Assessment of mesenchymal stem/stromal cell-based therapy in K/BxN serum transfer-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia and cartilage/bone destruction with systemic comorbidities. Despite advances in understanding the aetiology of RA and novel biologic drugs, a substantial number of individuals with RA remain intolerant or resistant to these therapies. In this context, mesenchymal stem/stromal cell (MSC)-based therapy has emerged as an innovative therapeutic alternative to address unresolved treatment issues for patients with RA thanks to the immunomodulatory properties of these cells. The majority of preclinical studies in MSC-based therapy have been conducted using the well-known collagen-induced arthritis (CIA) mouse model however due to its low incidence, the mouse strain restriction and the prolonged induction phase of collagen-induced arthritis, alternative experimental models of RA have been developed such as K/BxN serum transfer-induced arthritis (STIA), which mimics many of human RA features. In this study, we evaluate whether the K/BxN STIA model could be used as an alternative model to study the immunomodulatory potential of MSC-based therapy. Unexpectedly, our data suggest that adipose-derived MSC-based therapy is unsuitable for modulating the progression of K/BxN serum-transfer arthritis in mice despite the various experimental parameters tested. Based on the differences in the immune status and monocytic/macrophage balance among the different arthritic models, these results could help to identify the cellular targets of the MSCs and, most importantly to predict the RA patients that will respond positively to MSC-based therapy.

Improving the Efficacy of Mesenchymal Stem/Stromal-Based Therapy for Treatment of Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) consisting of persistent and relapsing inflammatory processes of the intestinal mucosa are caused by genetic, environmental, and commensal microbiota factors. Despite recent advances in clinical treatments aiming to decrease inflammation, nearly 30% of patients treated with biologicals experienced drawbacks including loss of response, while others can develop severe side effects. Hence, novel effective treatments are highly needed. Mesenchymal stem/stromal cell (MSCs) therapy is an innovative therapeutic alternative currently under investigation for IBD. MSCs have the inherent capacity of modulating inflammatory immune responses as well as regenerating damaged tissues and are therefore a prime candidate to use as cell therapy in patients with IBD. At present, MSC-based therapy has been shown preclinically to modulate intestinal inflammation, whilst the safety of MSC-based therapy has been demonstrated in clinical trials. However, the successful results in preclinical studies have not been replicated in clinical trials. In this review, we will summarize the protocols used in preclinical and clinical trials and the novel approaches currently under investigation which aim to increase the beneficial effects of MSC-based therapy for IBD.

Enhanced Susceptibility of Galectin-1 Deficient Mice to Experimental Colitis.

Galectin-1 is a ß-galactoside-binding lectin, ubiquitously expressed in stromal, epithelial, and different subsets of immune cells. Galectin-1 is the prototype member of the galectin family which shares specificity with ß-galactoside containing proteins and lipids. Immunomodulatory functions have been ascribed to endogenous galectin-1 due to its induction of T cell apoptosis, inhibitory effects of neutrophils and T cell trafficking. Several studies have demonstrated that administration of recombinant galectin-1 suppressed experimental colitis by modulating adaptive immune responses altering the fate and phenotype of T cells. However, the role of endogenous galectin-1 in intestinal inflammation is poorly defined. In the present study, the well-characterized acute dextran sulfate sodium (DSS)-induced model of ulcerative colitis was used to study the function of endogenous galectin-1 during the development of intestinal inflammation. We found that galectin-1 deficient mice (Lgals1-/- mice) displayed a more severe intestinal inflammation, characterized by significantly elevated clinical scores, than their wild type counterparts. The mechanisms underlying the enhanced inflammatory response in colitic Lgals1-/- mice involved an altered Th17/Th1 profile of effector CD4+ T cells. Furthermore, increased frequencies of Foxp3+CD4+ regulatory T cells in colon lamina propria in Lgals1-/- mice were found. Strikingly, the exacerbated intestinal inflammatory response observed in Lgals1-/- mice was alleviated by adoptive transfer of wild type Foxp3+CD4+ regulatory T cells at induction of colitis. Altogether, these data highlight the importance of endogenous galectin-1 as a novel determinant in regulating T cell reactivity during the development of intestinal inflammation.

Mesenchymal stem/stromal cell-based therapy for the treatment of rheumatoid arthritis: An update on preclinical studies.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and progressive joint destruction and is a primary cause of disability worldwide. Despite the existence of numerous anti-rheumatic drugs, a significant number of patients with RA do not respond or are intolerant to current treatments. Mesenchymal stem/stromal cell (MSCs) therapy represents a promising therapeutic tool to treat RA, mainly attributable to the immunomodulatory effects of these cells. This review comprises a comprehensive analysis of the scientific literature related to preclinical studies of MSC-based therapy in RA to analyse key aspects of current protocols as well as novel approaches which aim to improve the efficacy of MSC-based therapy.

Mesenchymal Stem/Stromal Cells for Rheumatoid Arthritis Treatment: An Update on Clinical Applications.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that affects the lining of the synovial joints leading to stiffness, pain, inflammation, loss of mobility, and erosion of joints. Its pathogenesis is related to aberrant immune responses against the synovium. Dysfunction of innate and adaptive immunity, including dysregulated cytokine networks and immune complex-mediated complement activation, are involved in the progression of RA. At present, drug treatments, including corticosteroids, antirheumatic drugs, and biological agents, are used in order to modulate the altered immune responses. Chronic use of these drugs may cause adverse effects to a significant number of RA patients. Additionally, some RA patients are resistant to these therapies. In recent years, mesenchymal stem/stromal cell (MSCs)-based therapies have been largely proposed as a novel and promising stem cell therapeutic approach in the treatment of RA. MSCs are multipotent progenitor cells that have immunomodulatory properties and can be obtained and expanded easily. Today, nearly one hundred studies in preclinical models of RA have shown promising trends for clinical application. Proof-of-concept clinical studies have demonstrated satisfactory safety profile of MSC therapy in RA patients. The present review discusses MSC-based therapy approaches with a focus on published clinical data, as well as on clinical trials, for treatment of RA that are currently underway.

Cell Therapy With Mesenchymal Stem Cells Induces an Innate Immune Memory Response That Attenuates Experimental Colitis in the Long Term.

BACKGROUND AND AIMS: Mesenchymal stem cells [MSCs] are used in preclinical and clinical studies for treatment of immune-mediated disorders, thanks to their immunomodulatory properties. Cell therapy with MSCs induces multiple effects in the immune system which ultimately lead to increase in the number of immune cells with regulatory phenotype. In this study, we investigated whether the beneficial effects of MSC therapy are maintained in the long term in a clinically relevant mouse model of colitis. METHODS: A single dose of adipose-derived MSCs [aMSCs] was infused into dextran sulphate sodium [DSS]-induced colitic mice during the induction phase of the disease. Following a latency period of 12 weeks, mice were re-challenged with a second 7-day cycle of DSS. RESULTS: DSS-induced colitic mice treated with aMSCs showed significant reduction in their colitic disease activity index during the second DSS challenge when compared with non-aMSC treated DSS-induced colitic mice. Strikingly, the long-term protection induced by aMSC therapy was also observed in Rag-1-/- mice where no adaptive immune memory cell responses take place. Increased percentages of Ly6G+CD11b+ myeloid cells were observed 12 weeks after the first inflammatory challenge in the peritoneal cavity, spleen, and bone marrow of DSS-induced colitic mice that were infused with aMSCs. Interestingly, upon re-challenge with DSS, these animals showed a concomitant increase in the regulatory/inflammatory macrophage ratio in the colon lamina propria. CONCLUSIONS: Our findings demonstrate for the first time that MSC therapy can imprint an innate immune memory-like response in mice which confers sustained protection against acute inflammation in the long term.

Elongated Flexuous Plant Virus-Derived Nanoparticles Functionalized for Autoantibody Detection.

Nanoparticles derived from the elongated flexuous capsids of Turnip mosaic virus (TuMV) have been shown to be efficient tools for antibody sensing with a very high sensitivity if adequately functionalized with the corresponding epitopes. Taking advantage of this possibility, TuMV virus-like particles (VLPs) have been genetically derivatized with a peptide from the chaperonin Hsp60, a protein described to be involved in inflammation processes and autoimmune diseases. Antibodies against the peptide have been previously shown to have a diagnostic value in at least one autoimmune disease, multiple sclerosis. The functionalized Hsp60-VLPs showed their significant increase in sensing potency when compared to monoclonal antibody detection of the peptide in a conventional immunoassay. Additionally, the developed Hsp60-VLPs allowed the detection of autoantibodies against the Hsp60 peptide in an in vivo mouse model of dextran sodium sulfate (DSS)-induced colitis. The detection of minute amounts of the autoantibodies allowed us to perform the analysis of their evolution during the progression of the disease. The anti-Hsp60 autoantibody levels in the sera of the inflamed mice went down during the induction phase of the disease. Increased levels of the anti-HSP60 autoantibodies were detected during the resolution phase of the disease. An extension of a previously proposed model for the involvement of Hsp60 in inflammatory processes is considered, incorporating a role for Hsp60 autoantibodies. This, and related models, can now be experimentally tested thanks to the autoantibody detection hypersensitivity provided by the functionalized VLPs.

Inhibitory Role of Growth Hormone in the Induction and Progression Phases of Collagen-Induced Arthritis.

Evidence indicates an intimate connection between the neuroendocrine and the immune systems. A number of in vitro and in vivo studies have demonstrated growth hormone (GH) involvement in immune regulation. The GH receptor is expressed by several leukocyte subpopulations, and GH modulates immune cell proliferation and activity. Here, we found that sustained GH expression protected against collagen-induced arthritis (CIA); in GH-transgenic C57BL/6 (GHTg) mice, disease onset was delayed, and its overall severity was decreased. The anti-collagen response was impaired in these mice, as were inflammatory cytokine levels. Compared to control arthritic littermates, immunized GHTg mice showed significantly lower RORγt (retinoic acid receptor-related orphan receptor gamma 2), IL-17, GM-CSF, IL-22, and IFNγ mRNA expression in draining lymph nodes, whereas there were no differences in IL-21, IL-6, or IL-2 mRNA levels. Data thus suggest that Th17/Th1 cell plasticity toward a pathological phenotype is reduced in these mice. Exogenous GH administration in arthritic DBA/1J mice reduced the severity of established CIA as well as the inflammatory environment, which also shows a GH effect on arthritis progression. These results indicate that GH prevents inflammatory joint destruction in CIA. Our findings demonstrate a modulatory GH role in immune system function that contributes to alleviating CIA symptoms and underlines the importance of endocrine regulation of the immune response.

Comparative Analysis between the In Vivo Biodistribution and Therapeutic Efficacy of Adipose-Derived Mesenchymal Stromal Cells Administered Intraperitoneally in Experimental Colitis.

Mesenchymal stem cells (MSCs) have emerged as a promising treatment for inflammatory diseases. The immunomodulatory effect of MSCs takes place both by direct cell-to-cell contact and by means of soluble factors that leads to an increased accumulation of regulatory immune cells at the sites of inflammation. Similar efficacy of MSCs has been described regardless of the route of administration used, the inflammation conditions and the major histocompatibility complex context. These observations raise the question of whether the migration of the MSCs to the inflamed tissues is a pre-requisite to achieve their beneficial effect. To address this, we examined the biodistribution and the efficacy of intraperitoneal luciferase-expressing human expanded adipose-derived stem cells (Luci-eASCs) in a mouse model of colitis. Luci-eASC-infused mice were stratified according to their response to the Luci-eASC treatment. According to the stratification criteria, there was a tendency to increase the bioluminescence signal in the intestine at the expense of a decrease in the bioluminescence signal in the liver in the "responder" mice. These data thus suggest that the accumulation of the eASCs to the inflamed tissues is beneficial for achieving an optimal modulation of inflammation.

Biodistribution and Efficacy of Human Adipose-Derived Mesenchymal Stem Cells Following Intranodal Administration in Experimental Colitis.

Mesenchymal stem cells (MSCs) have a large potential in cell therapy for treatment of inflammatory and autoimmune diseases, thanks to their immunomodulatory properties. The encouraging results in animal models have initiated the translation of MSC therapy to clinical trials. In cell therapy protocols with MSCs, administered intravenously, several studies have shown that a small proportion of infused MSCs can traffic to the draining lymph nodes (LNs). This is accompanied with an increase of different types of regulatory immune cells in the LNs, suggesting the importance of migration of MSCs to the LNs in order to contribute to immunomodulatory response. Intranodal (IN), also referred as intralymphatic, injection of cells, like dendritic cells, is being proposed in the clinic for the treatment of cancer and allergy, showing that this route of administration is clinically safe and efficient. In this study, we investigated, for the first time, the biodistribution and the efficacy of Luciferase+ adipose-derived MSCs (Luci-eASCs), infused through the inguinal LNs (iLNs), in normal mice and in inflamed mice with colitis. Most of the Luci-eASCs remain in the iLNs and in the adipose tissue surrounding the inguinal LNs. A small proportion of Luci-eASCs can migrate to other locations within the lymphatic system and to other tissues and organs, having a preferential migration toward the intestine in colitic mice. Our results show that the infused Luci-eASCs protected 58% of the mice against induced colitis. Importantly, a correlation between the response to eASC treatment and a higher accumulation of eASCs in popliteal, parathymic, parathyroid, and mesenteric LNs were found. Altogether, these results suggest that IN administration of eASCs is feasible and may represent an effective strategy for cell therapy protocols with human adipose-derived MSCs in the clinic for the treatment of immune-mediated disorders.

Intralymphatic Administration of Adipose Mesenchymal Stem Cells Reduces the Severity of Collagen-Induced Experimental Arthritis.

Mesenchymal stem cells (MSCs) are multipotent stromal cells with immunomodulatory properties. They have emerged as a very promising treatment for autoimmunity and inflammatory diseases such as rheumatoid arthritis. Previous studies have demonstrated that MSCs, administered systemically, migrate to lymphoid tissues associated with the inflammatory site where functional MSC-induced immune cells with a regulatory phenotype were increased mediating the immunomodulatory effects of MSCs. These results suggest that homing of MSCs to the lymphatic system plays an important role in the mechanism of action of MSCs in vivo. Thus, we hypothesized that direct intralymphatic (IL) (also referred as intranodal) administration of MSCs could be an alternative and effective route of administration for MSC-based therapy. Here, we report the feasibility and efficacy of the IL administration of human expanded adipose mesenchymal stem cells (eASCs) in a mouse model of collagen-induced arthritis (CIA). IL administration of eASCs attenuated the severity and progression of arthritis, reduced bone destruction and increased the levels of regulatory T cells (CD25+Foxp3+CD4+ cells) and Tr1 cells (IL10+CD4+), in spleen and draining lymph nodes. Taken together, these results indicate that IL administration of eASCs is very effective in modulating established CIA and may represent an alternative treatment modality for cell therapy with eASCs.

Adipose-derived mesenchymal stromal cells modulate experimental autoimmune arthritis by inducing an early regulatory innate cell signature.

Modulation of innate immune responses in rheumatoid arthritis and other immune-mediated disorders is of critical importance in the clinic since a growing body of information has shown the key contribution of dysregulated innate responses in the progression of the disease. Mesenchymal stromal cells (MSCs) are the focus of intensive efforts worldwide due to their key role in tissue regeneration and modulation of inflammation. In this study, we define innate immune responses occurring during the early course of treatment with a single dose of expanded adipose-derived MSCs (eASCs) in established collagen-induced arthritis. eASCs delay the progression of the disease during the early phase of the disease. This is accompanied by a transient induction of Ly6C+ monocytes that differentiate into IL10+F4/80+ cells in arthritic mice. Strikingly, the induced IL10+F4/80+ myeloid cells preferentially accumulated in the draining lymph nodes. This effect was accompanied with a concomitant declining of their frequencies in the spleens. Our results show that eASCs attenuate the arthritic process by inducing an early innate cell signature that involves a transient induction of Ly6C+ monocytes in periphery that differentiate into IL10+F4/80+ macrophages. Our findings demonstrate that early regulatory innate cell responses, involving the monocyte compartment, are targeted by the eASCs during the onset of collagen-induced inflammation.

The Minor Allele of rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression.

OBJECTIVE: The T allele of rs7574865 in STAT4 confers risk of developing autoimmune disorders. However, its functional significance remains unclear. Here we analyze how rs7574865 affects the transcription of STAT4 and its protein expression. METHODS: We studied 201 patients (80% female; median age, 54 years; median disease duration, 5.4 months) from PEARL study. Demographic, clinical, laboratory and therapeutic data were collected at each visit. IL-6 serum levels were measured by enzyme immune assay. The rs7574865 was genotyped using TaqMan probes. The expression levels of STAT4 mRNA were determined at 182 visits from 69 patients using quantitative real-time polymerase chain reaction. STAT4 protein was assessed by western blot in 62 samples from 34 patients. To determine the effect of different variables on the expression of STAT4 mRNA and protein, we performed multivariate longitudinal analyses using generalized linear models. RESULTS: After adjustment for age, disease activity and glucocorticoid dose as confounders, the presence of at least one copy of the T allele of rs7574865 was significantly associated with higher levels of STAT4 mRNA. Similarly, TT patients showed significantly higher levels of STAT4 protein than GG patients. IL-6 induced STAT4 and STAT5 phosphorylation in peripheral blood lymphocytes. Patients carrying at least one T allele of rs7574865 displayed lower levels of serum IL-6 compared to GG homozygous; by contrast the production of C-reactive protein was similar in both populations. CONCLUSION: Our data suggest that the presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. It may enhance the signaling of molecules depending on the STAT4 pathway.

Human Adipose-Derived Mesenchymal Stem Cells Modulate Experimental Autoimmune Arthritis by Modifying Early Adaptive T Cell Responses.

Mesenchymal stem cells (MSCs) are multipotent stromal cells with immunosuppressive properties. They have emerged as a very promising treatment for autoimmunity and inflammatory diseases such as rheumatoid arthritis. Recent data have identified that GM-CSF-expressing CD4 T cells and Th17 cells have critical roles in the pathogenesis of arthritis and other inflammatory diseases. Although many studies have demonstrated that MSCs can either prevent or suppress inflammation, no studies have addressed their modulation on GM-CSF-expressing CD4 T cells and on the plasticity of Th17 cells. To address this, a single dose of human expanded adipose-derived mesenchymal stem cells (eASCs) was administered to mice with established collagen-induced arthritis. A beneficial effect was observed soon after the infusion of the eASCs as shown by a significant decrease in the severity of arthritis. This was accompanied by reduced number of pathogenic GM-CSF(+) CD4(+) T cells in the spleen and peripheral blood and by an increase in the number of different subsets of regulatory T cells like FOXP3(+) CD4(+) T cells and IL10(+) IL17(-) CD4(+) T cells in the draining lymph nodes (LNs). Interestingly, increased numbers of Th17 cells coexpressing IL10 were also found in draining LNs. These results demonstrate that eASCs ameliorated arthritis after the onset of the disease by reducing the total number of pathogenic GM-CSF(+) CD4(+) T and by increasing the number of different subsets of regulatory T cells in draining LNs, including Th17 cells expressing IL10. All these cellular responses, ultimately, lead to the reestablishment of the regulatory/inflammatory balance in the draining LNs.

Tyr³²³-dependent p38 activation is associated with rheumatoid arthritis and correlates with disease activity.

OBJECTIVE: The p38 MAPK is important in the pathogenic immune response in rheumatoid arthritis (RA). The p38 molecule can be activated through phosphorylation on Thr¹8°-Tyr¹8² by upstream MAPK kinases and via an alternative pathway through phosphorylation on Tyr³²³. We undertook this study to quantify the phosphorylation of Tyr³²³ p38 and of Thr¹8°-Tyr¹8² p38 on T cells from healthy controls and patients with RA or ankylosing spondylitis (AS) to identify variables associated with p38 phosphorylation and disease activity. METHODS: We measured p38 phosphorylation on Tyr³²³ and Thr¹8°-Tyr¹8² by flow cytometry and Western blotting on T cells from 30 control subjects, 33 AS patients, 30 patients with RA in remission, and 79 patients with active RA. We collected the clinical characteristics and analyzed correlations between clinical variables, the Disease Activity Score in 28 joints (DAS28), and p38 phosphorylation levels. Multivariate regression analysis was performed to identify variables associated with p38 phosphorylation on Tyr³²³ and Thr¹8°-Tyr¹8². RESULTS: Phosphorylation of p38 on Tyr³²³ was higher in T cells from patients with active RA (P = 0.008 versus healthy controls) than in patients with RA in remission or in patients with AS. Tyr³²³ p38 phosphorylation was associated with disease activity determined by the DAS28 (P = 0.017). Enhanced p38 phosphorylation was linked to Lck-mediated activation of the Tyr³²³-dependent pathway in the absence of upstream MAPKK activation. CONCLUSION: Our results indicate that phosphorylation status on Tyr³²³ p38 correlates with RA disease activity and suggest that the Tyr³²³-dependent pathway is an attractive target for down-regulation of p38 activity in RA patients.

Herpesvirus saimiri transformation may help disclose inherent functional defects of mucosal T lymphocytes in patients with gastric adenocarcinoma.

To dissect the phenotypic and functional features of mucosal T lymphocytes in patients with gastric adenocarcinoma, we have used the Herpesvirus saimiri transformation procedure to achieve T-cell lines from gastric origin. Once achieved, cell function was assessed by in vitro stimulation with mitogens. CD2-specific monoclonal antibodies (alpha-CD2), alone or in combination with interleukin (IL)-2, rendered fewer counts in patients (34 408+/-3965 and 52 157+/-6473 c.p.m., respectively) than in controls (67 471+/-11 755 c.p.m., P<0.01 and 77 864+/-12 545 c.p.m., P<0.05, respectively). Likewise, CD3-based responses were defective in cancer cell lines: alpha-CD3 (54 794+/-9269 vs 86 104+/-10 341 c.p.m., P<0.01), alpha-CD3+IL-2 (57 789+/-8590 vs 88855+/-8516 c.p.m., P<0.01) and alpha-CD3+alpha-CD2 (52 130+/-7559 vs 120 852+/-16 552 c.p.m., P<0.01). Finally, IL-2 failed to adequately stimulate patient cell lines (39 310+/-4023 vs 60 945+/-9463 c.p.m., P<0.05). These results suggest that mucosal T lymphocytes in cancer patients are inherently impaired in their proliferative ability. This may be crucial in the control of tumour growth.

FcRgamma chain does not replace CD3zeta chain in CD3zeta-deficient T lymphocytes of patients with gastric adenocarcinoma.

Defective CD3zeta chain expression has been reported in T lymphocytes of patients with inflammatory diseases, such as systemic lupus erythematosus or osteoarthritis, and with cancer. In lupus, the absent CD3zeta chain is replaced by the FcRgamma chain, rendering the T cells hyper responsive. However, there are no data on T lymphocytes from patients with cancer. In this study, the presence of the FcRgamma chain and its associated kinase, Syk, was analysed in patients with gastric adenocarcinoma and healthy subjects. Western blot and immunoprecipitation experiments were carried out with total cell or lipid raft extracts from fresh peripheral blood mononuclear cells or T lymphocytes, and Herpesvirus saimiri-derived T-cell lines (of blood or tissue origin). Our results revealed that the absent CD3zeta chain in cancer T lymphocytes was not replaced by FcRgamma either in fresh T cells or T-cell lines, in contrast to lupus T cells. This altered expression of signalling molecules in T lymphocytes of cancer patients, would explain their low proliferative capacity. Our T-cell lines represent tools to unveil the signalling abnormalities of cancer T lymphocytes.

Expression of CD45 and proliferative response to CD3 as suitable classification markers of patients with gastric adenocarcinoma.

25 patients with resectable gastric adenocarcinoma, subdivided according to the absence or presence of residual neoplasic disease (RND- or RND+, respectively), were studied. Cytofluorometric analysis and proliferative responses to mitogens was performed in peripheral blood mononuclear cells of patients. When compared to healthy subjects, the percentage of CD3-expressing cells was significantly reduced in both groups of patients studied (p < 0.0001 in all instances). However, when CD45 is considered instead of (CD3, its expression is found to be significantly reduced only in the RND+ patients (72% +/- 11), when compared with the control group (96 +/- 1%, p < 0.0001). Likewise, cells from these patients significantly less proliferated when stimulated with monoclonal antibodies to CD3 than control cells (18,920 +/- 6,019 cpm vs. 42,697 +/- 1,798 cpm, p = 0.0036); a difference not found if RND- patients (33,619 +/- 11,733 cpm) were considered. We propose that the low expression of CD45 and the poor response to CD3 are markers that are able to identify the subgroup of patients in whom the disease will tend to progress more rapidly. We also suggest the use of such markers as additional criteria for the classification of patients with gastric adenocarcinoma or to identify patients who require more aggressive therapeutic strategies.

Defective CD3zeta chain expression in Herpesvirus saimiri (HVS)-derived T-cell lines in gastric adenocarcinoma.

Low expression of the CD3zeta chain has been reported in patients with cancer and it has been suggested that tumor-derived factors are involved in its downregulation. The expression of CD3zeta chain was measured in T-cell lines from patients with gastric adenocarcinoma and healthy volunteers and grown in vitro for several months and, hence, in the absence of any tumor-derived factors. T-cell lines of mucosal origin were obtained by Herpesvirus saimiri transformation from gastric cancer patients. The expression of CD3zeta and CD3epsilon was measured by flow cytometry and Western-blot analysis. Calcium mobilization and apoptosis rate were also measured. The levels of CD3zeta, but not CD3epsilon, chain on the cell surface were significantly reduced in T-cell lines derived from patients with gastric cancer when cultured in the absence of IL-2. Western-blot analysis of total cell extracts or lipid raft fractions confirmed this finding. Calcium mobilization, a measure of signal transduction, was reduced in T cell lines from patients with gastric cancer. We conclude that T cells from patients with cancer express lower levels of CD3zeta. This downregulation is not caused by a direct effect of tumor-derived factors but, rather, it appears to be inherent to the patient cells. The low CD3zeta expression would render T lymphocytes unable to control the growth of tumor cells.