The T cell compartment is phenotypically and functionally heterogeneous; subsets of naive and memory cells have different functional properties, and also differ with respect to homeostatic potential and the ability to persist in vivo. Human stem cell memory T (T(SCM)) cells, which possess superior immune reconstitution and antitumor response capabilities, can be identified by polychromatic flow cytometry on the basis of the simultaneous expression of several naive markers together with the memory marker CD95. We describe here a protocol based on the minimum set of markers required for optimal identification of human and nonhuman primate (NHP) T(SCM) cells with commonly available flow cytometers. By using flow sorters, T(SCM) cells can thereby be isolated efficiently at high yield and purity. With the use of the 5.5-h isolation procedure, depending on the number of cells needed, the sorting procedure can last for 2-15 h. We also indicate multiple strategies for their efficient expansion in vitro at consistent numbers for functional characterization or adoptive transfer experiments.

Lung carcinoma represents the most frequent cause of cancer death worldwide because of tumor metastases. The objective of the current study was to analyze the immunologic response during the progress of lung carcinoma metastasis.
The authors established two tumor cell lines that were derived from primary and metastatic lesions in a patient with lung carcinoma (Patient G603). One cell line (G603L) was established from the primary lesion, and the other cell line (G603AD) was established from a metastatic lesion in the right adrenal gland 7 months after the patient underwent surgery for the primary lesion. Autologous regional lymph node lymphocytes were stimulated with CD80-transfected G603L cells, then cytotoxic T lymphocytes (CTLs) were induced against both lung carcinoma cell lines.
Both G603L cells and G603AD cells expressed Class I human leukocyte antigen, intracellular cell adhesion molecule 1, and lymphocyte-associated antigen type 3 (LFA-3), but not Fas or Fas ligand on their surfaces. By stimulation with CD80-transfected G603L cells, 2 CTL clones (H2/17 and H2/36) were established from the bulk CTLs. CTL clone H2/17 lysed G603L cells but not G603AD cells, suggesting that the antigen recognized by CTL clone H2/17 was abrogated during the process of metastasis. In contrast, CTL clone H2/36 lysed both G603L cells and G603AD cells, indicating that the antigen recognized by CTL clone H2/36 was maintained in the tumor cells throughout tumor progression.
The results demonstrated the possibility that some tumor-associated antigens may be abrogated during the process of metastasis, although others are maintained. The identification of these antigens will lead to a better understanding of their immunologic role during disease progression in patients with lung carcinoma.

Many solid tumours have been shown to lack expression of either of the immune co-stimulatory molecules CD80 (B7.1) or CD86 (B7.2), which is thought to be one of the ways in which tumours may escape immune recognition. We have examined the surface expression of CD80, CD86, human leucocyte antigen (HLA) class I and II, CD11a, CD54, and CD58 on the blast cells from patients with acute myeloid leukaemia (AML) at presentation. CD80 was only rarely expressed on AML blasts and, in those leukaemic cells expressing CD80, the level of expression was low. In contrast, expression of CD86 was detected on the AML blasts in more than half of the samples tested and, in some cases, the level of expression was equivalent to that of mature monocytes and activated B lymphocytes. The percentage of leukaemic blasts expressing CD86 was higher in the M4 and M5 French-American-British (FAB) types, and expression of CD11a and HLA class II was higher in the M4 FAB type. There was no difference in expression of CD80, CD54, CD58, or HLA Class I between different FAB subgroups. There was no significant difference in duration of first remission with expression of CD80, CD86, CD11a, CD54 or HLA class II. However, when expression of CD80 and CD86 were considered together, a significantly longer duration of remission was found. We suggest that these molecules may play a role in immunosurveillance, resulting in prolonged remission in some patients treated for AML.

Childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, collected from bone marrow (BM) at diagnosis, were cultured, after thawing, on allogeneic human bone marrow stroma (HBMS) for 48 h in the presence of a soluble trimeric CD40 ligand (stCD40L) molecule. HBMS maintained leukemic cells viability in all tested cases (mean viability 85%). Under these culture conditions we noticed upregulation or de novo expression of costimulatory molecules CD40, CD80 (B7-1) and CD86 (B7-2) in 22/22, 15/23 and 21/23 cases, respectively. Upregulation, in terms of fluorescence intensity, was also observed in the expression of MHC I, MHC II, CD54 (ICAM 1) and CD58 (LFA 3) molecules. HBMS alone, although to a lesser extent, was able to induce modulation of these molecules, but not CD80, in a similar proportion of cases. Neither stCD40L nor HBMS induced modulation of CD10 and CD34 molecules. Moreover, in 4/4 tested cases, stCD40L-stimulated ALL cells were able to induce allogeneic T cells proliferation. To evaluate whether leukemia-reactive T cells were detectable in the BM of ALL patients at diagnosis, stCD40L-stimulated ALL cells were co-cultured with autologous T cells (ratio 1:1), isolated from BM at diagnosis, for 4 days and a 24 h ELISPOT assay was applied to detect the presence of interferon-gamma (IFN-gamma)-producing cells. In four of seven cases IFN-gamma-producing cells were detected with frequencies of 1/900, 1/1560, 1/2150 and 1/1575 autologous T cells. These data confirm that stCD40L exposure can activate the antigen-presenting cell (APC) capacity of BCP-ALL cells cultured on HBMS and that ELISPOT assay can be used to measure the frequency of leukemia-reactive autologous T cells in the BM of ALL patients even after short-term culture with stCD40L-stimulated ALL cells.

Aluminum adjuvants are widely used in human vaccines based on their ability to enhance antibody production. However, the mechanisms underlying these effects remain unknown. In the present study we assessed the direct in vitro effect of aluminum hydroxide on human peripheral blood monocytes, specifically with regard to its impact on the phenotype and functional properties of this cell population. Our results revealed significant changes in the accessory properties of monocytes following short-term exposure of cultured cells to aluminum hydroxide. Thus, flow cytometry analyses showed an increase in the expression of major histocompatibility complex (MHC) class II, CD40, CD54, CD58, CD83, and CD86 molecules on the monocytes. In addition, many cells in the cultures containing aluminum hydroxide acquired typical dendritic morphology. Increased synthesis of interleukin-4 (IL-4) mRNA, but not gamma interferon mRNA, was also noted after exposure to aluminum hydroxide. The increase in cell surface expression of MHC class II did not occur in the presence of neutralizing IL-4 antibody or in cultures of highly purified monocytes or CD4-depleted mononuclear cells. Our findings suggest that aluminum hydroxide directly stimulates monocytes to produce proinflammatory cytokines activating T cells. Activated Th2 cells release IL-4, which in turn can induce an increase in the expression of MHC class II molecules on monocytes. The increase in the expression of antigen-presenting and costimulatory molecules leads to enhanced accessory functions of monocytes. These properties of aluminum hydroxide observed in vitro may explain its potent in vivo adjuvant effect.