Differential effects of physiologically relevant hypoxic conditions on T lymphocyte development and effector functions.

Direct measurements revealed low oxygen tensions (0.5-4.5% oxygen) in murine lymphoid organs in vivo. To test whether adaptation to changes in oxygen tension may have an effect on lymphocyte functions, T cell differentiation and functions at varying oxygen tensions were studied. These studies show: 1) differentiated CTL deliver Fas ligand- and perforin-dependent lethal hit equally well at all redox conditions; 2) CTL development is delayed at 2.5% oxygen as compared with 20% oxygen. Remarkably, development of CTL at 2.5% oxygen is more sustained and the CTL much more lytic; and 3) hypoxic exposure and TCR-mediated activation are additive in enhancing levels of hypoxia response element-containing gene products in lymphocyte supernatants. In contrast, hypoxia inhibited the accumulation of nonhypoxia response element-containing gene products (e.g., IL-2 and IFN-gamma) in the same cultures. This suggests that T cell activation in hypoxic conditions in vivo may lead to different patterns of lymphokine secretion and accumulation of cytokines (e.g., vascular endothelial growth factor) affecting endothelial cells and vascular permeabilization. Thus, although higher numbers of cells survive and are activated during 20% oxygen incubation in vitro, the CTL which develop at 2.5% oxygen are more lytic with higher levels of activation markers. It is concluded that the ambient 20% oxygen tension (plus 2-ME) is remarkably well suited for immunologic specificity and cytotoxicity studies, but oxygen dependence should be taken into account during the design and interpretation of results of in vitro T cell development assays and gene expression studies in vivo.

Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling.

Adenosine deaminase (ADA) deficiency in humans results in a severe combined immunodeficiency (SCID). This immunodeficiency is associated with severe disturbances in purine metabolism that are thought to mediate lymphotoxicity. The recent generation of ADA-deficient (ADA(-/-)) mice has enabled the in vivo examination of mechanisms that may underlie the SCID resulting from ADA deficiency. We demonstrate severe depletion of T and B lymphocytes and defects in T and B cell development in ADA(-/-) mice. T cell apoptosis was abundant in thymi of ADA(-/-) mice, but no increase in apoptosis was detected in the spleen and lymph nodes of these animals, suggesting that the defect is specific to developing thymocytes. Studies of mature T cells recovered from spleens of ADA(-/-) mice revealed that ADA deficiency is accompanied by TCR activation defects of T cells in vivo. Furthermore, ex vivo experiments on ADA(-/-) T cells demonstrated that elevated adenosine is responsible for this abnormal TCR signaling. These findings suggest that the metabolic disturbances seen in ADA(-/-) mice affect various signaling pathways that regulate thymocyte survival and function. Experiments with thymocytes ex vivo confirmed that ADA deficiency reduces tyrosine phosphorylation of TCR-associated signaling molecules and blocks TCR-triggered calcium increases.

Gene dose effect reveals no Gs-coupled A2A adenosine receptor reserve in murine T-lymphocytes: studies of cells from A2A-receptor-gene-deficient mice.

Agonist binding to extracellular A2A adenosine receptors (A2ARs) inhibits the activation of virtually all tested functions of T-cells and can induce apoptosis in thymocytes. The evaluation of levels of expression of these immunosuppressive receptors is expected to clarify whether the absence of spare A2ARs (no 'receptor reserve') might be one of the mechanisms of attenuation of the effects of extracellular adenosine on T-cells. A2A transcript is found in T-cells and functional receptors can be demonstrated, but the density of receptor on T-cells is too low to be detected by radioligand binding. Studies of direct radioligand binding to murine brain with the selective A2AR agonist [3H]CGS21680 (2-(4-[(2-carboxyethyl)-phenyl]ethylamino)-5'-N-ethylcarboxamidoadenosine) established that striata levels of A2AR are virtually absent from A2A knock-out mice. Mice that are heterozygous (A2AR+/-) for the A2AR express significantly decreased levels of A2AR. To test for the presence of spare receptors in T-cells we took advantage of this gene dose effect and examined whether the decrease in the number of receptors in thymocytes from A2AR+/- mice was proportionately reflected in a decrease in the functional cAMP response of T-cells to adenosine. cAMP accumulation and apoptosis induced by adenosine and by A2AR agonist are of a lower magnitude in T-cells from A2AR+/- heterozygous mice than in T-cells from A2AR+/+ littermate control mice. These results indicate that there is no A2AR reserve in murine T-cells. Strongly decreased adenosine-triggered cAMP increases were detected in thymocytes from A2AR-/- mice, suggesting that A2B adenosine receptors cannot fully compensate for the loss of A2ARs in murine T-cells. We conclude that the number of A2ARs is the limiting factor in determining the maximal cAMP response of T-lymphocytes to extracellular adenosine, thereby minimizing the immunosuppressive effects of extracellular adenosine.

Study of A(2A) adenosine receptor gene deficient mice reveals that adenosine analogue CGS 21680 possesses no A(2A) receptor-unrelated lymphotoxicity.

Cell surface A(2A) adenosine receptor (A(2A)R) mediated signalling affects a variety of important processes and adenosine analogues possess promising pharmacological properties. Demonstrating the receptor specificity of potentially lymphotoxic adenosine-based drugs facilitates their development for clinical applications. To distinguish between the receptor-dependent and -independent lymphotoxicity and apoptotic activity of adenosine and its analogues we used lymphocytes from A(2A)R-deficient mice. Comparison of A(2A)R-expressing (+/+) and A(2A)R-deficient (-/-) cells in cyclic AMP accumulation assays confirmed that the A(2A)R agonist CGS 21680 is indeed selective for A(2A) receptors in T-lymphocytes. Incubation of A(2A)R-expressing thymocytes with extracellular adenosine or CGS 21680 in vitro results in the death of about 7-15% of thymocytes. In contrast, no death was induced in parallel assays in cells from A(2A)R-deficient mice, providing genetic evidence that CGS 21680 does not display adenosine receptor-independent intracellular cytotoxicity. The A(2A) receptor-specific lymphotoxicity of CGS 21680 is also demonstrated in a long-term (6-day) in vitro model of thymocyte positive selection where addition of A(2A)R antagonist ZM 241,385 did block the effects of CGS 21680, allowing the survival of T cells. The use of cells from adenosine receptor-deficient animals is proposed as a part of the screening process for potential adenosine-based drugs for their receptor-independent cytotoxicity and lymphotoxicity.

A(2A) receptor dependent and A(2A) receptor independent effects of extracellular adenosine on murine thymocytes in conditions of adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) and is accompanied by T-cell depletion and accumulation of both intracellular and extracellular adenosine (extAdo) and deoxyadenosine. To better understand the causes of T-cell depletion in vivo and to discriminate between extracellular and intracellular effects of exogenously added adenosine in vitro, we investigated mechanisms of 2 different effects of adenosine on murine thymocytes. These effects of adenosine include direct induction of apoptosis in about 6% to 15% thymocytes and inhibition of T-cell receptor (TCR)-induced activation of the majority of thymocytes with inhibited ADA. A(2A) adenosine receptors, but not A(2B), A(1), or A(3) receptors, are shown to be mostly responsible for extAdo-triggered signaling (cyclic adenosine monophosphate [cAMP] accumulation) in murine thymocytes and this prompted studies of the effects of extAdo on thymocytes from A(2A)R gene-deficient mice. It is found that direct apoptotic effects of extAdo on CD4(+)CD8(+) double positive (DP) thymocytes are completely accounted for by signaling through A(2A)R, with no contribution of intracellular lymphotoxicity or of compensating A(2B)Rs because only A(2A)R +/+, but not A(2A)R -/- thymocytes were susceptible to apoptotic effects of extAdo. Studies of the effects of cAMP-raising agents support observations of extAdo/A(2A)R/cAMP-triggered apoptosis in DP thymocytes. Unexpectedly, the extAdo strongly inhibited TCR-triggered activation of both A(2A)R +/+ and A(2A)R -/- thymocytes in the presence of ADA inhibitors. This was confirmed with thymocytes from ADA gene-deficient mice, suggesting the existence of A(2A)R-independent effects of extAdo on thymocytes. The presented data raises questions about the identity and functional role of A(2A)R-expressing thymocytes in T-cell differentiation and of the role of TCR-antagonizing effects of extAdo in conditions of ADA SCID. (Blood. 2000;95:3859-3867)

The extracellular versus intracellular mechanisms of inhibition of TCR-triggered activation in thymocytes by adenosine under conditions of inhibited adenosine deaminase.

The absence or low levels of adenosine deaminase (ADA) in humans result in severe combined immunodeficiency (SCID), which is characterized by hypoplastic thymus, T lymphocyte depletion and autoimmunity. Deficiency of ADA causes increased levels of both intracellular and extracellular adenosine, although only the intracellular lymphotoxicity of accumulated adenosine is considered in the pathogenesis of ADA SCID. It is shown that extracellular but not intracellular adenosine selectively inhibits TCR-triggered up-regulation of activation markers and apoptotic events in thymocytes under conditions of ADA deficiency. The effects of intracellular adenosine are dissociated from effects of extracellular adenosine in experiments using an adenosine transporter blocker. We found that prevention of toxicity of intracellular adenosine led to survival of TCR-cross-linked thymocytes in long-term (4 days) assays, but it was not sufficient for normal T cell differentiation under conditions of inhibited ADA. Surviving TCR-cross-linked thymocytes had a non-activated phenotype due to extracellular adenosine-mediated, TCR-antagonizing signaling. Taken together the data suggest that both intracellular toxicity and signaling by extracellular adenosine may contribute to pathogenesis of ADA SCID. Accordingly, extracellular adenosine may act on thymocytes, which survived intracellular toxicity of adenosine during ADA deficiency by counteracting TCR signaling. This, in turn, could lead to failure of positive and negative selection of thymocytes, and to additional elimination of thymocytes or autoimmunity of surviving T cells.

Memory of extracellular adenosine A2A purinergic receptor-mediated signaling in murine T cells.

Accumulation of extracellular and intracellular adenosine (Ado) under hypoxic conditions or in the absence of adenosine deaminase results in lymphocyte depletion and in severe combined immunodeficiency, which are currently explained by direct intracellular lymphotoxicity of Ado metabolites. In support of the alternative, "signaling" mechanism, we show that extracellular Ado (extAdo) suppresses all tested T cell receptor (TCR)-triggered effector functions of T lymphocytes including the TCR-triggered FasL mRNA up-regulation in cytotoxic T lymphocytes. Strong evidence against the intracellular lymphotoxicity of Ado (and in support of the signaling model) is provided by abrogation of TCR-triggered growth inhibition in Ado-exposed T cells. The brief exposure to Ado was sufficient to observe inhibition of TCR-triggered effector functions. The "memory" of T cells to exposure to extAdo is best explained by sustained increases in cAMP. Selective agonist (CGS21680) and antagonist (ZM241385) of A2A adenosine receptor were used in functional assays and cDNA probes for different sybtypes of adenosine receptors were used in Northern blot studies. A2A receptors are identified as the predominantly expressed subtype of Gs-coupled Ado receptors in T cells. The demonstration of cross-talk between the A2A receptors and TCR in both directions support the possible role of A2A receptors in mechanisms of extAdo-mediated immunosuppression in vivo under adenosine deaminase deficiency and hypoxic conditions in, e.g., solid tumors.

Phosphorylation of T-lymphocyte plasma membrane-associated proteins by ectoprotein kinases: implications for a possible role for ectophosphorylation in T-cell effector functions.

Extracellular adenosine triphosphate (ATPo) has been suggested to play a role in lymphocyte effector functions. Recently, it has been suggested that MgATP2- may be the molecular species which is involved in modulating the lytic interaction between cytotoxic T-lymphocytes (CTL) and their target cells. In this study, we provide evidence that ATPo mediates the phosphorylation of extracellular proteins on T-lymphocytes through the action of ectoprotein kinases. The ectophosphorylation is temperature-dependent, supported by Mg2+ and Mn2+, and both ATP and GTP, whereas kinase activity and/or substrates were removed by pretreatment of intact lymphocytes with trypsin. We show the presence of extracellular ATP/GTP-binding sites, indicating the presence of ectoenzymes on intact lymphocytes. The major ectoprotein kinase was identified as a casein kinase II-like protein kinase and could be inhibited by heparin, whereas its activity was enhanced by spermine. The ectoprotein kinase showed remarkable substrate specificity, phosphorylating the serum protein vitronectin, but not fibronectin. In experiments with the cell-impermeable protein kinase inhibitor K-252b, we demonstrate the possible functional importance of ectoprotein kinase in CTL-mediated cytotoxicity, i.e., target cell death was completely blocked by K-252b without affecting intracellular phosphorylation. These results suggest that ectoprotein phosphorylation may possibly be an important event in immunologically relevant cell-cell interactions.

Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion.

Accumulation of adenosine and of deoxyadenosine in the absence of adenosine deaminase activity (ADA) activity results in lymphocyte depletion and in severe combined immunodeficiency (ADA SCID), which is currently explained by direct cell death-causing effects of intracellular products of adenosine metabolism. We explored the alternative mechanisms of peripheral T-cell depletion as due to inhibition of T-cell expansion by extracellular adenosine-mediated signaling through purinergic receptors. The strong inhibition of the T-cell receptor (TCR)-triggered proliferation and of upregulation of interleukin-2 receptor alpha chain (CD25) molecules, but not the direct lymphotoxicity, were observed at low concentrations of extracellular adenosine. These effects of extracellular adenosine (Ado) are likely to be mediated by A2a receptor-mediated signaling rather than by intracellular toxicity of adenosine catabolites, because (1) poorly metabolized adenosine analogs cause the accumulation of cAMP and strong inhibition of TCR-triggered CD25 upregulation; (2) the A2a, but not the A1 or A3, receptors are the major expressed and functionally coupled adenosine receptors in mouse peripheral T and B lymphocytes, and the adenosine-induced cAMP accumulation in lymphocytes correlates with the expression of A2a receptors; (3) the specific agonist of A2a receptor, CGS21680, induces increases in [cAMP]i in lymphocytes, whereas the specific antagonist of A2a receptor, CSC, inhibits the effects of Ado and CGS21680; and (4) the increases in [cAMP]i mimic the adenosine-induced inhibition of TCR-triggered CD25 upregulation and splenocyte proliferation. These studies suggest the possible role of adenosine receptors in the regulation of lymphocyte expansion and point to the downregulation of A2a purinergic receptors on T cells as a potentially attractive pharmacologic target.

Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor.

To explore the possible role of purinergic receptors in thymocyte development and in pathogenesis of adenosine deaminase SCID, we studied effects of extracellular adenosine triphosphate (ATP(ext)) and adenosine on TCR- and steroid hormone-triggered processes in mouse thymocytes. Reverse transcriptase-PCR analysis confirms the mRNA expression of several types of purinergic receptors, while the functioning of ATP receptors in thymocytes is reflected by ATP(ext)-induced intracellular calcium increases and by thymocyte subset-specific sensitivity to the effects of ATP(ext) and adenosine. Only ATP(ext), but not the ATP catabolites, adenosine, dexamethasone, or TCR cross-linking, was efficient in triggering rapid protein synthesis independent lysis of CD4+8- thymocytes and peripheral CD4+ T cells. In contrast, extracellular adenosine specifically induced the apoptosis of CD4+8+ thymocytes. ATP(ext) also induced a slower process of DNA fragmentation and protein synthesis-dependent apoptosis in all thymocyte subsets. ATP(ext) had an additive effect with TCR cross-linking in the induction of thymocyte death, but, unexpectedly, the effects of ATP(ext) at high concentration were antagonistic to steroid-induced apoptosis. Described here, the properties of ATP(ext) and adenosine are consistent with their involvement in the regulation of T cell development due to differential expression and signaling through purinergic receptors in different thymocyte subsets. The possible role of purinergic receptor signaling in T cell differentiation and adenosine deaminase SCID is discussed.

Transient up-regulation of P2Y2 nucleotide receptor mRNA expression is an immediate early gene response in activated thymocytes.

In studies designed to understand the roles of P2 nucleotide receptors in differentiation of T lymphocytes, we observed a transient and protein synthesis-independent enhancement of mRNA expression for the G protein-coupled P2Y2 receptor in mouse thymocytes after the addition of steroid hormone or T cell receptor (TCR) crosslinking by anti-TCR mAb. Conversely, dexamethasone-induced increases in mRNA expression for the ligand-gated ion channel P2X1 receptor was detected in rat, but not mouse, thymocytes, raising questions about the previously suggested role of P2X1 receptors in thymocyte apoptosis. Flow cytometry analysis of thymocyte subsets excluded the possibility that the observed increases in P2Y2 receptor mRNA expression were due to the enrichment of steroid-treated cells with an P2Y2 mRNA-rich thymocyte subset. Triggering of TCR-mediated intracellular signaling pathways through crosslinking of TCR or by addition of phorbol ester and Ca2+ ionophore also resulted in the up-regulation of P2Y2, but not P2X1, receptor mRNA. It is proposed that the rapid increase of P2Y2 receptor mRNA expression could be a common early event in responses of T cells to different activating stimuli. Taken together with the recently discovered ability of nucleotide receptor-initiated signaling to antagonize or enhance the effects of TCR crosslinking or steroids on thymocytes, the observed rapid up-regulation of P2Y2 receptor mRNA expression may reflect an immediate early gene response where newly expressed cell surface nucleotide receptors provide regulatory feedback signaling from extracellular ATP in the T cell differentiation process.

Phosphorylation of extracellular domains of T-lymphocyte surface proteins. Constitutive serine and threonine phosphorylation of the T cell antigen receptor ectodomains.

The extracellular accumulation of ATP after activation of T-lymphocytes, as well as the presence of ecto-protein kinases in these cells, led us to propose that T cell surface receptors could be regulated through the reversible phosphorylation of their extracellular domains (ectodomains). Here, in a model system, we used T cell transfectants which express T cell antigen receptor chains lacking intracellular and transmembrane protein domains and 32Pi metabolic labeling of cells to definitively demonstrate phosphorylation of ectodomains of T cell surface proteins. We show that alphabetaTCR ectodomains were phosphorylated intracellularly and constitutively on serine and threonine residues and were then expressed on the T cell surface in phosphorylated form. TCR ectodomains also could be phosphorylated at the cell surface when extracellular [gamma-32P]ATP or [gamma-32P]GTP were used as phosphate donors with the same cells. Consensus phosphorylation sites for serine and threonine protein kinases were found to be strongly evolutionary conserved in both alpha and beta TCR chains constant regions. These results are consistent with the hypothesis, where T cell surface proteins which are phosphorylated intracellularly on their ectodomains, could subsequently be expressed at the cell surface and then be reversibly modified by ectoprotein phosphatase(s) and by ectokinase(s). Such modifications may change T cells cognate interactions by, e.g. affecting TCR-multimolecular complex formation and antigen binding affinity. It is suggested that alphabetaTCR ectodomain phosphorylation could serve as a potential mechanism for regulation of alphabetaTCR-mediated T-lymphocytes response.

Murine T lymphocytes modulate activity of an ATP-activated P2Z-type purinoceptor during differentiation.

Murine T, but not B, lymphocytes constitutively express a membrane receptor for adenosine nucleotides that opens a nonspecific pore that admits Ca2+ and ethidium (314 Da), but not propidium (415 Da) ions. ATP, ADP, and AMP show decreasing potency; UTP and adenosine are inactive. Nonhydrolyzable ATP analogues are completely ineffective. Oxidized ATP inhibits the response. Activity is detectable at ATP concentrations of 125 microM and peaks at 1 mM. The intracellular free Ca2+ ([Ca2+]i) rise is not reversed by removing ATP by centrifugation or apyrase. The kinetics, agonist and antagonist profiles, and the passage of ions as large as ethidium are the characteristics of a P2z-type purinoceptor. No expression of classical P2x-, P2u-, or P2Y-type purinoceptors can be detected. The [Ca2+]i elevating activity of the ATP receptor is modulated during T cell differentiation. CD4+8+ double-positive thymocytes are the least responsive. CD4-8+ single-positive thymocytes, CD8+ splenic T cells, CD4+8- single-positive thymocytes, and CD4+ splenic T cells show increasing reactivity. Measurement of P2Z expression by the rate of ethidium ion uptake correlates with the [Ca2+]i. The trimodal expression of P2Z by splenic CD4+ T cells correlates with the subsets defined by CD44 and CD45RB, differentiation Ags that distinguish memory cells: P2Zlow cells are CD44brightCD45RBbright; P2Zint are CD44dullCD45RBint; P2Zhigh are CD44brightCD45RBdull. It is suggested that P2Z receptor-mediated signaling could be involved in the regulation of differentiation and cell death in the thymus and peripheral T lymphocytes.

Development and antigen specificity of CD8+ cytotoxic T lymphocytes in beta 2-microglobulin-negative, MHC class I-deficient mice in response to immunization with tumor cells.

beta 2-Microglobulin knockout mice (beta 2-m-/-) with MHC class I expression deficiency are able to develop functional TCR(+)-alpha beta, CD8+ CTLs in response to tumor cell injection. The i.p. injection of beta 2-m-/- mice with tumor results in the massive accumulation of highly lytic CD8+ CTLs in the peritoneum and causes the local recruitment of CD8+ T cells into lymph nodes and spleens of immune animals. The accumulation of CD8+ CTLs in peritoneum is accompanied by the rejection of tumor cells and the survival of animals. The deficiency in MHC class I expression in beta 2-m/- mice is reflected in the delayed tumor rejection and CD8+ cell accumulation during the primary anti-tumor response in comparison with normal mice. The secondary response, however, is identical in normal and MHC class I-deficient mice. The rejection of tumor cells appears to be MHC class I directed because no rejection of tumors, no accumulation of CD8+ CTLs, and no survival of animals were observed when syngeneic tumor cells were used for injection with the notable exception of anti-minor Ag response. The Ag specificity of CD8+ CTLs in beta 2-m-/- mice is demonstrated using a panel of tumor target cells and class I transfectants. Although no substantial differences were found in the number and specificity of peritoneal CD8+ CTLs in beta 2-m-/- and normal mice using tumor rejection studies, the analysis of TCR-V beta phenotype using the panel of mAbs revealed the reduction in proportion of TCR-V beta 5 and TCR-V beta 6 used by CD8+ cell population from beta 2-m-/- mice. Development of lytic and H-2-directed CD8+ cells in regional lymph nodes was also observed after footpad immunization of beta 2-m-/- mice with TNP-labeled C57BL/6 splenocytes, suggesting anti-minor Ag reaction.

Cell-mediated cytotoxicity: contact and secreted factors.

The list of cells with cytotoxic potential now may include small resting T cells, but the exact nature of 'lethal hit delivery' by cytotoxic T lymphocytes remains elusive. Cell-mediated cytotoxicity by cytotoxic T lymphocytes is a complex, multistep process which seems likely to be mediated by several different pathways. Recent experimental evidence for the functioning of a novel cytotoxic mechanism through a target cell's surface receptor illustrates and emphasizes the necessity to study the interactions of cytotoxic T lymphocytes and target cells as a whole. Progress is evident in the description of molecular requirements for triggering cytotoxicity, cell-cell contacts and the regulation of the effector responses of cytotoxic T lymphocytes by extracellular, intracellular and granular proteins. Extracellular Ca(2+)-dependent secretion of perforin and protease(s) may explain several aspects of cellular cytotoxicity, whereas the apoptosis-mediating cell surface Fas protein is now implicated in Ca(2+)-independent cytotoxicity.

Highly lytic CD8+, alpha beta T-cell receptor cytotoxic T cells with major histocompatibility complex (MHC) class I antigen-directed cytotoxicity in beta 2-microglobulin, MHC class I-deficient mice.

Targeted disruption of the beta 2-microglobulin (beta 2m) gene results in major histocompatibility complex (MHC) class I deficiency and virtual disappearance of functional CD8+ cytotoxic T lymphocytes (CTLs) in beta 2m-deficient (beta 2m-/-) mice. We asked whether the beta 2m-/- mice are able to reject tumor cells injected i.p. and what is the cellular composition of peritoneal exudate leukocytes (PELs) from such mice. We found that beta 2m-/- mice do reject MHC class I-bearing tumor cells injected i.p. Surprisingly, analysis of PEL CTLs obtained from i.p. tumor-injected beta 2m -/- mice revealed the presence of a large proportion of functional, tumor-destroying CD8+, CD4-, alpha beta T-cell receptor-positive, CD3+, Thy-1+, MHC class I-negative CTLs with strong MHC class I-directed cytotoxic activity. These results call for careful studies of local accumulation of CD8+ CTLs in beta 2m -/- mouse models and suggest that the dramatic decrease in MHC class I expression caused by beta 2m gene disruption does not prevent CD8+/CD4- cell selection and expansion.

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection.

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.

[In vivo induction of cytotoxic T lymphocytes in H-2Kbm mutant mice and cross-reactivity of narrowly specific killer clones]. / Induktsiia in vivo tsitotoksicheskikh T-limfotsitov u myshei mutantnoi linii H-2Kbm i perekrestnaia reaktivnost' uzkospetsificheskikh killernykh klonov.

Anti-wild-type (B6) H-2Kbm mutant (bm) CTL were induced in the regional lymph nodes by 2 injections (with 2 week interval) of bm mice into foot-pads with B6 irradiated splenocytes. CTL were tested 7 days after the boost, including 3 days precultivation in monoculture (required for high CTL activity in bm). Active bm4 CTL inducible in vivo but not in the mixed lymphocyte culture (MLC), while bm1, bm3 and their F1 hybrids with BALB/c were equally active in both models. In vivo induced bm3 CTL were cloned with B6 irradiated splenocytes stimulators in the presence of rat interleukine-2. Of 9 Thy1.2 positive narrow-specific CTL clones 2 displayed cross-reactivity to allogeneic target cells (TC): the 1st lysed H-2Kk [TC B10.A(2R)] and the 2nd H-2Kd [TC B10.D2(R101)]. The results witness for non-identity of the in vivo and in vitro induced CTL. The variable cross-reactivity of the narrow-specific CTL clones possibly occur because of receptors' affinity difference.