Lytic Effector Research Articles

Control of Syngeneic Tumor Growth by Activation of CD8+ T Cells: Efficacy Is Limited by Migration Away from the Site and Induction of Nonresponsiveness

Activation of Ag-specific CD8+ T cells in response to syngeneic tumor has been visualized by adoptive transfer of CD8+ T cells from OT-I mice, with a transgenic TCR specific for H-2Kb and an OVA peptide, into Thy-1 congenic recipients. Intraperitoneal challenge with E.G7, the EL-4 thymoma transfected with OVA, results in activation and clonal expansion of the OT-I cells in the peritoneal cavity and transient control of tumor growth. However, within 2 days after becoming activated, the OT-I cells migrate out of the peritoneal cavity into the spleen and lymph nodes, and tumor growth resumes in the peritoneal cavity. The OT-I cells in lymph nodes and spleen have lytic effector activity, but exhibit split anergy in that they cannot proliferate in response to Ag unless exogenous IL-2 is provided. The failure to remain at the tumor site and continue to control tumor growth is not due to selection of Ag loss variants or development of suppression. These results suggest that effective CD8-targeted immunotherapy may depend less on enhancing the initial activation and more on sustaining the response at the appropriate location and/or reactivating cells that have left the site of tumor growth and become nonresponsive.

Distinct kinetics of cytokine production and cytolysis in effector and memory T cells after viral infection.

In the present study, naive T cells were compared with in vivo generated effector and memory T cells expressing the same TCR specific for lymphocytic choriomeningitis virus. Upon restimulation in vitro, the same minimal concentrations of the full agonist peptide p33 and also of weak and partial agonist peptides were required for proliferation of naive, effector and memory T cells, indicating no difference in threshold of activation. However, activation kinetics were distinct. While effector cytotoxic T cells exhibited immediate ex vivo lytic effector function, naive and memory T cells required 12 h and more exposure to antigen to develop lytic activity. However, both effector and memory T cells contained IFN-gamma mRNA in vivo and required less than 3 h for secretion of cytokines upon restimulation in vitro. In contrast, naive T cells did not contain IFN-gamma mRNA and required more than 12 h for cytokine secretion. Our results show that memory T cells exhibit a unique phenotype in that they produce cytokines and commit to proliferation as rapidly as effector cells, whereas they resemble naive T cells in the time requirement for development of cytolytic function.

Activation Requirements, Lytic Mechanism, and Development of a Novel Anti-CD8-Resistant CTL Population

Almost all conventional CD8+ CTL and their CD8+ precursors are inhibited by anti-CD8 mAb. This requirement for CD8 function reflects both an avidity-augmentation role and a signal-transduction role for CD8 on T cells. We have, however, previously identified and partially characterized a novel functional population of CD8+, but anti-CD8-resistant, MHC class I-allospecific CTL. These CTL have unusual activation requirements in that their efficient generation in vitro requires inhibition of the CD8 avidity contribution (but not the CD8 signaling contribution), by anti-CD8 mAb. In this study, we have investigated the relationship of anti-CD8-sensitive and anti-CD8-resistant CTL by several criteria. These CTL populations share the phenotypic markers we have tested to date, they have similar but not identical Ag-specific repertoires, and they both appear to be generated from naive unprimed T cells. However, anti-CD8-sensitive and anti-CD8-resistant CTL populations exhibit important functional differences. They differ in their kinetics of activation in vitro, their dependence on exogenous cytokines, their use of lytic effector mechanisms, and their tissue distribution during ontogeny. Based on these results, we favor the hypothesis that these CTL populations represent distinct T cell lineages or subsets, and not merely different TCR avidity ranges within a single T cell lineage or subset.

A functional and kinetic comparison of antiviral effector and memory cytotoxic T lymphocyte populations in vivo and in vitro.

To analyze the critical parameters for effective antiviral cytotoxic T lymphocyte (CTL) activity in vivo, control of lymphocytic choriomeningitis virus (LCMV) infection in the spleen was studied after adoptive transfer of different spleen cell populations into preinfected recipients. The quantitative, qualitative and kinetic requirements for virus control were defined and related to in vitro assays to compare the antiviral protective function of CTL from naive, acutely infected and memory mice. Treatment of mice with an established but limited LCMV infection by adoptive transfer of spleen cells from acutely LCMV-infected mice led to complete virus elimination mainly mediated by donor-derived CD8+ T cell-mediated, perforin-dependent cytotoxicity. Since virus is continuously spreading and the number of infected target cells rapidly increases, the time until target cell lysis is achieved was critical: if release of viral progeny was not prevented early, additional time to perform effector function did not improve overall virus control. When the function of various cell populations was compared in this model, we found that CTL from naive and memory mice perform considerably less well than CTL from acutely infected mice. In vitro studies indicated that this is probably due to the fact that they can not fulfill the limiting time requirements for immediate antiviral protection: while CTL from acutely infected mice can perform lytic effector function immediately, memory CTL require a considerable reactivation time before they can lyse infected target cells. This reactivation does not necessarily involve cell division. These findings illustrate how critical time limitations are for CTL to mediate early control of a dynamic virus infection in vivo.

Distinct Roles for LFA-1 and CD28 during Activation of Naive T Cells: Adhesion versus Costimulation

Efficient T cell activation requires the engagement of a variety of ligand/receptor molecules in addition to T cell receptor (TCR)–major histocompatibility complex (MHC)/peptide interactions. The leukocyte function antigen 1 (LFA-1) and the CD28 glycoprotein have both been implicated in T cell activation. The present study dissects the roles of LFA-1 and CD28 in the activation of naive virus-specific CD8+ T cells. We demonstrate that LFA-1 facilitates T cell activation by lowering the amounts of antigen necessary for T cell activation. In the absence of LFA-1, 100-fold more antigen was required for T cell–antigen-presenting cell (APC) conjugation and all subsequent events of T cell activation, including TCR down-regulation, Ca2+-flux, T cell proliferation, and lytic effector cell induction. Thus, LFA-1 facilitates the functional triggering of TCRs by promoting adhesion of T cells to APCs but does not affect T cell activation otherwise. In contrast, CD28 played an entirely different role during T cell activation. CD28 reduced the number of TCRs that had to be triggered for T cell activation and allowed activation of T cells by low affinity ligands. CD28 but not LFA-1 prevented induction of T cell unresponsiveness after stimulation of TCRs. These results demonstrate that LFA-1 and CD28 exhibit distinct, nonoverlapping ways to influence T cell activation and suggest that the terms costimulation and signal 2 should be revisited.

B7-1-dependent co-stimulation results in qualitatively and quantitatively different responses by CD4+ and CD8+ T cells.

To characterize better the co-stimulatory activity of native B7-1 in the absence of other receptor/ligand interactions that might contribute to the response, B7-1 was purified by monoclonal antibody (mAb) affinity chromatography. Immobilization of purified B7-1 with anti-T cell receptor (TCR) mAb on cell-sized latex microspheres provided an effective stimulus for activation of both CD4+ and CD8+ T cells as measured by proliferation, development of effector function, and changes in motility and adhesion. The CD4+ T cell response was prolonged and resulted in efficient interleukin-2 production and clonal expansion. In contrast, CD8+ responses were transient. Proliferation and clonal expansion peaked on days 3 and 4, coincident with maximal expression of lytic effector function, and the cells then died. These results demonstrate that B7-1 mediated co-stimulation is sufficient for the induction of effector function in both helper and cytotoxic T cell precursors, but suggest that B7-1 co-stimulation is not sufficient to sustain helper-independent CD8+ CTL responses. When the dose responses of CD4+ and CD8+ T cells to B7-1 were compared, CD8+ T cells were found to require higher densities of B7-1 to attain an equivalent level of activation, suggesting that the level of expression of B7-1 by APC may influence the development of helper or CTL responses. Finally, in contrast to results obtained by others with B7-1 transfectants, purified B7-1 did not provide co-stimulation when presented on a surface separate from the TCR stimulus.

Supplementation with selenium augments the functions of natural killer and lymphokine-activated killer cells.

This study examined the effects of dietary (2.0 ppm for 8 wk) and in vitro (1 x 10(-7)M) supplementation with selenium (Se, as sodium selenite) on the activity of spleen natural killer (NK) cells and plastic-adherent lymphokine-activated killer (A-LAK) cells from C57B1/6J male mice. Dietary supplementation with Se resulted in a significant increase in the lytic activity of activated NK cells, and cells from these highly lytic effector cell populations expressed significantly higher numbers of intermediate affinity interleukin-2 receptors (II-2R)/cell. In the presence of high concentrations of II-2 and 1 x 10(-7)M Se, resting populations of spleen NK cells developed into A-LAK cells that had a significantly enhanced ability to proliferate, as indicated by the significantly higher amounts of nuclear 3H-thymidine incorporation, and a significantly augmented cytolytic activity against both NK-sensitive and NK-resistant target cells. Se appears to enhance the lytic activity of activated NK cells and to augment the proliferation, expansion, and lytic activity of A-LAK cells in the presence of high concentrations of II-2 through its ability to enhance the expression of intermediate affinity II-2R on these cells.

Cytokine and chemokine expression in tumors of mice receiving systemic therapy with IL-12.

The cellular and molecular mechanisms of IL-12-mediated anti-tumor activity have been examined. BALB/c mice bearing established s.c. RENCA or CT26 tumors that were treated daily with IL-12 showed essentially complete tumor regression while tumors in untreated animals grew progressively. Examination of inflammatory gene expression in tumor tissue from treated vs untreated mice revealed the selective expression of IFN-gamma and the IFN-gamma-inducible CXC chemokine IP-10. Immunohistologic analysis demonstrated that tumors from treated mice were heavily infiltrated with CD8+ T cells and Mac-1+ mononuclear cells. Tumor regression in IL-12-treated mice was associated with expression of the lytic effector molecules perforin and granzyme B. These findings support the hypothesis that the anti-tumor function of IL-12 treatment depends upon the induced expression of IFN-gamma by T cells and/or NK cells, the amplification of the immune response mediated by IFN-gamma-induced expression of chemoattractant cytokines, and the IL-12-dependent potentiation of the cytolytic effector function of recruited CD8+ T cells.

Transforming growth factor beta 1 costimulated growth and regulatory function of staphylococcal enterotoxin B-responsive CD8+ T cells.

Transforming growth factor beta (TGF-beta) exhibits diverse effects on growth and differentiation of a wide range of cell types. In the immune system, TGF-beta 1 is a potent inhibitor of T cell proliferation and certain T cell effector functions. However, TGF-beta 1 also enhances growth of T cells, predominantly of naive phenotype, and induces their expression of selected cytokines. We have previously demonstrated that TGF-beta 1 costimulates growth of highly purified murine CD8+ T cells activated by immobilized anti-CD3 Ab. TGF-beta 1-costimulated CD8+ T cells rapidly express a memory phenotype, lose lytic function, and express a mixed cytokine pattern with IL-2, IFN-gamma, and appreciable IL-10, as well as TGF-beta 1. The present work examines the possibility that TGF-beta 1 similarly costimulates response of murine CD8+ T cells to the microbial superantigen staphylococcal enterotoxin B (SEB) and characterizes their effector and regulatory functions. TGF-beta 1 significantly enhances CD8+ T cell proliferation to SEB in the presence of MHC class II-positive APC and TGF-beta 1-primed CD8+ T cells are enriched for SEB-reactive V beta 8+ TCR expression. TGF-beta 1 priming also up-regulates a memory-like CD45RBlowCD44highMEL-14low phenotype. TGF-beta 1 priming inhibits development of SEB-specific lytic effector function by more than 90%. However, TGF-beta 1-primed CD8+ effector T cells express elevated levels of IL-10 and TGF-beta 1, variable IFN-gamma, and undetectable IL-4. Additionally, they exhibit growth inhibitory effector function of SEB-induced proliferation of other CD4+ and CD8+ T cells. Growth inhibition by TGF-beta 1-primed CD8+ T cells is reversed in part by anti-IL-10 Ab. Thus, in the context of SEB response, TGF-beta 1 promotes the outgrowth and induces the effector function of CD8+ T cells that have the capacity to impair T cell clonal growth.

Lytic effector cell activity and major depressive disorder in patients with breast cancer: a prospective study

The study examined lymphokine-activated killer cell (LAK) and natural killer (NK) cell activity in breast cancer patients prior to surgery as compared to effector cell lysis in patients with non-malignant breast tumors, further in connection with lifetime diagnosis of major depression, severity of current depression, anxiety and coping styles. Follow up studies covered a period of 6 and 12 months. Prior to surgery, life time diagnosis of major depressive disorder, trait anxiety and coping styles did not discriminate patients as far as effector cell lysis is concerned. LAK activity but not NK activity was reduced in patients with actual depressive symptoms ( P < 0.01) and high state anxiety ( P < 0.05). These findings support the hypothesis that LAK activity is a state marker of actually existing depression and anxiety prior to surgery. Affective rather than coping measures showed significant differences in LAK activity. For LAK activity, 1 year after surgery the only predicting factors were Tamoxifen therapy and chemotherapy.

Natural killer cell activity in long-term survivors of testicular cancer. Influence of cytostatic therapy and initial stage of disease.

Patients with testicular cancer can be cured by cisplatin-based chemotherapy in many cases. Thus, concern about possible late toxicities of treatment is warranted. In this investigation, the absolute number of natural killer (NK) cells according to their CD56+ phenotype, NK cell activity, and antibody dependent cellular cytotoxicity (ADCC) were investigated in 29 patients with seminomas or nonseminomatous germ cell tumors (NSGCT) a median of 31 months (range, 5-73 months) after termination of chemotherapeutic treatment. No difference in the absolute number of NK cells, NK cell activity, and ADCC was found between patients with testicular cancer after either standard polychemotherapy consisting of bleomycin, etoposide, and cisplatin (BEP) or monotherapy with carboplatin and healthy control subjects. When patients were analyzed further using multivariate analysis, a significant (P < 0.05) detrimental influence of BEP polychemotherapy versus carboplatin monotherapy on NK cell activity was found. Moreover, NK cell activity also depended significantly (P < 0.05) on the time elapsed after chemotherapy was administered, but normalized with time. Because the absolute number of NK cells was not affected, is was assumed that polychemotherapy induced a functional defect. In a subanalysis of patients with NSGCTs, metastases at diagnosis resulted in a significant (p < 0.05) and persistent stimulation of NK cell activity but not of ADCC. Cytostatic chemotherapy in patients with testicular cancer did not lead to compromised lytic effector cell function as assessed by NK cell activity and ADCC. However, a short, time-dependent reduction was found that also depended on the intensity of chemotherapeutic treatment. This finding related to the observation of a long-lasting stimulus of NK cell activity by initial metastases in patients with NSGCTs.

In vivo capsaicin treatment inhibits rat NK cell cytotoxic functions.

The direct and indirect interactions between the nervous system and its transmitters with NK cell cytotoxic functions has been evaluated in the rat by using the neurotoxin capsaicin (8-methyl-N-vanillyl-6-nonenamide). When administered to neonatal rats, capsaicin (50 mg/Kg in 10% ethanol and 10% tween 80 at 2 days of age) interferes with the synthesis and intraneuronal transport of peptides by causing irreversible degeneration of c fiber afferent nerves. Capsaicin treatment resulted in a marked inhibition of NK and ADCC activities both in the spleen and peripheral blood. Inhibition was already evident on day 15 after treatment and persisted until day 90 in the spleen; at this time NK cytotoxicity in the peripheral blood returned to control levels. The inhibitory effect of capsaicin treatment on peripheral blood NK and ADCC activities was associated with changes in NK cell number evaluated as percentage of cells with an LGL morphology and expressing the NK-RP1 cell surface receptor. LGL numbers did not always correlate with the percentage of NK-RP1+ cells suggesting that capsaicin may interfere with maturation of lytic effector cells. Overall these results indicate a direct influence of the nervous system on natural immune cytotoxic functions.

Role of Enhanced Cellular Adhesion in IL‐6‐Augmented Lymphokine‐Activated Killer‐Cell Function

The authors demonstrated previously that a short-term treatment with IL-6 of lymphokine-activated killer (LAK) cells produces an increase in cytotoxic activity of CD56+/CD3- effector cells generated from human PBL as well as from human thymocytes. In the study described here, the mechanisms by which IL-6 enhances LAK cytotoxicity were examined. Like untreated LAK cells, IL-6-treated LAK cells require Ca++ to initiate cytolysis. However, IL-6 treatment of LAK cells does not alter the rate of programming for lysis. Instead, IL-6 increases target-binding capacity of CD56+/CD3- LAK cells in association with the increased cytotoxicity. Similar to target-binding of untreated LAK cells, the binding between IL-6-treated LAK cells and target cells is dependent on Mg++. Cellular adhesion molecules (CAM), CD11a-c, CD18, CD54, CD56, CD58 and CD2 (T11(1) epitope), are up-regulated in LAK cells by culture with IL-2. Among MoAbs to these CAMs, only Abs to CD11a/CD18 (LFA-1) and CD54 (ICAM-1) decrease both target-binding and cytolysis by LAK cells. IL-6 treatment changes neither the proportion nor the intensity of CAM positive cells. However, MoAbs to CD11a/CD18 and CD54 reduce both target-conjugation and cytotoxicity of IL-6-enhanced LAK cells to the same level as control LAK cells treated with the MoAbs. IL-6-enhanced LAK functions (both target-conjugation and target-lysis) are not abrogated by MoAbs to other CAM which do not inhibit standard LAK functions. These results indicate that IL-6 up-regulates cellular events mediated by CD11a/CD18 and CD54 molecules which are involved in standard LAK functions. These events may result in activation of lytic effector cells, associated with an increase in target-binding and an increase in cytotoxicity.

Association of lytic effector cell function with high expression of HER-2 expression in breast cancer patients

Association of lytic effector cell function with high expression of HER-2 expression in breast cancer patients

Activation status of the CD4-8- gamma delta-T cells recovered from mice with influenza pneumonia.

The role that gamma delta-T lymphocytes play in virus infections is yet to be defined. The TCR-gamma delta + cell population found late in the course of influenza pneumonia has been analyzed for ligand-dependent lytic function. These gamma delta-T cells are not constitutively cytotoxic when recovered directly from the site of virus-induced damage in the respiratory tract, although the TCR-alpha beta + population that is present concurrently contains such lytic effectors. Both sets of lymphocytes mediate cytotoxic activity after further in vitro stimulation in the presence of mAb to CD3 and low concentrations of rIL-2. Secondary stimulation in vivo with a cross-reactive influenza A virus does not lead to the emergence of a cytotoxic gamma delta-T cell population, although substantial numbers of these gamma delta-T cells express mRNA for a variety of lymphokines and cytokines. Analysis of DNA content indicates that many of the gamma delta-T cells isolated directly from the pneumonic lung are cycling. This could reflect continuing stimulation by a specific ligand, perhaps a self-component expressed at abnormally high levels in the site of virus-induced pathology. However, we could find no evidence to indicate that the gamma delta-T cells are acting to eliminate redundant components of the host response. The percentage of inflammatory macrophages and nonphagocytic cells expressing mRNA for a 65-kDa heat-shock protein (the proposed target for at least a subset of these gamma delta-T cells) is not reduced during the time that lymphocytes with mRNA for the TCR-gamma delta are present in greatest numbers. Possible alternative functions for the gamma delta-T cells are discussed.

Functional consequences of cAMP accumulation in human natural killer cells. Implications for IL-2 and IL-4 signal transduction.

To approach the mechanisms whereby IL-2 activates human NK cells, we have compared the effects of IL-4 and of Bt2cAMP on this activation. Both agents block completely the proliferation induced by IL-2 on highly purified CD3-negative human NK cells. We also report that the net LAK response of PBL is inhibited by IL-4 and cAMP. However, kinetics analysis showed that IL-4 appears to inhibit an early stage of IL-2-induced activation of NK cells. IL-4 does not affect the cytotoxicity of freshly isolated NK cells against the K562 target and is ineffective on IL-2-preactivated cells. In contrast, cAMP primarily blocks the lytic effector phase, whether cells have been cultured in IL-2 or not, and its effect appears independent of time of addition. These differences between the activity of IL-4 and cAMP suggested that cAMP was not directly involved in IL-4 signal transduction in human NK cells. Consistent with this interpretation, we did not observe any variation in the level of intracellular cAMP concentrations when NK cells were stimulated with IL-4, or when they are stimulated with IL-2 or IL-2 plus IL-4. In addition, we also demonstrate that NK cell cytotoxic activation induced by IL-2 is still demonstrable in the presence of Bt2cAMP under conditions in which NK cell proliferation is blocked. These results clearly indicate that the differentiative effect of IL-2 on NK cells is independent of cell proliferation. Furthermore, the p70-75 IL-2R-initiated signal transduction pathway that leads to increased cytotoxicity appears not to be susceptible to inhibition by cAMP in human NK cells.

A Murine Model of NK Cell Mediated Resorption

There is increasing evidence that some models of immunologically mediated murine embryo demise involve nonspecific lytic effector cells. In this paper, we use two double stranded synthetic RNAs, known as potent interferon inducers and NK cell activators, the Poly (I). Poly (C) and the less toxic Poly (I). Poly (C12U). These polynucleotides enhance fetal resorption rates in both resorption prone and none-resorption prone strains of mice. We have studied the kinetics of the phenomenon, and observed an anti-implantation-like effect of early injection during early pregnancy. The abortifacient effects can be adoptively transferred to naive recipients by spleen cells from Ds RNA injected donors. Such effects are abrogated if the cells are pretreated with anti-NK cell antiserum. The relevance of these findings to the survival of the conceptus is suggested.

Murine trophoblast cells are not killed by tumor necrosis factor-α

Purified midgestation murine trophoblast cannot be killed by a variety of cell-mediated effector mechanisms, with the exception of highly lytic effectors such as lymphokine-activated killer cells. We now report that this trophoblast population is also resistant to tumor necrosis factor (TNF)-α.

Cytotoxic effectors in human peripheral blood mononuclear cells induced by a mannoprotein complex of Candida albicans: A comparison with interleukin 2-activated killer cells

In this study, the major histocompatibility complex-unrestricted cytotoxic effectors elicited in human peripheral blood mononuclear cells (PBMC) by a mannoprotein (MP) component from the cell wall of the human indigenous microrganism Candida albicans have been compared with those obtained by stimulation with interleukin 2. (Interleukin 2-activated killer cells: LAK). It has been found that MP-induced lytic effectors were substantially similar to LAK in potency, target specificity, and type of precursor/effector cells. In both cases, natural killer (NK)-susceptible and NK-resistant targets as well as fresh tumor (glioma) cells were efficiently killed by a population of effectors showing a predominant CD3−, CD16+ phenotype. However, the precursors of MP-induced killers were highly sensitive to the lysosomotropic toxic drug l-leucine methyl ester (Leu-OME) whereas the generation of LAK cells was unaffected by this drug. The Leu-OME sensitivity of MP-induced cytotoxicity generation was not due to a nonspecific effect on antigen-presenting cells or inhibition of cell proliferation. In addition, the generation of MP-induced killer cells was totally abrogated by treatment with CD 16 antibodies and complement, whereas a minor but significant fraction of LAK precursors was not susceptible to the above treatment. These results indicate that a defined component(s) of the cell wall of C. albicans has some properties of biological response modifiers in cultures of human PBMC in vitro.

Association of increased lytic effector cell function with high estrogen receptor levels in tumor-bearing patients with breast cancer

Tumor-bearing patients with breast cancer were assayed for their natural killer (NK) cell activity and for the function of activated cytotoxic T-cells, as assessed by lectin-dependent cellular cytotoxicity (LDCC). Tumor-bearing patients with breast cancer had a significant increase in NK activity and in LDCC, as compared with healthy control individuals. Although the enhanced NK cell activity and LDCC were closely associated with high levels (greater than 31 fmol/mg) of estrogen receptor (ER) content in the primary tumor, no other clinical or histologic correlation between the increase in either parameter of cytotoxic effector cell function could be found. Thus, ER levels greater than 31 fmol/mg might be associated with increased cytotoxic effector cell function in tumor-bearing patients with breast cancer.