CD1d expression on B-precursor acute lymphoblastic leukemia subsets with poor prognosis.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Although therapeutical advances have been achieved, some ALL subgroups still fare poorly. CD1d is a monomorphic molecule that provides a suitable target for immunotherapy in view of the characterization of a glycolipid, alpha-galactosylceramide (alpha-GalCer), capable of being presented to CD1d-restricted T cells with cytotoxic potential. We investigated CD1d expression in 80 pediatric B-cell precursor (BCP) ALL cases defined according to immunophenotype, cytogenetic features and age at onset. CD1d was detected on ALL cells in 15% of the patients. CD1d+ ALLs were significantly associated with infant leukemia, pro-B phenotype and mixed-lineage leukemia (MLL)/AF4 gene rearrangement. Accordingly, overall survival of patients with CD1d+ ALL was significantly shorter. CD1d+ leukemic blasts were able to present alpha-GalCer via CD1d to cytotoxic CD1d-restricted T cells, which induced apoptosis of ALL cells that was inhibited by mAb to CD1d. CD1d+ blasts loaded with alpha-GalCer elicited cytokine secretion by CD1d-restricted T cells. Analysis of bone marrow (BM) cells derived from normal donors revealed that CD19+/CD1d+ cells were mostly mature B lymphocytes. However, a minority of BCPs expressed CD1d. Thus, expression of CD1d in ALL cases heralds an adverse prognosis but may provide a therapeutic tool.

CD85/LIR-1/ILT2 and CD152 (cytotoxic T lymphocyte antigen 4) inhibitory molecules down-regulate the cytolytic activity of human CD4+ T-cell clones specific for Mycobacterium tuberculosis.

Antigen-specific cytolytic CD4+ T lymphocytes control Mycobacterium tuberculosis infection by secreting cytokines and by killing macrophages that have phagocytosed the pathogen. However, lysis of the latter cells promotes microbial dissemination, and other macrophages engulf the released bacteria. Subsequently, CD4+ T-cell-mediated killing of macrophages goes on, and this persistent process may hamper control of infection, unless regulatory mechanisms maintain a subtle balance between lysis of macrophages by cytolytic CD4+ cells and activation of cytolytic CD4+ cells by infected macrophages. We asked whether inhibitory molecules expressed by CD4+ cytolytic T lymphocytes could play a role in such a balance. To this end, human CD4+ T-cell clones specific for M. tuberculosis were produced that displayed an autologous major histocompatibility complex class II-restricted lytic ability against purified protein derivative (PPD)-pulsed antigen-presenting cells. All T-cell clones expressed CD152 (cytotoxic T-lymphocyte antigen 4 [CTLA-4]) and CD85/leukocyte immunoglobulin-like receptor 1 (LIR-1)/immunoglobulin-like transcript 2 (ILT2) inhibitory receptors, but not CD94 and the killer inhibitory receptor (or killer immunoglobulin-like receptor [KIR]) p58.2. CD3-mediated activation of the clones was inhibited in a redirected killing assay in which CD152 and CD85/LIR-1/ILT2 were cross-linked. Specific antigen-mediated proliferation of the clones was also sharply reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked by specific monoclonal antibody (MAb) followed by goat anti-mouse antiserum. In contrast, blockade of the receptors by specific MAb only increased their proliferation. Production of interleukin 2 (IL-2) and gamma interferon (IFN-gamma) by the T-cell clones was also strongly reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked. The lytic activity of the T-cell clones against PPD-pulsed autologous monocytes or Epstein-Barr virus-activated B cells was increased by blockade and decreased by cross-linking of the receptors. These results indicate that CD152 and CD85/LIR-1/ILT2 play a role in the regulation of the antigen-specific activity of CD4+ cytolytic T lymphocytes against PPD-presenting cells.

Adhesion molecule expression, clinical features and therapy outcome in childhood acute lymphoblastic leukemia.

In view of the relevance of adhesion molecule expression for the mechanisms of homing, trafficking and spreading of malignant cells, we have investigated the expression of surface adhesion molecules in lymphoblasts from 57 acute lymphoblastic leukemia (ALL) cases and tried to correlate the adhesive phenotype with immunological typing, prognostic factors at diagnosis and clinical follow-up. Blasts from all cases expressed adhesion molecules at high rates. Beta1 integrin chain (CD18) was consistently found on blasts from most ALL cases: among integrins of the beta2 family. LFA-1 was detected in 58% of cases, in the virtual absence of other alpha chains. CD54 and CD58 were expressed in variable proportions by ALL blasts and CD44 was detected in the majority of the malignant cells, whereas the CD62L selectin was only present in 24% of cases. B-lineage ALL's displayed similar adhesion molecule phenotypes irrespective of maturational stages of the leukemic cells. We found a significantly reduced expression of beta2 alphaL integrins in the hybrid ALL cases (CD13 and/or CD33 positive). However, these cases did not show differences in clinical presentation and behaviour in comparison with patients of other groups. We did not find a significant correlation between adhesion molecule expression and well established risk factors (age, white blood cell count, central nervous system involvement, chromosomal abnormalities), with the exception of splenomegaly, that was significantly associated with CD18 expression. In the follow-up, no evidence of significant correlation between adhesive phenotype and adverse events such as leukemic relapse and death was found. In conclusion, although expression of adhesion molecules on lymphoblasts confirms the phenotypic heterogeneity of ALL, it appears that this is not relevant for the clinical aspects of the disease and for prognosis.

Cytokine gene expression and T-cell proliferative responses in lymph node mononuclear cells from children with early stage human immunodeficiency virus infection.

BACKGROUND AND OBJECTIVES: The immunologic events taking place in secondary lymphoid tissue from children with early stage human immunodeficiency virus (HIV) infection are poorly understood. The aim of this study was to investigate cytokine gene expression and proliferative responses in lymph node (LN) biopsies from five children with early stage HIV infection, in the context of LN morphology and viral load. DESIGN AND METHODS: The design of the study was approved by the local Ethical Committee. Cytokine gene expression was studied in LN biopsies and in paired peripheral blood (PB) samples from HIV-infected children by reverse transcriptase-polymerase chain reaction. T-cell proliferation was assessed by 3H-thymidine incorporation. Viral burden in germinal centers was assessed by video densitometric analysis following immunohistochemical staining for HIV p24. RESULTS: Interleukin (IL)-2, IL-4 and IL-5 mRNA were not detected in any LN or PB sample from HIV-infected children. Interferon (IFN)-gamma mRNA was found only in CD8+ cells. IL-12 p35, IL-10, transforming growth factor-(TGF)-beta1, regulated on activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and IL-16 transcripts were detected in all samples. Proliferation of LN and PB mononuclear cells to polyclonal mitogens and soluble (recall and HIV-related) antigens was impaired as compared with the responses in a group of age-matched healthy controls. INTERPRETATION AND CONCLUSIONS: Changes in cytokine gene expression and T-cell proliferative responses are already detectable in lymph nodes from HIV-infected children at an early stage of disease.

The CD85/LIR-1/ILT2 inhibitory receptor is expressed by all human T lymphocytes and down-regulates their functions.

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3+ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4+ and CD8+ clones, and it down-regulates Ag recognition by CD8+ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca2+ mobilization.

A video densitometric analysis of viral burden and follicular dendritic cell damage in lymph nodes in the latency phase of HIV infection.

BACKGROUND: The aim of this study was to collect quantitative data on damage to follicular dendritic cells (FDC) and on the structure of germinal centers (GC) in the early phase of HIV infection. METHODS: Lymph node sections from 10 HIV(+) subjects and from 5 HIV(-) control subjects were stained by immunohistochemistry for CD21, an FDC marker; gp24, to assess the HIV load; and IgM, to measure antibodies within the GC. RESULTS: The volume fraction (V(area)) and the logarithm of the inverse gray value (ArLIGV) of CD21 areas showed a highly significant decrease in HIV(+) specimens. The mean ArLIGV values ranged between 0.0916 +/- 0.01 and 0.3826 +/- 0.11 versus 0.6856 +/- 0.19 on average in controls (P < 0.001 for both). Six of 10 HIV(+) specimens were positive for gp24. Staining was limited to GC and showed a distribution pattern similar to that of CD21. CONCLUSIONS: FDC already undergo considerable damage during the latency phase of HIV infection. The pattern of CD21 indicates that, although FDC decrease in number, the staining intensity of positive cells is basically preserved. Video densitometric analysis, an approach requiring a strict standardized protocol, may help monitor disease course and evaluate response to therapy by quantifying viral burden and lymph node damage.

Molecules that inhibit T-cell functions: cytochemical localization and shuttling.

Adaptive immune responses to antigens are mediated by specific receptors expressed on B cells (BCR's) and T cells (TCR's). Effector cells and memory cells are produced following a proliferative wave that accounts for clonal expansion. If not down-regulated, clonal expansion might lead to uncontrolled lymphoproliferation that would be harmful for the organism. Several mechanisms that account for the down-sizing of activated lymphocyte clones are briefly reviewed here. We next consider in detail one such mechanism that deals with the functional characterization and the immunocytochemical localization of two T-cell inhibitory molecules, namely the Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) and the HP-F1 antigen, both present in all T lymphocytes. CTLA-4 and HP-F1 inhibit CD4+ T-helper cell proliferation and the lytic ability of CD8+ T-cytotoxic cells in non-specific and in antigen-specific cytolytic assays. Interestingly, a clonal distribution exists as for the ability of CTLA-4 and HP-F1 to inhibit T-cell functions. In resting and activated T cells, both molecules are largely confined in the endosomal compartment, as shown by immunofluorescence analyses. However, upon interaction of T cells with Antigen-Presenting Cells (APC's) or with target cells that must be killed, CTLA-4 molecules are transported to the plasma membrane, at the site of cell-to-cell contact where, following interaction with ligands, they trigger inhibitory signals.

Cell activation via CD44 occurs in advanced stages of squamous cell carcinogenesis.

Squamous cell carcinoma (SCC) derives from dysplastic or metaplastic stratified epithelia. The process of squamous cell carcinogenesis has been investigated for the potential role of the adhesion molecule CD44, whose standard form (CD44s) and isoforms generated by alternative splicing of variant exons are known to display altered expression during tumorigenesis in other systems. We have utilized an in vitro correlate of squamous cell carcinogenesis, in which progression stages from normal squamous epithelium to dysplastic lesions and to SCC are represented by primary cultures of normal keratinocytes, by human papilloma virus-immortalized keratinocytes (UP) and by HPVimmortalized/v-Ha-ras transfected tumorigenic keratinocytes (UPR). We investigated expression of CD44 and of variant isoforms, from mRNA to intracellular and surface protein levels, and found no relationship between expression of CD44 and stages of squamous cell carcinogenesis. However, when the function of CD44 was analyzed as Ca(2+) mobilization ability upon monoclonal antibody binding and crosslinking, signal transduction via CD44 was found only for the neoplastic stage (UPR cells). Ca(2+) mobilization was completely independent of density of surface CD44. We have performed similar analyses in an in vitro model of SCC in which four squamous tumor cell lines and UPR cells were sorted according to increasing resistance to external cytotoxic stimuli, i.e. starving conditions, treatment with the retinoid N-(4-hydroxyphenyl)retinamide and cytolytic activity of effector lymphokine-activated killer cells. No relationship between expression of CD44 and level of cell resistance against external cell death-inducing stimuli was found, while CD44-mediated Ca(2+) mobilization ability was restricted to the highly resistant tumor cell lines. Our results indicate that the role(s) of CD44 in squamous cell proliferative disorders can be evinced from the functional features of the molecule, rather than from its phenotypic repertoire.

Anatomy of the immune system: facts and problems.

In the introductory section of this report, the anatomy of the immune system, from organs and tissues to molecules, will be reviewed briefly. Cell proliferation and differentiation in the central lymphoid organs (thymus and bone marrow) yield a repertoire of T- and B-cell clones that seed into peripheral lymphoid organs (spleen, lymph nodes and Mucosa-Associated Lymphoid Tissue, MALT), where humoral and cell-mediated antigen-specific immune responses occur. The stringent process of clonal selection in the central lymphoid organs implies deletion of inappropriate cells via apoptosis. In the peripheral lymphoid organs, the potential of unlimited activation and expansion of lymphocytes in response to antigens is primarily regulated by apoptosis and anergy. These events, on the one hand, are relevant to prevent autoimmunity and lymphoproliferative disorders; on the other hand, clonal deletion and anergy provide a detrimental escape to immune recognition of malignant cells. Two major inhibitory mechanisms of the immune response have emerged recently. One is linked to the existence of bona fide suppressor cells and cytokines; the other relies on the existence of inhibitory molecules expressed by T, B and NK cells, as well as by other leukocytes. In the studies herein reported, emphasis will be given to surface membrane molecules that down-regulate T-cell-mediated immune responses. These molecules control interactions between T cells and antigen presenting cells (APC's) or target (virus-infected or mutated) cells that have to be killed. Two sets of molecules exist that either upregulate (coactivation molecules) or down-regulate (inhibitory molecules) T-cell mediated responses. The latter aspect of the immune regulation, i.e. molecules that limit the expansion of T-cell clones following specific recognition of antigens will be considered in depth. Two inhibitory molecules, CD152 (CTLA-4) and CD85/LIR-1/ILT2 are expressed in all T cells, being largely confined within intracellular compartments of these lymphocytes when they are in a resting state, but ready to be shuttled to and from the plasma membrane when cells are activated following encounter with antigen. Membrane expression of the two inhibitory molecules is transient and is regulated by an internalization process directed to endosomal compartments and to receptor degradation and/or recycling. CTLA-4 and CD85/LIR-1/ILT2 play a pivotal role in T-cell homeostasis that follows any cell-mediated immune response; their localization and functional role will be thoroughly analyzed. In the last part of this study a major question will be faced, i.e. is the containment of the possibly unlimited expansion of the immune system due to a blockade of the cell cycle? Or, else, could be apoptosis the sole mechanism responsible? Experimental data in support of the latter contention will be provided.

Ultrastructural and functional studies of the interaction between IL-12 and IL-2 for the generation of lymphokine-activated killer cells.

IL-12 promotes generation of LAK activity in short-term-cultured NK cells, but information on the structure and function of IL-12-induced LAK cells is not yet available. The latter issues have been here investigated with emphasis on interactions between IL-12 and IL-2. Peripheral blood mononuclear cells (MNC) exposed to IL-12 for 5-7 days displayed a decrease in the amount and density of the matrix of large granular lymphocyte (LGL)-associated granules. In cells cultured with IL-12 and IL-2 for 5-7 days, empty vacuoles were predominant and the electron-dense matrix was scanty. In MNC incubated with IL-2 for 5-7 days, most granules were loaded with electron-dense matrix. IL-12 and IL-2 displayed an additive effect on LAK cell cytotoxicity until approximately 48 h in culture which was followed by a sharp decline. Immunocytochemical and biochemical studies demonstrated that MNC cultured for 5-7 days with IL-12 and IL-2 displayed downregulated perforin expression and upregulated granzyme B expression. Fas ligand expression was virtually undetectable in MNC cultured for 5-7 days with or without cytokines. It appears that perforin downregulation plays a major role in the reduced cytotoxicity of MNC cultured with IL-12 and IL-2 for 5-7 days.

IL4 production and increased CD30 expression by a unique CD8+ T-cell subset in B-cell chronic lymphocytic leukaemia.

Phenotypic and functional abnormalities within the residual non-B-cell compartment of B-cell chronic lymphocytic leukaemia (CLL) suggest an interaction between tumour cells and host immune effectors. To explore the possibility of a polarized Th1/Th2 response we have studied CD30 antigen expression and the pattern of cytokine production by purified CLL T cells. Activated T cells from CLL patients showed a significant increase in the expression of CD30 compared to normal controls. Accordingly, high levels of soluble CD30 were detected in supernatants from activated T-cell cultures, as well as in CLL serum samples. Messenger RNA for IL4 was found in both resting and, to a greater extent, in activated CLL T lymphocytes. The latter cells were also capable of releasing IL4. Three-colour immunofluorescence analyses revealed a strong CD30 expression in the CD3+/CD8+/CD28- large granular lymphocyte subset, which is considerably expanded in CLL. Production of IL4, as well as expression and release of CD30 by these T cells, was conclusively demonstrated at the clonal level. These findings document an expansion of a peculiar subset of 'Th2-like' cells in CLL, with an increased IL4 production and CD30 expression and release, that are likely to contribute to both the B-cell accumulation and immune-defects characteristic of this disease.

CTLA-4 (CD152) inhibits the specific lysis mediated by human cytolytic T lymphocytes in a clonally distributed fashion.

Since the functional outcome of effector T lymphocytes depends on a balance between activatory and inhibitory receptors, we studied the ability of CTLA-4 (CD152) to inhibit the cytolytic function of CTL. In 22 TCR alpha/beta+ CD3+ 8+ CTL clones, activation induced by anti-CD3, anti-CD28, or anti-CD2 mAb was inhibited by anti-CD152 mAb in a redirected killing assay. In eight clones inhibition was >40%, in 10 it ranged between 20-40%, and in four it was <20%. This suggests the existence of a clonal heterogeneity as well as for the ability of CTLA-4 to inhibit CD3/TCR-, CD28-, or CD2-mediated CTL activation. To support further this contention, we used an experimental model based upon Ag-specific CTL. Eight Ag-specific T cell clones that lyse autologous EBV-infected B lymphocytes, but are unable to lyse allogeneic EBV-infected B cell lines, were used in a cytolytic assay in which anti-CD152 mAb or soluble recombinant receptor (i.e., CTLA-4 Ig) were included. In this system, at variance from the redirected killing assay, cross-linking of surface molecules by mAb does not occur. Thus, addition of anti-CD152 mAb or of CTLA-4 Ig and anti-CD80/CD86 mAb to the assay should result in a blockade of receptor/ligand interactions. As a consequence, inhibition of a negative signal, such as that delivered via CD152, should enhance lysis. A >40% increment of target cell lysis was achieved in three of eight clones studied. Since it is not equally shared by all CTL clones, this feature also appears to be clonally distributed.

Lymphoid complement of the human salivary glands: function and pathology.

In normal human salivary glands the Duct-Associated Lymphoid Tissue (DALT) is poorly developed. In contrast, in the course of autoimmune disorders, typified by Sjögren's syndrome (SS), organized lymphoid accumulations are formed around the ducts. B cell-dependent zones with secondary follicles and T cell-dependent zones with HEV are detected in these lymphoid structures. In addition, the duct epithelium is infiltrated by abundant lymphocytes. A persistent antigenic stimulation may lead to development of B-cell Mucosa-Associated Lymphoid Tissue (MALT) lymphomas that, in low-grade cases, maintain the lobular organization of normal and of SS salivary glands.

Functional and clinical relevance of CD44 variant isoform expression on B-cell chronic lymphocytic leukemia cells.

BACKGROUND AND OBJECTIVE: Recent studies have shown that expression of adhesion molecules of the Ig superfamily, of integrins and of selectins allows definition of high vs low risk B-cell chronic lymphocytic leukemia (B-CLL). The proteoglycan CD44 is an adhesion molecule that may be expressed as a standard form of 85-95 KD or as several variant isoforms. The presence of certain CD44 variant (v) isoforms on neoplastic cells indicates poor prognosis in epithelial and lymphoid malignancies, as it is associated with tumor progression and metastasis. DESIGN AND METHODS: The expression of CD44 v3, 4, 5, 6, 7, 9 and 10 was analyzed in cells from 85 B-CLL patients. Indirect immunofluorescence and flow cytometry were used to identify CD44v. Functional studies were performed by analysis of adhesion to hyaluronate (HA), one CD44 ligand, and HA-induced Ca2+ influx. A variety of statistical methods were used to define phenotypic and functional differences between the various clones, to calculate survival curves, and for multivariate analyses. RESULTS: In 17/85 B-CLL (20%), one or more CD44v were detectable by indirect immunofluorescence, whereas in 68/85 cases (80%) this technique yielded negative results. However, moAb "mixes" against CD44v and patching of surface molecules on B-CLL cells have shown that all B-CLL clones express CD44v. This has been confirmed by Western blot in a number of cases. Thus, two groups of patients whose cells bear CD44v at high or low density, are distinguished. Functions of the two clonotypes were investigated, namely their adhesion to a CD44 ligand and hyaluronate (HA), and effect on HA-induced Ca2+ influx. Cells expressing high density CD44v adhere to HA-coated substrates more efficiently than cells with low density CD44v. In all clones, HA-signaling via CD44 yields Ca2+ influx. This indicates that CD44 mediates activatory signals following interaction with the ligand. INTERPRETATION AND CONCLUSIONS: The clinical relevance of these findings has been ascertained. The 17/85 cases whose cells bore high density CD44v had significantly worse prognostic features than those of patients with low density CD44v, namely more advanced disease stage, LDT < 12 months and therapy requirement. Moreover, the median survival in the former group of patients was < 5 years as opposed to > 12 years in the latter. Therefore, analysis of CD44v expression provides indications of biological and clinical relevance also in low grade lymphoproliferative disorders.

HIV is trapped and masked in the cytoplasm of lymph node follicular dendritic cells.

To gain further insight into the pathogenesis of human immunodeficiency virus (HIV) infection, lymph nodes from seven asymptomatic HIV+ subjects were analyzed during the latent phase of disease. Both ultrastructural and immunohistochemical analyses revealed that, in all of the cases, plasma cells producing IgM/gamma were present in germinal centers. Secreted immunoglobulins formed extracellular deposits mimicking the follicular dendritic cell network. Immunoglobulin produced by germinal center plasma cells are specific for HIV because they bind the HIV env protein gp 120. Plasma cells producing antibodies with the same specificity were also abundant in the extrafollicular regions of lymph nodes. During the latent phase of infection, the virus largely accumulates within the germinal centers. Therefore, extracellular immunoglobulin may form immune complexes, as shown by the presence of HIV-specific antibodies, HIV particles, and complement components C3c, C3d, and C1q in the interdendritic spaces. When the ultrastructural localization of HIV in germinal centers was analyzed, abundant virus particles were found in the interdendritic spaces. In addition to this extracellular localization of HIV, receptor-mediated endocytosis of viral particles by follicular dendritic cells was observed. Complete HIV particles were found within the endosomal compartment of the follicular dendritic cells and, as complete viral particles, free in the cytoplasm, indicating that the virus may escape from the endocytic compartment. As the virus is abundant in the cytoplasm, this event leads to formation of a hidden reservoir within follicular dendritic cells. In this location, HIV escapes recognition by cytotoxic T lymphocytes. In contrast, virus budding indicating a productive infection of follicular dendritic cells that would render them susceptible to T-cell-mediated lysis has been seldom observed.

Increased serum levels of soluble CD44 standard, but not of variant isoforms v5 and v6, in B cell chronic lymphocytic leukemia.

The CD44 cell surface proteoglycan participates in a variety of functions including lymphohematopoiesis, lymphocyte homing and tumor metastasis. In addition to the standard form (CD44st), a large family of variant isoforms (CD44v) is generated by alternative splicing of a single gene. Certain CD44v (v5 and V6) are upregulated in the course of neoplastic progression and reflect the metastatic potential of tumor cells. CD44 v6 is expressed in high-grade non-Hodgkin's lymphoma cells and is released in the serum, thus providing a soluble marker that reflects tumor burden, disease progression and treatment response. Here we show that serum CD44st is elevated in approximately half of B-CLL patients. In contrast, CD44v5 and v6 are detected at normal levels in the large majority of the cases. CD44st serum levels correlate significantly with the number of circulating leukemic B cells and with the levels of another soluble B-CLL marker, beta2-microglobulin. Immunoprecipitation analyses of B-CLL sera allow detection of several high molecular weight bands and of a 78 kDa band that represents a soluble form of CD44st and is 4 kDa lower than a similar band (82 kDa) detected in B-CLL cell lysates. Elevated serum CD44st associates with a number of unfavorable prognostic factors such as high peripheral blood lymphocytosis, splenomegaly, advanced disease stage and therapy requirement. A follow-up study indicates that serum levels of CD44st are related to disease status, thus reinforcing our veiw that this molecule may represent a reliable tumor marker in B-CLL.

Immunohistochemical study of skin lesions in acute and chronic graft versus host disease following bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) is the therapy of choice for a variety of malignant and nonmalignant disorders; however, a major constraint to successful BMT is graft versus host disease (GVHD). Skin lesions are the earliest presentation of GVHD. Donor-derived cytotoxic T lymphocytes are the effector cells responsible for lesions in the skin and other tissues. Here we show that most skin-infiltrating lymphocytes, in all forms of GVHD, are memory T cells with a predominance of CD4+ cells in the dermis and CD8+ cells in the epidermis. Relatively little attention has been focused on the adhesive phenotype of keratinocytes in GVHD. In this study, immunohistochemical analyses of skin biopsies from BMT patients with acute or chronic GVHD were conducted, with particular emphasis on antigen-presenting cells (APCs) and on keratinocytes. The distribution of APCs in the epidermis (Langerhans' cells) was investigated. Keratinocytes were analyzed for the expression of human leukocyte antigen DR locus (HLA-DR) and of a novel integrin, alpha10.1.2 beta1, which is detected in the basal layer of normal epidermis. Langerhans' cells were decreased in all grades of acute GVHD, but the epidermal APC network was reconstituted in chronic GVHD. HLA-DR was expressed by keratinocytes in grade 2 and 3 acute GVHD lesions, but not in two of three chronic GVHD cases, and in the regression phase of acute GVHD. Integrin chains alpha10.1.2 and beta1 were detected in the epidermal basal cell layer of most GVHD cases but they were also expressed in suprabasal keratinocytes of both acute and chronic GVHD. This latter finding indicates that a proliferative response uncoupled from differentiation occurs in keratinocytes in the course of GVHD.