The Value of IgM Memory B-Cells in the Assessment of Splenic Function in Childhood Cancer Survivors at Risk for Splenic Dysfunction: A DCCSS-LATER Study.

Background: Childhood cancer survivors (CCS) who received radiotherapy involving the spleen or total body irradiation (TBI) might be at risk for splenic dysfunction. A comprehensive screening test for examining splenic dysfunction is lacking. Objective: We investigated whether IgM memory B-cells could be used to assess splenic dysfunction in CCS who received a splenectomy, radiotherapy involving the spleen, or TBI. Methods: All CCS were enrolled from the DCCSS-LATER cohort. We analyzed differences in IgM memory B-cells and Howell-Jolly bodies (HJB) in CCS who had a splenectomy (n = 9), received radiotherapy involving the spleen (n = 36), or TBI (n = 15). IgM memory B-cells < 9 cells/µL was defined as abnormal. Results: We observed a higher median number of IgM memory B-cells in CCS who received radiotherapy involving the spleen (31 cells/µL, p=0.06) or TBI (55 cells/µL, p = 0.03) compared to CCS who received splenectomy (20 cells/µL). However, only two CCS had IgM memory B-cells below the lower limit of normal. No difference in IgM memory B-cells was observed between CCS with HJB present and absent (35 cells/µL vs. 44 cells/µL). Conclusion: Although the number of IgM memory B-cells differed between splenectomized CCS and CCS who received radiotherapy involving the spleen or TBI, only two CCS showed abnormal values. Therefore, this assessment cannot be used to screen for splenic dysfunction.

Azathioprine Hypersensitivity Syndrome in a Cohort of Antineutrophil Cytoplasmic Antibody-Associated Vasculitis Patients.

BACKGROUND: Azathioprine hypersensitivity syndrome is a rare complication of azathioprine therapy. Its symptoms resemble infection or relapse of inflammatory disease, hindering correct diagnosis. Current literature is limited to sporadic case reports and reviews. OBJECTIVE: To estimate the incidence of azathioprine hypersensitivity syndrome and describe its characteristics in the context of an observational cohort of patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Also, to facilitate early recognition and awareness among clinicians. METHODS: Within a cohort of 290 patients with ANCA-associated vasculitis receiving azathioprine maintenance therapy, frequency of azathioprine hypersensitivity was described and characteristics were compared between hypersensitive and nonhypersensitive patients. Clinical picture, laboratory abnormalities, and concurrent medication of patients with azathioprine hypersensitivity were described. RESULTS: Of 290 patients, 25 (9%) experienced azathioprine hypersensitivity after a median of 14 (interquartile range [IQR] 12-18) days. Frequent symptoms were fever (100%), malaise (60%), arthralgia (36%), and rash (32%). All patients used prednisolone (median 10 mg/day, IQR 9.4-16.3 mg/day) at the time of the hypersensitivity reaction. Most patients had a rise in C-reactive protein (CRP), leukocyte counts, and neutrophil counts, but no eosinophilia. Thiopurine S-methyltransferase (TPMT) activity was significantly lower in hypersensitive patients (median 74.4 [IQR 58.0-80.1] nmol/gHb/L) compared with controls (median 81.4 [71.9-90.5] nmol/gHb/L), P = .01. Hypersensitive patients had a higher risk of relapse (hazard ratio 2.2, 95% confidence interval 1.2-4.2; P = .01). CONCLUSIONS: Azathioprine hypersensitivity syndrome is strikingly common in ANCA-associated vasculitis, might be associated with reduced TPMT activity, is accompanied by an increase in neutrophil counts, and may occur even during concomitant prednisolone therapy. Proper recognition may prevent unnecessary hospital procedures and damage to the patient.

Genetic defects in PI3Kδ affect B-cell differentiation and maturation leading to hypogammaglobulineamia and recurrent infections.

BACKGROUND: Mutations in PIK3CD and PIK3R1 cause activated PI3K-δ syndrome (APDS) by dysregulation of the PI3K-AKT pathway. METHODS: We studied precursor and peripheral B-cell differentiation and apoptosis via flowcytometry. Furthermore, we performed AKT-phosphorylation assays and somatic hypermutations (SHM) and class switch recombination (CSR) analysis. RESULTS: We identified 13 patients of whom 3 had new mutations in PIK3CD or PIK3R1. Patients had low total B-cell numbers with increased frequencies of transitional B cells and plasmablasts, while the precursor B-cell compartment in bone marrow was relatively normal. Basal AKT phosphorylation was increased in lymphocytes from APDS patients and natural effector B cells where most affected. PI3K mutations resulted in altered SHM and CSR and increased apoptosis. CONCLUSIONS: The B-cell compartment in APDS patients is affected by the mutations in PI3K. There is reduced differentiation beyond the transitional stage, increased AKT phosphorylation and increased apoptosis. This B-cell phenotype contributes to the clinical phenotype.

Human plasmacytoid dendritic cells efficiently cross-present exogenous Ags to CD8+ T cells despite lower Ag uptake than myeloid dendritic cell subsets.

In human peripheral blood, 4 populations of dendritic cells (DCs) can be distinguished, plasmacytoid dendritic cells (pDCs) and CD16(+), CD1c(+), and BDCA-3(+) myeloid DCs (mDCs), each with distinct functional characteristics. DCs have the unique capacity to cross-present exogenously encountered antigens (Ags) to CD8(+) T cells. Here we studied the ability of all 4 blood DC subsets to take up, process, and present tumor Ags to T cells. Although pDCs take up less Ags than CD1c(+) and BDCA3(+) mDCs, pDCs induce potent Ag-specific CD4(+) and CD8(+) T-cell responses. We show that pDCs can preserve Ags for prolonged periods of time and on stimulation show strong induction of both MHC class I and II, which explains their efficient activation of both CD4(+) and CD8(+) T cells. Furthermore, pDCs cross-present soluble and cell-associated tumor Ags to cytotoxic T lymphocytes equally well as BDCA3(+) mDCs. These findings, and the fact that pDCs outnumber BDCA3(+) mDCs, both in peripheral blood and lymph nodes, together with their potent IFN-I production, known to activate both components of the innate and adaptive immune system, put human pDCs forward as potent activators of CD8(+) T cells in antitumor responses. Our findings may therefore have important consequences for the development of antitumor immunotherapy.

Targeted delivery of TLR ligands to human and mouse dendritic cells strongly enhances adjuvanticity.

Effective vaccines consist of 2 components: immunodominant antigens and effective adjuvants. Whereas it has been demonstrated that targeted delivery of antigens to dendritic cells (DCs) improves vaccine efficacy, we report here that co-targeting of TLR ligands (TLRLs) to DCs strongly enhances adjuvanticity and immunity. We encapsulated ligands for intracellular TLRs within biodegradable nanoparticles coated with Abs recognizing DC-specific receptors. Targeted delivery of TLRLs to human DCs enhanced the maturation and production of immune stimulatory cytokines and the Ag-specific activation of naive CD8(+) T cells. In vivo studies demonstrated that nanoparticles carrying Ag induced cytotoxic T-lymphocyte responses at 100-fold lower adjuvant dose when TLRLs were co-encapsulated instead of administered in soluble form. Moreover, the efficacy of these targeted TLRLs reduced the serum cytokine storm and related toxicity that is associated with administration of soluble TLRLs. We conclude that the targeted delivery of adjuvants may improve the efficacy and safety of DC-based vaccines.

Route of administration modulates the induction of dendritic cell vaccine-induced antigen-specific T cells in advanced melanoma patients.

PURPOSE: It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine. EXPERIMENTAL DESIGN: HLA-A2.1(+) melanoma patients scheduled for regional lymph node dissection were vaccinated four times biweekly via intradermal or intranodal injection with 12 × 106 to 17 × 106 mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (KLH). Half of the patients also received low-dose IL-2 (9 MIU daily for 7 days starting 3 days after each vaccination). KLH-specific B- and T-cell responses were monitored in blood. gp100- and tyrosinase-specific T-cell responses were monitored in blood by tetramer analysis and in biopsies from delayed-type hypersensitivity (DTH) skin tests by tetramer and functional analyses with (51)Cr release assays or IFNγ release, following coculture with peptide-pulsed T2 cells or gp100- or tyrosinase-expressing tumor cells. RESULTS: In 19 of 43 vaccinated patients, functional tumor antigen-specific T cells could be detected. Although significantly more DCs migrated to adjacent lymph nodes upon intranodal vaccination, this was also highly variable with a complete absence of migration in 7 of 24 intranodally vaccinated patients. Intradermal vaccinations proved superior in inducing functional tumor antigen-specific T cells. Coadministration of IL-2 did not further augment the antigen-specific T-cell response but did result in higher regulatory T-cell frequencies. CONCLUSION: Intradermal vaccination resulted in superior antitumor T-cell induction when compared with intranodal vaccination. No advantage of additional IL-2 treatment could be shown.

Potential target antigens for a universal vaccine in epithelial ovarian cancer.

The prognosis of epithelial ovarian cancer (EOC), the primary cause of death from gynaecological malignancies, has only modestly improved over the last decades. Immunotherapeutic treatment using a cocktail of antigens has been proposed as a "universal" vaccine strategy. We determined the expression of tumor antigens in the context of MHC class I expression in 270 primary tumor samples using tissue microarray. Expression of tumor antigens p53, SP17, survivin, WT1, and NY-ESO-1 was observed in 120 (48.0%), 173 (68.9%), 208 (90.0%), 129 (56.3%), and 27 (11.0%) of 270 tumor specimens, respectively. In 93.2% of EOC, at least one of the investigated tumor antigens was (over)expressed. Expression of MHC class I was observed in 78.1% of EOC. In 3 out 4 primary tumors, (over)expression of a tumor antigen combined with MHC class I was observed. These results indicate that a multiepitope vaccine, comprising these antigens, could serve as a universal therapeutic vaccine for the vast majority of ovarian cancer patients.

Role of T cell competition in the induction of cytotoxic T lymphocyte activity during viral vector-based immunization regimens.

T cell competition between antigen- and vector-specific T cells may determine the outcome of viral vector-based immunization regimens, as we previously proposed. Here, we unravelled the interplay between antigen- and vector-specific immunity, using recombinant Semliki Forest virus (rSFV). Priming of mice with rSFV, encoding LacZ (SFVLacZ), or with empty rSFV strongly suppressed subsequent induction of ovalbumin or Human Papilloma virus (HPV) E6/E7-specific CTL activity by a booster with SFVeOVA or SFVeE6,7, respectively. Yet, priming with irradiated, i.e. replication-defective, SFVLacZ did not affect subsequent CTL induction, indicating that the interfering vector-specific immunity was directed against the viral replicase. However, immune responses against the strongly immunogenic nucleoprotein of influenza virus encoded by SFV were less severely affected by priming with SFVLacZ. Thus the outcome of heterologous prime-boost immunizations appears to depend on the immunogenicity of the respective antigens.

Commonly used prophylactic vaccines as an alternative for synthetically produced TLR ligands to mature monocyte-derived dendritic cells.

Currently dendritic cell (DC)-based vaccines are explored in clinical trials, predominantly in cancer patients. Murine studies showed that only maturation with Toll-like receptor (TLR) ligands generates mature DCs that produce interleukin-12 and promote optimal T-cell help. Unfortunately, the limited availability of clinical-grade TLR ligands significantly hampers the translation of these findings into DC-based vaccines. Therefore, we explored 15 commonly used preventive vaccines as a possible source of TLR ligands. We have identified a cocktail of the vaccines BCG-SSI, Influvac, and Typhim that contains TLR ligands and is capable of optimally maturing DCs. These DCs (vaccine DCs) showed high expression of CD80, CD86, and CD83 and secreted interleukin-12. Although vaccine DCs exhibited an impaired migratory capacity, this could be restored by addition of prostaglandin E(2) (PGE(2); vaccine PGE(2) DCs). Vaccine PGE(2) DCs are potent inducers of T-cell proliferation and induce Th1 polarization. In addition, vaccine PGE(2) DCs are potent inducers of tumor antigen-specific CD8(+) effector T cells. Finally, vaccine PGE(2)-induced DC maturation is compatible with different antigen-loading strategies, including RNA electroporation. These data thus identify a new clinical application for a mixture of commonly used preventive vaccines in the generation of Th1-inducing clinical-grade mature DCs.

Immunization with a P53 synthetic long peptide vaccine induces P53-specific immune responses in ovarian cancer patients, a phase II trial.

The prognosis of ovarian cancer, the primary cause of death from gynecological malignancies, has only modestly improved over the last decades. Immunotherapy is one of the new treatment modalities explored for this disease. To investigate safety, tolerability, immunogenicity and obtain an impression of clinical activity of a p53 synthetic long peptide (p53-SLP) vaccine, twenty patients with recurrent elevation of CA-125 were included, eighteen of whom were immunized 4 times with 10 overlapping p53-SLP in Montanide ISA51. The first 5 patients were extensively monitored for toxicity, but showed no > or = grade 3 toxicity, thus accrual was continued. Overall, toxicity was limited to grade 1 and 2, mostly locoregional, inflammatory reactions. IFN-gamma producing p53-specific T-cell responses were induced in all patients who received all 4 immunizations as measured by IFN-gamma ELISPOT. An IFN-gamma secretion assay showed that vaccine-induced p53-specific T-cells were CD4(+), produced both Th1 and Th2 cytokines as analyzed by cytokine bead array. Notably, Th2 cytokines dominated the p53-specific response. P53-specific T-cells were present in a biopsy of the last immunization site of at least 9/17 (53%) patients, reflecting the migratory capacity of p53-specific T-cells. As best clinical response, stable disease evaluated by CA-125 levels and CT-scans, was observed in 2/20 (10%) patients, but no relationship was found with vaccine-induced immunity. This study shows that the p53-SLP vaccine is safe, well tolerated and induces p53-specific T-cell responses in ovarian cancer patients. Upcoming trials will focus on improving T helper-1 polarization and clinical efficacy.

Survival of ovarian cancer patients overexpressing the tumour antigen p53 is diminished in case of MHC class I down-regulation.

OBJECTIVES: The adaptive immune system seems to play an essential role in the natural course of ovarian cancer. Aim of this study was to establish whether disease-specific survival for patients expressing the tumour antigen p53 is influenced by MHC class I expression or the presence of p53 autoantibodies (p53-Aab). METHODS: P53 and MHC class I expression were analysed in ovarian cancer tissue of 329 patients by immunohistochemistry using tissue microarrays. For 233 patients, pre-treatment serum samples were available to study the presence of p53 autoantibodies by ELISA. Data were linked to clinicopathological parameters and disease-specific survival. RESULTS: P53 overexpression, MHC class I down-regulation in neoplastic cells and serum p53 autoantibodies were observed in 49.4, 38.9 and 15.9% of patients, respectively. MHC class I down-regulation in p53-overexpressing tumours correlated with a 10-month reduced disease-specific survival in univariate analysis (log-rank 4.10; p=0.043). p53-Aab were strongly correlated with p53 overexpression (p<0.001), but did not influence disease-specific survival. CONCLUSIONS: As the prognosis of patients with p53-overexpressing ovarian cancer is affected by the MHC class I status of tumour cells and ovarian cancer patients can generate immune responses to the p53 tumour antigen, the further development of immunotherapy should evaluate strategies to improve MHC class I expression by tumour cells to facilitate antigen presentation in an attempt to increase clinical responses.

Serum cytokine profiling as a diagnostic and prognostic tool in ovarian cancer: a potential role for interleukin 7.

PURPOSE: To evaluate if serum cytokine levels could be used as diagnostic or prognostic markers in ovarian cancer. EXPERIMENTAL DESIGN: A cytokine bead array was done to simultaneously analyze 14 cytokines in the sera of 187 ovarian cancer patients with complete clinicopathologic data and follow-up, 45 patients with benign ovarian tumors, and 50 healthy controls. Serum levels of the well-known serum tumor marker CA-125 were routinely measured in all patients. RESULTS: Serum levels of CA-125, interleukin 6 (IL-6), IL-7, and IL-10 were elevated in ovarian cancer patients compared with patients with benign ovarian tumors. Analyzing the cytokines in combination with CA-125 showed that a combination of IL-7 and CA-125 serum levels could accurately predict 69% of the ovarian cancer patients, without falsely classifying patients with benign pelvic mass. The cytokines IL-6, IL-7, IL-8, IL-10, monocyte chemotactic protein-1 (MCP-1), and IP-10 and CA-125 were associated with disease-free and overall survival in univariate analysis. In multivariate analysis, IL-7 and IP-10 were independent predictors of overall survival, although after inclusion of the clinicopathologic parameters, only stage and residual disease remained as independent predictors of survival. CONCLUSIONS: IL-7 levels were found to be strongly associated with ovarian cancer and could be used in combination with CA-125 to distinguish between malignant and benign ovarian tumors.

P53-specific T cell responses in patients with malignant and benign ovarian tumors: implications for p53 based immunotherapy.

Despite intensive treatment, 70% of the ovarian cancer patients will develop recurrent disease, emphasizing the need for new approaches such as immunotherapy. A promising antigenic target for immunotherapy in ovarian cancer is the frequently overexpressed p53 protein. The aim of the study was to evaluate the nature and magnitude of the baseline anti-p53 immune response in ovarian cancer patients. P53-specific T cell responses were detected in both half of the ovarian cancer patients as in the group of control subjects, consisting of women with benign ovarian tumors and healthy controls. Importantly, while in the control group p53-specific immunity was detected among the CD45RA(+) naïve subset of T cells only, the p53-specific T-cell responses in ovarian cancer patients were also present in the CD45RO(+) memory T-cell subset, suggesting that in the cancer patients sufficient amounts of cancer-derived p53 was presented to induce the formation of a p53-specific memory T-cell response. Further characterization of the p53-specific memory T-cell responses revealed that in addition to the type 1 cytokine IFN-gamma also the type 2 cytokines IL-4 and IL-5, as well as the immunosuppressive cytokine IL-10 were produced. Notably, p53-specific T cells were not only detected in the peripheral blood, but also among tumor infiltrating lymphocytes and in tumor-draining lymph nodes. In conclusion, the existence of a weak mixed T-helper type 1 and 2 p53-specific T-cell repertoire supports the rationale of using p53 long peptides in vaccination strategies aiming at the induction of p53-specific Th1/CTL immunity.