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.     

CD4(+)CD25high regulatory T cells increase with tumor stage in patients with gastric and esophageal cancers

Purpose: Regulatory T cells (T regs) can inhibit immune responses mediated by T cells. It has been shown that there is an increased proportion of T regs in several different human malignancies, although the actual mechanism remains unclear. In the present study, we evaluated the prevalence of CD4(+)CD25^high T regs in PBMCs from patients with gastric and esophageal cancers in relation to the clinical outcome. Methods: PBMCs in 72 patients with gastric cancer and 42 patients with esophageal cancer were evaluated for the proportion of CD4(+)CD25^high T cells, as a percentage of the total CD4(+) cells, by flow cytometric analysis with triple-color staining. Actuarial overall survival rates of the patients were analyzed by the Kaplan–Meier method. Results: The percentages of CD4(+)CD25^high T cells for cases of gastric cancer (4.9±1.2%) and esophageal cancer (5.2±2.1%) were significantly higher than those for healthy donors (1.9±1.1%, P<0.01). There were significant differences in the prevalence of CD4(+)CD25^high T cells between the early and advanced disease stages, both in gastric cancer (stage I vs. III, P<0.05; stage I vs. IV, P<0.05) and esophageal cancer (stage I vs. IV, P<0.05). The patients with a high proportion of CD4(+)CD25^high T cells showed poorer survival rates in comparison to those with a low proportion, in both gastric and esophageal cancers. After patients received curative resections of gastric cancers (n=57), the increased proportions of CD4(+)CD25^high T cells were significantly reduced, and the levels were almost equal to those in normal healthy donors. In addition, studies of gastric cancer patients with postoperative recurrent tumors (n=6) revealed that the prevalence of CD4(+)CD25^high T cells individually increased compared to 2 months after the operations. CD4(+)CD25^high T cells expressed FOXP3 mRNA and had abundant CD45RO and intracellular CTLA-4 molecules. Conclusions: These results strongly suggest that tumor-related factors induce and expand CD4(+)CD25^high T regs.     

Study of naïve and memory cells in a cohort of Egyptian chronic granulomatous disease patients

Abstract

Context: Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder caused by inherited defects in the NADPH oxidase complex which may be involved in important pathways that connect innate and adaptive immunity. Objectives: Characterize the naive and memory compartment of B and T lymphocytes in patients with CGD. Methods: Twenty CGD patients and twenty healthy controls matched for age and sex were enrolled in this study. Flow cytometric assessment of the naïve and memory compartments of peripheral blood lymphocytes was done using cell surface markers CD45RO, CD45RA, CD27, CD3 and CD19. Results: There were 15 (79%) autosomal recessive CGD patients (8 females (53%) and 7 males (47%), 100% positive parental consanguinity) and four (21%) X-linked CGD patients. On comparing the 3 groups; AR CGD, X-linked CGD and controls, there was a positive statistical significant difference for the percentage and absolute count of CD19?+?CD27+ memory B cell (p?=?0.028 and p?=?0.047 respectively), CD45RA cells (with p values of p?=?0.000 and 0.033, respectively), the naïve compartment CD3?+?CD45RA+ cells percentage and absolute counts (p?=?0.005, 0.01respectively), CD3?+?CD27?+?cells percentage and absolute counts (p?=?0.001, 0.012 respectively), CD3?+?CD45RA?+?CD27+ cells percentage and absolute counts (p?=?0.015, 0.005, respectively). The significance was mainly attributed to the decrease in the X-linked group than control group. Conclusion: There was an altered naïve and memory B profile in CGD patients, this may increase susceptibility of the patients to opportunistic infections and autoimmune disorders. T-cell alterations have to be interpreted cautiously especially in the presence of infections.     

The most reliable surface marker for the identification of colorectal cancer stem-like cells: A systematic review and meta-analysis

Several surface markers have been proposed for the identification and characterization of colorectal cancer stem-like cells (CR-CSLCs). However, their reliability in CR-CSLCs identification remains controversial. This study evaluated the correlation between all candidate surface marker's expression and CSLCs properties (tumorigenicity) through monitoring in vivo tumor incidence and final tumor volume. PubMed, Web of Science, and Scopus databases were systematically searched until November 2017. A total of 27 studies were found that met the inclusion criteria for cluster of differentiation 133 (CD133) and CD44 markers. Results indicated that either CD133 or CD44 positive cells caused about twofold increase in tumor volume compared with the negative cells (p < 0.05). In two groups of cells derived from primary tumors and cell lines, CD133 ⁺ cells had 25 and 1.45 times higher tumor incidence potential than CD133 ⁻ cells, respectively ( p < 0.05). Also, cohort evaluation showed that CD133 overexpression at protein level is a marker of poor overall survival in colorectal cancer (CRC) patients. While CD44 ⁺ cells displayed twofold tumorigenicity compared with the negative cells ( p < 0.05), combination of CD44 and CD133 showed about sevenfold tumorigenicity potential ( p < 0.05). In conclusion, the present meta-analysis suggests that CD133 is a robust biomarker to identify primary tumor CSLCs and can be proposed as a prognostic marker of CRC patient whereas it should be used with caution in cell lines. It seems to be more reliable to use CD133 in combination with CD44 as target biomarkers for the isolation of CR-CSLCs in both cell line and primary tumor cells populations.     

CMV seropositivity and T‐cell senescence predict increased cardiovascular mortality in octogenarians: results from the Newcastle 85+ study

Although chronic infection with cytomegalovirus (CMV) is known to drive T lymphocytes toward a senescent phenotype, it remains controversial whether and how CMV can cause coronary heart disease (CHD). To explore whether CMV seropositivity or T‐cell populations associated with immunosenescence were informative for adverse cardiovascular outcome in the very old, we prospectively analyzed peripheral blood samples from 751 octogenarians (38% males) from the Newcastle 85+ study for their power to predict survival during a 65‐month follow‐up (47.3% survival rate). CMV‐seropositive participants showed a higher prevalence of CHD (37.7% vs. 26.7%, P = 0.030) compared to CMV‐seronegative participants together with lower CD4/CD8 ratio (1.7 vs. 4.1, P < 0.0001) and higher frequencies of senescence‐like CD4 memory cells (41.1% vs. 4.5%, P < 0.001) and senescence‐like CD8 memory cells (TEMRA, 28.1% vs. 6.7%, P < 0.001). CMV seropositivity was also associated with increased six‐year cardiovascular mortality (HR 1.75 [1.09–2.82], P = 0.021) or death from myocardial infarction and stroke (HR 1.89 [107–3.36], P = 0.029). Gender‐adjusted multivariate Cox regression analysis revealed that low percentages of senescence‐like CD4 T cells (HR 0.48 [0.32–0.72], P < 0.001) and near‐senescent (CD27 negative) CD8 T cells (HR 0.60 [0.41–0.88], P = 0.029) reduced the risk of cardiovascular death. For senescence‐like CD4, but not near‐senescent CD8 T cells, these associations remained robust after additional adjustment for CMV status, comorbidities, and inflammation markers. We conclude that CMV seropositivity is linked to a higher incidence of CHD in octogenarians and that senescence in both the CD4 and CD8 T‐cell compartments is a predictor of overall cardiovascular mortality as well as death from myocardial infarction and stroke.     

Effects of zinc deficiency and pinealectomy on cellular immunity in rats infected with Toxoplasma gondii

The effects of zinc and/or melatonin deficiencies on cellular immunity were investigated in rats infected with Toxoplasma gondii. A total of 50 adult male Sprague-Dawley rats were divided into 5 groups of 10 rats each. In group I, the rats were infected with T. gondii and fed a zinc-deficient diet; in group II, the rats were infected and their pineal gland was surgically removed. Group III included rats that were infected, pinealectomized, and fed a zinc-deficient diet. Group IV consisted of T. gondii-infested rats that received no treatment of any kind, and group V were normal controls. After 3 wk of treatment, all rats were sacrificed and the percentages of CD3, CD4, and CD8 lymphocytes, zinc, and melatonin levels in plasma and the percentage of lymphocyte in blood smears were analyzed. The CD3 ratios of groups I–III were significantly lower than those of groups IV and V (p<0.01). The CD4 lymphocytes were significantly higher in group IV than that in all other groups (p<0.05). In group IV, the CD8 lymphocytes were higher than in groups I–III (p<0.01) and those in group V were higher than for groups I and III (p<0.01). Lymphocyte incidence in group IV was higher than in the other four groups (p<0.01). The plasma zinc and plasma melatonin levels in groups I–III were significantly lower than those in the controls (p<0.01, both cases). These results suggest that zinc and/or melatonin deficiency have a negative influence on cellular immunity in rats with toxoplasmosis.     

Lung carcinomas do not induce T-cell apoptosis via the Fas/Fas ligand pathway but down-regulate CD3 epsilon expression

Background Non-small cell lung carcinoma (NSCLC) patients have impaired cellular immune responses. It has been hypothesized that tumor cells expressing Fas Ligand (FasL) induce in T lymphocytes: (a) apoptosis (tumor counterattack) and (b) down-regulation of CD3ζ expression. However, the hypothesis of tumor counterattack is still controversial. Methods We analyzed FasL expression on NSCLC cell lines and on tumor cells from lung adenocarcinoma patients by flow cytometry and immunocytochemistry. FasL mRNA expression was detected in NSCLC cell lines using RT-PCR, and functional FasL was evaluated on Fas-expressing Jurkat T-cells by annexin-V-FITC staining and by SubG₁ peak detection. Also, the proapoptotic effect of microvesicles released from NSCLC cell lines in Jurkat T-cells was studied. Alterations in the expression levels of CD3ζ, CD3ε, and CD28 [measured as mean fluorescence intensity (MFI)] were determined in Jurkat T-cells after co-culture with NSCLC cell lines or tumor-derived microvesicles. Furthermore, the expression levels of CD3ζ and CD3ε in CD4+T and CD8+T lymphocytes from lung adenocarcinoma patients was studied. Results Our results indicate that NSCLC cells neither FasL expressed nor induced apoptosis in Jurkat T-cells. Tumor-derived microvesicles did not induce apoptosis in Jurkat T-cells. In contrast, NSCLC cell lines down-regulated CD3ε but not CD3ζ chain expression in Jurkat T-cells; this effect was induced by soluble factors but not by microvesicles. In lung adenocarcinoma patients, significant decreases of MFI values for CD3ε, but not CD3ζ, were found in CD4+T and CD8+T cells from pleural effusion compared to peripheral blood and in peripheral blood of patients compared to healthy donors. Conclusions Our data do not support the tumor counterattack hypothesis for NSCLC. Nonetheless, down-regulation of CD3ε in T-cells induced by NSCLC cells might lead to T-cell dysfunction.     

The CD4+CD28null and the regulatory CD4+CD25High T-cell phenotypes in patients with ulcerative colitis during active and quiescent disease, and following colectomy

The CD4⁺CD25^High T-cell phenotype has an essential immunoregulatory role, while the CD4⁺CD28^null T-cell reflects immune pathology. We investigated the profiles of the CD4⁺CD25^High and the CD4⁺CD28^null T-cell phenotypes in patients with ulcerative colitis (UC) during active and quiescent phases as well as following colectomy. Fifty-nine UC patients, 34 active (UCa) and 25 quiescent (UCq) together with 19 healthy controls (HC) were included. Ten of 34 UCa patients underwent colectomy due to unremitting UC (UCo). Immunohistochemical phenotypic of the peripheral blood lymphocytes bearing CD4, CD25 or CD28 was done for analyzes by a multiparameter fluorescence activated cell sorting technique. The expression of the CD4⁺CD25^High phenotype was higher in UCq (P < 0.01) or UCo (P < 0.01) group vs UCa group. Further, the expression of the CD4⁺CD28^null phenotype in UCa or UCo group was higher than in the HC group (P < 0.05). However, the expression of the CD4⁺CD28^null phenotype up to 12 months after colectomy was not significantly different from the levels in the same patients during acute phase. Our impression is that a high CD4⁺CD25^High T-cell reflects alleviation of inflammation, while the expression of the CD4⁺CD28^null T-cell phenotype is an etiologic feature in UC patients, and is maintained after removing the affected colon.     

Enhanced expression of the complement regulatory protein CD55 predicts a poor prognosis in colorectal cancer patients

This study prospectively correlated the level of expression of CD55 on tumours with 7-year survival in 136 colorectal cancer patients. Patients with tumours expressing high levels of CD55 had a significantly worse survival (24%) than patients with low CD55 levels (50%, p<0.02). A similar difference was seen for patients (Duke's B or C) with a high risk of recurrence (29% vs 58%, p<0.05). Furthermore, there was a progressive deterioration in prognosis with increasing antigen expression (p=0.01). It remains unclear if CD55 is overexpressed by tumours to protect them from complement or if it is related to the recent observation that CD55 is a ligand for the T-cell activation antigen CD97. However, it is a marker of aggression, as colorectal cancer patients whose tumours overexpress CD55 have a significantly reduced 7-year survival.     

Characterisation of the immune response to type I collagen in scleroderma

This study was conducted to examine the frequency, phenotype, and functional profile of T lymphocytes that proliferate in response to type I collagen (CI) in patients with scleroderma (SSc). Peripheral blood mononuclear cells (PBMCs) from SSc patients, healthy controls, and rheumatoid arthritis disease controls were labeled with carboxy-fluorescein diacetate, succinimidyl ester (CFSE), cultured with or without antigen (bovine CI) for 14 days, and analysed by flow cytometry. Surface markers of proliferating cells were identified by multi-color flow cytometry. T-cell lines were derived after sorting for proliferating T cells (CFSE^low). Cytokine expression in CI-responsive T cells was detected by intracellular staining/flow cytometry and by multiplex cytokine bead assay (Bio-Plex). A T-cell proliferative response to CI was detected in 8 of 25 (32%) SSc patients, but was infrequent in healthy or disease controls (3.6%; p = 0.009). The proliferating T cells expressed a CD4⁺, activated (CD25⁺), memory (CD45RO⁺) phenotype. Proliferation to CI did not correlate with disease duration or extent of skin involvement. T-cell lines were generated using in vitro CI stimulation to study the functional profile of these cells. Following activation of CI-reactive T cells, we detected intracellular interferon (IFN)-γ but not interleukin (IL)-4 by flow cytometry. Supernatants from the T-cell lines generated in vitro contained IL-2, IFN-γ, GM-CSF (granulocyte macrophage-colony-stimulating factor), and tumour necrosis factor-α, but little or no IL-4 and IL-10, suggesting that CI-responsive T cells express a predominantly Th1 cytokine pattern. In conclusion, circulating memory CD4 T cells that proliferate to CI are present in a subset of patients with SSc, but are infrequent in healthy or disease controls.     

Tumor Necrosis Factor Receptor Expression in HIV-1-Infected CD4+ T Cells

We investigated whether HIV-1 can regulate tumor necrosis factor receptor (TNFR) expression in SupT-1, a CD4 + T-cell line. The cells were infected with HIV-1 containing 1,000 cpm RT activity, as early as day 3 after infection and all along the culture the supernatant level of core protein p24 was >250 pg/ml, and on days 6 and 9 after infection, p24 was found in 10 % of the cells as determined by indirect immunofluorescence assay. The cells were growing without loss of viability. The study of TNFR expression was based on a microassay for measurement of binding of ¹²⁵I-TNFα to cells, in which free and cell-bound ligand separation was performed by centrifugation through oil. Scatchard analysis of TNFα binding on days 6 and 9 after infection revealed a 90 % increase in the expression of high-affinity membrane receptors in HIV + SupT-1 culture compared with uninfected cells (mean +/-S.D. = 501 +/-148.5 vs. 263 +/-77.8 receptors/cell, n = 9, P< 0.001) with no change in dissociation constants (mean +/? S.D. = 4.36 +/?1.06 vs. 4.00 +/?1.12 × 10^?10 m ).     

Impairment of alternate pathway (CD2) of T cell activation in leprosy

Recent studies in basic immunology have been directed towards the understanding of the mechanism of T cell activation. T cells can be activated to proliferatevia the classical pathway through the antigen receptor (CD3-Ti) orvia the alternate pathway through the CD2 receptor. Since immunologic unresponsiveness in lepromatous leprosy is considered to be due to the inability of T cells to proliferate upon stimulation, we have been interested in the nature of these receptors and the activation pathways in lymphocytes of leprosy patients. In the present investigation we demonstrate: (i) CD2 receptor (E-receptor) is downregulated in bacterial index positive lepromatous leprosy patients. (ii) The alternate pathway of T cell activation is impaired in lepromatous patients as revealed by the inability of their lymphocytes to proliferate in response to a pair of mitogenic anti-CD2 monoclonals. (iii) The addition of recombinant interleukin 2in vitro restores the ability of lymphocytes from lepromatous patients to proliferate in response to anti-CD2 antibodies. (iv) Interestingly, CD2 modulation and the associated functional impairment could be brought about in peripheral blood lymphocytes from normal subjects by prior treatment withMycobacterium leprae in vitro. This approach would be useful in understanding the molecular events leading to the defective T cell functions in leprosy.     

Spatiotemporal regulation of galectin-1-induced T-cell death in lamina propria from Crohn’s disease and ulcerative colitis patients

Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by chronic, relapsing intestinal inflammation. Galectin-1 (Gal-1) is an endogenous lectin with key pro-resolving roles, including induction of T-cell apoptosis and secretion of immunosuppressive cytokines. Despite considerable progress, the relevance of Gal-1-induced T-cell death in inflamed tissue from human IBD patients has not been ascertained. Intestinal biopsies and surgical specimens from control patients (n?=?52) and patients with active or inactive IBD (n?=?97) were studied. Gal-1 expression was studied by RT-qPCR, immunoblotting, ELISA and immunohistochemistry. Gal-1-specific ligands and Gal-1-induced apoptosis of lamina propria (LP) T-cells were determined by TUNEL and flow cytometry. We found a transient expression of asialo core 1-O-glycans in LP T-cells from inflamed areas (p?<?0.05) as revealed by flow cytometry using peanut agglutinin (PNA) binding and assessing dysregulation of the core-2 β 1-6-N-acetylglucosaminyltransferase 1 (C2GNT1), an enzyme responsible for elongation of core 2 O-glycans. Consequently, Gal-1 binding was attenuated in CD3⁺CD4⁺ and CD3⁺CD8⁺ LP T-cells isolated from inflamed sites (p?<?0.05). Incubation with recombinant Gal-1 induced apoptosis of LP CD3⁺ T-cells isolated from control subjects and non-inflamed areas of IBD patients (p?<?0.05), but not from inflamed areas. In conclusion, our findings showed that transient regulation of the O-glycan profile during inflammation modulates Gal-1 binding and LP T-cell survival in IBD patients.

     

Clinical reagents of GM-CSF and IFN-α induce the generation of functional chronic myeloid leukemia dendritic cells in vitro

Dendritic cells (DCs) have been successfully induced in vitro from chronic myeloid leukemia (CML) cells, which may provide a promising immunotherapeutic protocol for CML. To facilitate the optimization of DCs-based vaccination protocols, we investigated the efficiency of in vitro generation of DCs from bone marrow mononuclear cells of CML patients by clinical reagents of GM-CSF and IFN-α. Bone marrow mononuclear cells were isolated from eight CML patients and CML-DCs were generated in the presence of different cytokines (Group A: GM-CSF for research and IL-4 for research; Group B: GM-CSF for injection and IFN-α for injection) in RMPI-1640 medium containing 10% human AB serum. After 8 days, the morphologic features of CML-DCs were observed and their immunophenotypes were analyzed by flow cytometry. The activity of CML-DCs was determined by evaluating their ability to stimulate allogeneic mixed lymphocyte reaction (allo-MLR) and anti-leukemic cytotoxic T lymphocytes (CTLs). The culture protocols were successful in generating functional CML-DCs from all the CML patients as evidenced by the significant upregulation of CD80, CD86, CD83 HLA-DR and CD1a compared to pre-cultured (p < 0.05), and increased allogeneic T cell stimulating proliferation capacity (p < 0.05). CML-DCs could stimulate a specific anti-leukemia response. In summary, we demonstrate that the combination of clinical reagents GM-CSF and IFN-α induced the generation of DCs that have the ability to stimulate a specific anti-leukemia CTLs response in vitro, indicating their feasibility for clinical vaccination protocols for CML patients.     

Genetic polymorphism of NK receptors and their ligands in melanoma patients: prevalence of inhibitory over activating signals

Antitumor cytotoxicity of NK cells and T cells expressing NK-associated receptors is regulated by interaction between their cell surface killer immunoglobulin-like receptors (KIRs) and CD94/NKG2 heterodimers with MHC class I ligands on target cells. To test the hypothesis that KIR and/or HLA polymorphisms, and KIR/HLA combinations could contribute to the tumorigenesis, association studies were performed in 50 patients with malignant melanoma (MM) in different stages of disease and 54 controls. Our data showed that the frequency of inhibitory and activating KIR genes and KIR genotypes did not differ significantly between healthy individuals and melanoma patients. HLA haplotype distribution showed statistically significant increased frequencies of A*01-B*35-Cw*04 (0.069 vs 0.000; pc<0.05; OR=19.9), A*01-B*08-DRB1*03 (0.079 vs 0.019; pc<0.05; OR=4.5), and A*24-B*40-DRB1*11 (0.026 vs 0.000; pc<0.05; OR=7.1) in melanoma patients compared with healthy controls. Individuals homozygous for group 2 HLA-C ligands were less frequent in the patient group compared with the control cohort (12% vs 31.5%; p<0.017). In addition, we observed an increased frequency (88.0% vs 68.5%; p=0.017; OR=2.80) of KIR2DL2/2DL3 in combination with their group 1 HLA-C ligands, while the presence of these KIRs in the absence of the putative ligands was decreased (12.0% vs 31.5%; p=0.017) in the patient group. Furthermore, an increased frequency of activating KIR2DS1 in the absence of the putative HLA-C^Lys80 ligands was found in melanoma patients (16.0% vs 9.2%). In contrast, KIR2DS2 was absent in patients more often (38.0% vs 25.9%) when the presumptive HLA-C^Asn80 ligands were present. A slightly higher incidence of KIR3DL1 in combination with the less effective Bw4^Thr80 ligands was seen in patients with primary (20.8%) compared with metastatic (4.2%) disease. The data obtained in this study imply that there may not be a direct association between KIR gene content in the genome and the presence of malignant melanoma, or melanoma progression. However, some HLA haplotypes could be predisposing to MM in the Bulgarian population. Furthermore, distinct KIR/HLA ligand combinations may be relevant to the development of malignancy whereby inhibition overrides activation of NK cells and T cells expressing NK-associated receptors, which in turn might facilitate tumor escape and progression.     

Dendritic cells are dysfunctional in patients with operable breast cancer

Background: Dendritic cells (DCs) play a crucial role in presenting antigens to T lymphocytes and inducing cytotoxic T cells. DCs have been studied in patients with breast cancer to define the factors leading to failure of an effective systemic and locoregional anticancer host response. Methods: Purified DCs were obtained from peripheral blood (PB) and lymph nodes (LNs) of women with operable breast cancer, using immunomagnetic bead selection. The stimulatory capacity of DCs in the allogeneic mixed leukocyte reaction (MLR) and autologous T cell proliferation test (purified protein derivative (PPD) as stimulator), the expression of surface markers on DCs and the production of cytokines in vitro by DCs from patients with operable breast cancer and from healthy donors (controls) were studied. Results: 70–75% purified DCs were isolated from PB and LNs. PBDCs and LNDCs from patients with operable breast cancer demonstrated a reduced capacity to stimulate in an MLR, compared with PBDCs from normal donors (p<0.01). Autologous T cell proliferation in patients had a decreased ability to respond to PPD, when compared with controls (p<0.01). However, T cells from patients responded as well as control T lymphocytes in the presence of control DCs. PBDCs and LNDCs from patients expressed low levels of HLA-DR and CD86, and induced decreased interleukin-12 (IL-12) secretion in vitro, compared with DCs from normal donors (p<0.01). Conclusion: These data suggest a defective DC function in patients with operable breast cancer. Switched-off DCs in patients with early breast cancer and decreased IL-12 production may be important factors for progressive tumour growth.     

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.     

Clinical and immunological effects of Rituximab in patients with lupus nephritis refractory to conventional therapy: a pilot study

We studied the clinical and immunological effects of Rituximab (anti-CD20) therapy in patients with lupus nephritis. In an open clinical trial, 22 patients with active systemic lupus erythematosis and renal involvement (mainly class III and IV according to the WHO classification) that was refractory to conventional therapy were studied. In all these patients, Rituximab (0.5 to 1.0 g at days 1 and 15) was added to the immunosuppressive therapy and its therapeutic effect was evaluated. In addition, the levels and function of regulatory T lymphocytes and the apoptosis of immune cells were assessed. We found a significant reduction in disease activity (p < 0.05, MEX-SLEDAI index), and proteinuria (p < 0.05) at days 60 and 90 of Rituximab therapy. Although most patients showed improvement in creatinine clearance and erythrocyturia, no significant changes in these parameters were detected. In most patients (20/22), B cell depletion was observed, but no clear-cut effect of Rituximab on complement levels or auto-antibody titers was detected (p > 0.05 in all cases). One patient died at day 70 with invasive histoplasmosis. No important adverse effects of Rituximab therapy were registered in other patients. A significant enhancement in the levels of different CD4+ regulatory cells (T_REG, Th3, Tr1), but not CD8+ Ts lymphocytes, was observed at day 30. This increase was sustained for T_REG cells at day 90, and accompanied by an improvement in their regulatory function. In addition, we observed an unexpected increase in the apoptosis of T cells at day 30. Interestingly, the enhancement in the suppressive function of T_REG cells was not observed in the two patients that showed the poorest clinical response to Rituximab. We conclude that the data obtained in this open clinical trial suggest that Rituximab is a promising candidate for randomized controlled trials in patients with lupus nephritis refractory to the conventional immunosuppressive therapy. The effects of Rituximab on regulatory cells and apoptosis of T lymphocytes are interesting and its possible role in the putative effect of this biological agent in systemic lupus erythematosis deserves additional studies.     

Immune responses to p53 in patients with cancer:enrichment in tetramer+ p53 peptide-specific T cells and regulatory T cells at tumor sites

Objective: A majority of human cancers, including head and neck cancer (HNC), overexpress p53. Although T cells specific for wild-type (wt) sequence p53 peptides are detectable in the peripheral blood of patients with HNC, it is unknown whether such T cells accumulate in tumor-involved tissues. Also, the localization of regulatory T cells (Treg) to tumor sites in HNC has not been investigated to date. Methods: Tumor infiltrating lymphocytes (TIL), tumor-involved or non-involved lymph node lymphocytes (LNL) and peripheral blood mononuclear cells (PBMC) were obtained from 24 HLA-A2.1+ patients with HNC. Using tetramers and four-color flow cytometry, the frequency of Treg and CD3+CD8+ T cells specific for wt p53 epitopes as well as their functional attributes were determined. Results: The CD3+CD8+ tetramer+ cell frequency was significantly higher (P<0.001) in TIL than autologous PBMC as was the percentage of CD4+CD25+ T cells (P<0.003). TIL were enriched in FOXp3+, GITR+ and CTLA-4+ Treg. CD8+ TIL had low expression and produced little IFN- after ex vivo stimulation relative to autologous PBMC or PBMC from NC. Conclusions: Anti-wt p53 epitope-specific T cells and Treg preferentially localize to tumor sites in patients with HNC. However, despite enrichment in tumor peptide-specific T cells, the effector cell population (CD3+CD8+) in TIL or PBMC was unresponsive to activation in the tumor microenvironment enriched in Treg.     

Podophyllum hexandrum modulates gamma radiation-induced immunosuppression in Balb/c mice: Implications in radioprotection

Aqueous extract of Podophyllum hexandrum (RP-1), which has been reported to render more than 82% survival against whole body lethal (10 Gy) gamma-irradiation in mice, was further investigated for its immunomodulatory potential. In this study, no significant change could be scored in peritoneal macrophages survival up to 8th day after whole body irradiation. RP-1 treatment (200 mg/kg body weight, i.p.) alone or 2 h before whole body irradiation enhanced macrophage survival significantly (p < 0.05) as compared to irradiated control mice. In irradiated animals, there was significant (p < 0.01) reduction in splenocyte survival and proliferation as revealed by 3H-TdR method. RP-1 treatment (200 mg/kg) alone or 2 h before irradiation countered the decrease in survival of splenocytes and proliferation significantly (p < 0.05) as compared to irradiated control group. Whole body irradiation also significantly (p < 0.05) reduced the population of CD4+ and CD8+ T cells and bone marrow GM-CFU at 24 h and 72 h post-irradiation intervals, respectively, as compared to unirradiated control. RP-1 treatment 2 h before whole body irradiation countered the decrease in CD4+ and CD8+ T cells populations and CGM-CFU. Nitric oxide free radicals generation was enhanced significantly (p < 0.05) in the supernatant of peritoneal macrophage cultures exposed to 2 Gy gamma radiation ex vivo in comparison to unirradiated control, which was reduced by pre-irradiation (−2 h) administration of RP-1. Whole body irradiation (10 Gy) also reduced the serum titres of IL-3, IL-1 and various IgG isotypes observed at different post-irradiation time interval. RP-1 treatment alone or before whole body irradiation countered radiation induced decrease in the titre of IL-1, IL-3 and IgG’s in the serum of mice. These findings indicate immunostimulatory potential of RP-1.