Sara Mariani

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 T cells 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 T cells with a regulatory rather than an effector phenotype.

Long-term follow-up of idiotype vaccination in human myeloma as a maintenance therapy after high-dose chemotherapy

The aim of this work was to evaluate the long-term immunological and clinical impact of idiotype (Id) vaccination in multiple myeloma (MM) patients in first remission after high-dose chemotherapy. A total of 15 patients received a series of subcutaneous (s.c.) injections of autologous Id, conjugated to keyhole limpet hemocyanin (KLH) and in association with low doses of GM-CSF. The median duration of follow-up was 110 months from diagnosis. The vaccine induced immune responses that lasted almost 2 years after the end of treatment. Antibody responses included anti-KLH IgM and IgG (90% of patients), anti-KLH IgE (30%), anti-GM-CSF IgG (20%), anti-Id IgG (20%), and anti-Id IgE (30%). Id-specific delayed type hypersensitivity skin tests were positive in 85% of tested patients. Following vaccination, a progressive recovery of T-cell receptor (TCR) diversity was observed and the loss of oligoclonality was significantly correlated with the remission duration. Although Id/KLH conjugates did not eliminate the residual tumor burden, the median progression-free survival, and overall survival were 40 and 82 months, respectively. A retrospective case-matched analysis showed similar results in patients treated with IFN-α alone or in association with steroids. This vaccine formulation can overcome Id-specific immune tolerance by inducing clinical responses that are worthy of further investigation.

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 13 months. 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.

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.

Technical limits of comparison of step-sectioning,immunohistochemistry and RT-PCR on breast cancer sentinel nodes: a study on methacarn-fixed tissue

The optimal pathological assessment of sentinel nodes (SLNs) in breast cancer is a matter of debate. Currently, multilevel histological evaluation and immunohistochemistry (IHC) are recommended, but alternative RT‐PCR procedures have been developed. To assess the reliability of these different procedures, we devised a step‐sectioning protocol at 100 micron‐intervals of 74 SLNs using methacarn fixation. mRNA was extracted from sections collected from levels 4 to 5. Mammaglobin, CEA and CK19 were used for RT‐PCR. mRNA extraction was successful in 69 SLNs. Of these, 7 showed macrometastases (>2mm), 2 showed micrometastases (<2 mm) and 7 showed isolated tumour cells (ITC) by IHC. RT‐PCR was positive for the three markers in 6 of 7 macrometastases and in 1 of 2 micrometastases. In the 2 RT‐PCR negative cases, metastases were detected only on sections distant from those analysed by RT‐PCR. CEA and/or CK19 were positive by RT‐PCR in 3 of 7 ITC and in 23 morphologically negative SLNs. In conclusion, the main goal of our study was to show that the use of alternate sections of the same sample for different procedures is the key reason for the discrepancies between molecular and morphological analyses of SLN. We believe that only prospective studies with quantitative mRNA analysis of specific metastatic markers on the whole lymph node can elucidate the utility of molecular assessments of SLN.

Comprehensive assessment of the TCRBV repertoire in small T-cell samples by means of an improved and convenient multiplex PCR method

OBJECTIVE Overall diversity of the T-cell receptor (TCR) repertoire can be regarded as a recapitulatory signature of a hosts immunocompetence status. We aimed to establish a time- and cost-saving multiplex polymerase chain reaction (PCR) method for determining the TCR repertoire of conventional alphabeta T cells in small T-cell samples. MATERIALS AND METHODS The method estimates the length distribution of the complementarity-determining regions 3 (CDR3) of beta variable (BV) gene segments (TCRBV repertoire) by multiplex PCR, followed by fluorescent run-off reactions to visualize BV-BC and/or BV-BJ rearrangements. Run-off products are separated on a capillary sequencer and subsequently analyzed with GeneScan or Genotyper programs. Detection-limit studies with normal T cells, KMS27 cells, and regulatory T cells were carried out to evaluate sensitivity and reproducibility. RESULTS Head-to-head comparison of the method with conventional immunoscope assay has shown that it is a time- and cost-saving approach to characterize TCRBV and TCRBJ repertoires, including the presence of oligoclonal T cells in samples containing as few as 1 x 10(5) T cells. CONCLUSION We have developed a multiplex PCR method that allows comprehensive assessment of the TCRBV repertoire at the BV-BC and BV-BJ levels, and saves a considerable amount of time, reagents, and cell input.

Clonal CD8+ TCR-Vβ expanded populations with effector memory phenotype in Churg Strauss Syndrome

Churg Strauss Syndrome (CSS) is a systemic vasculitis in which oligoclonal T cell expansions might be involved in the pathogenesis. Combined analysis of TCR-Vbeta expression profile by flow cytometry and of TCR gene rearrangement by heteroduplex PCR was used to detect and characterize T cell expansions in 8 CSS patients, 10 asthmatics and 42 healthy subjects. In all CSS patients one or two Vbeta families were expanded among CD8+ cells, with an effector memory phenotype apt to populate tissues and inflammatory sites. Heteroduplex PCR showed the presence of one or more clonal TCR rearrangements, which reveals monoclonal or oligoclonal T cells subpopulations. After purification with a Vbeta specific monoclonal antibody, each CD8+/Vbeta+ expanded family showed a single TCR rearrangement, clearly suggestive of monoclonality. All CD8+ expansions were detectable throughout the disease course. TCR-Vbeta expanded or deleted populations were not observed in asthmatic patients. Clonal CD8+/Vbeta+ T cell expansions might be useful as a disease marker.

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 (P < 0·0001). 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.

The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulomatosis with polyangiitis (EGPA)

In eosinophilic granulomatosis with polyangiitis (EGPA) clonally expanded T cells might concur in granuloma formation and vascular injury. The TCR β-variable (BV) chain repertoire and third complementarity determining region (CDR3) of peripheral CD4+ and CD8+ cells in EGPA patients and age-matched controls and the expression of cytokines and chemokine receptors were investigated. The CD8+ lymphocytes of EGPA patients showed an increased frequency of BV expansions with a skewed profile of BV CDR3 lengths, increased CCR5 and CXCR3 expression and increased INFγ and TNFα production. In two patients, the TCR CDR3 cDNA sequences of the expanded BV family were identified. The CD4+ lymphocytes of EGPA patients revealed a higher expression of CRTH2 and increased production of IL-5. In conclusion, CD4+ T cells display a Th2 profile and CD8+ T cells are clonally expanded in EGPA and have a proinflammatory phenotype, suggesting their pathogenic role in vasculitic damage.