Characterization of an in vitro model to study the possible role of polyomavirus BK in prostate cancer.

Prostate cancer (PCa) is the most common male neoplasms in the Western world. Various risk factors may lead to carcinogenesis, including infectious agents such as polyomavirus BK (BKPyV), which infects the human renourinary tract, establishes latency, and encodes oncoproteins. Previous studies suggested that BKPyV plays a role in PCa pathogenesis. However, the unspecific tropism of BKPyV and the lack of in vitro models of BKPyV-infected prostate cells cast doubt on this hypothesis. The aim of the present study was to determine whether BKPyV could (a) infect normal and/or tumoral epithelial prostate cells and (b) affect their phenotype. Normal epithelial prostate RWPE-1 cells and PCa PC-3 cells were infected with BKPyV for 21 days. Cell proliferation, cytokine production, adhesion, invasion ability, and epithelial-to-mesenchymal transition (EMT) markers were analyzed. Our results show that (a) RWPE-1 and PC-3 cells are both infectable with BKPyV, but the outcome of the infection varies, (b) cell proliferation and TNF-α production were increased in BKPyV-infected RWPE-1, but not in PC-3 cells, (c) adhesion to matrigel and invasion abilities were elevated in BKPyV-infected RWPE-1 cells, and (d) loss of E-cadherin and expression of vimentin occurred in both uninfected and infected RWPE-1 cells. In conclusion, BKPyV may change some features of the normal prostate cells but is not needed for maintaining the transformed phenotype in the PCa cells The fact that RWPE-1 cells exhibit some phenotype modifications related to EMT represents a limit of this in vitro model.

Polyomavirus BK and prostate cancer: a complex interaction of potential clinical relevance.

Several studies associating BK polyomavirus (BKPyV) and prostate cancer (PCa) suggested that this virus may exert its oncogenic activity at early stages of cancer development. The BKPyV oncogene, the large T antigen (LTag), has frequently been detected in areas of proliferative inflammatory atrophy, which is considered a precursor lesion leading to prostatic intraepithelial neoplasia and overt PCa. In a recently updated systematic review, the presence of BKPyV was significantly higher in PCa tissues than in healthy control tissues, providing an indication for a link between BKPyV infection and cancer risk. In addition, recent original investigations highlighted an association between expression of the virus and the clinical course of PCa. For example, by studying immune responses elicited against BKPyV LTag, a significant association between LTag positive cancer lesions and a peculiar regulatory profiling has been observed in PCa patients with evidence of disease recurrence after surgical radical prostatectomy. Lastly, a study carried out in a larger cohort of patients undergoing radical prostatectomy revealed the IgG response against LTag as an independent predictor of disease recurrence. Although a full picture of the mechanisms potentially responsible for the involvement of BKPyV in PCa is not available yet, continuing work on this topic should help to refine the potential role of BKPyV in PCa patients, perhaps revealing unsuspected associations with the clinical course of this disease.

New insights on the association between the prostate cancer and the small DNA tumour virus, BK polyomavirus.

In recent years the scientific literature in the field of the prostate carcinoma (PCa) pointed out on the genetic heterogeneity and mutations occurring in this tumour, while little attention was given to the causes of PCa onset, in particular infectious agents. In this brief commentary, we wish to point out recent advancements done on the role of the human polyomavirus BK (BKPyV) in the development of PCa by harnessing both humoral and cellular immune responses. Altogether, these new insights suggest that BKPyV is involved in the transforming activity during the multistep process of PCa development. Although these findings do not provide evidence for a causal relationship between BKPyV and PCa development, additional investigations with novel techniques will help to make it a concrete event.

High VEGF-D and Low MMP-2 Serum Levels Predict Nodal-Positive Disease in Invasive Bladder Cancer.

BACKGROUND: To investigate stromal variables including angiogenesis, lymphangiogenesis, and matrix metalloproteinase (MMP) in the serum of patients with urothelial carcinoma of the bladder (UCB) and to evaluate their association with histopathological characteristics and clinical outcome. MATERIAL AND METHODS: Protein levels of vascular endothelial growth factors-A, -C, -D (VEGF-A/-C/-D), their receptors- VEGF-R2 and -R3 (VEGF-R2/-R3), and matrix metalloproteinases 2, -3, and -7 (MMP-2, MMP-3, MMP-7) were quantified in the blood serum samples of 71 patients with UCB before radical cystectomy (RC). Samples of patients with non-invasive UCB or no history of UCB were investigated as controls (n=20). Protein levels in the serum were measured using a flow cytometric cytokine assay. RESULTS: A positive association for VEGF-D (p<0.001) and an inverse association for MMP-2 (p=0.017) were observed in patients with positive lymph node (LN) status at the time of RC. VEGF-A (p<0.001), VEGF-C (p<0.001), MMP-2 (p<0.001), and MMP-7 (p=0.005) serum levels were different in serum of patients with invasive UCB compared with non-invasive UCB or healthy individuals. None of the serum markers were associated with disease progression. CONCLUSIONS: High VEGF-D and low MMP-2 serum levels predict LN metastasis in patients with UCB at the time of RC. VEGF-A, VEGF-C, MMP-2, and MMP-7 serum levels varied significantly between invasive and non-invasive disease as well as in comparison with healthy individuals. Clinical implementation of these marker serum measurements may be valuable to select high-risk patients with more invasive or nodal-positive disease.

Differential patterns of large tumor antigen-specific immune responsiveness in patients with BK polyomavirus-positive prostate cancer or benign prostatic hyperplasia.

The role of the polyomavirus BK (BKV) large tumor antigen (L-Tag) as a target of immune response in patients with prostate cancer (PCa) has not been investigated thus far. In this study, we comparatively analyzed humoral and cellular L-Tag-specific responsiveness in age-matched patients bearing PCa or benign prostatic hyperplasia, expressing or not expressing BKV L-Tag-specific sequences in their tissue specimens, and in non-age-matched healthy individuals. Furthermore, results from patients with PCa were correlated to 5-year follow-up clinical data focusing on evidence of biochemical recurrence (BR) after surgery (prostate specific antigen level of ≥0.2 ng/ml). In peripheral blood mononuclear cells (PBMC) from patients with PCa with evidence of BR and BKV L-Tag-positive tumors, stimulation with peptides derived from the BKV L-Tag but not those derived from Epstein-Barr virus, influenza virus, or cytomegalovirus induced a peculiar cytokine gene expression profile, characterized by high expression of interleukin-10 (IL-10) and transforming growth factor ß1 and low expression of gamma interferon genes. This pattern was confirmed by protein secretion data and correlated with high levels of anti-BKV L-Tag IgG. Furthermore, in PBMC from these PCa-bearing patients, L-Tag-derived peptides significantly expanded an IL-10-secreting CD4(+) CD25(+(high)) CD127(-) FoxP3(+) T cell population with an effector memory phenotype (CD103(+)) capable of inhibiting proliferation of autologous anti-CD3/CD28-triggered CD4(+) CD25(-) T cells. Collectively, our findings indicate that potentially tolerogenic features of L-Tag-specific immune response are significantly associated with tumor progression in patients with BKV(+) PCa.

Myoblasts inhibit prostate cancer growth by paracrine secretion of tumor necrosis factor-α.

PURPOSE: Myoblasts can form muscle fibers after transplantation. Therefore, they are envisioned as a treatment for urinary incontinence after radical prostatectomy. However, to our knowledge the safety of this treatment and the interaction of myoblasts with any remaining neighboring cancer are unknown. We investigated the interactions between myoblasts and prostate carcinoma cells in vitro and in vivo. MATERIALS AND METHODS: Myoblasts isolated from the rectus abdominis were used in a series of co-culture experiments with prostate cancer cells and subcutaneously co-injected in vivo. Cell proliferation, cell cycle arrest and apoptosis of cancer in co-culture with myoblasts were assessed. Tumor volume and metastasis formation were evaluated in a mouse model. Tissue specific markers were assessed by immunohistochemistry, fluorescence activated cell sorting analysis, Western blot and real-time quantitative polymerase chain reaction. RESULTS: Myoblasts in proximity to tumor provided paracrine tumor necrosis factor-α to their microenvironment, decreasing the tumor growth of all prostate cancer cell lines examined. Co-culture experiments revealed induction of cell cycle arrest, tumor death by apoptosis and increased myoblast differentiation. This effect was largely blocked by tumor necrosis factor-α inhibition. The same outcome was noted in a mouse model, in which co-injected human myoblasts also inhibited the tumor growth and metastasis formation of all prostate cancer cell lines evaluated. CONCLUSIONS: Myoblasts restrict prostate cancer growth and limit metastasis formation by paracrine tumor necrosis factor-α secretion in vitro and in vivo.

Identification of Urine Biomarkers to Improve Eligibility for Prostate Biopsy and Detect High-Grade Prostate Cancer.

PCa screening is based on the measurements of the serum prostate specific antigen (PSA) to select men with higher risks for tumors and, thus, eligible for prostate biopsy. However, PSA testing has a low specificity, leading to unnecessary biopsies in 50-75% of cases. Therefore, more specific screening opportunities are needed to reduce the number of biopsies performed on healthy men and patients with indolent tumors. Urine samples from 45 patients with elevated PSA were collected prior to prostate biopsy, a mass spectrometry (MS) screening was performed to identify novel biomarkers and the best candidates were validated by ELISA. The urine quantification of PEDF, HPX, CD99, CANX, FCER2, HRNR, and KRT13 showed superior performance compared to PSA. Additionally, the combination of two biomarkers and patient age resulted in an AUC of 0.8196 (PSA = 0.6020) and 0.7801 (PSA = 0.5690) in detecting healthy men and high-grade PCa, respectively. In this study, we identified and validated novel urine biomarkers for the screening of PCa, showing that an upfront urine test, based on quantitative biomarkers and patient age, is a feasible method to reduce the number of unnecessary prostate biopsies and detect both healthy men and clinically significant PCa.

Elevated levels of circulating IL-7 and IL-15 in patients with early stage prostate cancer.

BACKGROUND: Chronic inflammation has been suggested to favour prostate cancer (PCA) development. Interleukins (IL) represent essential inflammation mediators. IL-2, IL-7, IL-15 and IL-21, sharing a common receptor γ chain (c-γ), control T lymphocyte homeostasis and proliferation and play major roles in regulating cancer-immune system interactions. We evaluated local IL-2, IL-7, IL-15 and IL-21 gene expression in prostate tissues from patients with early stage PCA or benign prostatic hyperplasia (BPH). As control, we used IL-6 gene, encoding an IL involved in PCA progression. IL-6, IL-7 and IL-15 titres were also measured in patients' sera. METHODS: Eighty patients with BPH and 79 with early (1 to 2c) stage PCA were enrolled. Gene expression in prostate tissues was analyzed by quantitative real-time PCR (qRT-PCR). Serum IL concentrations and acute phase protein titres were evaluated by ELISA. Mann-Whitney, Wilcoxon and χ(2) tests were used to compare IL gene expression and serum titers in the two groups of patients. Receiver operating characteristic (ROC) curves were constructed to evaluate the possibility to distinguish sera from different groups of patients based on IL titers. RESULTS: IL-2 and IL-21 gene expression was comparably detectable, with low frequency and at low extents, in PCA and BPH tissues. In contrast, IL-6, IL-7 and IL-15 genes were expressed more frequently (p < 0.0001, p = 0.0047 and p = 0.0085, respectively) and to significantly higher extents (p = 0.0051, p = 0.0310 and p = 0.0205, respectively) in early stage PCA than in BPH tissues. Corresponding proteins could be detected to significantly higher amounts in sera from patients with localized PCA, than in those from patients with BPH (p = 0.0153, p = 0.0174 and p = 0.0064, respectively). Analysis of ROC curves indicates that IL-7 (p = 0.0039), but not IL-6 (p = 0.2938) or IL-15 (p = 0.1804) titres were able to distinguish sera from patients with malignancy from those from patients with benign disease. Serum titres of C reactive (CRP), high mobility group B1 (HMGB1) and serum amyloid A (SAA) acute phase proteins were similar in both groups of patients. CONCLUSIONS: Expression IL-7 and IL-15 genes in prostate tissues and corresponding serum titres are significantly increased in patients with early stage PCA as compared with patients with BPH.

Insulin-like growth factor II mRNA binding protein 3 (IMP3) is overexpressed in prostate cancer and correlates with higher Gleason scores.

BACKGROUND: The oncofetal protein insulin-like growth factor II mRNA binding protein 3 (IMP3) is an important factor for cell-migration and adhesion in malignancies. Recent studies have shown a remarkable overexpression of IMP3 in different human malignant neoplasms and also revealed it as an important prognostic marker in some tumor entities. To our knowledge, IMP3 expression has not been investigated in prostate carcinomas so far. METHODS: Immunohistochemical stainings for IMP3 were performed on tissue microarray (TMA) organized samples from 507 patients: 31 normal prostate tissues, 425 primary carcinomas and 51 prostate cancer metastases or castration-resistant prostate cancers (CRPC). IMP3 immunoreactivity was semiquantitatively scored and correlated with clinical-pathologic parameters including survival. RESULTS: IMP3 is significantly stronger expressed in prostate carcinomas compared to normal prostate tissues (p < 0.0001), but did not show significant correlation with the pT-stage, the proliferation index (MIB1), preoperative serum PSA level and the margin status. Only a weak and slightly significant correlation was found with the Gleason score and IMP3 expression failed to show prognostic significance in clinico-pathological correlation-analyses. CONCLUSIONS: Although IMP3 is overexpressed in a significant proportion of prostate cancer cases, which might be of importance for novel therapeutic approaches, it does not appear to possess any immediate diagnostic or prognostic value, limiting its potential as a tissue biomarker for prostate cancer. These results might be corroborated by the fact, that two independent tumor cohorts were separately reviewed.

The Prognostic Value of Indoleamine-2,3-Dioxygenase Gene Expression in Urine of Prostate Cancer Patients Undergoing Radical Prostatectomy as First Treatment of Choice.

Prostate cancer (PCa) is a slow-growing tumor representing one of the major causes of all new cancer cases and cancer mortality in men worldwide. Although screening methods for PCa have substantially improved, the outcome for patients with advanced PCa remains poor. The elucidation of the molecular mechanism that drives the progression from a slow-growing, organ-confined tumor to a highly invasive and castration-resistant PCa (CRPC) is therefore important. We have already proved the diagnostic potential of indoleamine-2,3-dioxygenase (IDO) when detected in urine of individuals at risk of developing PCa. The aim of this study was to implement IDO as a prognostic marker for PCa patients undergoing surgical treatment. We have thus conducted an observational study by collecting 100 urine samples from patients undergoing radical prostatectomy as first treatment of choice. To test the integrity of our investigation, scale dilution cells of an established PC3 cell line were added to urine of healthy donors and used for gene expression analysis by a TaqMan assay on the catalytic part of IDO mRNA. Our data show that the quantification of IDO mRNA in urine of patients has a very promising ability to identify patients at high risk of cancer advancement, as defined by Gleason score. Our goal is to lay the groundwork to develop a superior test for PCa. The data generated are thus necessary (i) to strengthen the IDO-based diagnostic/prognostic test and (ii) to provide patients and clinicians with an affordable and easy screening test.

A HCMV pp65 polypeptide promotes the expansion of CD4+ and CD8+ T cells across a wide range of HLA specificities.

Human cytomegalovirus (HCMV) can cause life-threatening disease in infected hosts. Immunization with human leukocyte antigen (HLA)-restricted immunodominant synthetic peptides and adoptive transfer of epitope-specific T cells have been envisaged to generate or boost HCMV-specific cellular immunity, thereby preventing HCMV infection or reactivation. However, induction or expansion of T cells effective against HCMV are limited by the need of utilizing peptides with defined HLA restrictions. We took advantage of a combination of seven predictive algorithms to identify immunogenic peptides of potential use in the prevention or treatment of HCMV infection or reactivation. Here we describe a pp65-derived peptide (pp65(340-355), RQYDPVAALFFFDIDL: RQY16-mer), characterized by peculiar features. First, RQY-16mer is able to stimulate HCMV pp65 specific responses in both CD4(+) and CD8(+) T cells, restricted by a wide range of HLA class I and II determinants. Second, RQY-16mer is able to induce an unusually wide range of effector functions in CD4(+) T cells, including proliferation, killing of autologous HCMV-infected target cells and cytokine production. Third, and most importantly, the RQY-16mer is able to stimulate CD4(+) and CD8(+) T-cell responses in pharmacologically immunosuppressed patients. These data suggest that a single reagent might qualify as synthetic immunogen for potentially large populations exposed to HCMV infection or reactivation.

T cell epitope-mapping by cytokine gene expression assay.

The following method describes the identification of candidate immunogenic peptides through their ability to recall an immune T-cell activation from peripheral blood mononuclear cells (PBMCs) of individuals with defined HLA-peptide restrictions that have been previously exposed to the antigen. Isolated PBMCs are plated out at a concentration of 1 x 10(6) cells/ml in a 200 microl medium and incubated overnight to reduce cytokine gene expression due to cell manipulation. After starving, cells are either directly stimulated with individual peptides or not stimulated and incubated from 3 to 12 h according to experimental conditions. Quantitative real-time PCR (qrt-PCR) is performed on reverse-transcribed complementary DNA (cDNA) from total RNA that is isolated from peptide-cultured PBMCs. To perform high quality qrt-PCR, primers and probes are designed to span exon-intron junctions in order to prevent amplification of genomic DNA and to produce amplicons <150 base pairs (bp). Real-time monitoring of fluorescent emission from the cleavage of sequence specific probe by the nuclease activity of Taq polymerase (TaqMan method) defines threshold cycles during exponential phases of amplification. Standard curves of copy numbers of the gene of interest are normalized using as reference copy numbers of control genes.

Gene expression signatures of interleukin-2 in vivo and in vitro and their relation to anticancer therapy.

Treatment with human recombinant interleukin-2 (rIL-2) can successfully eradicate advanced cancer in humans; however, its utilization is limited by the unpredictability of its effectiveness and the excessive toxicity often associated with its use. The mechanisms responsible for immune-mediated tumor regression and those associated with limiting toxicity have not yet been sorted out. Thus, this review critically addresses what has been done in the past to understand this biologically and practically important question and discusses future strategies to enhance the understanding of this interesting model of immune-mediated tumor rejection. In particular, the first aim of this review is to discuss what is known about the mechanism(s) responsible for tumor rejection; the second aim is to review the relationship between the toxicity induced by rIL-2 treatment and its effectiveness; the third aim is to summarize novel insights into the possible mechanism of rIL-2 activity in vivo using high-throughput strategies aimed at the global assessment in real-time of events associated with rIL-2 therapy in humans. This information will not only lead to a better utilization of this biological agent in clinical practice but it may also provide important information about how immune-mediated tissue rejection occurs.

Expression of Indoleamine 2,3-Dioxygenase Induced by IFN-γ and TNF-α as Potential Biomarker of Prostate Cancer Progression.

Inflammation has been suggested to play an important role in onset and progression of prostate cancer (PCa). Histological analysis of prostatectomy specimens has revealed focal inflammation in early stage lesions of this malignancy. We addressed the role of inflammatory stimuli in the release of PCa-specific, tumor-derived soluble factors (PCa-TDSFs) already reported to be mediators of PCa morbidity, such as indoleamine 2,3-dioxygenase (IDO) and interleukin (IL)-6. Inflammation-driven production and functions of PCa-TDFSs were tested "in vitro" by stimulating established cell lines (CA-HPV-10 and PC3) with IFN-γ or TNF-α. Expression of genes encoding IDO, IL-6, IFN-γ, TNF-α, and their receptors was investigated in tumor tissues of PCa patients undergoing radical prostatectomy, in comparison with benign prostatic hyperplasia (BPH) specimens. IFN-γ and TNF-α-treatment resulted in the induction of IDO and IL-6 gene expression and release in established cell lines, suggesting that the elicitation of PCa-TDSFs by these cytokines might contribute to progression of cancer into an untreatable phenotype. An analysis based on timing of biochemical recurrence revealed the prognostic value of IDO but not IL-6 gene expression in predicting recurrence-free survival in patients (RFS) with PCa. In addition, a urine-based mRNA biomarker study revealed the diagnostic potential of IDO gene expression in urines of men at risk of PCa development.

MHC-peptide specificity and T-cell epitope mapping: where immunotherapy starts.

The evaluation and characterization of epitope-specific human leukocyte antigen (HLA)-restricted memory T-cell reactivity is an important step for the development of preventive vaccines and peptide-based immunotherapies for viral and tumor diseases. The past decade has witnessed the use of HLA-restricted peptides as tools to activate strong immune responses of naïve or memory T cells specifically. This has fuelled an active search for methodological approaches focusing on HLA and peptide associations. Here, we outline new perspective on the emerging opportunity of evaluating HLA and peptide restriction by using novel approaches, such as quantitative real-time PCR, that can identify epitope specificities that are potentially useful in clinical settings.

Complementary techniques: validation of gene expression data by quantitative real time PCR.

Microarray technology can be considered the most powerful tool for screening gene expression profiles of biological samples. After data mining, results need to be validated with highly reliable biotechniques allowing for precise quantitation of transcriptional abundance of identified genes. Quantitative real time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Currently, qrt-PCR is considered by most experts the most appropriate method to confirm or confute microarray-generated data. The knowledge of the biochemical principles underlying qrt-PCR as well as some related technical issues must be beard in mind when using this biotechnology.

Identification of HLA-A33-restricted CMV pp65 epitopes as common targets for CD8(+) CMV-specific cytotoxic T lymphocytes.

OBJECTIVE: To identify an immunodominant HLA-A33-restricted epitope within the CMV matrix phosphoprotein 65 (pp65) that could be used to elicit CMV-specific CTLs. METHODS: A computer algorithm was used to identify pp65 peptides that were expected to bind to HLA-A33. The peptides were screened for their ability to reactivate memory T lymphocytes from CMV-seropositive HLA-A33 donors by using quantitative real-time RT-PCR. The most promising peptides were then tested for their ability to expand a CD8(+) population of HLA-A33 CTLs that produced interferon-gamma (IFN-gamma) and were cytotoxic to either peptide-loaded or CMV-infected target cells. RESULTS: Sixteen out of 250 peptides were selected using a computer algorithm and peptide stimulation by 8 out of the 16 induced a significant quantity of IFN-gamma mRNA transcript from CMV-seropositive HLA-A33 peripheral blood mononuclear cells. All the eight peptides induced consistent T-cell reactivation. One specifically, the peptide pp65(91-100) (SVNVHNPTGR), proved to be more active. T cells in vitro sensitized for 2 or 3 weeks with pp65(91-100) were cytotoxic to both HLA-A33 peptide-loaded and CMV-infected target cells. CONCLUSIONS: CMV pp65(91-100) is a new HLA-A33-restricted peptide that broadens the list of antigenic determinants that can be used for CMV adoptive immunotherapy.

Implication of vascular endothelial growth factor A and C in revealing diagnostic lymphangiogenic markers in node-positive bladder cancer.

Several lymphangiogenic factors, such as vascular endothelial growth factors (VEGFs), have been found to drive the development of lymphatic metastasis in bladder cancer (BCa).Here, we have analyzed the gene expression of lymphangiogenic factors in tissue specimens from 12 non-muscle invasive bladder cancers (NMIBC) and 11 muscle invasive bladder cancers (MIBC), considering tumor and tumor-adjacent normal bladder areas obtained from the same organs. We then compared the results observed in patients with those obtained after treating human primary bladder microvascular endothelial cells (MEC) with either direct stimulation with VEGF-A or VEGF-C or by co-culturing (trans-well assay) MEC with bladder cancer cell lines varying in VEGF-A and VEGF-C production based on tumor grade.The genes of three markers of lymphatic endothelial commitment and development (PDPN, LYVE-1 and SLP-76) were significantly overexpressed in tissues of MIBC patients showing positive lymphovascular invasion (LVI+), lymph node metastasis (Ln+) and tumor progression. Their expression was also significantly enhanced either after direct stimulation of MEC by VEGF-A and VEGF-C or in the trans-well assay with each bladder cancer cell line.SLP-76 showed the highest gene expression. Both VEGF-A and VEGF-C also enhanced the expression of SLP-76 protein in MEC. However, a correlation between increase of SLP-76 gene expression and the ability of MEC to migrate could only be seen after induction by VEGF-C.The significant expression of SLP-76 in LVI+/Ln+ progressive MIBC and its overexpression in MEC after VEGF-A and VEGF-C stimulation suggest the need to develop this regulator of developmental lymphangiogenesis as a diagnostic tool in BCa.

Molecular signatures induced by interleukin-2 on peripheral blood mononuclear cells and T cell subsets.

Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear cells (PBMC) from 47 donors of different genetic background induced generalized T cell activation and anti-apoptotic effects. Most effects were dependent upon interactions among immune cells. Specialized functions of CD4 and CD8 T cells were less dependent upon and often dampened by the presence of other PBMC populations. In particular, cytotoxic T cell effector function was variably affected with a component strictly dependent upon the direct stimulation of CD8 T cells in the absence of other PBMC. This observation may provide a roadmap for the interpretation of the discrepant biological activities of rIL-2 observed in distinct pathological conditions or treatment modalities.