is enriched for effector antigen-specific CD8+ T cells. The protective effect of CD8+ T cells is supported by extensive clinical studies on HSCT patients. Analysis of immune reconstitution in both autologous or allogenic stem cell transplantation recipients showed that autologous recipients presented less viral reactivation associated with more vigouros CD8+-effector specific immune response [6]. Conversely, HIV-infected patients, as confirmed by our study, are characterized by the accumulation of pre-TD CTL, which phenomenon has been linked to the lack of control of the immune response on HIV replication in infected patients. It is therefore arguable that in a CD4+-lymphopenic setting, preser- vation of CD8+ effector function may be essential in the prevention of opportunistic infections. Our observations indicate that although overall T CD4+ cell number and function are significantly reduced in ASCT recipients, the CD8+ T cell compartment is relatively well preserved. As animal models have elegantly shown, memory CD8 T cells generated in the presence of CD4 T cells gave the same robust secondary response after transfer into CD4-deficient or CD4- competent hosts [30]. Given the low rate of infectious mortality in ASCT patients, our data suggest that a CD8+ effector function, originally imprinted by a preserved CD4+ T cells system, might provide a clinically meaningful degree of protective immunity even in the absence of actual T-helper supports. In conclusion, our study, even with limited data, confirms the substantial difference in both quantitative and functional regener- ation of CD4+ and CD8+ cells after ASCT, providing strong evidence of a protective role played by differentiated T CD8+ cells in combating opportunistic infections and viral reactivation. We think that this issue together with the reasons of the skewed representation of memory CD4 T cells and cytokine and proliferative defects present in ASCT recipients should be explored more extensively in larger studies. In progressive HIV-infection, interventions aimed at expanding CD8+ T cell activity in the early phase of infection, when CD4 and CD8 T cell function is relatively preserved, may be an effective approach to improving global immunity and preventing opportunistic infections in advanced phase of immune-deficiency. Overall, these findings could prove critical in the clinical follow-up of ASCT recipients and for the design of vaccines in this population. Materials and Methods Study design We performed a cross-sectional, observational, institutional review board-approved study of ASCT recipients and HIV- infected subjects attending the Chair of Infectious Diseases and Tropical Medicine–University of Milan, Italy. Ten age-matched patients with comparable CD4+ lymphopenia (CD4+,300/mL) were enrolled according with the following definition: (i) neoplastic patients (breast cancer and multiple myeloma) (n = 5) receiving ASCT 9 months before observation, without neoplasia’s relapse and free from opportunistic infections; (ii) HIV-infected patients (n = 5) na¨ıve for antiretroviral therapy, stage B3 or C3 according to CDC ‘93 classification. Exclusion criteria were patients with recurrence of tumor or re-introduction of chemotherapy within 9 months after ASCT, patients with primary HIV infection defined as confirmed, recent HIV-1 infection by documented seroconversion illness and incomplete Western blot or a negative test within the previous 6 months, and previous treatment with cytokines or immunomodulants. Comparison of lymphocyte subset phenotype and function was made to a data set obtained from 5 healthy controls aged 32 to 52 years (median 47 years) provided by the Chair of Infectious Disease and Tropical Medicine- University of Milan, Italy. Lymphocytes in ASCT and HIV Patients and healthy controls participating in this study gave written informed consent according to the Declaration of Helsinki. Flow cytometric analysis. Peripheral cells were washed in PBS and stained for 30 min at 4uC in the dark with the following antibodies: CD4 Cy5PE (mouse IgG2a isotype, Caltag); CD8 Cy5PE (mouse IgG2a isotype), CD8 FITC (mouse IgG2a isotype); CD45RA FITC (mouse IgG2b isotype); CCR7 PE (mouse IgG2a isotype, R&D Systems, Minneapolis, MN). Cells used for intracellular molecule staining were washed and fixed in Reagent A solution (FIX & PERM cell permabilization Kits; Caltag) for 10 min at room temperature in the dark. The cells were washed once again in PBS and resuspended in Reagent B (FIX & PERM cell permabilization Kits; Caltag) with cytokine- or perforin/granzyme- specific monoclonal antibodies (IL-2 PE; mouse IgG2a isotype; IFNc FITC; mouse IgG1 isotype; TNF-a PE; mouse IgG2a isotype; granzyme B PE, mouse IgG1 isotype; Caltag Laboratories; perforin FITC; mouse IgG2b isotype; Ancell, Bayport, MN). The cells were then fixed in 1% paraformaldehyde in PBS. Cytometric analyses were performed using an EPICS XL flow cytometer (Beckman-Coulter Inc., Miami, FL) equipped with a single 15 mW argon ion laser operating at 488 nm interfaced with 486 DX2 IBM computer (IBM, Cambridge, UK). For each analysis, 200,000 events were acquired. Green florescence from FITC (FL1) was collected through 525-nm bandpass filter and deep-red fluorescence from Cy5PE (FL4) was collected through 670-nm bandpass filter. Data were collected using linear amplifiers for forward and side scatter and logarithmic amplifiers for FL1 and FL4. Samples were first run using isotype controls or single fluorochrome-stained preparations for color compensation. The EPICS XL Coulter is 4-colour flow cytometer and a CD3 ECD mAb was used to gate T cells. Moreover, to be sure to identify only CD8+ T cells we analysed only CD8bright cells that are CTLs, excluding CD8dim cells which include NK cells. Cytokine and perforin/granzyme production. PBMC were incubated for 18 hours with medium or Staphylococcal b enterotoxin (SEB) (5 mg/ml) (Sigma, St. Louis, MO). CD28 antibody (Clone 37407.111; R&D Systems, Minneapolis, Minnesota, USA) was added during incubation (1 mg/well) to facilitate co-stimulation. For cytokine and peforin/granzyme analyses 10 mg/ml Brefeldin A (Sigma, St. Louis, Missouri, USA) were added to the cell cultures during the last 6h of stimulation to block protein secretion. Analyses were performed on freshly collected cells. SEB concentration utilized in this study was tested to not induce cell death after overnight incubation. However, cells were analysed for vitality (blue trypan exclusion method) before ICC staining procedure. Proliferative tests. PBMCs (36105) were placed in round- bottom wells of a microtiter culture plate (COSTAR, Cambridge, MA) in a final volume of 0,2 ml in the presence/absence of influenza virus vaccine prepared with a mixture of A/Taiwan, A/ Shangai, and B/Victoria (FLU) (24 mg/ml); phytoemoagglutin (PHA) (2,5 mg/ml) (Sigma, St. Louis, MO); or Candida Albicans antigen (CAN) (1,6 mg/ml) (Green Laboratories, USA). Three replicate cultures were performed for each stimulation. Pooled human plasma (10%) were added to each well. We stimulated cells for 3 days to analyze lymphoproliferative responses to PHA and for 6 days to FLU vaccine prep and Candida albicans. After incubation at 37uC and 7% CO2, cultures were pulsed with 1 mCi of [3H]-thymidine and harvested 18 h later. Incorporated [3H]thymidine was measured with a liquid scintil- lation beta counter. Results were expressed as mean counts per minute. The stimulation index represents the ratio of mean counts obtained in the presence of antigen to mean counts obtained without antigen. PLoS ONE | www.plosone.org 7 October 2008 | Volume 3 | Issue 10 | e3616