Fariba Mozaffari

Signalling molecules and cytokine production in T cells of multiple myeloma‐increased abnormalities with advancing stage

T‐cell immune dysfunction in patients with malignant tumours has been attributed to the altered expression of components of the T‐cell receptor (TCR)/CD3 complex and their associated intracellular protein tyrosine kinases. In this study, four‐colour flow cytometry was applied to study the surface bound molecules TCRαβ, CD28, CD152 and CD154 involved in T‐cell signalling and the signal transduction molecules CD3ζ, p56lck, p59fyn, ZAP‐70 and phosphatidyl‐inositol‐3 kinase (PI3‐k) as well as the intracellular cytokines interferon‐γ (IFN‐γ), interleukin (IL)‐4 and IL‐2 as a functional read‐out of non‐stimulated and superantigen (staphylococcus enterotoxin B)‐stimulated blood T cells of multiple myeloma (MM) patients at different stages of the disease. Multiple abnormalities were demonstrated in the CD4 and CD8 populations, both under non‐stimulated and superantigen‐stimulated conditions. There was a marked reduction, particular in advanced stage MM, in the proportion of CD4 and CD8 cells expressing CD28, CD152, CD3ζ, p56lck, ZAP‐70 and PI3‐k. The level of intracellular T‐cell cytokines (IFN‐γ, IL‐2 and IL‐4) was normal or increased in non‐stimulated cells but activation‐induced cytokine production was impaired. These results illustrated profound and multiple T‐cell signalling defects, from the surface and down‐stream, consistent with involvement of a master T‐cell function, especially in advanced stage MM. These data should be taken into consideration when developing immune‐based therapeutic approaches and when applying new emerging technologies that aim to restore T‐cell functions.

NK-cell and T-cell functions in patients with breast cancer: effects of surgery and adjuvant chemo- and radiotherapy

Breast cancer is globally the most common malignancy in women. Her2-targeted monoclonal antibodies are established treatment modalities, and vaccines are in late-stage clinical testing in patients with breast cancer and known to promote tumour-killing through mechanisms like antibody-dependent cellular cytotoxicity. It is therefore increasingly important to study immunological consequences of conventional treatment strategies. In this study, functional tests and four-colour flow cytometry were used to detect natural killer (NK)-cell functions and receptors as well as T-cell signal transduction molecules and intracellular cytokines in preoperative breast cancer patients, and patients who had received adjuvant radiotherapy or adjuvant combined chemo-radiotherapy as well as in age-matched healthy controls. The absolute number of NK cells, the density of NK receptors as well as in vitro quantitation of functional NK cytotoxicity were significantly higher in preoperative patients than the post-treatments group and controls. A similar pattern was seen with regard to T-cell signalling molecules, and preoperative patients produced significantly higher amounts of cytokines in NK and T cells compared to other groups. The results indicate that functions of NK and T cells are well preserved before surgery but decrease following adjuvant therapy, which may speak in favour of early rather than late use of immunotherapeutic agents such as trastuzumab that may depend on intact immune effector functions.

A Phase I safety study of plasmid DNA immunization targeting carcinoembryonic antigen in colorectal cancer patients.

A plasmid DNA vaccine, encoding a truncated form of human CEA fused to a T-helper epitope (CEA66 DNA) was delivered three times intradermally at 2 mg or intramuscularly at 8 mg by Biojector® to patients with colorectal cancer. Prior to the first vaccination, all patients received cyclophosphamide (300 mg/m²) intravenously. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered subcutaneously with each vaccination. All patients completed the vaccine schedule. There were no grade 3 or 4 adverse events (AE). The most frequently reported AE grades 1 and 2 were injection site reactions, fatigue, headache, arthralgia, chest tightness and myalgia. Vaccination with CEA66 DNA in combination with GM-CSF was well tolerated and no signs of autoimmunity have been detected.

Telomerase (GV1001) vaccination together with gemcitabine in advanced pancreatic cancer patients

Telomerase is expressed in 85-90 % of pancreatic adenocarcinomas and might be a target for active cancer immunotherapy. A study was conducted to investigate safety and immunogenicity in non-resectable pancreatic carcinoma patients using a 16-amino acid telomerase peptide (GV1001) for vaccination in combination with GM-CSF and gemcitabine as first line treatment. Three different vaccine treatment schedules were used; [A (n=6), B (n=6) and C (n=5)]. Groups A/B received GV1001, GM-CSF and gemcitabine concurrently. Group C received initially GV1001 and GM-CSF while gemcitabine was added at disease progression. Group D (n=4) was treated with gemcitabine alone. Adverse events (AE) related to vaccination were mild (grades I-II). Grade III AEs were few and transient. An induced GV 1001‑specific immune response was defined as an increase ≥2 above the baseline value in one of the assays (DTH, proliferation, ELISPOT and cytokine secretion assays, respectively). A telomerase‑specific immune response was noted in 4/6 patients in group A, 4/6 patients in group B and 2/5 patients in group C. An induced ras‑specific immune response (antigenic spreading) was seen in 5 of the 17 patients. The cytokine pattern was that of a Th1-like profile. A treatment induced telomerase or ras response was also noted in group D. All responses were weak and transient. A significant decrease in regulatory T-cells over time was noted in patients in groups A and B (p<0.05). Telomerase vaccination (GV1001) in combination with chemotherapy appeared to be safe but the immune responses were weak and transient. Measures have to be taken to optimize immune responses of GV1001 for it to be considered of clinical interest.

Signaling molecules and cytokine production in T cells of patients with B-cell chronic lymphocytic leukemia (B-CLL): comparison of indolent and progressive disease.

T-cell dysfunction in B-CLL patients might be attributed to altered expression of components of the TCR/CD3 complex and associated intracellular tyrosine kinases. Four-color flow cytometry was applied to the expression of these molecules as well as the T-cell regulatory cytokines (IFN-γ and IL-4) in B-CLL patients with indolent and progressive disease. Intracellular levels [mean fluorescent intensity (MFI)] of IFN-γ and IL-4 in both CD4 and CD8 T cells of both patient groups were significantly higher than in healthy donors. Absolute number of IL-4 producing CD4 T cells in patients with indolent was significantly higher than in healthy donors. The expression level (MFI) of the CD3-ξ chain was higher in patients than in normal donors as well as ZAP-70 in patients with indolent disease as compared to healthy donors and progressive patients. No significant difference was noted in the expression of p56lck, p59fyn, and PI3-kinase between healthy donors and patients or between the patient subgroups. The results indicate multiple T-cell abnormalities especially in indolent-stage B-CLL suggesting a state of chronic and aberrant activation. This information might be of significance when studying the immunobiology of B-CLL as well as developing new therapeutic approaches.

Systemic immune effects of adjuvant chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide and/or radiotherapy in breast cancer: a longitudinal study

Immunotherapy is being increasingly utilized for adjuvant treatment for breast cancer (BC). We have previously described immune functions during primary therapy for BC. The present study describes immune recovery patterns during long-term, unmaintained follow-up after completion of adjuvant therapy.A group of patients with primary BC had been treated with adjuvant radio-chemotherapy (RT + CT) 5-fluorouracil, epirubicin and cyclophosphamide (FEC) (n = 21) and another group with radiotherapy (RT) (n = 20) alone. Immunological testing of NK and T-cell functions was performed initially at the end of adjuvant treatment and repeated after 2, 6 and 12 months. NK cell cytotoxicity was significantly higher (P < 0.05) at all time-points in patients than in age-matched controls and did not differ between the two treatments groups during one year observation. In contrast, lower numbers of CD4 T-cells and lower expression of CD28 on T-cells was observed particularly in RT + CT patients and did not normalize during the observation period. The numbers of Treg cells (CD4+CD25high) were low in the RT + CT group during follow-up, as well as expression of TCRξ, Zap70, p56lck, P59fyn and PI3 k in CD4+ cells. In contrast, expression of intracellular cytokines (IFN-γ, IL-2, IL-4) in CD4 and CD8 T cells were significantly higher in RT + CT patients than in the RT group and the difference increased during follow-up. In conclusion, NK-cell cytotoxicity increased during unmaintained long-term follow-up whereas CD4 and regulatory T cells as well as signal transduction molecules remained low following adjuvant radio-chemotherapy.

Signaling molecules and cytokine production in T cells of patients with B-cell chronic lymphocytic leukemia: long-term effects of fludarabine and alemtuzumab treatment

Fludarabine and alemtuzumab are routinely used for treatment of B-cell chronic lymphocytic leukemia (B-CLL). The present study aimed to compare the expression of signaling molecules and cytokine production by T cells of B-CLL patients in long-term unmaintained remission/plateau phase following fludarabine or alemtuzumab treatment with that of indolent/untreated B-CLL patients and healthy donors. The frequency and intensity of TCR-CD3ζ chain, p56lck, p59fyn, ZAP-70, PI3-kinase and interferon (IFN)-γ/interleukin (IL)-4 production in CD4 and CD8 T cells was examined by flow cytometry. T-cell function was assessed by stimulation with purified protein derivative (PPD) and phytohemagglutinin (PHA). Despite a reduction in number, the expression of IFN-γ/IL-4 in T-cells in patients was significantly higher than in healthy donors. The intensity of most signaling molecules in treated patients was relatively unaffected vs. healthy donors but lower than untreated-indolent patients. However, the total number of T cells which expressed each of the signaling molecules was decreased in patients, with no difference between fludarabine- and alemtuzumab-treated patients. The T-cell response to PHA but not PPD was reduced in treated patients. The results suggest that, despite some alterations in signaling molecules and a reduction in T-cell number, overall T-cell functions may be relatively well preserved long-term after treatment with fludarabine and alemtuzumab.

Overcoming immunosuppressive mechanisms

Clinical trials have demonstrated from time to time that cancer vaccines can elicit effective antitumour cellular immunity that may translate to clinical benefit for cancer patients. Additionally, several monoclonal antibodies currently used for treating cancer patients mediate their effects through mechanisms like antibody-dependent cellular cytotoxicity (ADCC) and therefore rely on an effective immune system. Patients with advanced tumours, however, are known to have aberrations in their immune function. Improving the clinical effectiveness of immunotherapy therefore depends on having a better understanding of the mechanisms and developing novel strategies to overcome tumour-specific immune suppression.

Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia

Background ROR1 is a receptor tyrosine kinase expressed in chronic lymphocytic leukemia (CLL) and several other malignancies but absent in most adult normal tissues. ROR1 is considered an onco-fetal antigen. In the present study we analysed spontaneous humoral and cellular immunity against ROR1 in CLL patients. Materials and Methods Antibodies against ROR1 were analysed in 23 patients and 20 healthy donors by ELISA and Western blot. Purified serum IgG from patients was tested for cytotoxicity against CLL cells using the MTT viability assay. A cellular immune response against ROR1 derived HLA-A2 restricted 9 aa and 16 aa long peptides were analysed using peptide loaded dendritic cells co-cultured with autologous T cells from CLL patients (n = 9) and healthy donors (n = 6). IFN-γ, IL-5 and IL-17A-secreting T cells were assessed by ELISPOT and a proliferative response using a H3-thymidine incorporation assay. Results The majority of CLL patients had antibodies against ROR1. Significantly higher titers of anti-ROR1 antibodies were noted in patients with non-progressive as compared to progressive disease. The extracellular membrane-close ROR1 KNG domain seemed to be an immunodominant epitope. Ten patients with high titers of anti-ROR1 binding antibodies were tested for cytotoxicity. Five of those had cytotoxic anti-ROR1 antibodies against CLL cells. ROR1-specific IFN-γ and IL-17A producing T cells could be detected in CLL patients, preferentially in non-progressive as compared to patients with progressive disease (p<0.05). Conclusion ROR1 seemed to spontaneously induce a humoral as well as a T cell response in CLL patients. The data support the notion that ROR1 might be a specific neo-antigen and may serve as a target for immunotherapy.

First-in-class oral small molecule inhibitor of the tyrosine kinase ROR1 (KAN0439834) induced significant apoptosis of chronic lymphocytic leukemia cells

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