Signal transduction via phosphorylated adhesion molecule, LFA-1{beta} (CD18), is increased by culture of natural killer cells with IL-2 in the generation of lymphokine-activated killer cells

It is well known that IL-2 stimulates natural killer (NK) cellsto express lymphokine activated killer (LAK) activity and thatthis stimulation prompts the acquisition of the ability to lysepreviously insensitive target cells. The possible role of adhesionmolecules in the IL-2 activation process was probed by focussingon a lymphocyte function-associated antigen (LFA)-1-dependentmodel system. A mAb to the LFA-1ß chain abrogatedLAK activity, but only moderately suppressed NK activity, suggestinga differential role for LFA-1ß In LAK compared withNK mediated lysis. Orthophosphate labeling demonstrated thatthe LFA-1ß chain was strongly phosphorylated in LAKbut not NK cells; in contrast, the chain was phosphorylatedsimilarlyin both effector cell types. At least a portion ofthe phosphorylation of the ß chain was on tyrosineresidues, as shown by Western blotting with anti-phosphotyrosineantibody of LFA-1ß immunoprecipitates. Crosslinkingof the LFA-1ß chain with plastic-adhered antibodystimulated Ca²⁺-dependent release of cytoplasmic lytic granulesand induced phosphatidyl inositol turnover in LAK but not NKcells. We conclude that the IL-2-induced phosphorylation oftheß chain of the LFA-1 adhesion molecule in LAK cellsand associated alteration in signal transduction may be importantin the stimulation of LAK cell activity in NK cells.     

Suppression by delta-9-tetrahydrocannabinol of interleukin 2-induced lymphocyte proliferation and lymphokine-activated killer cell activity

The major psychoactive marijuana component, delta-9-tetrahydrocannabinol (THC), suppressed proliferation of murine spleen cells stimulated with recombinant human interleukin 2 (IL-2) and also suppressed the appearance of the lymphokine-activated killer (LAK) cell phenomenon in IL-2-treated spleen cell preparations. Cell function was depressed in a dose-dependent manner with as little as 2.5 μg/ml THC (8μM). In addition, spleen cells previously stimulated in culture with IL-2 and then incubated with THC for 4 h prior to target cell addition, displayed suppressed cytolytic activity against both YAC-1 and EL-4 tumor targets. Killing of EL 4 cells was suppressd at lower drug doses than the killing of YAC-1 targets. These results suggest that THC can suppress several important functions of IL-2 including clonal expansion of lymphocytes, expansion of killer cell populations and stimulation of killer cell cytotoxic activity.     

Differential dependence of TH-0, TH-1 and TH-2 CD4 4+ T cells on co-stimulatory activity provided by the accessory molecule LFA-1

Background The adhesion molecule LFA-1 contributes to the activation response of peripheral blood human CD4+ T cells. Less is known of its contribution to stimulation of long-term CD4+ T cell lines and clones or of its potential to co-stimulate CD4+ T cells of different functional phenotype. Objective This study was therefore performed to investigate co-stimulatory properties of the LFA-1 (CD11a/CD 18) complex in the activation of human CD4+ T cell lines and clones of TH-0. TH-1 and TH-2 subsets. Methods Co-stimulatory activity was measured by cross-linking antibodies to CD 11a or CD18 with anti-CD3 antibodies to plastic and then measuring the proliferative response of CD4+ T cells to these antibodies. Results A house duct mite allergen-specific CD4+ T cell line (TH-2) demonstrated much greater dependence on both C'DI la and CD IK than a mycobacterial antigen-specific CD4+ T cell line (TH-1). Co-stimulatory activity through LFA-1 was also provided to a house dust mite-specific CD4+ T cell clone (DE-9; TH-2) but not to an influenza haemagglutinin-specific CD4+ T cell clone (HA 1.7: TH-0). In contrast, soluble antibodies to CD 18 inhibited proliferativc responses of both DE-9 and HA1.7 to an immunogenic challenge of antigen and to stimulation by unti-CD3 antibodies. However, the allergen-specific T cells were more susceptible to inhibition. Signal transduction was also observed from the T-cell receptor to LFA-1. Ligation of the T-cell receptor modulated the phenotypic expression of LFA-1 and ICAM-1 on both HA1-7 and DE-9). Phenotypic modulation was observed as a result of both activation and the induction of non-responsiveness. Conclusion These experiments indicate that CD4+ T cells of TH-2 functional phenotype may have a greater requirement for the co-stimulatory activity of LFA-1 than CD4+ T cells of TH-0 or TH-1 phenotypes.     

Adhesion molecule-mediated signals regulate major histocompatibility complex-unrestricted and CD3/T cell receptor-triggered cytotoxicity.

Appropriate experimental conditions were devised to demonstrate that CD58 (LFA-3), CD54 (ICAM-1) and CD11a/CD18 (LFA-1) adhesion molecules are the source of signals that regulate nonspecific major histocompatibility complex-unrestricted and CD3/T cell receptor (TcR)-triggered cytotoxicity. Using anti-LFA-3 monoclonal antibody (mAb)-treated, interleukin-2 (IL-2)-cultured peripheral blood lymphocytes (PBL) or cloned CD3+/CD8+ cells as lymphocyte-activated killer (LAK) effectors, and ligand (CD2)-negative tumor cell lines as targets, a down-regulation of CD3- and CD3+ cell-mediated LAK activity was consistently observed. Anti-LFA-3 mAb also down-regulated tumor cell lysis when T cell clones were triggered to kill P815 cells through stimulation of the CD3/TcR complex by an anti-CD3 mAb. The inhibitory effect of anti-LFA-3 mAb was not prevented by stimulatory anti-CD2 mAb. Anti-ICAM-1 mAb treatment of IL-2-cultured PBL consistently up-regulated LAK cytotoxicity against tumor target cells. However, this effect was only exerted on CD3- LAK effectors. Anti-LFA-1 mAb blocked conjugate formation between effector cells and tumor target cells, thus rendering this model unsuitable to evaluate the regulatory role of LFA-1. Therefore, a cytotoxicity model system was applied in which a hybrid anti-CD3/anti-human red blood cell (HuRBC) mAb triggers cytolytic T cells to lyse HuRBC. In these experiments, anti-LFA-1 mAb markedly up-regulated the lytic ability of IL-2-cultured PBL. We conclude that mAb against LFA-3, ICAM-1 and LFA-1 molecules deliver regulatory signals for LAK cells and cytotoxic T lymphocytes. As these stimuli may be delivered by ligands expressed on tumor targets as well as on other immune competent and inflammatory cells, the present observations are relevant in the context of both the host's immune response against tumors and the general functioning of the immune system.     

Human T lymphocytes expressing a defined T-cell antigen receptor family specifically kill the hybridoma that makes the anti-receptor monoclonal antibody.

Human T lymphocytes expressing a family of T-cell antigen receptors defined by a particular monoclonal antibody can be induced to grow with the Mab and recombinant IL-2. The resulting population consists largely of cells that have the phenotype T3+, T8+, T4-, and that react with the inducing anti-TcR Mab. The ability of such T cells to kill the hybridoma which produces the inducing Mab has been investigated. Specific killing is demonstrated which is inhibited by Mab to T3, TcR, and LFA-1. Further, lytic activity is rapidly lost when cultures are deprived of lymphokines. Cytolytic activity is restored by incubating cells in recombinant IL-2.     

Inhibition of proliferative and cytotoxic activities of human T lymphocytes with rabbit antibodies directed against leucoagglutinin-reactive T cell surface components

Three rabbit antisera (870, 872 and 873) were raised against leucoagglutinin-reactive components from the surface of human T cells. The antibodies reacted with two major glycoproteins of 175 kDa and 105 kDa. None of the antibodies triggered peripheral blood lymphocytes or T cells to proliferation when tested under different culture conditions. All antibodies inhibited the proliferative response to concanavalin A or to allogeneic lymphocytes in mixed lymphocyte culture when whole IgG fractions were used. Complete inhibition of cytotoxic activity was obtained in cell-mediated lympholysis and in natural killer cell cytotoxicity (NK) when fresh peripheral blood lymphocytes were used as effector cells. Weak inhibition was also obtained in NK when mixed lymphocyte culture-activated effector cells were used. The inhibition was stronger, when NK activity was determined against MOLT4 target cells as compared to K562. Whereas F(ab')2 fragments of 873 IgG inhibited cytotoxic T lymphocyte activity completely, Fab fragments of 873 IgG neither inhibited proliferation nor cytotoxic T lymphocyte activity, but gave some inhibition of NK against MOLT4 targets. The results indicate that antibodies against these leucoagglutinin-reactive structures reacted with polypeptides similar to or identical with the human "leukocyte function-associated antigen-1" (LFA-1) considered to be an important mediator of cell-cell interactions and nonspecific adherence.     

Role of adhesion molecules in lymphokine-activated killer cell killing of bladder cancer cells: further evidence for a third ligand for leucocyte function-associated antigen-1.

The lysis of eight human bladder cancer cell lines by lymphokine-activated killer cells (LAK) was found to be partially dependent upon the expression by the target cell of either intercellular adhesion molecule-1 (ICAM-1) or intercellular adhesion molecule-2 (ICAM-2). Using adhesion blockade studies these molecules were found to contribute towards sensitivity to lysis. Tumour lines of low grade (G1) did not constitutively express ICAM-1, but were found to express ICAM-2. High grade cells (G2, G3), however, only constitutively expressed ICAM-1 on their cell surface. Interferon-gamma (IFN-gamma) induced and augmented the expression of ICAM-1 by all except one of the cell lines (UMUC3) in a dose- and time-dependent manner. This was accompanied by an increased susceptibility to lymphokine-activated killer mediated cytolysis. Anti-ICAM-1 antibodies partially inhibited the increase in cell lysis due to IFN-gamma. However, this inhibition was not complete. When effector cells were treated with antibodies to leucocyte function-associated antigen-1 (LFA-1) the inhibition of lysis was greater and ranged from 72 to 96% with a mean of 87% inhibition. These results suggest that the increased sensitivity of IFN-gamma-treated bladder cancer cell lines to LAK cells is partially attributable to the induction of ICAM-1. However, blocking of ICAM-1 with antibodies could only partially inhibit the increased LFA-1-dependent lysis. This supports recent evidence for the existence of an additional ligand for LFA-1, other than ICAM-1 and ICAM-2.     

Adhesion mediated by LFA-1 is required for efficient IL-12-induced NK and NKT cell cytotoxicity

Interleukin 12 (IL-12)-activated NK1.1+TCRalpha beta+ (NKT2) and NK1.1+TCRalpha beta- (NK) cells exhibit cytotoxic activity against a wide variety of tumor cells in the absence of prior sensitization. Here we demonstrate that the integrin adhesion receptor LFA-1 (CD11a/CD18) regulates the cytotoxic activity of IL-12-activated NKT and NK cells against YAC-1 and EL-4 tumor cells. Differentiation in vivo and the expression of the cytolytic effector molecules perforin and Fas-L were comparable in both IL-12-activated NKT and NK cells from LFA-1-/ - and LFA-1+/+ mice. However, LFA-1-/-IL-12-activated NKT and NK cells showed impaired conjugate formation with target cells. These results provide the first genetic evidence for a role for an adhesion receptor in killing by IL-12-activated NK cells.     

Interleukin-4 differentially regulates interleukin-2-mediated and CD2-mediated induction of human lymphokine-activated killer effectors.

Natural killer (NK) cells can be differentiated into lymphokine-activated killer (LAK) effectors following stimulation with interleukin (IL)-2. This induction can be negatively regulated by IL-4. In this study, we demonstrate that the stimulation of NK cells through the CD2 pathway with (9-1 + 9.6) monoclonal antibodies can also induce these cells to secrete tumor necrosis factor-alpha (TNF-alpha) and to differentiate into LAK effectors. More importantly, our data indicate that, in contrast to the IL-2-induced LAK generation, the anti-CD2-triggered LAK activity was not regulated by IL-4. IL-4 was found to enhance the LAK activity as well as NK cell proliferation following activation with anti-CD2 by a mechanism involving, at least in part, an increased TNF-alpha production. Using immobilized monoclonal antibodies against the Fc receptor (Fc gamma RIII or CD16) for NK stimulation, we also observed that the anti-CD16-induced LAK activity was not inhibited by IL-4. These data further point to a pivotal role of TNF-alpha as a regulatory cytokine in anti-CD2-induced LAK generation, and suggest that IL-4 could serve as a discriminatory factor between two distinct pathways involved in the activation of non-MHC-restricted cytotoxicity.     

Adhesion receptors involved in clustering of blood dendritic cells and T lymphocytes.

The relationship of dendritic cells (DC) isolated from the peripheral blood to those of lymphoid tissue is, in terms of maturation and function, incompletely understood. In our present study, we have explored the molecular basis of adhesion of T cells to blood DC. Analysis of the expression of adhesion receptors on the cell surface of blood DC revealed that these cells express lymphocyte function-associated antigen (LFA)-1 (CD11a/18), ICAM-1 (CD54), LFA-3 (CD58) and CD44, but are very late antigen (VLA)-4 (CD49d) and vascular cell-adhesion molecule (VCAM)-1 negative. The LFA-1 pathway was found to play a key role in T cells-blood DC adhesion; monoclonal antibodies (mAb) against both LFA-1 and ICAM-1 strongly inhibited adhesion between those cells. Moreover, a T cell clone from an LFA-1-deficient patient showed poor binding to blood DC. The important role of LFA-1 in T cell-blood DC adhesion was also supported by the metabolic energy and divalent cation dependence of the interaction. mAb against LFA-3 and CD2 did not inhibit T cell-blood DC binding. In contrast to the strong inhibition by antibodies to LFA-1 and ICAM-1, antibodies to CD44 enhanced conjugate formation between T cells and blood DC. Together, our results show that the LFA-1/ICAM-1 pathway plays a central role in T cell-blood DC adhesion, a situation like that in T cell adhesion to lymphoid DC. However, unlike lymphoid DC, blood DC do not express VCAM-1 nor use LFA-3 for T cell binding.     

TGF beta down-regulates TLiSA1 expression and inhibits the differentiation of precursor lymphocytes into CTL and LAK cells.

This study analysed the regulatory effects of transforming growth factor beta (TGF beta) on the expression of a 70,000 MW cell surface activation antigen, TLiSA1, involved in the differentiation of cytotoxic T lymphocytes (CTL) and lymphokine-activated killer (LAK) cells from their precursor(s), and also examined the role of TGF beta in the generation of these functional cells. TGF beta was shown to suppress the expression of TLiSA1 and to inhibit, in a dose-dependent manner, the generation of both CTL and LAK cells when present from the beginning of mixed lymphocyte culture; the same inhibitory effect upon the development of cytotoxic effector cells was observed with a monoclonal antibody and with monospecific rabbit antibodies against the TLiSA1 protein. Antibody to TGF beta reversed the inhibitory effect of the cytokine on differentiation and on TLiSA1 expression. Exogenous IL-2 or, to a lesser extent, tumour necrosis factor alpha (TNF alpha) added to mixed lymphocyte cultures (MLC) augmented both TLiSA1 antigen expression and cytotoxic function by the resulting blast cells; the co-addition of TGF beta inhibited both of these cytokine-mediated effects. Similarly, it was shown that phytohaemagglutini (PHA)-induced lymphoblasts up-regulate their surface expression of TLiSA1 and exhibit increased LAK activity in response to IL-2, and TGF beta inhibited both of these events; this IL-2-induced increase in LAK cell function was also inhibited by antibodies to TLiSA1. It is suggested that TLiSA1 antigen expression is intimately linked to the differentiation of cytotoxic effector cells and that such differentiation may be a distinct process from IL-2-induced proliferation, although both events can be regulated by TGF-beta.     

Interleukin 4 induces cellular adhesion among B lymphocytes

We here report that interleukin 4 (IL-4) alone is able to induce cellular adhesion among mouse lymphocytes, and together with lipopolysaccharide (LPS), it increases the adhesion induced by LPS. The adhesion was inhibited by antibodies against IL-4. IL-4 appears to be acting mainly on B lymphocytes, since the response caused by IL-4 alone was much less sensitive to depletion of adherent cells than the LPS response. Depletion of T cells had no effect on IL-4- or LPS-induced adhesion. IL-4 could together with Con A, but not alone, induce adhesion among T cells. Cell clusters, which were formed after 2-3 days of LPS plus IL-4 stimulation, could be completely dissociated, and when the cells were recultured in medium, they readily started to reaggregate. The adhesion molecule lymphocyte function-associated antigen 1 (LFA-1) is, at least in part, involved in LPS plus IL-4-induced adhesion. Antibodies against LFA-1 inhibited the adhesion, but antibodies against other cell surface molecules were without inhibitory effect. Adhesion induced by IL-4 alone may involve other adhesion molecules than LFA-1.     

Human tonsillar dendritic cell-induced T cell responses: analysis of molecular mechanisms using monoclonal antibodies

Dendritic cells, isolated from human tonsillar tissue, were found to be potent stimulators of the sodium periodate T cell oxidative mitogenesis reaction. Monoclonal antibodies against CD2, CD4, CD11a, CD18, LFA-3, ICAM-1, class I and class II major histocompatibility complex (MHC) inhibited T cell proliferation in this response, whereas antibodies against CD8, CD11b, CD11c and CD16 had no effect. Further, antibodies against CD2, CD11a, CD18, LFA-3 and ICAM-1 inhibited the early dendritic cell-T cell clustering event which occurs in this cell interaction. In contrast, antibodies against CD4, class I and class II MHC did not inhibit clustering. Studies examining the expression of the respective molecules upon isolated dendritic cells and T cells suggest that anti-LFA-3 and anti-class II MHC antibodies inhibit at the level of the dendritic cell, whereas anti-CD2 and anti-CD4 antibodies inhibit at the level of the T cell. However, antibodies against CD11a, CD18, ICAM-1 and class I MHC may inhibit at either or both cell levels. These findings have enabled us to propose a molecular mechanism for dendritic cell-T cell interaction in oxidative mitogenesis. Dendritic cell-T cell clustering is mediated by bidirectional binding of LFA-1 (CD11a and CD18) and ICAM-1 (involving both molecules on both cell types) and unidirectional binding of CD2 and LFA-3 (involving T cell CD2 and dendritic cell LFA-3). This initial event permits a second interaction of dendritic cell and T cell molecules, involving T cell CD4, class I MHC (possibly at both cellular levels) and dendritic cell class II MHC, which deliver the signal for proliferation.     

Interleukin 4 Induces Cellular Adhesion Among B Lymphocytes

Abstract

We here report that interleukin 4 (IL-4) alone is able to induce cellular adhesion among mouse lymphocytes, and together with lipopolysaccharide (LPS), it increases the adhesion induced by LPS. The adhesion was inhibited by antibodies against IL-4. IL-4 appears to be acting mainly on B lymphocytes, since the response caused by IL-4 alone was much less sensitive to depletion of adherent cells than the LPS response. Depletion of T cells had no effect on IL-4- or LPS-induced adhesion. IL-4 could together with Con A, but not alone, induce adhesion among T cells. Cell clusters, which were formed after 2-3 days of LPS plus IL-4 stimulation, could be completely dissociated, and when the cells were recultured in medium, they readily started to reaggregate. The adhesion molecule lymphocyte function-associated antigen 1 (LFA-1) is, at least in part, involved in LPS plus IL-4-induced adhesion. Antibodies against LFA-1 inhibited the adhesion, but antibodies against other cell surface molecules were without inhibitory effect. Adhesion induced by IL-4 alone may involve other adhesion molecules than LFA-1.     

Interleukin-2 modulates the expression of lymphocyte function-associated antigen-one (LFA-1) and p150,95 during the generation of lymphokine-activated killer (LAK) cells

Lymphocyte function-associated antigen-one (LFA-1), Mac1 and p150,95 represent a family of heterodimeric cell surface molecules with a common beta subunit and distinct alpha subunits. LFA-1 is known to be functionally important in cell-cell interactions between immune cells. In the present study, a mouse monoclonal antibody (mAb), RH1-38, which recognizes an epitope on the beta-chain of LFA-1 was used to study the function and expression of LFA-1 on lymphokine-activated killer (LAK) cells. This mAb has been shown previously to block, in the absence of complement, cytolytic activity mediated by natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and a monocyte-like cell (phorbol diester-stimulated HL-60 cells). LAK cells were generated by culturing in vitro human peripheral blood lymphocytes (PBL) in the presence of human recombinant interleukin-2 (rIL-2), and cytotoxic activity was measured by a 51Cr-release assay using the human NK-resistant Daudi cell line. Addition of RH1-38 ascites supernatant, purified RH1-38 mAb, or F(ab')2 fragment of RH1-38 markedly reduced (>80) LAK cytolytic activity, whereas NS-1 (parent hybridoma) ascites supernatant, normal mouse IgG, and monoclonal anti-HLA had no effect on LAK-mediated killing. Equivalent inhibition of NK and CTL activity by purified RH1-38 required 10-100-fold more antibody. Appreciable inhibition occurred if the mAb was added up to 2 hr after LAK cells were mixed with targets. Indirect immunofluorescence flow cytometry and immunoprecipitation studies revealed that LFA-1 and p150,95 expression were dramatically enhanced in PBL populations cultured with rIL-2 compared with PBL cultured without rIL-2; Daudi cells expressed no detectable LFA-1 family heterodimers. Time-course experiments demonstrated that during culture of PBL in the presence of rIL-2, development of enhanced expression of LFA-1 and p150,95 correlated closely with LAK cytolytic activity. These studies (i) demonstrate that LFA-1 and/or p150,95 are functionally important effector cell surface molecules expressed by LAK cells and that some homology to NK and CTL mechanisms of cell-mediated lysis may exist; and (ii) suggest that enhanced LFA-1 and/or p150,95 expression are important for development of the fully differentiated LAK effector cell in the presence of rIL-2.     

Inhibition of the activity of cytotoxic murine T lymphocytes by antibodies to idiotypic determinants

The nature of the receptors on the surface of cytotoxic T lymphocytes (CTL), which enable these cells to recognize antigens on allogeneic targets, is still a matter of controversy. In the present study various mouse alloantisera were tested for their capacity to inhibit, in the absence of complement, the cytotoxic activity of sensitized peritoneal T lymphocytes. The only antiserum which, even after heat inactivation, consistently inhibited cytotoxic T lymphocytes was an antiserum elicited in (C3H X C57B1/6)F1 mice by immunization with AKR/Cum thymus cells. The serum inhibited the cytotoxic reaction of either AKR/J or AKR/Cum CTL on EL-4 target cells but had no inhibitory activity on the cytotoxic reaction of AKR/J cells against P-815 target cells. Thus the inhibitory activity of the serum could not be attributed to antibodies against Ly-3 determinants present in the serum. This conclusion was strengthened by the finding that the inhibitory activity of the serum could be removed by absorption, not only with AKR/J thymus cells but also with AKR/J bone-marrow cells, a procedure which did not affect the titre of Ly-3 antibodies. The serum failed to exert any inhibition on cytotoxic T lymphocytes of BALB/c and C3H mice reacting against EL-4 target cells, indicating that the inhibitory activity of the antiserum did not result from contamination by antibodies against C57B1 antigenic determinants. It was concluded that the inhibitory activity of the antiserum resulted from the presence of antibodies against idiotypic determinants expressed on AKR/Cum thymus cells reacting against the hybrid hosts. It seems, therefore, that idiotypic determinants expressed on the surface of cytotoxic T lymphocytes may be directly involved in their cytotoxic activity.     

ICAM-1-, -2- and LFA-1-independent homotypic T cell aggregation induced by a novel activating monoclonal antibody targeting the murine Thy-1 molecule

We have previously reported that the murine T cell line EL-4 has an aggregating phenotype, displaying homotypic aggregation (HTA) when exposed to monoclonal antibodies targeting specific cell surface molecules such as leukocyte function-associated antigen-1 (LFA-1). We have used this property of EL-4 cells to isolate additional HTA-inducing MAb by screening a panel of hybridomas that were generated from rats immunized with EL-4 cells. We have isolated a novel anti-Thy-1 MAb (termed FF-10) that is a powerful inducer of HTA in EL-4 cells. In addition to induction of HTA, FF-10 also induces splenocyte proliferation but inhibits anti-CD3-driven T cell proliferation. Thy-1-induced HTA cannot be blocked with MAb targeting intercellular adhesion molecule-1 and -2 (ICAM-1, ICAM-2) or LFA1. Thus, the FF-10 MAb represents a novel and unique tool to investigate the diverse roles of the murine Thy-1 molecule in T cell activation, proliferation and apoptosis.     

Allosensitization in IL-2-containing limiting dilution cultures generates cytotoxic T lymphocytes (CTL) rather than LAK cells reactive with a syngeneic nonimmunogenic murine tumor

We have previously demonstrated that high frequency (1/20) of potent cytotoxic cells reactive with the nonimmunogenic lymphoma PIR-2 of C57BL/6 (B6, H-2b) origin, can be obtained by allosensitization of syngeneic B6 splenocytes against BALB/c (H-2d) splenocytes in limiting dilution cultures (LDC). Since a high concentration (250 U/ml) of exogenous interleukin 2 (IL-2), sufficient for the elicitation of lymphokine-activated killer (LAK) cells, was used in the LDC, and because the LDC-derived cytotoxic cells were active against a wide spectrum of target cells, we investigated whether the anti PIR-2 effector cells are LAK cells or cytotoxic T lymphocytes (CTL). We found that depletion from the B6 responder cell population of Lyt2+ (CTL precursors), but not of asialo GM1+ (LAK cell precursors), prior to LDC, results in the ablation of anti PIR-2 activity. When B6 splenocytes were plated in LDC with IL-2, in the absence of allogeneic stimulating cells, the resulting anti PIR-2 activity was greater than 10- to 500-fold lower than that obtained in LDC in the presence of allogeneic stimulating cells and IL-2. These and other observations suggest that the cytotoxic response against syngeneic tumors elicited by alloantigens in LDC is mediated by CTL rather than LAK cells, and that allogeneic sensitization in LDC can provide a means for the generation of CTL against syngeneic, nonimmunogenic tumors.     

Terminal differentiation of T cells specific for mutant H-2K antigens. Conversion of Lyt-1,2 cells into Lyt-2 but not Lyt-1 cells,in vitro

The Lyt phenotype of T cells at different stages of response to mutant H-2K antigens was determined by immunofluorescence using monoclonal rat anti-Lyt antibodies. Previous observations indicated a differential expression of the two allelic forms of Lyt-1 antigen on these cells. Since the rat antibodies recognize nonpolymorphic framework determinants of Lyt antigens, in our approach the expression of both Lyt-1 alleles was analyzed with the same antibody. It was found that cells reacting to three different H-2K mutants have the Lyt-1,2 phenotype, regardless of the Lyt-1 allele carried by the responder strain. The Lyt phenotype of responder cells remained unchanged after priming in vivo. However, cells recovered from cultures after secondary stimulation in vitro were mainly Lyt-2, with few Lyt-1,2 and virtually no Lyt-1 cells present. This change of Lyt phenotype ran in parallel with the loss of proliferative capacity to the priming antigen, but cytolytic activity of the cells remained unimpaired. Long-term proliferation of T cells induced against mutant H-2K antigens could only be maintained in the presence of a T cell growth factor. Cultures with growth factor contained almost exclusively Lyt-2 cells and exerted strong cytolytic activity. These results demonstrate that the Lyt differentiation pathway of anti-mutant T cells is from Lyt-1,2 to Lyt-2. Furthermore, the data suggest that no helper cells are induced in response to mutant H-2K antigens. A model which incorporates these findings into current concepts of T cell differentiation is discussed.     

Differential cytokine regulation of natural killer cell-mediated necrotic and apoptotic cytotoxicity.

Natural killer (NK) cells can kill target cells by either necrotic or apoptotic mechanisms. Using the 51Cr-release assay to measure necrotic death of target cells, neonatal NK cells had low NK activity (K562 targets) and high lymphokine-activated killer (LAK) activity (Daudi targets) compared with adult cells, as has been previously reported. Using a 125I-deoxyuridine (125I-UdR) release assay, cord cells were shown to also have higher apoptotic LAK activity against YAC-1 target cells. Interleukin-4 (IL-4) inhibited interleukin-2 (IL-2)-induced necrotic killing of target cells by adult effectors but had no such inhibitory effect on cord cells. In contrast, IL-4 inhibited both adult and cord LAK cytotoxicity of YAC-1 target cells by apoptotic mechanisms with higher suppression observed in cord cell preparations. Using a colorimetric substrate conversion assay, IL-2 induced higher, and IL-4 had a more significant suppressive effect on, cord cell granzyme B enzyme activity compared with adult cells, paralleling apoptosis cytotoxicity data. Co-culture of either adult or cord LAK cells with IL-4 had a similar inhibitory effect on granzyme B protein expression, as detected by Western blotting. In contrast, IL-4 did not inhibit perforin expression, thereby defining IL-4 as a cytokine that can differentially regulate the NK cell-mediated cytotoxicity processes of apoptosis and necrosis. The differential sensitivity of cord cells to cytokine regulation of cytotoxicity may also have implications for cord blood transplantations, as NK cells are known to function as an effector cell in both graft-versus-host disease and in the graft-versus-leukaemia phenomena.