Polyfunctionality of Allospecific Cytotoxic T-Lymphocytes in in vitro Systems

The data presented demonstrate the capacity of allospecific cytotoxic lymphocytes to fulfill noncytolytic regulatory functions. Cytotoxic lymphocytes suppress cell proliferation induced during development of a response in reactions of a mixed culture of lymphocytes and blast transformation irrespective of haplotypes of the cells involved in these reactions. At the same time, cytotoxic lymphocytes do not practically affect spontaneous proliferation of T-cells. The inhibitory effect of cytotoxic lymphocytes is expressed only upon direct contact with activated T-lymphocytes and is not relate to their apoptosis and death. The results obtained suggest polyfunctionality of allospecific cytotoxic lymphocytes expressed in the capacity of these lymphocytes to fulfill both effector (target lysis) and regulatory functions.     

Cloned "anomalous" killer cells derived from allogeneic mixed leukocyte culture

In addition to allospecific cytotoxic lymphocytes, cytolytic effector cells capable of killing a broad range of targets are generated during mixed leukocyte culture (MLC). These cells, which have been previously called anomalous killer cells, are a distinct functional subset separate from natural killer cells or allospecific cytotoxic lymphocytes but display many characteristics of lymphokine-activated killers. In order to isolate anomalous killer cells for detailed analysis, we generated the cytolytic effectors from an allogeneic MLC using heat-inactivated stimulators. This treatment of the stimulator population abrogated the generation of classical allospecific cytotoxic lymphocytes but allowed the generation of anomalous killer cells which were subsequently cloned via limiting dilution. The clones derived by this method displayed the functional properties of anomalous killers seen in bulk MLCs. The clones demonstrated potent cytolytic activity against both NK-sensitive and NK-resistant tumor targets in vitro and also suppressed tumor growth in vivo. Ultrastructural studies revealed features similar to those of cloned antigen-specific cytolytic cells and clones with NK-like function. The cells expressed surface glycoproteins associated with both NK and T lymphocytes including Thy-1, Ly-2, T200, Qa-5, asialo GM1, and the antigens defined by the NK alloantisera NK-2.1 and NK-3.1. These cells may play an important role during early phases of the immune response, since cytolytic cells of broad specificity may protect the host until classical cytotoxic lymphocytes with restricted specificity are generated.     

Effects of humoral factors of the mastocytoma P815 cells on the formation of allospecific killers in mixed culture of lymphocytes and their cytotoxic activity

The influence of the P-815 mastocytoma cells and their humoral factors, contained in culture supernatant and ascitic fluids on the generation of allospecific cytotoxic T-cells (alloCTL) in mixed culture of lymphocytes was studied. Both tumor cells and humoral factors were shown to inhibit generation of allospecific killers. Normal spleen cells and peptonic ascites of DBA/2 mice did not indicate immunosuppressive activity. Immunosuppressive factors did not affect CTL effector function (lysis of target cells). Both tumor cells and immunosuppressive factors did not exert toxic effect on mouse splenocyte. The suppressive effect of tumor cells and humoral factors was not associated with their cytotoxic action on lymphocytes.     

Inhibition of proliferation without affecting the generation of cytotoxicity in the human mixed lymphocyte reaction

Phosphoramide mustard (PM) is considered to be the major tumoricidal metabolite of cyclophosphamide in vivo. The effects of this metabolite in vitro on several immune functions of human lymphocytes have been investigated. Very low concentrations (10(-7) to 10(-9) M) of PM added to lymphocyte cultures inhibited proliferation of the lymphocytes in response to mitogens and alloantigens. At these concentrations, inhibition of proliferation appeared to be due to a direct action of PM on the proliferative cells. Thus, concanavalin A-stimulated lymphocytes still acquired IL-2 receptors (Tac antigen) normally in the presence of PM (10(-6) to 10(-9) M). Only exceedingly high concentrations of PM (10(-5) M or greater) prevented the acquisition of Tac antigen. Similarly, the inhibition of proliferation was probably not related to endogenous IL-2 levels: addition of exogenous IL-2 to PM-containing cultures did not result in any restoration of proliferation. Further evidence that PM directly affected proliferative cells was that low concentrations of PM inhibited the proliferation of T cells continuously growing in IL-2. The exposure time to PM necessary for inhibition was essentially identical to those for lymphoproliferative responses to mitogens and alloantigens. Paradoxically, however, the generation of cytotoxic lymphocytes in mixed lymphocyte reactions (MLRs) and mixed lymphocyte tumor cell cultures (MLTCs) was very resistant to PM. In parallel MLRs and MLTCs the cytotoxic responses were resistant to approximately 1000-fold more PM than were the proliferative responses. Only at 10(-5) M PM were these inhibited. These data suggest that clonal expansion of cytotoxic lymphocytes or their precursors by proliferation is not an absolute requirement for the generation of cytolytic activity.     

Studies on cytotoxicity generated in human mixed lymphocyte culture

Summary High levels of cytotoxic activity against the natural killer (NK) cell-sensitive target K562 and the NK-resistant target UCLA-SO-M14 (M14) can be generated in vitro either by mixed lymphocyte culture (MLC) or by culture of lymphocytes in interleukin 2 (IL2) (lymphokine activated killer (LAK) cells). The purpose of this study was to identify similarities and differences between MLC-LAK and IL2-LAK cells and allospecific cytotoxic T cells. Induction of cytotoxicity against K562 and M14 in both culture systems was inhibited by antibodies specific either for IL2 or the Tac IL2 receptor. Like NK effector cells, the precursors for the MLC-LAK cells were low density large lymphocytes. However these precursors differed from the large granular lymphocytes that mediated NK cytolysis in sensitivity to the toxic lysosomotropic agent L-leucine methyl ester (LME). The resistance of the MLC-LAK precursors to LME indicated that the precursors included large agranular lymphocytes. Although interferon-gamma (IFN-gamma) is produced in MLC and in IL2 containing cultures, it is not required for induction of either type of cytotoxic activity. Neutralization of IFN-gamma in MLC-and IL2-containing cultures with specific antibodies had no effect on the induction of cytotoxic activities. Both allospecific cytotoxic T lymphocyte (CTL) and LAK activities were enhanced by IL2 and IFN-gamma at the effector cell stage. However, the mechanism of cytolysis was different in the two systems. NK- and MLC-induced LAK activities were independent of CD3-T cell receptor complex while CTL activity was blocked by monoclonal antibodies specific for the CD3 antigen. These results suggest that NK and the in vitro induced LAK cytotoxicities are a family of related functions that differ from CTL. Furthermore, MLC-induced and IL2-induced cytotoxicities against K562 and M14 appear to be identical.This work was supported by NIH grant CA34442     

Antigen-specific human T lymphocyte clones: mechanisms of inhibition of proliferative responses by xenoantiserum to human nonpolymorphic HLA-DR antigens

We studied the effects of xenoantiserum to human nonpolymorphic Ia-like antigens upon in vitro antigen-specific T cell proliferative responses in unfractionated PBL populations and at the monoclonal level. Our findings suggest that the xenoantiserum, although it inhibits the antigen-specific response of unfractionated PBL and allospecific T cell clones, does not inhibit the proliferative response to cloned influenza virus immune human T lymphocytes, and therefore may be mediating inhibition by dual mechanisms: direct inhibition of alloantigen recognition and induction of nonspecific suppression. Kinetic differences may explain these phenomena. In cocultivation experiments with a virus-specific clone, the RaIa antiserum appears to induce an OKT3+,8+,4-, radiosensitive regulatory subset of lymphocytes. When adoptively transferred, these induced cells inhibit the TLC response in an antigen-nonspecific and genetically nonrestricted manner. We discuss the various modes and levels of inhibition of antigen-specific proliferation by anti-Ia antisera and their multiple activities.     

Transforming growth factor-beta inhibits the in vitro generation of lymphokine-activated killer cells and cytotoxic T cells

Summary The effect(s) of purified transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) on the induction and function of lymphokine-activated killer (LAK) cells and cytotoxic T lymphocytes (CTL) was examined. The addition of TGF-beta, but not PDGF, to cultures containing fresh C57BL/6 mouse splenocytes or human peripheral blood lymphocytes plus recombinant interleukin-2 markedly inhibited the development of mouse and human LAK cell activity (measured after 3 days for cytotoxicity against cultured or fresh tumor targets in 4-h ⁵¹Cr release assays). The addition of TGF-beta, but not PDGF, to a one-way, C57BL/6 anti-DBA/2, mixed lymphocyte reaction effectively blocked the generation of allospecific CTL as well. However, TGF-beta did not inhibit the effector function of LAK cells or of allospecific CTL when added directly to the short-term cytolytic assay. A second form of homodimeric TGF-beta, type 2, was also found to be suppressive on the development of murine LAK cells and allospecific CTL. Collectively, these data demonstrate that the peptide TGF-beta is a potent inhibitor of LAK cell and CTL generation in vitro.     

The functional capabilities of cells leaving the thymus

There has been a controversy for many years over the functional status of cells that leave the thymus (thymus migrants) to populate the peripheral lymphoid organs. Are they immunoincompetent like cortical thymocytes and so probably derived from them, or are they functionally mature like medullary thymocytes? Until recently the techniques used to assess putative thymus migrants have been indirect, but it is now possible to measure the function of recent thymus migrants directly. We used intrathymic injection of a solution of fluorescein isothiocyanate to label thymocytes, and used electronic cell sorting to purify the fluorescent cells that accumulate in the periphery over the following 3 to 4 hr. The migrants have been enriched from an original frequency of about 1:1000 in lymph nodes and spleen, to greater than 98% purity. These cells have been compared with normal peripheral T cells for proliferative and cytotoxic precursor activity in a high cloning efficiency, lectin-induced, limit dilution culture system and in an allospecific limit dilution system. The frequency of precursors of proliferative lymphocytes and cytotoxic lymphocytes and the size of the clones produced is the same for recent migrants and peripheral T cells. Thus by the criteria of proliferation and cytotoxic responses to mitogens and generation of allospecific CTL, thymus migrants, a few hours after their emigration from the thymus, are fully immunocompetent; we therefore see no evidence of a post-thymic precursor-type cell that requires major maturation steps after leaving the thymus.     

Spontaneous in vitro evolution of lytic specificity of cytotoxic T lymphocyte clones isolated from murine intestinal epithelium

Three long-term clonally derived cytotoxic lines have been established from isolates of murine intraepithelial lymphocytes (IEL). All three lines were selected for with antigen and represent two allospecific cytotoxic T lymphocyte (CTL) clones and a major histocompatibility complex (MHC)-restricted clone specific for a murine minor histocompatibility antigen. On long-term in vitro culture, IEL clones gradually lost antigen-specific lytic activity and simultaneously acquired the capacity to lyse natural killer (NK)-sensitive target cells which, in some cases, required high-level lymphokine activation. Of interest was the finding that, despite changes in lytic specificity, IEL clones remained strictly antigen-dependent for proliferation. A murine CTL clone of splenic origin, which was propagated under culture conditions identical to those used for IEL, did not exhibit changes in lytic specificity, suggesting that acquired changes in IEL function cannot be attributed solely to the influence of in vitro culture. Phenotypic analyses of IEL clones with altered lytic specificity revealed that all lines remained Thy-1+, Lyt-2+, L3T4-, with or without lytic activation by lymphokines. The expression of CT-1, a murine CTL activation antigen, and asialo GM1, a murine NK cell marker, were variable on IEL clones, and their presence did not correlate with the changes in lytic behavior. Collectively, these findings provide evidence, at the clonal level, that at least some NK activity present in isolates of murine IEL may originate from antigen-specific CTL. The data also indicate that, on binding antigen, different signals are conveyed to T cells, resulting in proliferation or target cell lysis.     

Phorbol esters inhibit the functional activation of cytotoxic precursors in mixed lymphocyte cultures

We have investigated the effect of phorbol esters on T cell activation and generation of suppressor and cytotoxic activity in mixed lymphocyte cultures (MLC). The presence of 30 nM P(Bu)2 during the sensitization phase inhibited the generation of allospecific cytotoxicity and also decreased the killing potential against NK-sensitive targets. The inhibition was not mediated by direct blocking of the lytic capacity nor by suppression of clonal expansion of cytotoxic cells through modulation of lymphokine production. The presence of P(Bu)2 enhanced cell proliferation, but inhibited the functional activation of lymphocytes and consequent generation of Dr antigen-positive T cells. Because the presence of the compound did not affect the MLC-induced generation of suppressor activity, it is likely that P(Bu)2 selectively blocks the maturation of cytotoxic precursors. Surface-marker analysis with OKT monoclonal antibodies revealed that the effects on lymphocyte activation were associated with a decrease in OKT3 and OKT4 reactivity and an increase in the percentage of OKT8-positive cells. The decrease in OKT4 reactivity was not due to selective loss of this lymphocyte subpopulation, because P(Bu)2 was equally mitogenic for the purified OKT4- and OKT8-positive subsets. The results suggest that the effect of P(Bu)2 on cell differentiation and its ability to modulate the expression of functional markers in lymphocyte subsets may interfere with T-T cell cooperation that controls the functional maturation of cytotoxic precursors.     

Predominant Th2/Tc2 polarity of tumor-infiltrating lymphocytes in human cervical cancer

Cytotoxic T lymphocytes (Tc) play a central role in cellular immunity against cancers. The cytotoxic potential of freshly isolated tumor-infiltrating lymphocytes (TILs) is usually not expressed. This suggests the possible existence of as yet unspecified and perhaps complex immunosuppressive factors or cytokines that affect the anti-tumor capacity of these TILs in the tumor milieu. In the present study, we demonstrated for the first time that TILs derived from human cervical cancer tissue consist mainly of Th2/Tc2 phenotypes. In vitro kinetic assays further revealed that cancer cells could direct the tumor-encountered T cells toward the Th2/Tc2 polarity. Cancer cells promote the production of IL-4 and down-regulate the production of IFN-gamma in cancer-encountered T cells. The regulatory effects of cervical cancer cells are mediated mainly by IL-10, and TGF-beta plays only a synergistic role. The cancer-derived effects can be reversed by neutralizing anti-IL-10 and anti-TGF-beta Abs. IL-10 and TGF-beta are present in cancer tissue and weakly expressed in precancerous tissue, but not in normal cervical epithelial cells. Our study strongly suggests important regulatory roles of IL-10 and TGF-beta in cancer-mediated immunosuppression.     

Cytohesin-associated scaffolding protein (CASP) is a substrate for granzyme B and ubiquitination

Natural killer (NK) cells are a sub-population of cytotoxic lymphocytes that can kill tumor or infected cells without prior exposure, by secreting the contents of preformed cytotoxic vesicles, containing perforin and granzymes, at the immune synapse. Cytohesin-associated scaffolding protein (CASP) is an adaptor molecule uniquely expressed in lymphocytes that forms complexes with both vesicle-initiating and sorting proteins, and has roles in NK cell migration, cytotoxicity, and cytokine secretion. In this study, we show that CASP contains a conserved granzyme B cleavage site that could modify its intracellular localization and interaction with sorting nexin 27. We also provide evidence that CASP is modified by ubiquitination. Both of these post-translational modifications could rapidly modify CASP function and highlight the dynamic regulatory mechanisms that direct its role in NK cell functions.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

mdr1a-encoded P-glycoprotein is not required for peripheral T cell proliferation, cytokine release, or cytotoxic effector function in mice.

The plasma membrane transport protein P-glycoprotein (P-gp) is expressed by subsets of both CD4+ and CD8+ T cells in mice. The proportion of T cells that express P-gp goes up with age, and the P-gp-expressing subset of the CD4 memory population is hyporesponsive in many in vitro assays. The significance of P-gp expression for T cell function has not been well established, although several reports have suggested that it may promote cytokine export and/or cytotoxic T cell function. To elucidate which T cell functions may require P-gp, we have compared a variety of responses using T cells from wt and P-gp knockout mice. Protein expression and rhodamine-123 efflux studies revealed that peripheral T cells exclusively utilize the mdr1a-encoded isoform rather than the homologous mdr1b or mdr2 isoforms. Comparisons of T cells from mdr1a+/+ and mdr1a-/- mice showed no differences in proliferation or in secretion of IL-2, IL-4, IL-5, IL-10, or IFN-gamma in response to polyclonal stimulation. Moreover, mdr1a-/- T cells produced strong allospecific cytotoxic responses comparable to those of wt T cells. Our results show that P-gp is not a necessary component of peripheral T cell functional responses. Further investigation will be needed to determine the significance of P-gp expression in T lymphocytes.     

Relationship among function, phenotype, and specificity in primary allospecific T cell populations: identification of phenotypically identical but functionally distinct primary T cell subsets that differ in their recognition of MHC class I and class II allodeterminants

The goal of the present study was to evaluate the relationship among function, Lyt phenotype, and MHC recognition specificity in primary allospecific T cell populations. By using Lyt-2+ and L3T4+ T cells obtained from the same responder populations, we assessed the ability of T cells of each phenotype to generate cytotoxic effector cells (CTL) and IL 2-secreting helper T cells in response to either class I or class II MHC allodeterminants. It was found that a discordance between Lyt phenotype and MHC recognition specificity does exist in primary allospecific T cells, but only in one T cell subpopulation with limited functional potential: namely, Lyt-2+ T cells with cytotoxic, but not helper, function that recognize class II MHC alloantigens. Target cell lysis by these Lyt-2+ class II-allospecific CTL was inhibited by anti-Ia monoclonal antibodies (mAb), but not anti-Lyt-2 mAb, indicating that they recognized class II MHC determinants as their "restriction" specificity and not as their "nominal" specificity even though they were Lyt-2+. A second allospecific T cell subset with limited functional potential was also identified but whose Lyt phenotype and MHC restriction specificity were not discordant: namely, an L3T4+ T cell subset with helper, but not cytotoxic, function specific for class I MHC allodeterminants presented in the context of self-Ia. Thus, the present study demonstrates that primary allospecific T cell populations contain phenotypically identical subpopulations of helper and effector cells that express fundamentally different MHC recognition specificities. Because the recognition specificities expressed by mature T cells reflect the selection pressures they encountered during their differentiation into functional competence, these findings suggest that functionally distinct but phenotypically identical T cell subsets may be selected independently of one another during ontogeny. Thus, the existence of Lyt-2+ CTL specific for class II allodeterminants can be explained by the hypothesis that the association of Lyt phenotype with MHC recognition specificity results from the process of thymic selection that these Lyt-2+ effector cells avoid.     

Veto cell H-2 antigens: veto cell activity is restricted by determinants encoded by K, D, and I MHC regions

Responder cells from primary syngeneic and allogeneic one-way mixed-lymphocyte cultures (MLC) specifically inhibit the development of cytotoxic T lymphocytes (CTL) directed against the major histocompatibility complex (MHC) antigens of the MLC responder cells. This special kind of suppressor activity is known as veto suppression. Ia+ cells with veto activity obtained from H-2 recombinant mouse strains were shown to downregulate alloantigen (class II)-specific helper activity for class I-specific CTL development in a primary MLC provided that the veto cells expressed the same I-E alpha subregion as the MLC stimulator cells. The veto-induced suppression of allo-help was prevented by the addition of supernatant from concanavalin A-stimulated spleen cells (Con A-SN) and was inhibited considerably by very high amounts of recombinant interleukin-2 (IL-2). In the presence of Con A-SN, CTL precursors recognizing either the K end or the D end of the veto cell MHC were found to be inactivated. Thus, our results indicate that MLC responder cells include active veto cells expressing Ia region-encoded restriction elements for allospecific T helper cells, as well as K- or D-encoded restriction elements for allospecific T cytotoxic cells.     

Stimulator cell requirements for allospecific T cell subsets: specialized accessory cells are required to activate helper but not cytolytic T lymphocyte precursors

Murine cortisone-resistant thymocytes were separated by staining with monoclonal anti-Lyt-2 antibody and FMF into Lyt-2- and Lyt-2+ subsets in order to analyze the nature of stimulator accessory cells required to activate each of these functionally distinct T cell subpopulations. The Lyt-2- fraction was able to proliferate but not to generate cytotoxic cells when stimulated by irradiated allogeneic spleen cells. Fractionation of the stimulator population showed that low numbers of dendritic cells and splenic macrophages, but not equivalent numbers of whole spleen cells or peritoneal macrophages, were able to stimulate the Lyt-2- population. On the other hand, the Lyt-2+ population, which showed little if any proliferation in response to irradiated spleen cells, contained all the precursors of cytolytic T lymphocytes. In contrast to the highly specific stimulator requirement of the Lyt-2- fraction, allospecific cytotoxic cells were generated from Lyt-2+ cells by any alloantigen-bearing stimulator cell provided interleukin 2 was present. This was confirmed by limiting dilution analysis: alloreactive CTL-P frequencies in spleen and thymus were not influenced by the nature of the stimulator cell. These data collectively indicate that heterogeneous Ia+ accessory cells are required to stimulate helper but not cytolytic T cell precursors.     

A novel negative regulator molecule, Cho-1, is involved in the cytotoxicity by human natural killer cells but not in cytotoxic T lymphocytes.

We previously reported the cytotoxic negative regulatory molecule, Cho-1, that was expressed on the cell surface of rat fetal fibroblast cells in the cytotoxicity by natural killer (NK) cells. This molecule was IFN-gamma-inducible, but appeared to be different from MHC class I. It was expressed on NK-resistant cells but not on NK-sensitive murine target cells such as YAC-1. In this paper, first we determined whether Cho-1 could also act as the negative regulatory molecule in a human NK-resistant HEPM line. Our data strongly suggested that Cho-1 could act as such a negative regulatory molecule in human NK cytotoxicity. The immunoprecipitates made with HEPM cell lysate and anti-MHC class I monoclonal antibody (mAb) did not react against anti-Cho-1 mAb, indicating that Cho-I was different from MHC class I. Second, an assessment was made as to whether or not this molecule is involved in the cytotoxicity of CD8 (+) cytotoxic T lymphocytes (CTL) against human autologous tumor cells. The data indicated that although this cell surface molecule was expressed on certain tumor lines, it was not involved in the cytotoxic mechanism of CTL. Thus, Cho-1 appeared to be the novel regulatory molecule in the NK cytotoxic mechanism.     

Phenotypic and functional characterization of allospecific and nonspecific (NK- and K-like) cytotoxic T lymphocytes generated in human mixed-lymphocyte cultures from noncytotoxic precursors

Highly efficient cytotoxic cells able to lyse unsensitized, antibody-coated, and relevant targets were generated de novo following sensitization in allogeneic human mixed lymphocyte cultures (MLC) of noncytotoxic IgG-Fc receptor (FcR)-negative T lymphocytes. The MLC-induced killers maintained the surface markers of T cells, as demonstrated by their reactivity with two monoclonal anti-T-cell antibodies. None of them reacted with a monoclonal antibody specific for myelomonocytic cells and up to 30% expressed DR antigens. The nonspecific (NK-and K-like) effector cells had lower affinity for sheep erythrocytes than the allospecific killers and appeared to have low-affinity FcR as compared to freshly isolated NK and K cells. Unlike fresh NK cells, the MLC-induced NK-like cells were not sensitive to trypsin treatment. However, both fresh NK and MLC-induced NK-like cells were sensitive to interferon treatment and were completely inactivated after preincubation with unlabeled K562 cells at 37 °C for 4 hr. The latter observation suggests the possible use of this system to deplete NK-like effects from the MLC-generated killers and to discriminate between nonspecific and allospecific cytotoxic T lymphocytes.     

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.