Tumour-associated B cells in urothelial urinary bladder cancer

Tumour infiltrating B cells and CD38⁺ plasma cells have been correlated with survival in different malignancies but their role in urinary bladder cancer is unclear. IL-10 is a multifunctional cytokine with both anti-inflammatory and immunostimulatory properties, that can be released by regulatory B cells (Bregs). We have stained paraffin-embedded tumour sections from 31 patients with invasive urothelial urinary bladder cancer with respect to CD20⁺ B cells, CD38⁺ cells, IL-10-expressing cells, IgG, C1q and C3a and analysed the impact of these markers on survival. Interestingly, we observe tumour-associated CD20⁺ B cells forming follicle-like structures in tumours of some patients. We demonstrate that follicle-like structures, tumour-associated CD38⁺ cells, IL-10 produced by non-B cells, tumour infiltrating IgG and activation of the complement system, may associate to longer survival of urinary bladder cancer patients. IL-10 expression by tumour-associated Bregs may instead negatively affect prognosis. More research is needed to fully understand the role of B cells and IL-10 in urinary bladder cancer.     

Bcl-2 immunoreactivity in salivary gland neoplasms is unrelated to the expression of mRNA for natural killer cell stimulatory cytokines interleukin (IL)-2 and IL-12

Certain cytokines are involved in the generation of natural killer (NK) cells and participate in the regulation of the proto-oncogene bcl-2. We aimed to study the mRNA expression of interleukin (IL) -2, IL-4 and IL-5, the composition of the tumour infiltrating lymphocytes (TIL), and the expression of bcl-2 in 14 benign and malignant human parotid tumours. T IL were predominantly composed of T lymphocytes and NK cells. We found evidence for the homing of T cells, and for generation of NK cells in the vicinity of the tumours. mRNA for IL-2 and IL-12, were identified but IL-4 mRNA was not found. The cytokine profiles and the composition of TIL of the two tumour categories were indistinguishable, suggesting that these host-response variables do not explain the differences in biological behaviour of these particular tumours. The results support a shift towards Th1 (T helper 1) cells and interferon- production, and that IL-12 also in vivo may play an important role in the regulatory interaction between innate resistance and adaptive immunity in tumour diseases. Most infiltrating lymphocytes showed strong expression of bcl-2; an interesting observation with regard to lymphocytic apoptosis in neoplastic diseases. The immunoreactivity fot the bcl-2 protein varied considerably between and within tumours, and almost all benign tumours showed strong bcl-2 positively whereas several of the malignant tumours showed weak or absent staining. The variable expression of bcl-2 protein suggests a different susceptibility of tumour cells to apoptosis. The results also indicate that bcl-2 cannot play a major role as protective agent in the specific apoptotic pathway induced by NK cells.     

Antigen processing and immune regulation in the response to tumours

The MHC class I and II antigen processing and presentation pathways display peptides to circulating CD8⁺ cytotoxic and CD4⁺ helper T cells respectively to enable pathogens and transformed cells to be identified. Once detected, T cells become activated and either directly kill the infected / transformed cells (CD8⁺ cytotoxic T lymphocytes) or orchestrate the activation of the adaptive immune response (CD4⁺ T cells). The immune surveillance of transformed/tumour cells drives alteration of the antigen processing and presentation pathways to evade detection and hence the immune response. Evasion of the immune response is a significant event tumour development and considered one of the hallmarks of cancer. To avoid immune recognition, tumours employ a multitude of strategies with most resulting in a down‐regulation of the MHC class I expression at the cell surface, significantly impairing the ability of CD8⁺ cytotoxic T lymphocytes to recognize the tumour. Alteration of the expression of key players in antigen processing not only affects MHC class I expression but also significantly alters the repertoire of peptides being presented. These modified peptide repertoires may serve to further reduce the presentation of tumour‐specific/associated antigenic epitopes to aid immune evasion and tumour progression. Here we review the modifications to the antigen processing and presentation pathway in tumours and how it affects the anti‐tumour immune response, considering the role of tumour‐infiltrating cell populations and highlighting possible future therapeutic targets.     

Spontaneous Cytokine Production and Its Effect on Induced Production

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) by peripheral-blood B lymphocytes, T cells, CD8⁻ T cells, CD8⁺ T cells, CD3⁻ CD16/56⁺ lymphocytes (natural killer [NK] cells), CD3⁺ CD16/56⁺ lymphocytes (natural T [NT] cells), and/or monocytes of 316 acutely ill hospitalized persons and 62 healthy adults in Malawi, Africa. We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-α production occurred most frequently, followed in descending order by IFN-γ, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-α; for 12 of these patients, TNF-α was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-α and IFN-γ, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states.     

Expression and location of IL-17A,E, F and their receptors in colorectal adenocarcinoma: Comparison with benign intestinal disease

The research aimed to investigate secretion, expression and location of IL-17 relative ligands, IL-17 relative receptors, infiltrating inflammatory cells and parenchymal structural cells in colorectal cancer (CRC) compared with ulcerative colitis (UC) and benign hyperplastic polyp. 29 human intestinal tissues with CRC, 17 with UC and 7 with polyp were stained using immunohistochemistry to evaluate immunoreactivity for IL-17 family relative ligands including IL-17A, E, F and their respective relative receptors such as IL-17RA, IL-17RB and IL-17RC. At the same time the infiltration of inflammatory cells including lymphocytes, phagocytes, mast cells and neutrophils and parenchymal structural cell changes involving vascular endothelial cells and CD90⁺ fibroblast cells were also evaluated using the same methods The immunoreactivity or positive inflammatory cells of all the sections were analyzed using professional image analysis software to determine statistical significance. The immunoreactivity for IL-17A, IL-17RA, IL-17E, IL-17RB and IL-17F showed significant decrease in CRC tissue when compared to UC (p?=?0.00001. respectively). The reduction of above IL-17 relative ligands and receptors was accompanied by an obvious decrease in the number of infiltrating neutrophils and mast cells in CRC (p?=?0.00001 and p?=?0.007, respectively) but accompanied by a marked increase of CD31⁺ blood vessels (p?=?0.001). The immunoreactivity of IL-17A, IL-17RA, IL-17E, IL-17RB and IL-17F and the numbers of infiltrating neutrophils and mast cells showed significant decrease in CRC tissues when compared to those in polyp (p?<?0.05). In contrast, the immunoreactivity of IL-17RC and the numbers of CD3⁺ 1ymphocytes were elevated in CRC when compared with those in polyp (p?=?0.0001, p?=?0.007, respectively). In CRC tissues, positive correlations between IL-17A, IL-17RA with CD68⁺ macrophages were observed respectively (r?=?0.621, p?=?0.0001; r?=?0.75, p?=?0.0001). IL-17 cytokine family including ligands and their corresponding receptors were secreted and expressed by infiltrating inflammatory cells. Not only infiltrating lymphocytes but also increased blood endothelial cells were relative significantly to genesis and progression of CRC.     

Combined CD4+ Donor Lymphocyte Infusion and Low-Dose Recombinant IL-2 Expand FOXP3+ Regulatory T Cells following Allogeneic Hematopoietic Stem Cell Transplantation

CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Treg) successfully control graft-versus-host-disease (GVHD) in animal models. In humans, incomplete reconstitution of Treg after allogeneic hematopoietic stem cell transplantation (HSCT) has been associated with chronic GVHD (cGVHD). Recent studies have demonstrated that interleukin (IL)-2 infusions expand Treg in vivo. However, the effectiveness of this therapy depends on the number of cells capable of responding to IL-2. We examined the effect of low-dose IL-2 infusions on Treg populations after HSCT in patients who also received infusions of donor CD4⁺ lymphocytes. Utilizing FOXP3 as a Treg marker, we found that patients who received CD4+DLI concomitantly with IL-2 had greater expansion of Treg compared to patients who received IL-2 (P = .03) or CD4⁺DLI alone (P = .001). FOXP3 expression correlated with absolute CD4⁺CD25⁺ cell counts. Moreover, expanded CD4⁺CD25⁺ T cells displayed normal suppressive function and treatment with CD4⁺DLI and IL-2 was not associated with GVHD. This study suggests that administration of low-dose IL-2 combined with adoptive CD4⁺ cellular therapy may provide a mechanism to expand Treg in vivo.     

Prognostic significance of CD8+ T lymphocytes in breast cancer depends upon both oestrogen receptor status and histological grade

Baker K, Lachapelle J, Zlobec I, Bismar T A, Terracciano L & Foulkes W D (2011) Histopathology 58, 1107–1116 Prognostic significance of CD8 ⁺ T lymphocytes in breast cancer depends upon both oestrogen receptor status and histological grade Aims: Results of previous studies on the influence of tumour infiltrating lymphocytes on prognosis of women with breast cancer have been mixed. This study re‐evaluates the role of tumour‐infiltrating lymphocytes as a prognostic marker in women with breast cancer. Methods and results: Immunochemistry staining of CD8⁺ T cells was performed on a tissue microarray of 1953 breast carcinomas. When all tumours were considered, no association between the lymphocyte count and patient survival was found. In univariate analysis, there was a reduced disease‐specific survival for women with oestrogen receptor (ER)‐positive tumours with high intraepithelial lymphocyte count (P = 0.004). In those with ER‐negative tumours, the disease‐specific survival was improved when the intraepithelial, stromal and total lymphocyte counts were high, the total lymphocyte count also being an independent prognostic marker on multivariate analysis (P = 0.031). When stratified by histological grade, on univariate analysis, the previously observed inferior outcome in women with high lymphocyte count and ER‐positive tumours remained significant only if tumours were also of low grade, and the superior outcome in those with ER‐negative tumours remained significant if tumours were also of high grade. Conclusions: Our results raise the possibility of different immune–tumour interactions based on ER status and histological grade.     

Evidence of a role for CD200 in regulation of immune rejection of leukaemic tumour cells in C57BL/6 mice.

Increased expression of the molecule CD200 in mice receiving renal allografts is associated with immunosuppression leading to increased graft survival, and altered cytokine production in lymphocytes harvested from the transplanted animals. Preferential production of IL-4, IL-10 and TGFbeta occurs on donor-specific restimulation in vitro, with decreased production of IL-2, IFNgamma and TNFalpha. These effects are enhanced by simultaneous infusion of CD200 immunoadhesin (CD200Fc) and donor CD200 receptor (CD200r) bearing macrophages to transplanted mice. C57BL/6 mice do not normally resist growth of EL4 or C1498 leukaemia tumour cells. Following transplantation of cyclophosphamide-treated C57BL/6 with T-depleted C3H bone marrow cells, or for the EL4 tumour, immunization of C57BL/6 mice with tumour cells transfected with a vector encoding the co-stimulatory molecule CD80 (EL4-CD80), mice resist growth of tumour challenge. Immunization of C57BL/6 mice with EL4 cells overexpressing CD86 (EL4-CD86) is ineffective. Protection from tumour growth in either model is suppressed by infusion of CD200Fc, an effect enhanced by co-infusion of CD200r+ macrophages. CD200Fc acts on both CD4+ and CD8+ cells to produce this suppression. These data are consistent with the hypothesis that immunosuppression following CD200-CD200r interaction can regulate a functionally important tumour growth inhibition response in mice.     

Interleukin-10 promotes B16-melanoma growth by inhibition of macrophage functions and induction of tumour and vascular cell proliferation

The aim of this study was to investigate the mechanisms by which interleukin-10 (IL-10) induces tumour growth in a mouse-melanoma model. A B16-melanoma cell line (B16-0) was transfected with IL-10 cDNA and three clones that secreted high (B16-10), medium and low amounts of IL-10 were selected. Cell proliferation and IL-10 production were compared in vitro, and tumour growth, percentages of necrotic areas, tumour cells positive for proliferating cell nuclear antigen (PCNA), IL-10 receptor (IL-10R) and major histocompatibility complex type I (MHC-I) and II (MHC-II), as well as infiltration of macrophages, CD4+ and CD8+ lymphocytes and blood vessels were compared in vivo among IL-10-transfected and non-transfected tumours. Proliferation and tumour growth were greater for IL-10-transfected than for non-transfected cells (P < 0.001), and correlated with IL-10 concentration (r > or =0.79, P < 0.006). Percentages of tumour cells positive for PCNA and IL-10R were 4.4- and 16.7-fold higher, respectively, in B16-10 than in B16-0 tumours (P < 0.001). Macrophage distribution changed from a diffuse pattern in non-transfected (6.4 +/- 1.7%) to a peripheral pattern in IL-10-transfected (3.8 +/- 1.7%) tumours. The percentage of CD4+ lymphocytes was 7.6 times higher in B16-10 than in B16-0 tumours (P = 0.002). The expression of MHC-I molecules was present in all B16-0 tumour cells and completely negative in B16-10 tumour cells. In B16-0 tumours, 89 +/- 4% of the whole tumour area was necrotic, whereas tumours produced by B16-10 cells showed only 4.3 +/- 6% of necrotic areas. IL-10-transfected tumours had 17-fold more blood vessels than non-transfected tumours (61.8 +/- 8% versus 3.5 +/- 1.7% blood vessels/tumour; P < 0.001). All the effects induced by IL-10 were prevented in mice treated with a neutralizing anti-IL-10 monoclonal antibody. These data indicate that IL-10 could induce tumour growth in this B16-melanoma model by stimulation of tumour-cell proliferation, angiogenesis and immunosuppression.     

Lymphocyte infiltration in oesophageal carcinoma: lack of correlation with MHC antigens,ICAM-1, and tumour stage and grade.

Infiltration by T lymphocytes into oesophageal carcinomas was assessed immunohistochemically, total T lymphocyte numbers by staining for CD3 and activated T lymphocytes by staining for CD25. Five squamous carcinomas and seven adenocarcinomas, resected without neoadjuvant treatment, were studied. Computer aided quantitation showed that total numbers of tumour infiltrating CD3 positive cells were highly variable (range 48-1673 cells/mm2). They were located largely in the stromal (87.9-99.2%) rather than intratumoral regions. Up to 84% of tumour infiltrating T lymphocytes were CD25 positive, although the median figure was 33%. There was no correlation between T lymphocyte infiltration or activation and expression of class I and II histocompatibility antigens, intercellular adhesion molecule-1, tumour stage or grade. These results imply that the local inflammatory response in oesophageal carcinomas is deregulated, which may be a factor contributing to the aggressive nature of the tumours.     

T lymphocytes that infiltrate nasal polyps have a specialized phenotype and produce a mixed TH1 /TH2 pattern of cytokines

Background: Nasal polyps (NPs) are inflammatory reactions in the nasal mucosa the etiology and pathogenesis of which remains unknown. Objective: The purpose of this study was to study in detail the phenotype and function of T lymphocytes infiltrating NPs by analyzing the expression of surface markers and cytokine secretion. Methods: NP tissue samples and peripheral blood were obtained from 18 patients. Mononuclear cells were purified from these samples, and their phenotype was investigated by triple-color immunofluorescence and flow cytometric analysis. Cytokine production was determined in cultures by using an ELISA technique. Results: NP lymphoid cells mainly consisted of T lymphocytes. These T lymphocytes showed a CD45RO+CD45RA– phenotype and expressed pan-T cell molecules; the CD8+ subset was predominant. NP T cells showed a lower density of CD28, CD3, and TCR-αβ compared with T cells from peripheral blood. NP T lymphocytes expressed the activation markers DR and CD69 and exhibited the adhesion molecule profile CD54+, CD62L–, and CD103+ CD49d^low . Virtually all NP T cells bore CD95 (FAS), but they did not undergo apoptosis, either spontaneously or induced by CD95 cross-linking with the mAb CH11. The pattern of cytokines secreted by NP T lymphocytes was characterized by the spontaneous and simultaneous production of IFN-γ and IL-5. Neither IL-2 nor IL-4 were detectable in nonstimulated cultures. Conclusion: This study defines the T lymphocytes that infiltrate NPs as memory T cells in an activated status, with homing properties related to the mucosal immune system. They are resistant to anti-CD95-mediated apoptosis and produced a mixed T_H1 /T_H2 cytokine pattern as defined by the simultaneous production of IFN-γ and IL-5. (J Allergy Clin Immunol 1998;102:953-60.)     

The density of infiltrating T cells and macrophages in the parental tumour correlates with growth rate of tumoroids established from colorectal adenocarcinoma

The aim of the present study was to investigate the correlation between the density of infiltrating T cells and macrophages in the parental colorectal cancer (CRC) and the growth rate of tumoroids (i.e. a patient‐derived in vitro 3D model). Tumoroids were established from fresh specimens of primary and metastatic CRC from 29 patients. The in vitro growth rate of tumoroids was monitored by automated imaging. The density of infiltrating T cells and macrophages was determined in the centre of the tumour (CT) and at the invasive margin (IM) of the parental tumours. This was performed by digital image analysis on the whole‐slide scanned images using Visiopharm^® software. Tumoroids with higher density of infiltrating CD3+ lymphocytes in the IM of their parental tumour showed a higher growth rate (P < .0005). The average relative growth rate (log10) during the period from day 1 to day 11 was 0.364 ± 0.006 (mean ± SD) for the CD3+ (IM)‐high group and 0.273 ± 0.008 (mean ± SD) for the CD3+ (IM)‐low group. In contrast, the density of CD68+ infiltrating macrophages in the parental tumours showed significant inverse effect on the growth rate of the tumoroids (P < .0005). The present study showed that the density of immune cells in the parental CRC correlates with the growth rate of the tumoroids. The future perspective for such a 3D model could be in vitro investigations of the tumour‐associated inflammatory microenvironment as well as personalized cancer immunotherapy.     

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) induced in human CD4⁺ and CD8⁺ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4⁺ cells can make IL-2 than the other two cytokines, and that there are more TNF-α-positive CD4⁺ cells than cells with IFN-γ. For normal CD8⁺ cells the highest production of cytokine is of IFN-γ (up to 50% of the cells) especially at longer times (10–20h) of stimulation. For CD8⁺ cells, IL-2-positive cells exceed those with TNF-α. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4⁺ or CD8⁺ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-α but significantly raised levels of production of IFN-γ in both CD4⁺ and CD8⁺ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.     

Phenotype, cytokine production and cytolytic capacity of fresh (uncultured) tumour-infiltrating T lymphocytes in human renal cell carcinoma

We investigated the phenotype and functional capacities of tumour-infiltrating lymphocytes (TIL), freshly isolated from primary renal cell carcinoma (RCC) specimens (n = 20). Three-colour flow cytometry immunophenotyping revealed that RCC TIL consist mainly of CD3⁺ T cells, with a clear predominance of CD4⁻CD8⁺ over CD4⁺ CD8⁻ T cells, and a marked population of CD4⁺ CD8⁺ T cells. Natural killer (NK) cells were also strongly represented (> 25% in 15 of 20 tumour samples), while B cells constituted a minor TIL subset (< 5% in 18 of 20 tumour samples). More importantly, the T and NK cells within the tumour displayed a significantly higher expression of the early activation marker CD69 than their counterparts in adjacent normal renal tissue and in peripheral blood. Expression of CD54 and of HLA-DR was also elevated on CD3⁺ TIL, and HLA-DR expression was further vigorously up-regulated following ex vivo stimulation with anti-CD3, all suggesting enhanced immune activity within the tumour microenvironment. CD3⁺ CD4⁺ TIL displayed a normal capacity to up-regulate CD25 expression and to secrete both Th1-type (IL-2, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ)) and Th2-type (IL-4, IL-5 and IL-10) cytokines upon triggering with anti-CD3. Furthermore, cytokine production was susceptible to modulation by CD28 costimulation. CD3⁺ CD8⁺ TIL, on the other hand, consistently demonstrated a poor up-regulation of CD25 upon triggering with anti-CD3, and displayed poor ex vivo cytolytic activity in an anti-CD3-redirected 4-h cytotoxicity assay against murine P815 cells. Collectively, our findings indicate that the CD3⁺ CD4⁺ TIL in RCC have normal functional capacities, whereas the proportionally major CD3⁺ CD8⁺ TIL are functionally impaired. The relevance of these findings to the in vivo local immune response in RCC is discussed.     

Opposite role for interleukin-4 and interferon-γ on CD30 and lymphocyte activation gene-3 (LAG-3) expression by activated naive T cells

Polarized human type 1 and type 2 T helper cells not only produce different sets of cytokines, but they also preferentially express certain activation markers, such as lymphocyte activation gene-3 (LAG-3) and CD30, respectively. In this study we have examined the LAG-3 and CD30 expression in relation to the lineage commitment of human naive CD4⁺ T cells, as assessed at the single-cell level of committed T cells. Purified CD45RA⁺ umbilical cord blood T lymphocytes were activated with phytohemagglutinin and interleukin (IL)-2 in the absence or presence of interleukin IL-4 or IL-12 and assessed for CD30 and LAG-3 expression, as well as for intracellular cytokine synthesis. Significant numbers of CD30⁺ cells were only found in CD4⁺ and CD8⁺ T lymphocytes of cultures primed with IL-4, which developed into cells able to produce IL-4 and IL-13 in addition to interferon (IFN)-γ. By contrast, LAG-3 expression was strongly up-regulated in CD4⁺ and CD8⁺ T cells from cultures primed with IL-12, which developed into high numbers of IFN-γ producers. The addition of a neutralizing anti-IFN-γ antibody to IL-12-primed CD4⁺ T cell cultures virtually abolished the development of LAG-3-expressing CD4⁺ T cells. Taken together, these data suggest that CD30 expression is dependent on the presence of IL-4, whereas LAG-3 expression is dependent on the production of IFN-γ during the lineage commitment of human naive T cells.     

Maturation of CD4+ Regulatory T Lymphocytes and of Cytokine Secretions in Infants Born Prematurely

Purpose

To characterize the maturation of CD4⁺ regulatory T lymphocytes (Treg) and of cytokine productions in preterm infants during their first 16 months of life.

Methods

The proportions of CD4⁺ Treg cells, their phenotypic characteristics, and the mitogen-induced cytokine productions by peripheral blood mononuclear cells (PBMC) were analysed in 26 very-preterm infants from 2 to 16 months of age, and compared to results obtained for 17 cord blood mononuclear cells (CBMC) from very-preterm infants, 12 from term infants and to blood samples from 40 adults.

Results

High proportion of CD25^+/highCD4⁺ Treg cells was found at birth in preterm CB with a gradual decreased afterwards. However, their percentage at 16 months of age was still higher than in term CB. In contrast to adults, preterm infants were characterized by excellent linear correlations between the proportions of CD25^+/highCD4⁺ and CD25^+/highFoxP3⁺ CD4⁺ or CD25^+/highCD127^low CD4⁺ or CD25^+/highFoxP3⁺CD127^low CD4⁺T lymphocytes. CD45RO⁺ and HLA-DR⁺ expressions were very low on preterm Treg and progressively increased with age. Functionally, preterm compared to term CBMC secreted in response to phytohaemagglutinin lower IFN-γ, higher IL-5 and similar IL-12p70, IL-10, IL-2 and IL-13 concentrations. IFN-γ, IL-12p70 and IL-10 productions were at 16 months still lower than those obtained for adults

Conclusion

Preterm differed from term CBMC both by their proportion and phenotype of CD4⁺ Treg lymphocytes and by their cytokine secretions. Maturation occurred during infancy with similar IFN-γ secretion but with persistently higher proportion of CD4⁺ Treg cells in 1 year preterm infants compared to term neonates.     

Phenotype analysis of tumour-infiltrating lymphocytes and lymphocytes in peripheral blood in patients with renal carcinoma

INTRODUCTION: When checking tumour growth, a number of observations indicate that the immune system plays a significant role in patients with renal cell carcinoma (RCC). Infiltration by lymphocytes (tumour infiltrating lymphocytes, TILs) is more prevalent in RCC than any other tumours. T lymphocytes are the dominant population of TIL cells. Views concerning the role of T lymphocytic subpopulations, B lymphocytes and NK cells in an anti-tumour response are not established. AIM: The aim is to determine the phenotype and activation of T and B lymphocytic subpopulations and NK cells and to compare their representation in tumour stroma and peripheral blood lymphocytes (PBL) in patients with RCC. MATERIAL AND METHODS: Samples of peripheral blood taken from the cubital and renal veins and tumour stroma cells were obtained from 44 patients in the course of their surgeries carried out due to primary RCC. TILs were isolated from mechanically disintegrated tumour tissue. Immunophenotype multiparametric analysis of PBL and TILs was carried out. Their surface and activation characteristics were determined by means of flow cytometer. RESULTS: CD3+ T lymphocytes (69.7%) were the main population of TILs. The number of CD3+/CD8+ T lymphocytes was significantly higher in TILs, 42.6% (p < 0.01), while CD4+ T lymphocytes were the majority population in peripheral blood, 41.35% (p < 0.001). The representation of CD3+/69+ T lymphocytes was significantly higher in TILs, 32.9%, compared to PBL (p < 0.001). On the contrary, the numbers of CD3+/CD25+, CD8+/57+ and CD4+/RA+ (naive CD4+ T lymphocytes) were higher in PBL (p < 0.001). The differences in representation of (CD3-/16+56+) NK cells and CD3+/DR+ T cells in TILs and PBL were not significant. CONCLUSION: The above-mentioned results prove that the characteristics and intensity of anti-tumour responses are different in compared compartments (tumour/PBL). CD3+/CD8+ T lymphocytes are the dominant lymphocytic population of TILs. The knowledge of the phenotype and functions of effector cells, which are responsible for anti-tumour response, are the basic precondition for understanding the anti-tumour immune response and the cause of its failure.