Nedocromil sodium inhibits T-cell function in vitro and in vivo

Nedocromil sodium, a topical antiinflammatory agent recommended as a prophylactic regimen for asthma, is known to inhibit both allergic and nonallergic inflammatory processes in which an essential role for T cells has been implicated. Therefore the direct effects of this drug on several aspects of T-cell activity were analyzed in the present study. By using murine lymphocytes we found that NS at concentrations of 10(-8) to 10(-6) mol/L inhibited the mitogen- or antigen-induced proliferative responses of these cells. It is interesting to note that higher concentrations were ineffective. Preincubation of immune lymph node cells from contact sensitized mice with the drug abrogated their ability to transfer contact sensitivity to naive recipients, an effect that was found to be specific for the treated cells. Nedocromil sodium also interfered with the mitogen-induced interleukin-2 and tumor necrosis factor production by T cells and with their ability to adhere to the bound protein components of the extracellular matrix laminin and fibronectin. All these effects may be attributed to the inhibition of the increase of cytosolic calcium, which accompanies the early phase of T-cell activation and which is an essential step in inducing the aforementioned phenomena.     

Aberrant T-cell function in vitro and impaired T-cell dependent antibody response in vivo in vitamin A-deficient rats

We have previously reported that vitamin A deficiency resulted in a reduced IgA antibody response to cholera toxin (CT) after per-oral immunization. In the present investigation we have studied the in vivo and in vitro immune response in vitamin A-deficient rats to two parenterally applied antigens, beta-lactoglobulin (beta-LG) and picrylsulphonic acid (TNP)-Ficoll. The serum IgG and IgM antibody responses to the T-cell dependent antigen beta-LG were significantly lower in the vitamin A-deficient rats than in the pair-fed control rats. No such differences were seen with the IgG and IgM responses to the T-cell independent antigen TNP-Ficoll. However, the biliary IgA and the serum IgE antibodies against both antigens were decreased in the vitamin A-deficient rats. In vitro lymphocyte stimulation with concanavalin A (Con A) or beta-LG gave higher T-cell proliferation rates in the vitamin A-deficient than in the control rats. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) levels in supernatants from Con A-stimulated mesenteric lymph node cells were also higher in the vitamin A-deficient rats, while IL-6 levels were decreased, which is consistent with an up-regulated Th1 activity. Proliferation studies on purified accessory cells and T cells from the deficient and the control rats, mixed in different combinations, showed that the T cells, but not the accessory cells, were disturbed in the vitamin A-deficient rats. Despite the increased T-cell activity in vitro the vitamin A-deficient rats had a lower delayed-type hypersensitivity (DTH) reaction than the pair-fed control rats. In conclusion, the increased IL-2 and IFN-gamma levels may reflect an up-regulation of Th1 cell function, while the decreased IgA, IgE and IL-6 levels indicate a suppression of Th2 cells. The disturbed T-lymphocyte function is manifested in vivo as a decreased DTH reaction and suppressed antibody production, the latter possibly due to a lack of B-cell switching and proliferation factors in vitamin A-deficient rats.     

Enkephalins modulate differentiation of normal human keratinocytes in vitro

Opioid peptides are a group of neuropeptides which include enkephalins, endorphins and dynorphins. In addition to their central and peripheral antinociceptive function, opioids can modulate immune activity and cell proliferation. Previously, we have shown that enkephalins are present in macrophages infiltrating the dermal papillae in involved psoriatic skin and that the amount of enkephalin is significantly increased in involved psoriatic skin. Because enkephalins were detected close to the epidermis, we examined the effects of opioid peptides on the differentiation (transglutaminase type 1 activity and cytokeratin 10 expression) and proliferation (MTT assay) of cultured human keratinocytes. Enkephalins (methionine-enkephalin, leucine-enkephalin and the synthetic DADL) inhibited cell differentiation dose-dependently, while beta-endorphin had no effect. The opioid receptor antagonist naltrexone completely antagonized the inhibitory effect of methionine-enkephalin and leucine-enkephalin, but not that of DADL. Furthermore, methionine-enkephalin had a slight inhibitory effect on the proliferation of keratinocytes. Enkephalin was detected in unstimulated keratinocyte cultures, and naltrexone alone stimulated keratinocyte differentiation. These results indicate that enkephalins may play a role in the differentiation of epidermal keratinocytes. It remains to be determined whether the enkephalin detected in psoriatic skin are sufficient to affect epidermal differentiation in vivo.     

Macrophage-inflammatory protein-1alpha regulates preosteoclast differentiation in vitro

A validated in vitro system was used to investigate the nature of osteoclast-inducing growth factors (OGF) present in fetal rat calvarial conditioned medium (RCCM). Evidence is presented here that macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, is an essential factor for the induction of osteoclast differentiation in this system. Specific polyclonal antibodies against MIP-1alpha significantly inhibited development of TRAP-positive osteoclast precursors and multinucleated osteoclasts induced by RCCM. Anti-MIP-1alpha antibody treatment was accompanied by an increase in the number of macrophage-like cells, suggesting that bone-derived MIP-1alpha is involved in the direction of preosteoclast formation with an inhibitory action on progenitor cell proliferation. Reverse-phase HPLC of RCCM resolved multiple fractions with OGF activity. OGF fractions separated at low acetonitrile (AcN) concentrations (相似文献   

Requirements for differentiation of an immature CD4+8+ T-cell line

The CD3 epsilon and zeta chains of the TCR have been shown to possess independent signaling capabilities. Studies with chimeric molecules containing the cytoplasmic domains of either zeta or epsilon have suggested that these two structurally distinct members of the TCR-CD3 complex are able to function autonomously and have redundant features in the context of TCR-signal transduction in mature T cells. Expression of a chimeric human IL-2-receptor-zeta-chain molecule in the CD4+8+ T-cell line, DPK, has enabled us to directly analyze responses initiated by the zeta-chain-signaling module alone within the context of immature T-cell differentiation. In this paper, we show that antibody crosslinking of the chimeric zeta chain delivers only a limited activation signal as measured by Ca[2+] flux, induction of low-level CD5 expression, and minimal differentiation as assessed by loss of cell-surface CD8 expression. TCR-induced activation through antibody crosslinking of the endogenous CD3 epsilon receptor in the absence of costimulation was also relatively inefficient in initiating activation and differentiation. However, co-crosslinking of the CD4 coreceptor with CD3 resulted in a synergistic response, where as there was little effect of co-crosslinking of CD4 and the zeta-chain chimera. Striking differences were also observed in the substrate pattern of tyrosine phosphorylation, as well as lymphokine secretion following triggering through the intact TCR versus the zeta chain alone. These results indicate that although the zeta-chain may possess some signaling capacities similar to that of the intact TCR, it appears to have limited function as an autonomous subunit in initiating CD4+8+ T-cell differentiation.     

Modulation of in vitro and in vivo T-cell responses by transferrin-gallium and gallium nitrate

Gallium is a group IIIa metal that has efficacy in the therapy of malignant disorders such as lymphoma and urothelial tract tumors. Preclinical studies also indicate a role for gallium in autoimmune disorders, suggesting that gallium is able to modulate T-cell immune reactivity. The purpose of this study was to examine the in vitro and in vivo immunomodulatory action of gallium on T-cell function. Since gallium binds to transferrin in vivo, in vitro studies evaluated the effect of transferrin-gallium (Tf-Ga) on human T cells. Tf-Ga inhibited the mitogen-induced proliferative response of peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion. Alloantigen-induced proliferation was also potently suppressed when evaluated in a mixed lymphocyte culture assay. Tf-Ga affected a significant reduction in the density of IL-2 receptors on activated T cells and a slight reduction in the number of CD3+/CD25+ T cells in PHA-stimulated cultures. Neither secretion of interleukin-2 (IL-2) nor the induction of IL-2-stimulated lymphokine-activated killer activity, however, was inhibited by Tf-Ga. Tf-Ga produced significant upregulation of the transferrin receptor (CD71) in T cells as determined by flow cytometric analysis and northern blot assay, but did not affect the percentage of CD3+/ CD71+ T cells after mitogen stimulation. To assess the in vivo effects of gallium on alloreactive T cells, we evaluated the immunosuppressive effect of gallium in a murine model of graft-versus-host disease (GVHD). Administration of gallium significantly prolonged survival in mice undergoing severe GVHD, suggesting that gallium can ameliorate GVH reactivity. Collectively, these data demonstrate that, at clinically achievable concentrations, Tf-Ga potently inhibits T-cell activation and that this immunosuppressive property of gallium may be of adjunctive therapeutic value in the management of disorders characterized by the presence of autoreactive or alloreactive T-cell populations.     

Lipids from Mycobacterium leprae cell wall suppress T-cell activation in vivo and in vitro

The need for an ethics of medical justice in Latin America is asserted in the context of a review of concepts of justice throughout history and of changing governmental perspectives on provision of health care in the US and other developed countries. The current view that individuals are primarily human resources is at odds with a long tradition asserting the intrinsic dignity of human beings. English-speaking bioethicists began in the 1960s to stress the principal of autonomy of patients, recognizing their right to make decisions on their own lives and medical care equally with the physician. At the same time, the US has approved no legislation establishing a right to health care, which is rather regarded as a private good. Governments are increasingly inclined to renounce their role as direct providers of health care. The liberal democratic state until recently understood that it fulfilled its ethical commitment to promoting social justice through provision of health care. Nevertheless, societies that stress the importance of the individual in decision-making and that conceive of health as a private good are confronted with the contradiction of apparently irreconcilable visions. With infinite demand for health services and limited health resources, the discourse of autonomy has slowly been replaced by a discourse of distributive justice. The most appropriate version of distributive justice for Latin America is probably that which affirms the duty of assisting those most in need. The prevalence of malnutrition, misery, and premature death in the world is a clear sign of imbalance. If the essential dignity of all human beings and not just of the elite is to be affirmed, medical justice must become the most urgent priority of Latin America.     

The role of vitamin E in T-cell differentiation and the decrease of cellular immunity with aging

Spontaneously hypertensive rats (SHR) as a model for aging were used in this experiment and fed a regular (50 IU/Kg diet) or high vitamin E (500 IU/Kg diet) diet for 6 weeks. At 12 weeks old, they were killed and assayed. Although proliferation of thymic lymphocytes was significantly decreased in SHR fed the regular diet compared to Wistar Kyoto rats (WKY) fed the same diet, high vitamin E diet enhanced proliferation of thymic lymphocytes in SHR to almost the levels in WKY fed the regular diet. In addition, the expressions of both CD4 and CD8 antigens on CD+CD8+ T cells, immature T cells existing in thymic cortex, were also decreased in SHR, and significantly improved by high vitamin E diet. These results suggest that high vitamin E diet enhances thymic lymphocyte proliferation through increased T-cell differentiation in thymus. Then, the effect of vitamin E on T-cell differentiation in thymus was investigated by using male Fisher rats. Rats were divided into three groups; vitamin E-free, regular and high vitamin E groups and fed a diet containing various levels of vitamin E (0, 50 and 500 IU/Kg diet) for 7 weeks. Although the percentages of CD4+CD8- and CD4-CD8+ T cells in thymocytes were significantly greater in the high vitamin E group, the percentage of CD4+CD8- T cells inversely decreased in the vitamin E-free group compared to the regular group. We have tried to investigate the mechanism of the increased T-cell differentiation in thymus of rats fed the high vitamin E diet through cytokine production, and thymic epithelial cell (TEC) and macrophage functions. We have found that vitamin E enhances T-cell differentiation through the increase of not macrophage but TEC function in thymus, which is associated with the increased binding capacity of TEC to immature T cells via increased expression of adhesion molecule, ICAM-1. These results suggest that vitamin E is a potent nutrient for promoting health in the aged via the improvement of cellular immunity decreased with aging.     

Uncoupling between proliferation and differentiation of ovine granulosa cells in vitro

Granulosa cells of ovarian follicles both proliferate and undergo differentiation. In vivo, an inverse relationship between proliferation and steroidogenesis is observed. However, both processes can be enhanced by insulin-like growth factor-I (IGF-I) in vitro. Studies were undertaken in the ewe to understand the mechanisms controlling the balance between proliferation and differentiation in cultured granulosa cells from antral follicles better. For this purpose, granulosa cells from ovine small follicles (1-3 mm in diameter) and large follicles (5-7 mm in diameter) were compared for progesterone secretion, cytochrome P450 side-chain cleavage (P450scc) expression and their proportions of non-proliferating (G0) cells, in response to IGF-I and FSH stimulation in vitro. IGF-I mainly enhanced the proliferation of granulosa cells from small follicles but it strongly increased progesterone secretion and P450scc expression in granulosa cells from large follicles, in synergy with FSH. Blocking granulosa cell proliferation by the administration of colcemid or aphidicolin had no effect or a weak stimulating effect on progesterone secretion. At the beginning of the culture period, the proportion of non-proliferating cells, estimated by continuous [3H]thymidine labelling experiments, was clearly higher in large than in small follicles (91% vs 30%, P < 0.001). For both cell types, treatment with IGF-I in vitro reduced the proportion of non-proliferating cells at 72 h of culture (40% vs 70% respectively in IGF-I-stimulated and unstimulated cells from large follicles, P < 0.001, and 17% vs 30% respectively in IGF-I-stimulated and unstimulated cells from small follicles, P < 0.001). Treatment with FSH had no effect on the proportion of non-proliferating cells. As revealed by immunohistochemistry experiments, IGF-I, in synergy with FSH, clearly increased the percentage of cells expressing P450scc enzyme and the intensity of staining in granulosa cells from large follicles. Unexpectedly, heavily stained cells in mitosis were observed in IGF-I-stimulated cells from large follicles after 96 h of culture, suggesting that dividing cells might also produce progesterone. Overall, these results support the hypothesis that the growth-promoting and the cytodifferentiative effects of IGF-I are clearly distinct. Moreover, they suggest that uncoupling between proliferation and steroidogenesis may occur in cultured ovine granulosa cells. The loss of proliferative activity accompanying terminal follicular growth in vivo could be reversed in vitro. During terminal follicular growth in vivo, the existence of an active mechanism inhibiting granulosa cell proliferation, and unrelated to terminal differentiation, is therefore strongly suspected.     

Low-energy laser irradiation affects satellite cell proliferation and differentiation in vitro

Low-energy laser (He-Ne) irradiation was found to promote skeletal muscle regeneration in vivo. In this study, its effect on the proliferation and differentiation of satellite cells in vitro was evaluated. Primary rat satellite cells were irradiated for various time periods immediately after preparation, and thymidine incorporation was determined after 2 days in culture. Laser irradiation affected thymidine incorporation in a bell-shaped manner, with a peak at 3 s of irradiation. Three seconds of irradiation caused an induction of cell-cycle regulatory proteins: cyclin D1, cyclin E and cyclin A in an established line of mouse satellite cells, pmi28, and proliferating cell nuclear antigen (PCNA) in primary rat satellite cells. The induction of cyclins by laser irradiation was compatible with their induction by serum refeeding of the cells. Laser irradiation effect on cell proliferation was dependent on the rat's age. At 3 weeks of age, thymidine incorporation in the irradiated cells was more than twofold higher than that in the controls, while at 6 weeks of age this difference had almost disappeared. Myosin heavy chain (MHC) protein levels were twofold lower in the irradiated than in the control cells, whereas the proliferation of the irradiated cells was twofold higher. Fusion percentage was lower in the irradiated compared to non-irradiated cells. In light of these data, the promoting effect of laser irradiation on skeletal muscle regeneration in vivo may be due to its effect on the activation of early cell-cycle regulatory genes in satellite cells, leading to increased proliferation and to a delay in cell differentiation.     

Proliferation and differentiation of porcine inner cell mass and epiblast in vitro

The proliferation rate and differentiation state were investigated in porcine inner cell masses (ICMs) and epiblasts in vitro. ICMs isolated from early blastocysts (Day 7 of pregnancy) and epiblasts isolated from preelongated blastocysts (Day 11 of pregnancy) were cultured for up to 5 days in the presence of human leukemia inhibitory factor (hLIF) (1000 U/ml). The proliferation rate was evaluated by determination of the percentage of cells in S-phase. The differentiation state was determined by studying the expression of the stage-specific embryonic antigen-1 (SSEA-1), a marker for undifferentiated murine embryonic stem (ES) cells, and the expression of laminin and cytokeratins 8/18, markers of ES cell differentiation. The staining pattern showed that freshly collected Day 11 epiblasts appeared undifferentiated but rapidly lost this characteristic in vitro. A decrease in the proliferation rate was also observed during culture. This decrease was reduced in the presence of high concentrations of hLIF (optimal concentrations: 5000 U/ml). Conversely, treatment of Day 11 epiblast cells with retinoic acid, an agent known to induce differentiation in murine ES cells, caused a dramatic decrease in the proliferation rate in vitro. In contrast to Day 11 epiblasts, Day 7 ICMs expressed SSEA-1 in vitro and showed a higher proliferation rate (p < 0.01). However, their proliferation rate also decreased during culture and following trypsinization. These results indicate that the undifferentiated characteristics of Day 7 ICMs are more likely to be maintained in vitro than are those of Day 11 epiblasts, which are rapidly committed into early differentiation.     

An in vitro three-dimensional coculture model of cerebral microvascular angiogenesis and differentiation

The microvasculature of the developing brain is plastic and responds differently to the many insults associated with preterm birth. We developed three-dimensional in vitro culture models for the study of the responses of the developing cerebral microvasculature. Beagle brain microvascular endothelial cells (BBMEC) were isolated by differential centrifugation from newborn beagle pups on postnatal Day 1 and placed in three-dimensional culture dispersed in a collagen gel. Alternatively, BBMEC were placed in a three-dimensional coculture with neonatal rat forebrain astrocytes. Cultures were analyzed for extracellular matrix components at 1 and 6 d, and total RNA was extracted for Northern analyses. Urokinase plasminogen activator activity was assayed in both mono- and cocultures of the two cell types. Studies of three-dimensional BBMEC/astrocyte cocultures demonstrated progressive tube formation with only low levels of endothelial proliferation. By 6 d in three-dimensional coculture, the BBMEC formed capillarylike tubes with a wrapping of glial processes, and basement membrane protein synthesis was noted. Urokinase plasminogen zymography suggested intercellular signaling by the two cell types. These data suggest that the three-dimensional beagle brain germinal matrix microvascular endothelial cell/neonatal rat astrocyte coculture provides a good model for the investigation of microvascular responses in the developing brain.     

In vitro induction of differentiation by ginsenoides in F9 teratocarcinoma cells

The aim of this study was to determine the ability of the ginsenosides, extracts of Panax ginseng C.A. Meyer, to cause differentiation of F9 teratocarcinoma stem cells as a model system. F9 stem cells cultured in the presence of the ginsenosides together with dibutyryl cyclic AMP (dbcAMP) became parietal endoderm-like cells. Moreover, the expression of differentiation marker genes, such as laminin B1 and type IV collagen, was increased after treatment with the ginsenosides. Among the various purified ginsenosides, Rh1 and Rh2 were the most effective at causing differentiation of F9 cells. Since ginsenosides and glucocorticoid hormone have similar chemical structures, we examined the possibility of the involvement of a glucocorticoid receptor (GR) in the differentiation process induced by the ginsenosides. According to Southwestern blot analysis, a 94 kDa protein regarded as a GR was detected in F9 cells cultured in the medium containing the ginsenosides Rh1 or Rh2. In addition, F9 stem cells treated with the ginsenosides Rh1 or Rh2 and with RU486, a glucocorticoid antagonist with a high affinity for the GR, did not differentiate into endoderm cells morphologically, and the expression of laminin B1 gene was not induced in these cells. In a gel mobility shift assay, protein factors capable of binding to the glucocorticoid responsive element (GRE) specifically were detected in nuclear extracts of the ginsenoside-treated F9 cells. Moreover, overexpression of GR by cotransfection of GR expression vector and GRE-luciferase vector enhanced the transactivation activity of GRE promoter in the presence of ginsenosides Rh1 or Rh2 and was further augmented by dbcAMP. In addition, ginsenosides Rh1 and Rh2 bound to a GR assessed by whole-cell binding assay, even though the specific binding affinity was weaker compared to dexamethasone. Based on these data, we suggest that the ginsenosides Rh1 and Rh2 cause the differentiation of F9 cells and the effects of ginsenosides might be exerted via binding with a GR or its analogous nuclear receptor.     

Evidence that agrin directly influences presynaptic differentiation at neuromuscular junctions in vitro

The synaptic protein agrin is required for aspects of both pre- and postsynaptic differentiation at neuromuscular junctions. Although a direct effect of agrin on postsynaptic differentiation, presumably through the MuSK receptor, is established, it is not clear whether agrin directly affects the presynaptic nerve. To provide evidence on this point, we used anti-agrin IgG to disrupt agrin function in chick ciliary ganglion (CG) neuron/myotube cocultures. In cocultures grown in the presence of 200 microg/ml anti-agrin IgG, clustering of acetylcholine receptors (AChRs), extracellular matrix proteins, and the synaptic vesicle protein synaptotagmin (syt) at nerve-muscle contacts was inhibited. Syt clustering was still inhibited in the presence of 100 microg/ml blocking antibody, while the postsynaptic clustering of AChRs, heparan sulphate proteoglycan, and s-laminin was retained. Additionally, in CG neurons cultured with COS cells expressing agrin A0B0, which lacks the ability to signal postsynaptic differentiation, syt clustering was induced and this clustering was also blocked by anti-agrin IgG. Our results demonstrate that agrin function is acutely required for pre- and postsynaptic differentiation in vitro, and strongly suggest that agrin is directly involved in the induction of presynaptic differentiation.     

Regulation of cathepsin E expression during human B cell differentiation in vitro

Cathepsin E is an aspartic proteinase which has been implicated in antigen processing in the class II major histocompatibility complex pathway. In this study we show that cathepsin E, measured at both the protein and message level, is up-regulated late in human B cell activation. The implications of this observation in terms of cathepsin E function are discussed.     

Clonal lines of teratocarcinoma cells in vitro: differentiation and cytogenetic characteristics

Clonal lines of embryonal carcinoma cells have been established in culture from four independently-derived transplantable teratocarcinomas of mice: three from strain C3H and one from strain 129/Sv. Cells from all lines retain the capacity to differentiate into a variety of tissue types both in tumors formed following the injection of cells into syngeneic animals and in vitro under appropriate culture conditions. Analysis of their G-banded chromosomes indicated that the four lines have near-diploid but not absolutely normal karyotypes. The same chromosomal abnormalities were often present in more than one line. Tetraploid embryonal carcinoma cells made by Colcemid or cytochalasin B treatment were also pluripotential in spite of chromosomal instability. Hybrid cells were readily obtained between diploid or tetraploid embryonal carcinoma cells and mouse 3T3 fibroblasts. Hybrid cells failed to differentiate and were contact inhibited like the 3T3 parent.     

Contrasting role of presenilin-1 and presenilin-2 in neuronal differentiation in vitro

Presenilin-1 (PS1) and presenilin-2 (PS2), the major genes of familial Alzheimer's disease, are homologous to sel-12, a Caenorhabditis elegans gene involved in cell fate decision during development. Recently, wild-type and mutant presenilins have been associated also with apoptotic cell death. By using stable transfection of antisense cDNAs, we studied the functions of PS1 and PS2 during neuronal differentiation in the NTera2 human teratocarcinoma (NT2) cell line. Expression of antisense PS1 resulted in a failure of the clones to differentiate into neurons after retinoic acid induction, whereas cells transfected with antisense PS2 differentiated normally. Concomitantly, antisense PS1 clones were associated with increased apoptosis both under basal conditions and during the early period of neuronal differentiation after retinoic acid treatment. Overexpression of bcl-2 in antisense PS1 clones reduced cell death and resulted in a recovery of neuronal differentiation. These studies suggest that PS1 plays a role in differentiation and cell death and that PS1 and PS2 have differing physiological roles in this experimental paradigm.     

HLA class I expression on human ovarian carcinoma cells correlates with T-cell infiltration in vivo and T-cell expansion in vitro in low concentrations of recombinant interleukin-2

This study was carried out to determine whether HLA class I or class II expression on ovarian tumor cells and lymphocytic infiltration of the epithelial ovarian carcinoma (EOC) tissues were responsible for the ability to expand tumor-infiltrating lymphocytes (TIL) in vitro in low concentrations of recombinant interleukin-2 (rIL-2). Immunohistochemical analysis was performed using monoclonal antibodies that recognize framework determinants of either HLA class I or HLA class II or leukocyte differentiation antigens (LCA, CD3, CD4, and CD8). Cryostat sections of EOC had HLA class I and HLA class II expression on at least 5% of tumor cells in 18 of 20 specimens (90%). From another portion of the same tumor specimens T-cell lines were developed from TIL in low concentrations of rIL-2 (200-600 IU/ml) in 7 of 17 patients. Tumors from which TIL were expanded in vitro with rIL-2 had significantly higher proportions of HLA class I-positive tumor cells (73 +/- 10%) compared to tumors from which TIL failed to grow (40 +/- 10%) (P = 0.036). However, there was no difference in the proportions of HLA class II-positive tumor cells between the two groups. Tumor specimens of patients whose TIL were expanded in rIL-2 had significantly higher numbers per field (423 +/- 114 vs 154 +/- 20; P = 0.005) and proportions (90 +/- 3% vs 77 +/- 4%; P = 0.023) of infiltrating CD3+ cells, significantly higher numbers per field (115 +/- 44 vs 19 +/- 5; P = 0.003) and proportions (25 +/- 5% vs 11 +/- 2%; P = 0.017) of CD8+ cells and significantly higher numbers per field of CD4+ cells (318 +/- 101 vs 113 +/- 18; P = 0.025), in comparison to tumor specimens from patients whose TIL did not grow in vitro. Significant correlations were observed between the proportions of HLA class I-positive EOC tumor cells and the numbers of infiltrating LCA-positive cells (r = 0.67; P = 0.005) CD3+ cells (r = 0.70; P = 0.002), CD4+ cells (r = 0.69; P = 0.003), and CD8+ cells (r = 0.82; P = 0.001). The proportions of HLA class II-positive tumor cells correlated positively (r = 0.45; P = 0.049) only with the numbers of CD8+-infiltrating cells. In conclusion, we report here that HLA class I expression on EOC cells correlates with T-cell infiltration in vivo and T-cell expansion in vitro, in low concentrations of rIL-2.