine phosphatase, it is likely that this molecule regulates differentiation of NK and other lymphoid cells.

The monoclonal antibody CZ-1 identifies a mouse CD45-associated epitope expressed on interleukin-2-responsive cells.

We have previously described a monoclonal antibody (mAb), CZ-1, which reacts with an epitope expressed on most peripheral basophils, natural killer cells, B cells, and CD8+ T cells, but not with most thymocytes or peripheral CD4+ T cells. Here we show that mAb CZ-1 defines a sialic acid-dependent epitope associated with a subpopulation of CD45 molecules. This conclusion is based on the ability to block binding of mAb CZ-1 by sialic acid, neuramin-lactose, neuraminidase, and mAb to CD45RB, and by expression of the epitope on transfected psi 2 cells expressing exon B of CD45. The results suggest that the CZ-1 epitope is a post-translational modification expressed on a subpopulation of the CD45 molecules also expressing the B exon. Expression of the CZ-1 epitope was required for freshly isolated lymphocytes to respond to interleukin-2 (IL-2). Depletion of CZ-1+ cells by C' or by cell sorting of thymocytes or splenocytes eliminated the IL-2 responsive cells. The subpopulations of thymocytes and CD4+ splenocytes responding to IL-2 were exclusively within the small CZ-1+ subpopulation. mAb CZ-1 was also used to subdivide CD45+ and CD45RB+ splenocytes into IL-2-responsive and -nonresponsive subpopulations. The CZ-1 epitope was also expressed on virtually all lymphokine-activated killer cell precursors. These data, thus, indicate that cells responsive to IL-2 express this sialated modification of CD45.

Generation of "natural killer cell-escape" variants of Pichinde virus during acute and persistent infections.

Pichinde virus (PV) strain AN 3739 was determined to be sensitive to natural killer (NK) cells in vivo by enhanced replication in NK-cell-depleted mice. An NK-sensitive subclone (PV-NKs1) was serially passed in mice whose NK cells had previously been activated by an interferon inducer, and two plaque isolates were shown to be resistant to NK cells but not to interferon. Inoculation of severe-combined-immunodeficient mice with PV-NKs1 led to a persistent infection resulting in an NK-resistant viral population. This is the first demonstration of the isolation of viral "NK-escape" variants, as defined by the ability of the virus to replicate in vivo.

A lymphocyte differentiation and activation antigen, CZ-1, that distinguishes between CD8+ and unstimulated CD4+ T lymphocytes.

We report the generation and cellular reactivity of a novel rat IgM monoclonal antibody (mAb), CZ-1, made against mouse natural killer (NK) cells activated in vivo. mAb CZ-1 recognizes a molecule whose properties are consistent with that of a trypsin-sensitive, non-phosphatidyl inositol-linked sialoglycoprotein. The expression of the antigen recognized by mAb CZ-1 is restricted mostly to cells of the lymphoid lineage. The antigen is expressed on 10%-25% of bone marrow cells and 3%-5% of thymocytes. Analysis of thymocyte subpopulations indicates expression of the CZ-1 antigen on 100% of the NK1.1+, 27% of the CD4-CD8-, 1.1% of the CD4+CD8+, 1.1% of the CD4+CD8-, and 33% of the CD4-CD8+ cells. In the spleen, the CZ-1 antigen is expressed on B lymphocytes, NK cells, and virtually all CD8+ T lymphocytes. Most unstimulated CD4+ splenic T lymphocytes, monocytes and polymorphonuclear cells, with the notable exception of basophils, do not react with mAb CZ-1. CD4+ T cells activated in vivo by virus infection or in vitro by anti-CD3 and interleukin-2 express the CZ-1 antigen. These results indicate that mAb CZ-1 identifies a novel inducible lymphocyte activation/differentiation antigen that distinguishes between thymic and unstimulated splenic CD4+ and CD8+ T lymphocytes. This mAb will be a useful tool in the identification of lymphocyte subpopulations and in the study of the ontogeny and activation of these cells.

Natural killer (NK) cell response to virus infections in mice with severe combined immunodeficiency. The stimulation of NK cells and the NK cell-dependent control of virus infections occur independently of T and B cell function.

The activation, proliferation, and antiviral properties of natural killer (NK) cells were examined in severe combined immunodeficiency (SCID) mice to determine the influence of mature T or B cells on virus-induced NK cell functions and to more conclusively determine the antiviral properties of prototypical CD3- NK cells. NK cells were activated to high levels of cytotoxicity 3 d after infection of mice with lymphocytic choriomeningitis virus (LCMV) or murine cytomegalovirus (MCMV). Analyses of spleen leukocytes from LCMV-infected mice by a variety of techniques indicated that the NK cells proliferated and increased in number during infection. Propidium iodide staining of the DNA of cycling cells revealed that the great majority of proliferating spleen leukocytes 3 d after LCMV infection was of the NK cell phenotype (CD3-, Ig-, Mac-1+, CZ1+, 50% Thy-1+), in contrast to uninfected mice, whose proliferating cells were predominantly of other lineages. Analyses of the NK cell responses over a 2 wk period in control CB17 mice infected with MCMV indicated a sharp rise in serum interferon (IFN) and spleen NK cell activity early (days 3-5) in infection, followed by sharp declines at later stages. In SCID mice the IFN levels continued to rise over a 10-d period, whereas the NK cell response peaked on day 3-5 and gradually tapered. In contrast to the immunocompetent CB17 mice, SCID mice did not clear the MCMV infection and eventually succumbed. SCID mice, again in contrast to immunocompetent CB17 mice, also failed to clear infections with LCMV and Pichinde virus (PV); these mice, infected as adults, did not die but instead developed long-term persistent infections. Depletion of the NK cells in vivo with antiserum to asialo GM1 rendered both SCID and CB17 control mice much more sensitive to MCMV infection, as shown by titers of virus in organs and by survival curves. In contrast, similar depletions of NK cells did not enhance the titers of the NK cell-resistant virus, LCMV. Two variants of PV, one sensitive to NK cells and the other selected for resistance to NK cells by in vivo passage, were also tested in NK cell-depleted SCID mice. The NK-sensitive PV replicated to higher titers in NK cell-depleted SCID mice, whereas the titers of the NK cell-resistant PV were the same, whether or not the mice had NK cells. These experiments support the concept that CD3- prototypical NK cells mediate resistance to NK cell-sensitive viruses via a mechanism independent of antiviral or "natural" antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibodies against leucoagglutinin-reactive human T-lymphocyte surface components. II. Studies on the mechanism of K46M-induced activation and determination of the frequency of responding cells.

A monoclonal antibody, K46M (IgM kappa), obtained after immunization with leucoagglutinin (La)-reactive T-cell surface components, stimulated human lymphocytes to proliferate. It induced maximal proliferation at greater than 20 micrograms IgM/ml after 3-4 days of culture. Cells stimulated by K46M produced interleukin 2 (IL-2) and gamma interferon (IFN-gamma) and expressed receptors for IL-2 and transferrin. The majority of the activated cells were phenotypically T cells as defined by monoclonal antibodies against CD3 and CD2, and an increase in the K46M-positive cells was also observed during the activation period. K46M-activated cells display major histocompatibility complex (MHC)-unrestricted cytotoxicity against several cultured target cells. The frequencies of the cytotoxic and of the proliferative precursor cells were determined using a limiting dilution assay. K46M seems to activate a larger fraction of cytotoxic precursor cells against Molt 4 than against K562, but the statistical significance of these observations requires further exploration. Both K46M or La activated 40% of PBL to proliferate, whereas 70% of PBL were induced by OKT3. However, the frequency of K46M-activated cells was 40% only when the lymphocytes were plated at low cell densities, i.e. less than 0.5 cells per well. At higher densities an inhibition of proliferation was seen that resulted in a biphasic response curve, indicating that the activation of PBL by K46M was not a single hit event. This was not found with either La or OKT3. Whether K46M, in contrast to OKT3 and La, activates a subpopulation with suppressor activity remains to be established.

Characterization of human CD4+ T-cell clones that secrete helper factor(s) for B-cell proliferation and maturation.

Human peripheral blood lymphocytes (PBL) were activated with K46M, a m mitogenic monoclonal antibody against La-reactive T lymphocyte surface structures. The cultures were expanded in the presence of interleukin 2 (IL-2). After 1 month of culture, the activated T cells were cloned by limiting dilution at 0.5 cells/well. Five clones with the CD3+CD4+ phenotype and one clone with the CD3+CD8+ phenotype were obtained. The CD3+CD8+ clone (K99) displayed a strong major histocompatibility complex (MHC)-unrestricted cytolytic activity against MOLT-4 and a weaker reactivity against the bladder tumour cell lines T24 and RT4. The natural killer (NK)-susceptible K562 cells were not lysed. Two of the CD3+CD4+ clones (K91 and K914) showed a helper activity in pokeweed mitogen (PWM)-induced IgG production by B cells. These cells differed in the expression of CD45R and CDw29 antigens, as defined by the monoclonal antibodies 2H4 or D10D11 and 4B4. When stimulated with PWM for 48 or 72 h, clone K91 and an additional CD4-positive clone (K913) secreted a factor into the supernatants which helped B cells to produce IgG. The K913 supernatant also induced some IgM production. The supernatant obtained after similar stimulation of K914 cells was inactive. None of these supernatants induced B cells to proliferate when tested together with phorbol myristate acetate (PMA). However, when K91 and K914 cells were activated with phytohaemagglutinin (PHA) for 48 or 72 h, the supernatant from K91 was strongly helpful in B-cell proliferation, whereas the supernatant from K914 cultures was only moderately active. In conclusion, we have established human T helper clones that release different factors supporting either B-cell proliferation or maturation when stimulated with PWM or PHA.

Enhancement of human spontaneous cell-mediated cytotoxicity by a monoclonal antibody against the large sialoglycoprotein (CD 43) on peripheral blood lymphocytes.

A murine monoclonal antibody, MoAb B1B6 (IgG1 chi), which recognizes the large sialoglycoprotein (LSGP) on human peripheral blood lymphocytes (PBL) effectively enhanced the spontaneous cytotoxicity of these cells against the natural killer (NK)-sensitive target cells K562 and Molt-4. Whereas preincubation of the lymphocytes with MoAb B1B6 resulted in increased cytotoxicity, preincubation of the target cells had no effect, indicating that the MoAb amplified cytotoxicity at the effector cell level. Kinetic analysis of the data revealed no differences between the control and the MoAb-treated lymphocytes with regard to Vmax, usually considered to reflect the overall lytic potential of the cells. The slopes of the saturation curves, however, differed significantly for the two cell populations, indicating a substantial increment in the activity of the MoAb-treated cells. When studied at the single cell level and with K562 as targets, treatment of PBL with the MoAb resulted in the recruitment of new effector lymphocytes from the pool of non-binding cells. In contrast, when Molt-4 cells were employed as targets, no additional effector cells were recruited. These results indicate that the enhanced cytotoxicity induced by MoAb B1B6 is the result of either recruitment of new effector lymphocytes or of an increased recycling capacity of preexisting effector cells. Together with previous observations, these findings support the conclusion that LSGP belongs to the set of surface molecules which regulate human lymphocyte activation.

MHC nonrestricted cytotoxic T cell clones with selective specificity from patients with transitional cell carcinoma (TCC) of the urinary bladder.

Lymphocytes from patients with transitional cell carcinoma (TCC) of the urinary bladder are more cytotoxic to bladder tumor cells than to a variety of control cells. This disease-related cytotoxicity has previously been shown to involve several mechanisms and different types of effector cells. To analyze further the nature of the effector cells operative in this system, peripheral blood lymphocytes from eight TCC patients were stimulated in vitro with TCC extract and cultured in the presence of interleukin 2 and allogeneic feeder cells. When tested for cytotoxicity in vitro on a target cell panel including both adherent and nonadherent cell lines, the lymphocytes killed a broad spectrum of targets in a major histocompatibility complex (MHC)-unrestricted fashion. When cloned by limiting dilution, clones were obtained which displayed a more restricted pattern of target cell killing. Some of the clones were highly but not exclusively selective for TCC-derived target cells. Phenotypically, these cells resembled mature T cells of CTL-type (CD8+/CD4-). They also expressed the CD3/5 T cell antigen receptor complex but target cell killing was not MHC-restricted. The results of various inhibition experiments suggested that the CD3/TCR complex was involved in the cytotoxicity exhibited by these effector cells. However, its precise role in target cell recognition and the identification of the tumor cell structures recognised by the effector cells require further studies.

T lymphocytes displaying major histocompatibility complex-unrestricted cytotoxicity after activation by K46M, a mitogenic monoclonal antibody against leucoagglutinin-reactive human T lymphocyte surface components.

Activation of human peripheral blood lymphocytes (PBL) with the mitogenic monoclonal antibody (MoAb) K46M, which recognizes 1-5% of PBL, resulted in the expansion of cells with cytolytic activity. Thus, after culture of the activated lymphocytes in medium containing interleukin 2 (IL-2), they lysed a variety of cultured cell lines. The majority of the activated lymphocytes reacted with MoAb to CD8, CD3, and to the T cell antigen receptor heterodimer (Ti) but not with antibodies to antigens expressed on natural killer (NK) cells. The cytotoxicity was not inhibited by MoAb to CD3 or Ti. However, the killing of K562, but not of other cell lines, was enhanced by three to four times in the presence of anti-Ti antibodies. Anti-CD3 or other control antibodies had no effect. Cold target inhibition experiments indicated that the cytolytic lymphocytes recognized closely related structures on the target cells. Phenotypically and functionally similar effector cells emerged after activation of PBL with the anti-CD3 MoAb OKT3. Taken together, the results indicate that activation of PBL with MoAb K46M induces cytotoxic cells that differ from classical NK cells but that resemble mature cytotoxic T lymphocytes (CTL). However, unlike CTL, cytotoxicity was not MHC-restricted and the conventional T-cell receptor complex (CD3/Ti) appeared not to be involved in target cell recognition and cytolysis.

Monoclonal antibodies against leucoagglutinin-reactive human T lymphocyte surface components. Two antibodies which inhibit cell-mediated cytotoxicity at a post-binding stage.

Two out of 20 monoclonal antibodies (IgM, kappa), mAb 3192 and mAb K3G, raised against leucoagglutinin-reactive components on human T cells, effectively blocked lymphocyte-mediated cytotoxicity in vitro. No antigenic polypeptide reactive with these antibodies has been identified thus far. However, they have previously been shown to react specifically with certain neutral glycolipids obtained from spleen. Both mAb inhibited the cytotoxicity of natural killer (NK) cells against K562 cells, antibody-dependent cellular cytotoxicity (ADCC) towards antibody-coated bovine erythrocytes and cytotoxic T lymphocyte activity against allogeneic target cells. In both NK and ADCC, preincubation of the lymphocytes with different antibody concentrations resulted in a dose-dependent reduction of cytotoxicity. In contrast, preincubation of the target cells had no effect indicating that the mAb inhibited cytotoxicity at the effector cell level. When studied at the single-cell level, the mAb did not alter the number of lymphocytes forming conjugates with K562 but significantly reduced the frequency of conjugates containing dead target cells. Addition of the mAb to preformed conjugates resulted in a dose-dependent reduction in the proportion of conjugates containing dead target cells. Furthermore, mAb 3192 did not reduce the number of lymphocytes forming rosettes with bovine erythrocytes, indicating that inhibition of ADCC was not due to blocking of the effector cell-target cell interaction mediated by the Fc receptor of the effector cells. Taken together, these results suggest that the mAb inhibited cytotoxicity by interfering with a post-binding step common for the different cytotoxicity systems.

Surface markers of human natural killer cells as analyzed in a modified single cell cytotoxicity assay on poly-L-lysine coated cover slips.

A modified single cell cytotoxicity assay using poly-L-lysine coated cover slips (PLL-SCCA) was employed to study the frequency and surface marker profile of human peripheral blood lymphocytes (PBL) with NK reactivity against K 562 target cells. When compared with the previously described agarose single cell cytotoxicity assay (A-SCCA) identical results were obtained. For 13 donors tested 18.1 +/- 4.4% of the PBL formed conjugates with K 562 and 2.7 +/- 1.6% displayed NK reactivity. In contrast to the A-SCCA, the PLL-modified assay permits direct identification of both conjugate forming (TBC) and cytolytic PBL (NK) by means of surface markers. Indirect immunofluorescence studies with monoclonal anti-PBL antibodies revealed that neither the plating procedures nor the incubation conditions employed affected the expression of the antigens recognized by these reagents. This method of directly identifying NK cells showed that OKM1+ cells were enriched among the NK cells as compared to PBL and TBC (55% vs. 23% and 43%, respectively). In contrast, the OKT3+ or Leu1+ fraction of the NK cells was reduced as compared to PBL and TBC. However, using this method of identification at the effector cell level, a substantial proportion of the NK cells were OKT3+ or Leu1+ (57% or 58% respectively, 7 donors). Approximately 25% of the NK cells were Leu2a+ and 30% were Leu3a+, respectively. However, the size of the Leu3a+ fraction varied considerably with individual donors and the size of this fraction appeared to be inversely related to that of the donors NK pool.