T cell receptor (TCR) gene transfer with lentiviral vectors allows efficient redirection of tumor specificity in naive and memory T cells without prior stimulation of endogenous TCR.

We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

Ab-induced ectodomain shedding mediates hepatocyte growth factor receptor down-regulation and hampers biological activity.

Targeting tyrosine kinase receptors (RTKs) with specific Abs is a promising therapeutic approach for cancer treatment, although the molecular mechanism(s) responsible for the Abs' biological activity are not completely known. We targeted the transmembrane RTK for hepatocyte growth factor (HGF) with a monoclonal Ab (DN30). In vitro, chronic treatment of carcinoma cell lines resulted in impairment of HGF-induced signal transduction, anchorage-independent growth, and invasiveness. In vivo, administration of DN30 inhibited growth and metastatic spread to the lung of neoplastic cells s.c. transplanted into immunodeficient nu/nu mice. This Ab efficiently down-regulates HGF receptor through a molecular mechanism involving a double proteolytic cleavage: (i) cleavage of the extracellular portion, resulting in "shedding" of the ectodomain, and (ii) cleavage of the intracellular domain, which is rapidly degraded by the proteasome. Interestingly, the "decoy effect" generated by the shed ectodomain, acting as a dominant negative molecule, enhanced the inhibitory effect of the Ab.

CD100/Plexin-B1 interactions sustain proliferation and survival of normal and leukemic CD5+ B lymphocytes.

Growth and survival of chronic B-cell tumors are favored by the malignant cell's capacity to respond to selected microenvironmental stimuli provided by nontumoral bystander cells. To investigate which mechanisms operate in these crosstalks and whether they are malignancy-related or reproduce the mechanisms used by normal B cells we have studied the expression and functional role of semaphorin CD100 (now called Sema4D) in chronic lymphocytic leukemia (CLL) cells and normal CD5+ B cells. We demonstrate here that (1) leukemic and normal CD5+ B lymphocytes uniformly express CD100; (2) the CD100 high-affinity receptor Plexin-B1 is expressed by bone marrow stromal cells, follicular dendritic cells, and activated T lymphocytes, and is thus available to CD100+ lymphocytes in different specific microenvironments; and (3) upon interaction between CD100 and Plexin-B1 both CLL and normal CD5+ B cells increase their proliferative activity and extend their life span. These findings establish that Plexin-B1 is an easily accessible receptor for CD100 within the immune system. The encounter of CD100+ leukemic cells with Plexin-B1 may promote the proliferation and survival of malignant cells. The crosstalk operated by the CD100/Plexin-B1 interaction is not malignancy related but reproduces a mechanism used by normal CD5+ B cells.

Chronic lymphocytic leukaemia: a model for investigating potential new targets for the therapy of indolent lymphomas.

We address two key issues whose investigation may help to define new prognostic parameters and new potential targets for therapeutic intervention. First, which are the conceptual implications of the cellular origin of indolent lymphomas? Second, how may deciphering the biology of chronic lymphocytic leukaemia (CLL) lead to the development of new modalities of treatment? The latter issue is articulated in the following three key questions. (1) Which are the molecular pathways through which the microenvironment exerts its influence on the malignant clone? (2) What are the relationships between proliferation and defective apoptosis? (3)Is there any evidence of a role for antigenic stimulation?

Chronic lymphocytic leukemia B cells are endowed with the capacity to attract CD4+, CD40L+ T cells by producing CCL22.

The natural history of B-chronic lymphocytic leukemia (CLL) is not entirely explained by intrinsic defects of the neoplastic cell, but is also favored by microenvironmental signals. As CLL cells retain the capacity to respond to CD40 ligand (CD40L) and as CD4(+) T cells are always present in involved tissues, we asked whether malignant CLL cells might produce T cell-attracting chemokines. We studied the chemokine expression of CD19(+)/CD5(+) malignant B cells from peripheral blood (PB), lymph nodes (LN) or bone marrow (BM) of 32 patients and found a major difference. LN- and BM-, but not PB-derived cells, expressed a readily detectable reverse transcription-PCR band for CCL22 and one for CCL17 of variable intensity. CD40 ligation of PB cells induced the mRNA expression of both CCL22 and CCL17. CCL22 was also released in the culture supernatants. These supernatants induced the migration of activated CD4(+), CD40L(+) T cells expressing the CCL22 receptor, CCR4. T cell migration was abrogated by anti-CCL22 antibodies. Immunohistochemistry and cytofluorography studies revealed that a proportion of CD4(+) T cells in CLL LN and BM expressed CD40L. Our data demonstrate that malignant CLL cells chemo-attract CD4(+) T cells that in turn induce a strong chemokine production by the leukemic clone, suggesting a vicious circle, leading to the progressive accumulation of the neoplastic cells.

Chronic B cell malignancies and bone marrow microenvironment.

Chronic B-lymphoid malignancies depend upon supportive interactions within specific microenvironments. Follicular lymphoma (FL), chronic lymphocytic leukaemia (CLL) and multiple myeloma (MM) cells accumulate in the bone marrow (BM) where they receive survival or growth signals from by-stander cells. However, they deeply differ in their interaction with the microenvironment. We propose a model where FL and CLL recreate in the BM the microenvironment most suitable to their growth by 'importing' the normal cells that usually nurse them in secondary lymphoid organs. In contrast, MM takes advantage of the actual BM microenvironment by 'instructing' it through an abnormal activation state.