Purpose: Recent studies of Hodgkin's lymphoma (HL) have suggested that the presence of regulatory T cells in the reactive background may explain the inhibition of the antitumoral host immune response observed in these patients. This study aimed to assess the relevance of regulatory T cells and CTLs present in the background of HL samples in the prognosis of a series of classic HL (cHL) patients.Experimental Design: Expression of granzyme B and TIA-1 (markers for CTL) and FOXP3 (a marker for regulatory T cells) were evaluated independently by immunohistochemistry in tissue microarrays of 257 cHL patients and correlated with patient outcome.Results: The combined influence of the presence of FOXP3 + and TIA-1 + cells distinguished three risk groups of patients with 5-year overall survival of 100%, 88%, and 73%. The presence of a small number of FOXP3 + cells and a high proportion of TIA-1 + cells in the infiltrate represent an independent prognostic factor that negatively influenced event-free survival and disease-free survival in cHL. Compared with the features at diagnosis, relapsed samples tended to have more TIA-1 + cells and a lower proportion of FOXP3 + cells in the reactive background. Conclusions: These data suggest that low infiltration of FOXP3 + cells in conjunction with high infiltration of TIA-1 + cells in cHL may represent biological markers predicting an unfavorable outcome. Moreover, the variation of these markers over the course of the disease implies a possible role for them in the progression of HL cases.

Adenosine deaminase (ADA), a protein whose deficit leads to severe combined immunodeficiency, binds to the cell surface by means of either CD26, A 1 adenosine receptors, or A2B adenosine receptors. The physiological role of these interactions is not well understood. Our results show that by a 3-fold reduction in the EC 50 for the antigen, ADA potentiated T cell proliferation in autologous cocultures with antigen-pulsed immature or mature dendritic cells. Costimulation was not due to the enzymatic activity but to the interaction of ADA-CD26 complexes in T cells with an ADAanchoring protein in dendritic cells. From colocalization studies, it is deduced that ADA colocalizing with adenosine receptors on dendritic cells interact with CD26 expressed on lymphocytes. This costimulatory signal in the immunological synapse leads to a marked increase (3-to 34-fold) in the production of the T helper 1 and proimmflamatory cytokines IFN-␥, TNF-␣, and IL-6. adenosine deaminase ͉ costimulation ͉ immunosynapse A denosine deaminase (ADA; EC 3.5.4.4) an enzyme involved in purine metabolism, catalyzes the hydrolytic deamination of adenosine or 2Ј-deoxyadenosine to inosine or 2Ј-deoxyinosine and ammonia. Congenital defect of ADA causes severe combined immunodeficiency, which is characterized by the absence of functional T and B lymphocytes in affected individuals (1). For many years, ADA was considered to be cytosolic, but it has been found on the cell surface of many cell types; therefore, it can be considered an ectoenzyme. In addition, ecto-ADA has been proposed to have a catalytic-independent function as a costimulatory molecule in lymphocytes (2).So far, two types of surface anchoring proteins for ecto-ADA have been described. The first type, with only one member, is CD26, a multifunctional protein of 110 KDa strongly expressed on epithelial cells (kidney proximal tubules, intestine, and bile duct) and on several types of endothelial cells and fibroblasts and on leukocyte subsets (3-5). The second type of ecto-ADA-binding proteins includes the adenosine receptors (AR) A 1 (A 1 R) (6) and A 2B (A 2B R) (7). The association between ADA and CD26 on the T cell surface has been proposed to have a costimulatory function during T cell antigen receptor-CD3 complex engagement (2). Because CD26 has a short cytoplasmatic tail, it needs partners to transduce the signal. Ishii et al. (8) have described that CD26-mediated signaling occurs through its association with CD45RO. At present, it is not known whether ADA generates a signal when it binds to AR. However, we have previously demonstrated that ADA binding to A 1 R or A 2B R is required for high efficiency affinity binding of the agonist and for efficient agonist-dependent signaling (6, 7).Dendritic cells (DC) are the most potent antigen-presenting cells (APC) specialized in the initiation of immune responses by directing the activation and differentiation of naïve T lymphocytes (9, 10). Immature DC (iDC) reside in most tissues to uptake antigen; they are engaged when exposed to danger ...

Our results demonstrate the existence of two specific patterns in the reactive microenvironment of FL, an immunosurveillance pattern (T lymphocytes and macrophages) and an immune-escape pattern (CD57+ T cells), that were directly associated with the clinicobiologic features of these patients.

Therapeutic immunization with autologous monocyte-derived dendritic cells (DCs) loaded with heat-inactivated autologous human immunodeficiency virus type 1 (HIV-1) in 12 patients with chronic HIV-1 infection who were receiving highly active antiretroviral therapy (HAART) was feasible, safe, and well tolerated. Virus was obtained during an initial interruption of HAART (hereafter, "stop 1") so that DCs could be pulsed. After immunization and a second interruption of HAART (hereafter, "stop 2"), set-point plasma viral load (PVL; 24 weeks after stop 2) decreased > or =0.5 log(10) copies/mL relative to baseline PVL in 4 of 12 patients. We observed a significant lengthening in mean doubling time of PVL rebound and significant decreases in the area under the curve and the mean peak of PVL rebound after stop 2, compared with those after stop 1. This response was associated with changes in HIV-1-specific CD4(+) lymphoproliferative and CD8(+) T cell responses. These changes were not observed in a group of nonimmunized control patients.

Plasma stromal cell-derived factor (SDF)-1 levels, SDF1-3'A polymorphism, and CXCR4(+) T lymphocytes in relation to resistance to human immunodeficiency virus (HIV)-1 infection and its progression were investigated in a study of HIV-positive patients, exposed but uninfected (EU) subjects, and healthy control subjects, all lacking CCR5 Delta 32 homozygosity. SDF1-3'A homozygosity was associated with low plasma SDF-1 levels in uninfected persons and was not related to long-term nonprogression. HIV-1 infection involved increased plasma SDF-1 levels, which were not attributable to any kind of chronic viral infection, because all EU hemophiliacs were hepatitis C virus-positive but had normal SDF-1 levels. High plasma SDF-1 levels and low CXCR4 expression on T lymphocytes was associated with long-term nonprogression, whereas in advancing disease expression of CXCR4 increased, accompanied by a decrease in plasma SDF-1 during the more advanced stages of HIV-1 infection. EU subjects with sexual exposure to HIV-1, but not EU hemophiliacs, showed an underpresentation of SDF1-3'A allele frequency, which was coupled with high plasma SDF-1 levels and low CXCR4 expression.

The present study aimed to describe the general tissular composition of the immune infiltrate observed in Hodgkin's lymphoma (HL) and its possible relationship with clinical and survival prognostic factors. In this retrospective study of 267 HL patients, the relative proportions of infiltrating T lymphocytes (CD4+, CD8+), natural killer cells (CD 56+, CD 57+), cytotoxic cells (Granzyme B+, TIA-1+) and dendritic cells (CD 21+, S-100+) were quantified immunohistochemically with tissue microarray technology. Our results confirm the predominance of CD4 + T lymphocytes in the background of tumoral cells, in addition to a high number of cytotoxic lymphocytes (CD8, CD 57 and TIA-1). Patients with low numbers of infiltrating CD8, CD 56, CD 57+cells and high numbers of Granzyme B and TIA-1+cells presented a significantly unfavourable clinical course (presence of leukocytosis, B symptoms, advanced clinical stage (III/IV), non-responding patients). A reduced infiltration of CD4+T lymphocytes was related with the presence of Epstein - Barr virus. Significantly longer survival times were observed in patients with a high level of infiltrating CD 57, as well as a low level of Granzyme B and TIA-1+cells (log-rank test). When evaluated in a multivariate model, high levels of infiltrating TIA-1 and Granzyme B+cells were shown to be independent prognostic factors that negatively influenced overall survival. The presence of TIA-1+cells was found to be the only unfavorable prognostic factor of event-free survival and disease-free survival. The overall detection of tumor-infiltrating cells in HL confirms the importance of cytotoxic T lymphocyte infiltration (Granzyme B and TIA-1+cells) in these patients. Independently of the classical clinical and pathological features, these cells appear to be an unfavourable prognostic factor in HL and, more particularly, the presence of cytotoxic TIA-1+cells.

Tissue microarray technology and immunohistochemical techniques have become a routine and indispensable tool for current anatomical pathology diagnosis. However, manual quantification by eye is relatively slow and subjective, and the use of digital image analysis software to extract information of immunostained specimens is an area of ongoing research, especially when the immunohistochemical signals have different localization in the cells (nuclear, membrane, cytoplasm). To minimize critical aspects of manual quantitative data acquisition, we generated semiautomated image-processing steps for the quantification of individual stained cells with immunohistochemical staining of different subcellular location. The precision of these macros was evaluated in 196 digital colour images of different Hodgkin lymphoma biopsies stained for different nuclear (Ki67, p53), cytoplasmic (TIA-1, CD68) and membrane markers (CD4, CD8, CD56, HLA-Dr). Semi-automated counts were compared to those obtained manually by three separate observers. Paired t -tests demonstrated significant differences between intra-and inter-observer measurements, with more substantial variability when the cellular density of the digital images was > 100 positive cells/image. Overall, variability was more pronounced for intra-observer than for inter-observer comparisons, especially for cytoplasmic and membrane staining patterns ( P < 0.0001 and P = 0.050). The comparison between the semi-automated and manual microscopic measurement methods indicates significantly lower variability in the results yielded by the former method. Our semi-automated computerized method eliminates the major causes of observer variability and may be considered a valid alternative to manual microscopic quantification for diagnostic, prognostic and therapeutic purposes.

Manual quantification of immunohistochemically stained nuclear markers is still laborious and subjective and the use of computerized systems for digital image analysis have not yet resolved the problems of nuclear clustering. In this study, we designed a new automatic procedure for quantifying various immunohistochemical nuclear markers with variable clustering complexity. This procedure consisted of two combined macros. The first, developed with a commercial software, enabled the analysis of the digital images using color and morphological segmentation including a masking process. All information extracted with this first macro was automatically exported to an Excel datasheet, where a second macro composed of four different algorithms analyzed all the information and calculated the definitive number of positive nuclei for each image. One hundred and eighteen images with different levels of clustering complexity was analyzed and compared with the manual quantification obtained by a trained observer. Statistical analysis indicated a great reliability (intra-class correlation coefficient > 0.950) and no significant differences between the two methods. Bland-Altman plot and Kaplan-Meier curves indicated that the results of both methods were concordant around 90% of analyzed images. In conclusion, this new automated procedure is an objective, faster and reproducible method that has an excellent level of accuracy, even with digital images with a high complexity.