In vitro complement activation, adherence to red blood cells and induction of mononuclear cell cytokine production by four strains of Aggregatibacter actinomycetemcomitans with different fimbriation and expression of leukotoxin.

BACKGROUND AND OBJECTIVE: The periodontal pathogen Aggregatibacter actinomycetemcomitans has been proposed as pro-atherogenic, and complement-mediated adherence to red blood cells (RBCs) may facilitate its systemic spread. We investigated the ability of four strains of A. actinomycetemcomitans with differential expression of leukotoxin A (LtxA) and fimbriae to activate complement, adhere to RBCs and elicit cytokine responses by mononuclear cells (MNCs). MATERIAL AND METHODS: Aggregatibacter actinomycetemcomitans serotype b strains HK 921, HK 1651, HK 2092 and HK 2108 were fluorescence-labeled, incubated with human whole blood cells in the presence of autologous serum, and assessed for RBC adherence by flow cytometry and for capacity to induce cytokine production by cytometric bead array analysis. The levels of IgG to A. actinomycetemcomitans serotype b were quantified by ELISA, as was consumption of complement. RESULTS: The JP2 clone variants HK 1651 and, to a lesser extent, HK 2092, consumed complement efficiently, while HK 2108 (= strain Y4) consumed complement poorly. Nonetheless, the four tested strains adhered equally well to RBCs in the presence of autologous serum, without causing RBC lysis. The JP2 clone variant HK 2092, selectively lacking LtxA production, induced higher production of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-10 by MNCs than did the other three strains, while the four strains induced similar production of IL-12p70. RBCs facilitated the HK 2092-induced production of TNF-α and IL-1ß, and IL-6 was enhanced by RBCs, and this facilitation could be counteracted by blockade of complement receptor 3 (CD11b/CD18). CONCLUSION: Our data suggest that the JP2 clone of A. actinomycetemcomitans, most closely resembled by the variant HK 1651, activates complement well, while strain Y4, represented by HK 2108, activates complement poorly. However, all strains of A. actinomycetemcomitans adhere to RBCs and, when capable of producing LtxA, prevent production of inflammatory cytokines by MNCs. This "immunologically silent" immune adherence may facilitate systemic spread and atherogenesis.

5-Azacytidine treatment sensitizes tumor cells to T-cell mediated cytotoxicity and modulates NK cells in patients with myeloid malignancies.

Treatment with the demethylating agent 5-Azacytidine leads to prolonged survival for patients with myelodysplastic syndrome, and the demethylation induces upregulation of cancer-testis antigens. Cancer-testis antigens are well-known targets for immune recognition in cancer, and the immune system may have a role in this treatment regimen. We show here that 5-Azacytidine treatment leads to increased T-cell recognition of tumor cells. T-cell responses against a large panel of cancer-testis antigens were detected before treatment, and these responses were further induced upon initiation of treatment. These characteristics point to an ideal combination of 5-Azacytidine and immune therapy to preferentially boost T-cell responses against cancer-testis antigens. To initiate such combination therapy, essential knowledge is required about the general immune modulatory effect of 5-Azacytidine. We therefore examined potential treatment effects on both immune stimulatory (CD8 and CD4 T cells and Natural Killer (NK) cells) and immune inhibitory cell subsets (myeloid-derived suppressor cells and regulatory T cells). We observed a minor decrease and modulation of NK cells, but for all other populations no effects could be detected. Together, these data support a strategy for combining 5-Azacytidine treatment with immune therapy for potential clinical benefit.

Changes in peripheral blood level of regulatory T cells in patients with malignant melanoma during treatment with dendritic cell vaccination and low-dose IL-2.

In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell-based vaccine and concomitant low-dose IFN-α and IL-2. The regulatory subset of CD4 T cells, characterized by CD25(high) , was prospectively analysed in fresh blood, and treatment-associated quantitative and qualitative changes were analysed. By the 4th vaccine, patients showed a marked increase in CD4+ CD25(high) T cell subset from 6% to 22% (P<0.001). At the 6th vaccine, a general decline was observed and a significantly (P=0.01) lower level of CD4+ CD25(high) Treg cells was reached in the group of patients who attained disease stabilization (9.5%) compared to patients with continued progressive disease (14.5%). However, when FoxP3 was employed for retrospective analysis of Tregs on frozen blood, this difference did not reach significance (P=0.09). The vast majority of the Treg produced IL-10 and, to a varying extent, TGF-ß. In addition, sorted CD4+ CD25(high) CD127⁻ Tregs were able to suppress proliferation of peripheral blood mononuclear cells in a dose-dependent manner, thus suggesting a regulatory functionality. These findings emphasize the need for strategies to effectively eliminate Treg cells to optimize the clinical effectiveness of cancer immunotherapy.

Increased level of both CD4+FOXP3+ regulatory T cells and CD14+HLA-DR⁻/low myeloid-derived suppressor cells and decreased level of dendritic cells in patients with multiple myeloma.

Patients with multiple myeloma (MM) suffer from a general impaired immunity comprising deficiencies in humoral responses, T-cell responses as well as dendritic cell (DC) function. Thus, to achieve control of tumour growth through immune therapy constitutes a challenge. Careful evaluation of the immune status in patients with MM seems crucial prior to active immune therapy. We evaluated the proportion of both, DC, Treg cells and myeloid-derived suppressor cells (MDSC) in peripheral blood from patients with MM at diagnosis and in remission as well as patients with monoclonal gammopathy of undetermined significance (MGUS). We found that the proportion of both myeloid (m) DC and plasmacytoid (p) DC in patients at diagnosis was lowered compared to control donors, while only the proportion of pDC in patients in remission and with MGUS was significantly lower than in controls. The proportion of CD4+FOXP3+ Treg cells was increased in patients at diagnosis and not in patients in remission or with MGUS. Also, Treg cells from patients with MM were functionally intact as they were able to inhibit proliferation of both CD4 and CD8 T cells. Finally, we observed an increase in the proportion of CD14+HLA-DR⁻/low MDSC in patients with MM at diagnosis, illustrating that this cell fraction is also distorted in patients with MM. Taken together, our results illustrate that, both mDC, pDC, Treg cells and MDSC are affected in patients with MM underlining the fact that the immune system is dysregulated as a consequence of the disease.

A phase II study of thalidomide and temozolomide in patients with brain metastases from malignant melanoma: lymphopenia correlates with response.

BACKGROUND: Central nervous system (CNS) metastases develop in nearly half of patients with advanced melanoma and in 15-20% CNS is the first site of relapse. Median overall survival is short, ranging from two to four months, and one-year survival rate is only 10-15%. THA has been shown to have both anti-angiogenetic and immuno-modulating effects. TMZ is an oral alkylating agent with an excellent oral bioavailability and it is highly lipophillic with an ability to penetrate the blood-brain barrier. TMZ and THA in combination were tested in patients with brain metastases from malignant melanoma. METHODS: Between June 2004 and February 2007 patients with measurable metastatic melanoma in progression and PS ≤ 1 received TMZ in a dose of 150 mg/m(2) qd for seven days, followed by seven days off therapy and THA in 200 mg qd, both orally administered. Concomitant treatment with steroids was allowed. PBMCs were collected from the last 14 consecutive patients for evaluation of immune parameters. RESULTS: Forty screened patients were eligible and evaluable for response, and 39 were evaluable for toxicity. 25 patients had asymptomatic and 15 symptomatic brain metastases. The toxicity was primarily grade 1-2 with no grade 4 or treatment-related deaths. Four patients had thromboembolic events grade 3. One patient obtained a CR and five a PR in the CNS, while two had CR and four had PR outside CNS. Overall response rate was 17.5%. We found a significant positive correlation between lymphopenia and objective response. CONCLUSIONS: The combination treatment was well tolerated but with more frequent thromboembolic events compared to single drug TMZ or THA. The treatment demonstrated activity in CNS as well as outside CNS. The correlation between lymphopenia and objective response needs further investigation.

Impaired functionality and phenotypic profile of dendritic cells from patients with multiple myeloma.

Multiple myeloma (MM) is a B cell cancer characterized by clonal proliferation in the bone marrow and impaired immunity. Because MM is an incurable malignancy, efficient consolidation is needed urgently. Targeting clonotypic B cells by idiotype vaccination has proved the principle to be effective and indicated that future strategies, including dendritic cell-based vaccination, could be a suitable approach. However, as MM patients suffer from a general impaired immunity, which may include dendritic cells (DCs), a careful evaluation of phenotypic traits and functionality of DCs from MM patients is necessary before an efficient vaccine can be developed. This study determined the number, phenotypic profile and functionality of myeloid and plasmacytoid DCs purified directly from blood from MM patients at diagnosis. A reduced number and lower expression of human leucocyte antigen (HLA) molecules was observed on both myeloid and plasmacytoid DCs in MM patients compared to healthy controls. Also, the expression of CCR5, CCR7 and DEC205 was lower in MM patients compared to normal donors. In addition, the capacity to stimulate allogeneic T cell proliferation and to stimulate cytokine production was decreased, suggesting that DCs from these patients are functionally impaired. Finally, the analysis of samples following chemotherapy and transplantation demonstrated an increased expression of HLA molecules, suggesting that this time-point is optimal for harvest and use in vaccination.