Lymphocytes in ASCT and HIV Figure 3. Ratios of CD4+ and CD8+ lymphocytes subsets across ASCT recipients (ASCT), HIV-infected patients (HIV) and healthy controls. ASCT recipients and HIV-infected patients displayed similar ratios between CD4+ subpopulations, CD45RA2CCR7+(CM)/CD45RA2CCR72 (EM). CD45RA2CCR72CD8+(EM)/CD45RA+CCR72CD8+(TD) ratio was significantly lower in ASCT recipients compared to HIV+ patients, with no differences in CD45RA2CCR7+ CD8+ (CM)/CD45RA2CCR72 CD8+ (EM) and CD45RA2CCR7+ CD8+ (CM)/ CD45RA+CCR72CD8+ (TD) ratios among the two group of patients. doi:10.1371/journal.pone.0003616.g003 (ASCT 2.27%, HIV 0.45%, HC 2.56%, p,0.05 for HIV versus ASCT and HC), despite comparable IFN-c levels (ASCT 1.22%, HIV 3.05%, HC 4.04%, p = ns). Granzyme B expression To better characterize the functional properties of cytotoxic cells, we additionally assessed by flow cytometry granzyme B expression in CD8+ T cells from ASCT recipients and HIV-infected subjects compared to healthy controls. Our results were consistent with those obtained using CD45RA/CCR7 co-expression, since the amounts of CD8+ T cells expressing granzyme B were many fold higher in ASCT recipients than in HIV-infected patients (ASCT 73.9%, HIV 22.3%, p,0.05) both in basal conditions and after SEB stimulation (ASCT 67.4%,HIV 26.8%, p,0.05) with no significant differences compared to age-matched healthy controls (basal 89.7%, after SEB stimulation 88.7%, p,0.05 for HC versus HIV, p.0.05 for HC versus ASCT)(fig. 4C). These findings correlate nicely with the phenotypic pattern described in ASCT recipients, considering that expression of granzyme B is a specific characteristic of mature effector CD8+ T cells. Lymphocyte proliferation Finally, in order to investigate lymphocyte function, we analyzed lymphocyte proliferation in ASCT-recipients and in HIV-infected patients (fig. 4D). Antigen-induced proliferative responses were depressed in all patients with respect to age- matched healthy controls, reaching statistical significance for FLU stimulation (median stimulation index values: PHA ASCT 1.89, HIV 2.03, HC 2.69; FLU ASCT 1.55,HIV 1.02, HC 1.97, p,0.05 for HC versus HIV, Candida albicans ASCT 1.98,HIV 1.37, HC 1.40). One explanation for these observations could be that lymphoproliferation is primarily a function of T CD4+ cells, the numbers and functional capabilities of which were comparably depressed in both group of patients. Discussion Delayed immune reconstitution remains one of the least understood issues among the many clinical challenges facing ASCT clinicians, and the clinical significance of profound and persistent CD4+ T cells lymphopenia, although often unassociated with opportunistic infections, has yet to be fully defined [27]. In patients with HIV infection, on the other hand, the selective loss of CD4+ T cells apparently triggers profound immunodeficiency, often leading to fatal infection, AIDS-defining illnesses and, ultimately, death [11]. To verify if the differing qualitative aspects of immune response characterizing CD4+-lymphopenic patients subjected to ASCT, with respect to those of HIV-infected patients, could account for the differences in infectious risks between the two groups, we phenotypically and functionally analyzed CD4+ and CD8+ lymphocytes in ASCT or HIV patients with comparable CD4 counts. Our results demonstrate that patients undergoing high dose chemotherapy and ASCT have a persistent, marked deficit in CD4+ T-cell function. And yet, in contrast to HIV-infected subjects, such individuals are characterized by a CD8 T-cell compartment that is phenotipically normal and functionally active. An alteration of the CD4/CD8 ratio, secondary to CD4+ T cells decrease and largely sustained by a loss of CD4+ na¨ıve T cells, has often been described in different cohorts of ASCT recipients [3–5]. Our study confirms the profound loss of na¨ıve CD4+ T cells in patients treated with high-dose chemotherapy and ASCT. Moreover, we show that the remaining CD4+ T compartment is constituted by T cells which have a memory phenotype, but which are skewed toward the later stages of differentiation, a character- istic typical of HIV infection. Central memory and effector CD4+ cells in fact were decreased both in HAART- na¨ıve HIV-infected and in ASCT patients in whom a massive expansion of PLoS ONE | www.plosone.org 5 October 2008 | Volume 3 | Issue 10 | e3616