Silvia Peola

Effector γδ T cells and tumor cells as immune targets of zoledronic acid in multiple myeloma

The aim of this study was to investigate the in vitro immunomodulatory effects of zoledronic acid (Zol) on peripheral blood Vγ9/Vδ2 (γδ) T cells of normal donors and multiple myeloma (MM) patients. γδ T cells were stimulated with Zol and low doses of interleukin-2 (IL-2), and then analyzed for proliferation, cytokine production, and generation of effector activity against myeloma cell lines and primary myeloma cells. Proliferation of γδ T cells was observed in 100% of normal donors and 50% of MM patients. γδ T cells produced IFN-γ, surface mobilized the CD107a and CD107b antigens, and exerted direct cell-to-cell antimyeloma activity irrespective of the ability to proliferate to Zol and IL-2. The memory phenotype was predominant in the MM γδ T cells that proliferated in response to Zol (responders), whereas effector cells were predominant in those that did not (nonresponders). Zol induced antimyeloma activity through the monocyte-dependent activation of γδ T cells and by enhancing the immunosensitivity of myeloma cells to γδ T cells. Mevastatin, a specific inhibitor of hydroxy-methylglutaryl-CoA reductase, completely abrogated this antimyeloma activity.

Exposure to myeloma cell lysates affects the immune competence of dendritic cells and favors the induction of Tr1-like regulatory T cells

The aim of this work was to investigate the interactions of tumor cells with dendritic cells (DC) in normal donors and patients with multiple myeloma (MM). Normal and MM DC internalized necrotic lysates derived from myeloma cell lines (MCL) with high efficiency, whereas necrotic lysates from primary myeloma cells (PMC) were internalized with significantly lower efficiency. A positive correlation was found between susceptibility to internalization and the ability to induce DC maturation. After PMC exposure, DC produced large amounts of IL‐10 and measurable amounts of IL‐4 but no detectable IL‐12. Two rounds of exposure to PMC‐treated DC generated autologous Tu2004cells with low proliferative capacity, decreased IFN‐γ production and increased IL‐10 production in the absence of IL‐4 production. These data indicate that myeloma cells can affect host immunity by priming DC towards a maturation state favoring the generation of Tu2004cells with a regulatory rather than an effector phenotype.

Severe and long-lasting disruption of T-cell receptor diversity in human myeloma after high-dose chemotherapy and autologous peripheral blood progenitor cell infusion.

Vaccine‐based strategies are currently under investigation as a means of inducing tumour‐specific immune responses and improving the clinical outcome of multiple myeloma (MM) patients in remission after high‐dose chemotherapy and peripheral blood progenitor cell (PBPC) infusion. The immune competence of these patients was investigated by determining the overall diversity of the T‐cell receptor (TCR) repertoire in the peripheral blood (PB) and bone marrow (BM). The average time after transplantation was 13u2003months. The clonality and reciprocal usage of BV gene segments (TCRBV repertoire) was estimated at the cDNA level and membrane protein expression. The TCRBV repertoire of MM was severely disrupted compared with age‐matched normal donors. On average, one‐third of the total repertoire in both the PB and the BM consisted of T cells expressing oligoclonal TCRβ transcripts. Flow cytometry showed an increased frequency of abnormally expanded BV subfamilies at both sites. BV expansions were predominantly CD8+ and had the phenotype of antigen‐experienced memory T cells as well as T cells with the naive phenotype. Oligoclonality was not restricted to phenotypically expanded BV subfamilies, but also involved normally represented BV subfamilies. The TCR repertoire of MM in remission was then compared with monoclonal gammopathy of undetermined significance (MGUS) and MM patients at diagnosis. The degree of TCR diversity was similar in age‐matched normal donors and MGUS, but progressively decreased from MGUS to MM at diagnosis and then to MM in remission. These data indicate that: (1) there is a long‐lasting and severe disruption of TCR diversity after high‐dose chemotherapy and PBPC infusion, and (2) the extent of TCR disruption may affect the clinical outcome of vaccine‐based strategies delivered at the stage of minimal residual disease.

Immune Modulation by Zoledronic Acid in Human Myeloma: An Advantageous Cross-Talk between Vγ9Vδ2 T Cells, αβ CD8+ T Cells, Regulatory T Cells, and Dendritic Cells

Vγ9Vδ2 T cells play a major role as effector cells of innate immune responses against microbes, stressed cells, and tumor cells. They constitute <5% of PBLs but can be expanded by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC). Much less is known about their ability to act as cellular adjuvants bridging innate and adaptive immunity, especially in patients with cancer. We have addressed this issue in multiple myeloma (MM), a prototypic disease with several immune dysfunctions that also affect γδ T cells and DC. ZA-treated MM DC were highly effective in activating autologous γδ T cells, even in patients refractory to stimulation with ZA-treated monocytes. ZA inhibited the mevalonate pathway of MM DC and induced the intracellular accumulation and release into the supernatant of isopentenyl pyrophosphate, a selective γδ T cell activator, in sufficient amounts to induce the proliferation of γδ T cells. Immune responses against the tumor-associated Ag survivin (SRV) by MHC-restricted, SRV-specific CD8+ αβ T cells were amplified by the concurrent activation of γδ T cells driven by autologous DC copulsed with ZA and SRV-derived peptides. Ancillary to the isopentenyl pyrophosphate-induced γδ T cell proliferation was the mevalonate-independent ZA ability to directly antagonize regulatory T cells and downregulate PD-L2 expression on the DC cell surface. In conclusion, ZA has multiple immune modulatory activities that allow MM DC to effectively handle the concurrent activation of γδ T cells and MHC-restricted CD8+ αβ antitumor effector T cells.

IGHV unmutated CLL B cells are more prone to spontaneous apoptosis and subject to environmental prosurvival signals than mutated CLL B cells

Tumor cells in chronic lymphocytic leukemia (CLL) are more prone to apoptosis when cultured ex vivo, because they lack prosurvival signals furnished in vivo via B-cell receptor (BCR)-dependent and -independent pathways. This study compared the susceptibility of unmutated (UM) and mutated (M) CLL B cells to spontaneous apoptosis and prosurvival signals. UM CLL B cells showed a significantly higher rate of spontaneous apoptosis than M CLL B cells. Nuclear factor-kB (NF-kB) was rapidly inactivated, and B-cell leukemia/lymphoma 2 (Bcl-2) expression progressively down-regulated in the UM CLL B cells. CD40-Ligand, interleukin-4 and stromal cells significantly improved their viability and partially recovered Bcl-2, but not NF-kB expression. Peripheral blood mononuclear cells also offered protection of UM CLL B cells, and recovered both NF-kB and Bcl-2 expression. T cells, rather than nurse-like cells, were responsible for protecting UM CLL B cells by means of cell-to-cell contact and soluble factors. Despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions.

Dysfunctional Vγ9Vδ2 T cells are negative prognosticators and markers of dysregulated mevalonate pathway activity in chronic lymphocytic leukemia cells

The role of Vγ9Vδ2 T cells in chronic lymphocytic leukemia (CLL) is unexplored, although these cells have a natural inclination to react against B-cell malignancies. Proliferation induced by zoledronic acid was used as a surrogate of γδ TCR-dependent stimulation to functionally interrogate Vγ9Vδ2 T cells in 106 untreated CLL patients. This assay permitted the identification of responder and low-responder (LR) patients. The LR status was associated with greater baseline counts of Vγ9Vδ2 T cells and to the expansion of the effector memory and terminally differentiated effector memory subsets. The tumor immunoglobulin heavy chain variable region was more frequently unmutated in CLL cells of LR patients, and the mevalonate pathway, which generates Vγ9Vδ2 TCR ligands, was more active in unmutated CLL cells. In addition, greater numbers of circulating regulatory T cells were detected in LR patients. In multivariate analysis, the LR condition was an independent predictor of shorter time-to-first treatment. Accordingly, the time-to-first treatment was significantly shorter in patients with greater baseline numbers of total Vγ9Vδ2 T cells and effector memory and terminally differentiated effector memory subpopulations. These results unveil a clinically relevant in vivo relationship between the mevalonate pathway activity of CLL cells and dys-functional Vγ9Vδ2 T cells.

Distribution of T-cell signalling molecules in human myeloma

It is controversial whether altered levels of TCR/CD3‐associated signalling molecules play a role in the T‐cell dysfunction of cancer patients. In multiple myeloma (MM), peripheral blood T (PBT) lymphocytes are functionally impaired by prolonged exposure to tumour cells, and so we investigated the organization of the TCR/CD3‐associated signal transduction machinery. The aim of this study was two‐fold: first, to investigate the levels of CD3ζ, p56lck, p59fyn, ZAP‐70, protein kinase C‐α (PKC‐α) and phospholipase C‐γ (PLC‐γ) in MM PBT cells; second, to determine whether levels of expression were correlated with clinical or prognostic factors. Forty‐four MM patients were studied and 25 age‐matched normal donors served as controls. On average, PKC‐α was the only significantly decreased (P<0.001) signalling molecule, whereas levels of CD3ζ, p56lck, p59fyn, PLC‐γ and ZAP‐70 were not statistically different. However, there was wide variation between individual patients, and levels for each single protein also varied. A 75% or greater decrease in protein expression was observed, ranging from 8% (p59fyn) to 68% (PCK‐α) of MM patients. When patients were grouped according to the cut‐off values of prognostic factors such as the serum levels of C reactive protein (CRP), β2‐microglobulin (β2M), neopterin (NPT) and the labelling index (LI%) of bone marrow (BM) plasma cells, the only difference observed was the lower PKC‐α expression in patients with high serum NPT values. None of the T‐cell signalling molecule levels was affected by the duration of tumour exposure, calculated on the number of years and/or months that had elapsed since diagnosis, or by disease status. In conclusion, there was a significant decrease of PCK‐α in MM T cells; however, neither this decrease nor the heterogenous levels of the other T‐cell signalling molecules were clearly correlated with prognosis, duration of tumour exposure, and disease status.

Increased expression of non-functional killer inhibitory receptor CD94 in CD8+ cells of myeloma patients.

Different MHC class I‐specific killer inhibitory receptors (KIRs) are expressed in vivo by a minor fraction of activated memory CD8+ cells. It has been postulated that KIRs may ‘fine‐tune’ specific responses by altering their threshold of activation by the TCR–CD3 complex. We have previously shown that, in multiple myeloma (MM) patients, a large fraction of peripheral blood CD8+ cells display the phenotype of chronically activated memory T cells (CD38+, HLA‐DR+, CD25−, CD45R0+, CD28−). We investigated the expression of KIRs on MM T cells and determined their possible influence on cytolytic responses elicited via the CD3–TCR complex. The expression of CD94, a molecule that is part of a heterodimeric KIR recognizing the non‐classical MHC surface HLA‐E molecule, was almost threefold higher in MM T cells than in age‐matched normal control subjects (Pu2003<u20030·0001). CD94 expression was preferentially confined to CD8+ cells but not restricted to activated (HLA‐DR+) and/or memory (CD45R0+) T cells. Unlike normal T cells, in which CD94 is assembled with glycoproteins of the NKG2 family to form functional receptors with activating or inhibitory properties, most CD94+ MM T cells were devoid of both the NKG2‐A and NKG2‐C glycoproteins detected in the inhibitory or activating form respectively. CD94 blockade did not significantly affect either T‐cell proliferation or cytotoxic T‐lymphocyte generation induced by the myeloma‐derived cell lines NCI and RPMI 8226. Similarly, the cytolytic activity induced by direct anti‐CD3‐mediated targeting of MM T cells to FCR+ P815 target cells was unaffected by the addition of anti‐CD94 and/or anti‐NKG2‐A/C monoclonal antibodies (mAbs). These data indicate that the large majority of MM CD8+ cells do not express a functional CD94 receptor. Thus, their ability to ‘fine‐tune’ an appropriate immune response against tumour cells can be impaired.

Generation of anti‐tumour activity by OKT3‐stimulation in multiple myeloma: in vitro inhibition of autologous haemopoiesis

Summary. T cells in multiple myeloma (MM) patients are highly susceptible to activation with the anti‐CD3 monoclonal antibody (mAb) OKT3. When short‐term OKT3 stimulation is carried out on bone marrow mononuclear cells (BMMC), large numbers of CD3+CD25+HLA‐DR+ cells are rapidly generated and autologous malignant plasma cells are killed. OKT3 may thus be exploited in autologous bone marrow transplantation (ABMT) to purge residual plasma cells and simultaneously activate T cells to induce graftversus‐leukaemia‐like (GVL‐like) activity upon reinfusion. However, the possible impact of ex‐vivo short‐term OKT3 stimulation on haematological recovery is unknown. The aim of this work was to investigate the effect of OKT3 stimulation in vitro on autologous haemopoietic progenitor cells (HPC) of MM patients. Colony formation by granulocyte‐macrophage progenitor cells (granulocyte‐macrophage colony‐forming units, CFU‐GM) was highly suppressed, although supernatants of OKT3‐activated T cells contained up to 2500 pg/ml of granulocyte‐macrophage colonystimulating factor (GM‐CSF). T cell depletion completely prevented this suppression. Neutralizing antibodies against TNF‐α, TNF‐β and IFN‐γ (which are also produced by OKT3‐activated MM T cells) did not prevent it, and Transwell cultures showed that cell‐to‐cell contact was the main mechanism involved. OKT3‐activated T cells also suppressed erythroid burst‐forming units (BFU‐E) and CFU‐GM generation from HPC responsible for long‐term maintenance of in vitro myelopoiesis.

Sequential administration of OKT3 (anti-CD3) and interleukin-2 in two patients with chemoresistant hematological disease.

To the editor: Since tumor-derived antigen(s) can rarely be used to activate T cells in humans, stimulation with antiCD3 monoclonal antibodies (mAb) has been proposed as a nonspecific approach to trigger anti-tumor activity (1). CD3 engagement acts as a surrogate for antigen-specific stimulation and is effective in vitro and in vivo in unprimed and tumor-primed T cells (1-3). The T-cell activation state triggered by CD3 priming can be prolonged and enhanced by interleukin-2 (IL-2) (2, 3). Protocols using OKT3 (anti-CD3 mAb) followed or not by IL-2 or tumornecrosis factor-a (TNF-a) are currently being tested