Lymphocyte-mediated Cytotoxicity Research Articles

Granzyme A and B-deficient killer lymphocytes are defective in eliciting DNA fragmentation but retain potent in vivo anti-tumor capacity.

Recent studies have demonstrated that granzymes A and B make an important contribution to the clearance of the orthopoxvirus ectromelia, and in graft versus host disease. To test whether granzymes are generally necessary for lymphocyte-mediated cytotoxicity in vivo, we assessed the cytotoxic capacity of granzyme A and/or B-deficient lymphocytes in several perforin-dependent settings. Splenocytes and allogeneic CTL of granzyme A and/or B-deficient mice were defective for induction of DNA fragmentation, but induced significant membrane damage and target cell death. These results correlated well with the behavior of granzyme A/B-deficient CTL and NK cells in three different perforin-dependent tumor models. In a classical assay of NK cell-mediated rejection, granzyme A and/or B-deficient mice inoculated with RMA-S cells were as susceptible to tumor as wild-type mice. Perforin-deficient mice were also considerably more susceptible to tumor initiation by methylcholanthrene than granzyme A and/or B-deficient mice. Furthermore, rejection of the K1735-melanoma expressing MHC class I and II molecules was mediated by adoptively transferred H-2b anti-k CTL from immunized granzyme A and/or B-deficient mice. In summary, these data suggest that granzymes A and B are not critical for most anti-tumor effector functions of NK cells and CTL that are perforin mediated.

Regulation of p38 mitogen-activated protein kinase during NK cell activation.

The mitogen-activated protein kinase (MAPK) p38 modulates a variety of cellular functions, including proliferation, differentiation and cell death. However, we report here a novel function for p38, i.e. the regulation of cytotoxic lymphocyte-mediated cytotoxicity. Stimulation of NK cells by either cross-linking of their FcgammaRIII receptors or by binding to NK-sensitive target cells induces the phosphorylation and activation of p38, and also of its upstream regulators MKK3/MKK6. Pharmacologic analyses suggest that Src-family and Syk-family protein tyrosine kinases couple the NK cell surface receptors to p38 activation. The role of p38 in the cytotoxic function of NK cells was tested by treatment of NK cells with the cell-permeable, p38-specific inhibitor SB203580. Interestingly, exposure to the drug reduced both antibody-dependent cellular cytotoxicity and natural cytotoxicity, but maximal inhibitory concentrations resulted in only partial inhibition. Collectively, these results suggest that the p38 MAPK pathway is stimulated during the development of NK cell-mediated cytotoxicity and that efficient killing is influenced by both p38-dependent and p38-independent pathways. More broadly, this study identifies the regulation of cell-mediated killing as a novel role for p38 in cytotoxic lymphocytes.

FD-891, a structural analogue of concanamycin A that does not affect vacuolar acidification or perforin activity, yet potently prevents cytotoxic T lymphocyte-mediated cytotoxicity through the blockage of conjugate formation.

FD-891 belongs to a group of 18-membered macrolides, and is a structural analogue of a specific inhibitor of vacuolar type H+-ATPase, concanamycin A (CMA). In our previous work, we have shown that CMA specifically inhibits perforin-dependent cytotoxic T lymphocyte (CTL)-mediated cytotoxicity through the degradation and inactivation of perforin, although CMA does not affect Fas ligand (FasL)-dependent cytotoxicity. Here, we show that FD-891 potently prevents not only perforin-dependent but also FasL-dependent CTL-mediated killing pathways by blocking CTL-target conjugate formation. In contrast to CMA, FD-891 was unable to inhibit vacuolar acidification and only slightly decreased the perforin activity in lytic granules. FD-891 blocked granule exocytosis in response to anti-CD3, mainly owing to the lack of CTL binding to immobilized anti-CD3. The conjugate formation was markedly inhibited only when effector cells were pretreated with FD-891. Consistent with these observations, fluorescence-activated cell sorter (FACS) analysis for cell surface receptors revealed that FD-891 significantly reduced the expression of the T-cell receptor (TCR)/CD3 complex. These data suggest that the blockage of conjugate formation and subsequent target cell killing might be at least partly owing to FD-891-induced down-regulation of the TCR/CD3 complex.

Yogurt Consumption Does Not Enhance Immune Function in Healthy Premenopausal Women

Fermented milk products may protect against breast cancer by stimulating immunologic activity. Twenty- five women [24.0 ± 0.7 (SE) yr] were assigned randomly to two groups: control (n = 12) and yogurt treatment (n = 13). Controls refrained from yogurt products for three months, whereas the yogurt treatment group consumed two cups (454 g/day) of commercially produced yogurt for three consecutive months. Prior yogurt consumption did not exceed 4-6 cups/mo, and subjects consumed their usual diet during the study. Three-day diet records and fasting midluteal blood samples were obtained during subjects' first, second, and fourth menstrual cycles (baseline, Month 1, and Month 3, respectively). Macronutrient intakes differed between groups only for carbohydrate. Calcium intake increased for yogurt consumers during intervention. Lymphocyte proliferation induced by concanavalin A, phytohemagglutinin, and pokeweed mitogen, interleukin 2 production, and cytotoxic T lymphocyte-mediated cytotoxicity was assessed after baseline and Months 1 and 3 for both groups. No significant immune differences between the control and yogurt treatment group were observed for concanavalin A, phytohemagglutinin, pokeweed mitogen, interleukin-2, or cytotoxicity. In conclusion, three months of yogurt consumption did not enhance ex vivo cell-mediated immune function in young women.

Role of TNF in lymphocyte-mediated cytotoxicity.

We now know that tumor necrosis factor (TNF) family ligands regulate development of lymphoid tissue and coordinate cellular differentiation to defend against intracellular pathogens. In particular, TNF provides essential signals for the formation of secondary lymphoid tissue structures and plays an important role in several physiological and pathological conditions that relate to its action in inflammation and leukocyte movement. The TNF-related family of membrane-anchored and secreted ligands also represents a major mechanism regulating cell death and cell survival. TNF was first described as an endotoxin-induced and macrophage secreted factor that caused haemorrhagic necrosis of tumor cells. Over the past two decades we have come to appreciate that T lymphocytes and natural killer (NK) cells also produce TNF, yet no clear single role for lymphocyte-derived TNF has emerged. This review describes the key molecular details of the action of TNF and discusses the evidence for TNF-mediated cytotoxicity being critical to lymphocyte function and immunoregulation.

What are the immunologically active components of bacille Calmette-Guérin in therapy of superficial bladder cancer?

The subcomponents of bacille Calmette-Guérin (BCG) involved in the mechanism of action of intravesical BCG immunotherapy used for prophylaxis of superficial bladder cancer recurrences have been poorly investigated. We purified various BCG subcomponents and analyzed in vitro their ability to enhance a Th1 polarized immune response as well as to increase lymphocyte-mediated cytotoxicity against bladder tumors. Human peripheral blood mononuclear cells (PBMCs) from healthy purified protein derivative-positive subjects were incubated for 7 days with whole BCG and various fractions (BCG cell wall, plasma membrane, cytosol, purified polysaccharides as glucan or arabinomannan, purified native proteins from BCG culture filtrate, recombinant 22 kDa protein, phosphate transporter PstS-2 and -3 proteins). IFN-gamma, IL-12, IL-2, and IL-6 production by stimulated PBMCs was compared to unstimulated controls and the phenotype of expanded cells analyzed by flow cytometry (FACS analysis). A (51)Cr-release assay monitored the cytotoxicity of amplified effector cells against T24 bladder tumor cells. Live BCG and most of its subcomponents (with the exception of cytosol, PstS-2 and -3) significantly enhanced IFN-gamma and IL-12 secretion, expanded CD3(-)CD56(+) cells and the non-MHC-restricted cytotoxicity against bladder tumor cells compared to unstimulated controls (all P < 0.001, t-test). IL-2 receptor blockage resulted in a clear reduction in the cytotoxic activity of stimulated PBMCs. Numerous BCG subcomponents thus provide positive stimuli for Th1 cell differentiation and enhance in vitro, non-MHC-restricted cytotoxicity against bladder tumor cells. Our findings provide the basis for the therapeutic use of several of these subfractions in experimental animal models bearing bladder tumors.

Biological activities of granzyme K are conserved in the mouse and account for residual Z-Lys-SBzl activity in granzyme A-deficient mice

Tryptase-like activities of T and NK cells contribute to the induction of target cell apoptosis, but only granzyme A (GzmA) has been shown to exhibit Z-Lys-SBzl esterase activity in murine T cells. GzmA-deficient mice exhibit residual Z-Lys-SBzl hydrolyzing activity and almost normal levels of lymphocyte-mediated cytotoxicity. Here we report the cloning and biochemical characterization of recombinant mouse granzyme K (GzmK). The purified murine protein shows Z-Lys-SBzl hydrolyzing activity and is inhibited by bikunin, the light chain of inter-α-trypsin inhibitor, like the human homolog. We conclude that GzmK expressed by GzmA-deficient T cells accounts for the remaining Z-Lys-SBzl activity. Functional similarities between GzmA and GzmK may explain the subtle immunological deficits observed in GzmA-deficient mice.

Primary Ovarian Cancer Cultures are Resistant to Fas-Mediated Apoptosis

Objectives. Fas, a primary mediator of cellular apoptosis, is expressed by the normal ovarian epithelium. We analyzed the levels of Fas and soluble Fas (sFas) expression in ovarian cancer tissue and determined the susceptibility of primary ovarian cancer cell (CSOC) cultures to Fas-mediated apoptosis.Methods. Fas mRNA levels were detected by RT-PCR, and Fas protein levels were determined by immunohistochemistry and Western blot analysis. Secreted sFas levels were measured by ELISA. Localization of Fas to the cell surface was demonstrated by flow cytometry. The effect of Fas on cell proliferation was measured by MTT assay.Results. Intense Fas staining was detected on the cell surface and in the cytoplasm of ovarian carcinoma specimens. We also found that mean levels of sFas, which can function as a Fas agonist, were significantly increased in 18 sera from cancer patients (0.98 ng/ml) compared to those of 8 healthy individuals (0.61 ng/ml, P = 0.004). Fas mRNA and protein were expressed in all primary ovarian cancer cell cultures. Despite abundant Fas expression, CSOC cultures were significantly less sensitive to Fas-mediated apoptosis (11.3%) than primary cultures of normal ovarian epithelial cells (HOSE) (50.0%) (P = 0.00001). The sFas level in CSOC-conditioned medium was minimal (0.07 ng/ml) and not significantly different from that of HOSE-conditioned medium (0.09 ng/ml). The small amount of sFas secreted by CSOC does not likely account for the observed resistance to Fas-mediated apoptosis.Conclusion. Decreased sensitivity to Fas-mediated apoptosis could contribute to ovarian tumorigenesis through resistance to cytotoxic T lymphocyte-mediated cytotoxicity and may play a role in ovarian tumorigenesis.

Immunological changes in human immunodeficiency virus (HIV)-infected individuals during HIV-specific protease inhibitor treatment.

The present study examines the influence of effective anti-retroviral treatment on immune function, evaluated by a broad array of immunological tests. We followed 12 individuals infected with human immunodeficiency virus (HIV) for 6 months after initiation of combination anti-retroviral treatment including a protease inhibitor. Unstimulated and pokeweed mitogen (PWM)-, interleukin (IL)-2- and phytohaemagglutinin (PHA)-stimulated lymphocyte proliferative responses increased during follow-up reaching average levels from 1.3-fold (PHA) to 3.7-fold (PWM) above baseline values. The total CD4+ lymphocyte count increased mainly due to increases in numbers of CD4+ CD28+ and CD4+ CD45RO+ cells, whereas increases in numbers of CD4+ CD45RA+ cells contributed little to the increase in CD4+ cell count. The total cytotoxic T-cell (CTL) killing of autologous B cells infected with HIV-encoding recombinant Vaccinia virus was increased after 3-6 months, whereas the specific HIV-directed CTL activity and the concentration and lytic activity of natural killer (NK) cells were unchanged during follow-up. These results demonstrate that the initiation of a treatment including an HIV protease inhibitor is followed by an increase in lymphocyte proliferation and lymphocyte-mediated cytotoxicity. However, unchanged levels of specific HIV CTL and NK cell activity warn us that not all measures of immune function may respond simultaneously to anti-retroviral treatment.

HISTOLOGICAL MORPHOLOGY OF COLITIS ASSOCIATED WITH MYCOPHENOLATE MOFETIL (MMF) IN RENAL ALLOGRAFT RECIPIENTS: ACUTE GRAFT VERSUS HOST DISEASE (GVH)-LIKE FEATURES.

997 Diarrhea is the commonest complication of MMF in allograft recipients. Although colonic ulcers have been observed endoscopically, neither the underlying histology of the MMF induced colonic lesion has been described, nor the pathophysiologic mechanisms have been understood. In the current study we compared colon biopsies from patients with MMF associated diarrhea (where no other causes of pathology could be found and the diarrhea resolved with no other treatment than the interruption or reduction of MMF) to normal controls and other types of colonic pathology in transplant and non-transplant patients. 20 colonic biopsies from 11 kidney transplant recipients with proven MMF related diarrhea were analyzed quantitatively for the presence of: a) crypt epithelial cell apoptosis; b) epithelial mitoses; c) crypt and lamina propria inflammation, d) architectural distortion, and e) mucosal ulceration. The same parameters were measured in histological material from patients with GVH colitis (6), infectious colitis (IC) in transplant patients not receiving MMF (3), ulcerative colitis (UC) (5) and normal controls (NC) in resections for diverticulosis (4). (Table)TableCrypt cell apoptosis (typical feature of GVH) was significantly higher in MMF colitis than all the remaining groups (p <.0001). Also the pattern of inflammatory infiltration and architectural distortion bear marked resemblances between MMF and GVH, including direct lymphocyte contact cytotoxicity (satellitosis). Thus, a histological pattern of colitis associated with MMF emerges from this study that is remarkably similar to GVH colitis. These findings are of pathophysiological significance indicating a potential immunopathological mechanism of the MMF colitis. The identification of lymphocyte mediated cytotoxicity against intestinal cells could reflect either an altered antigenicity of the epithelium or an imbalance in the lymphocyte regulation leading to autoreactivity. There are also of diagnostic importance since a specific diagnosis can be made pathologically, both through the absence of another specific pathological entity such as infection, but also through the finding of GVH-like features.

Effect of anticancer agents on Fas-mediated cytotoxicity against bladder cancer cells

Fas and Fas ligand play an important role in cytotoxic T lymphocyte-mediated cytotoxicity. Like Fas ligand, anti-Fas monoclonal antibody (mAb) induces apoptosis of cells expressing Fas and mimics tumor necrosis factor-alpha (TNF-alpha) in its cytotoxic activity, but not in regard to other TNF-alpha-mediated activities. Since combination treatment with TNF-alpha and some anticancer chemotherapeutic agents results in synergistic cytotoxicity against various cancer cells, anti-Fas mAb may also synergize with anticancer agents in exerting cytotoxicity. The present study examined this hypothesis using bladder cancer cells. Cytotoxicity was examined by a 1-day microculture tetrazolium dye assay. Treatment of T24 cells with anti-Fas mAb in combination with mitomycin C, methotrexate, or 5-fluorouracil did not overcome their resistance to these agents. However, combination treatment with anti-Fas mAb and adriamycin (ADR) resulted in a synergistic cytotoxic effect on T24 cells, three other bladder cancer lines, and fresh bladder cancer cells derived from four patients. Treatment with ADR enhanced the expression of Fas on T24 cells. The expression of P-glycoprotein was not affected by the antibody-mediated sensitization. This study showed that combination treatment of bladder cancer cells with anti-Fas mAb and ADR can overcome their resistance and that the upregulation of Fas expression by ADR may play a role in the enhanced cytotoxicity.

Mechanisms of lymphocyte-mediated cytotoxicity

Mechanisms of lymphocyte-mediated cytotoxicity

Mechanisms of lymphocyte-mediated cytotoxicity in acute renal allograft rejection.

Graft-infiltrating T-cell (GIC) lines cultured from biopsies obtained during acute renal allograft rejection exhibit donor-specific cytotoxicity toward proximal tubular epithelial cell (PTEC) lines cultured from corresponding biopsies. This system allows for study of the relative contributions of perforin/granzyme B (GrB)- and Fas ligand (FasL)-based cytotoxicity to killing of PTEC. Expression of perforin, GrB and FasL was analyzed by immunocytochemical staining of cytocentrifuge preparations of GIC lines cultured from 10 renal allograft biopsies. Specific inhibitors of the perforin/GrB- and FasL-based pathways were used in 51Cr release and apoptosis assays to determine their relative contributions to cytotoxicity of GIC lines toward corresponding donor PTEC lines. Cells with a strong granular pattern were observed upon immunocytochemical staining of GIC lines with anti-perforin or anti-GrB monoclonal antibodies. A diffuse staining pattern was observed upon staining with anti-FasL polyclonal antibodies. Six of eight GIC lines cultured from biopsies with acute rejection showed cytotoxicity toward corresponding donor PTEC lines, whereas two GIC lines cultured from biopsies without rejection did not. Preincubation of cytotoxic GIC lines with concanamycin A, an inhibitor of the perforin/GrB-based pathway, caused inhibition of both lysis and apoptosis of PTEC. Inhibition was not observed upon incubation with monoclonal antibodies that inhibit Fas. The perforin/GrB-based pathway is mainly responsible for cytotoxicity of GIC lines toward corresponding donor PTEC lines, suggesting that this pathway predominates in tubular epithelial cell destruction by cytotoxic T lymphocytes during acute renal allograft rejection in vivo.

Docetaxel treatment of HT-29 colon carcinoma cells reinforces the adhesion and immunocytotoxicity of peripheral blood lymphocytes in vitro.

In vitro effects of docetaxel on the human colon carcinoma cell line HT-29 were studied with respect to the expression of different adhesion and surface marker molecules, the adhesion and immunocytotoxicity of peripheral blood lymphocytes and the secretion of IFN-gamma and TNF-alpha. Docetaxel, in a low concentration range (1-3x10-9 M), increased the expression of the adhesion molecules LFA-3, ICAM-1, CD44s, CD44v6, CD15, CD13 and VLA-4/5/6 on the tumor cells. Unstimulated and interleukin-2 (IL-2) activated killer (LAK) cells showed a better adherence to docetaxel treated HT-29 cells than to untreated cells. In neutralization experiments, anti-LFA-3, -CD44v6, -CD15, -VLA -4 and anti-CD13 mAb reduced the lymphocyte adhesion to untreated and docetaxel treated cells at different degrees, while CEA mAb increased the adhesion. Unstimulated and IL-2 activated lymphocytes exhibited significantly higher cytotoxicities against docetaxel treated cells than against untreated HT-29 cells. Unstimulated and IL-2 stimulated lymphocytes secreted more TNF-alpha and IFN-gamma when cocultured with docetaxel treated HT-29 cells than with untreated cells. These results suggest, that the increased lymphocyte mediated cytotoxicity against docetaxel treated HT-29 colon carcinoma cells may reflect an immunological process coupled with induction of differentiation, that may contribute to the clinically known cytostatic effects of the drug.

Activation of cytotoxic T cells by solid tumours?

Tumour-specific cytotoxic T cells (CTLs) are among the best-defined biological anticancer weapons. Nevertheless, they often fail to control tumour growth in vivo. Many reasons for this have been evoked: tumours may actively inhibit CTLs, or may protect themselves from CTL recognition by various means. However, one does not necessarily need to postulate such active immune evasion mechanisms specifically acquired by tumour cells. In this review we argue that the failure of immune protection is due to the intrinsic inability of tumours to activate an effective immune response, and that many tumours are similar to normal tissues in this respect. It is striking to see that the majority of the so-called immune escape mechanisms are not specifically acquired by selected tumour cells, but are common mechanisms shared between solid tumours and normal, healthy tissues. Immune responses are poor because tumour antigens do not efficiently localize to lymph follicles in lymphoid tissues, and are not efficiently presented to CTLs in an immunogenic context. The fact that tumours do not induce CTLs but are often susceptible to lymphocyte-mediated cytotoxicity indicates that more intensified immunization protocols should result in improved clinical outcome.

Taurine attenuates recombinant interleukin-2-activated, lymphocyte-mediated endothelial cell injury.

Recombinant interleukin-2 (rIL-2) immunotherapy is limited by microvascular endothelial cell (EC)-targeted injury. The interaction between rIL-2-activated lymphoid cells and EC is a possible mechanism of this systemic toxicity. Taurine, a beta-amino acid, is known to have several physiologic actions, including the modulation of calcium homeostasis. The aims of this study were to analyze the effects of taurine on rIL-2-activated, lymphocyte-mediated EC and tumor cell cytotoxicity and to investigate the mechanisms of its action. IL-2-activated cytotoxicity, mediated by peripheral blood mononuclear cells, against susceptible tumor cell lines and against EC (fresh EC and an EC cell line) in the presence of taurine was assessed. The effects of taurine on lymphocyte [Ca2+]i were assessed by flow cytometry, and the effects of taurine on granzyme activity were assessed by spectrophotometry. The authors' findings indicated that the addition of taurine significantly reduced rIL-2-activated EC cytotoxicity mediated by natural killer cells, without reducing antitumor response. Taurine was also shown to reduce significantly EC lysis mediated by lymphokine-activated killer (LAK) cells, while also significantly increasing tumor cytotoxicity. The authors demonstrated the importance of calcium in the role played by taurine in lymphocyte-mediated cytotoxicity and found that LAK [Ca2+]i following conjugation to EC was enhanced by taurine. They also found that taurine enhanced Ca2+-dependent granzyme exocytosis from LAK cells. These findings indicate that taurine may play a dual role in rIL-2 immunotherapy, due to its ability to reduce the vascular injury associated with this therapy while enhancing its antineoplastic activity.

Role of indirect allorecognition in experimental late acute rejection.

Late acute rejection affects up to 28% of renal allograft recipients and remains a major risk factor for late graft loss. As donor-origin antigen-presenting cells are depleted with time, T-cell recognition of donor-derived alloantigenic peptides presented by self antigen-presenting cells (the "indirect pathway" of allorecognition) may play a key role in the initiation of late acute rejection episodes. To test this hypothesis, we developed a clinically relevant experimental model in the rat (Wistar-Furth/Lewis) in which allograft recipients received cyclosporine for 1 month after transplantation and were then allowed to reject the graft upon discontinuation of immunosuppression. Lymphocyte proliferation assays to synthetic class II MHC allopeptides of donor origin and also to intact donor (Wistar-Furth) cells were performed at this time. The effector mechanisms studied included delayed-type hypersensitivity (DTH) responses, lymphocyte-mediated cytotoxicity, and alloantibody production. Lymphocytes from recipients undergoing late acute rejection had marked suppression of mixed lymphocyte reaction proliferation to intact donor cells. Significant proliferation to donor-derived 25-mer polymorphic class II MHC allopeptides was elicited, however. In vivo, significant DTH responses were observed to both MHC allopeptides and intact Wistar-Furth cells. Recipient lymphocytes also exhibited significant killing of donor cells, although not third-party cells, and anti-donor alloantibodies were detected by flow cytometry. Our results indicate that T cells primed via the indirect pathway are present during acute rejection that occurs after discontinuation of cyclosporine. Mixed lymphocyte reactivity is markedly reduced at this time. Furthermore, there is an association between such allopeptide-primed T cells and the elicitation of specific DTH responses and provision of help to B cells to produce alloantibodies and activation of CD8+ T cells to become effector cytotoxic cells.

CD28-B7 T-cell co-stimulatory blockade potentiates the effects of intrathymic immunomodulation in sensitized graft recipients.

Peripheral and central immune mechanisms contribute to the induction of tolerance in acute rejection rodent transplant models after systemic administration of CTLA4Ig and intrathymic infusion of donor alloantigen, respectively. We have investigated the effects of CTLA4Ig-induced blockade of CD28-B7 T-cell co-stimulation in conjunction with intrathymic immunomodulation on cellular and humoral immune responses leading to accelerated rejection of cardiac allografts in presensitized rats. Lewis rats were challenged with Wistar-Furth (WF) skin transplants, followed 7 days later by transplantation of WF hearts. These cardiac allografts were rejected in a fulminant manner in <24 hr. A single infusion of human CTLA4Ig (0.5 mg/rat i.v.) at the time of cardiac engraftment (day 0) did not affect accelerated rejection. Intrathymic injection of WF spleen cells (2x10[7]) at the time of skin transplantation (day -7) abrogated <24-hr rejection and extended cardiac allograft survival to 6.6+/-0.6 days. Moreover, intrathymic host immunomodulation combined with administration of human CTLA4Ig (days 0-14, every other day) extended cardiac allograft survival synergistically to 27.7+/-7.5 days, and immunomodulation combined with murine CTLA4Ig extended survival to >42.5+/-4.8 days. The prolongation of allograft survival required the blockade of both B7-1 and B7-2 ligands and was accompanied by reduction of host proliferative responses (mixed lymphocyte response) and depression of anti-donor cytotoxic T-cell generation/function (lymphocyte-mediated cytotoxicity). CTLA4Ig therapy did not affect the strong systemic IgM and IgG alloantibody response seen otherwise after intrathymic immunomodulation. CTLA4Ig enhances the effects of intrathymic donor-type cell infusion in sensitized rat recipients of cardiac allografts, indicating that "peripheral" blockade of CD28-B7 T-cell co-stimulation synergizes with the "central" immunosuppressive effects of intrathymic immunomodulation.

Characteristics of antigens from an HCGα secreting cell line (ElCo) derived from human breast carcinoma in a Native American patient

OBJECTIVE: Our purpose is to report a cell line derived from a Native American patient that could serve as a model for ethnic diversity in cell culture research. STUDY DESIGN: Tumor biopsy specimens from reproductive tract malignancies were explanted in the cell culture laboratory. Characterization of a successfully established line included a parameter commonly observed in minority populations in rapid moving Type A glucose-6-phosphate dehydrogenase enzyme (G6-PD). Multiple other standard characterization studies were carried out. RESULTS: A breast cancer cell line with a G6-PD-A enzyme was derived from an American Indian patient; this pattern is commonly observed in African-American populations. Results of lymphocyte-mediated cytotoxicity tests indicated that lymphocytes from patients with breast cancer were cytotoxic to ElCo cells. On the other hand, lymphocytes from patients with other types of cancer were not significantly different from healthy donor lymphocytes in their cytotoxicity. These studies indicate that ElCo cells express tumor-associated antigen(s). Many antigens of tumor cell origin were detected with use of antisera raised in rabbits in concentrates of ElCo cell culture fluid. One of these antigens was the alpha subunit of human chorionic gonadotropin. CONCLUSIONS: Chemotherapy testing, vaccine production, and various new treatment schemes are most commonly developed in cell culture systems. These systems should reflect characteristics of the target population so that desired outcomes best fit the population being served. Thus the cell line being reported, which was derived from a Native American woman, represents a tool that can be used in cell culture research as a model for diversity. (Am J Obstet Gynecol 1997;176:S246-54.)

Cell-Mediated Cytotoxicity in Hearts With Dilated Cardiomyopathy: Correlation With Interstitial Fibrosis and Foci of Activated T Lymphocytes

Objectives. The purpose of this study was to investigate the expression of perforin and T-cell intracellular antigen-1, two crucial components of lymphocyte-mediated cytotoxicity, in endomyocardial biopsies from patients with idiopathic dilated cardiomyopathy.Background. Previous reports have demonstrated the presence of myocardial interstitial fibrosis and increased infiltrating lymphocytes in patients with dilated cardiomyopathy. However, the pathogenic significance of these lymphocytic infiltrates remains unclear.Methods. Endomyocardial biopsies from 134 patients with idiopathic dilated cardiomyopathy were histologically and immunohistologically analyzed. Monoclonal antibodies against diverse T-lymphocyte antigens, perforin and T-cell intracellular antigen-1 were used with the highly sensitive avidin–biotin complex technique. Positive cells were counted in at least 10 high power fields.Results. Perforin and T-cell intracellular antigen-1 were immunohistologically detected in all biopsies. Immunoreactivity was restricted to the cytoplasm and was granular in nature, indicating specific staining of cytoplasmic granules. Correlations were established between the expression of perforin and T-cell intracellular antigen-1 and the abundance of foci of various T-lymphocyte subpopulations and, most importantly, the degree of interstitial fibrosis on routine histologic examination (p = 0.015).Conclusions. Cytotoxic activity is clearly present in endomyocardial biopsies from patients with idiopathic dilated cardiomyopathy. Local activation—that is, focal accumulation of T lymphocytes—seems to be important for the generation of lymphocyte-mediated cytotoxicity. The interstitial fibrosis commonly seen in dilated cardiomyopathy may be caused by cytotoxic T-lymphocyte damage to the myocardium.(J Am Coll Cardiol 1997;29:429–34)