Interferon-induced programmed death-ligand 1 (PD-L1/B7-H1) expression increases on human acute myeloid leukemia blast cells during treatment.

INTRODUCTION: While current treatment for acute myeloid leukemia is characterized by high response rates, patients' long-term outcome is still disappointing, due to frequent relapse and ineligibility of the often elderly patients for stem cell transplantation approaches. Considerable efforts have, thus, been made to incorporate immunotherapeutic approaches in the acute myeloid leukemia (AML) consolidation, with so far disappointing clinical benefit. The B7 family ligand programmed-death receptor-ligand 1 (PD-L1, B7-H1, CD274) has been recently described (with conflicting results) to be expressed on AML blast cells, and interaction with its receptor on T cells, programmed death receptor-1 (PD-1, CD279), has been shown to suppress T-cell functions and to allow survival of dormant AML cells in animal models. DESIGN AND METHODS: In this work, we analyzed freshly isolated myeloid precursor cells from healthy donors and from AML patients for PD-L1 expression with or without interferon-γ exposure at different time points during their treatment. RESULTS: While without IFN exposure, only minor differences were observed, we found IFN-γ-induced PD-L1 expression most prominent after initial treatment and independent of treatment outcome. CONCLUSIONS: Our observations support the recently suggested PD-L1-mediated adaptive immune resistance and argue for a targeting of the PD-L1/PD-1 pathway during the consolidation phase of AML treatment.

NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability.

The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy.

PD-1 expression on Melan-A-reactive T cells increases during progression to metastatic disease.

Programmed death 1 (PD-1) is known as an important factor for the development of tolerogenicity. This has been proven in chronic viral infections and different tumor models. To address the role of PD-1 and its ligand programmed death ligand 1 (PD-L1) in different stages of malignant melanoma, we investigated peripheral blood and tumor tissues in regard to overall survival (OS) and prognostic relevance. One hundred samples of peripheral blood mononuclear cells from HLA-A2(+) patients with malignant melanoma (Stages I-IV) were analyzed in seven color FACS combined with multimer analyses for the immunodominant epitope of Melan-A (peptide A2/Melan-A(p26-35mod) ). Corresponding formalin-fixed paraffin-embedded tissues of primary tumor and distant organ metastases from 37 cases were analyzed by immunohistochemistry for Melan-A, PD-L1 and PD-1 expression. Compared to the total CD8(+) T cell population, PD-1 expression by A2/Melan-A(+) CD8(+) T cells was over-represented in melanoma stages III and IV (p < 0.001). Although elevation of PD-1(+) Melan-A(+) CD8(+) T cells had no significant influence on OS, a positive correlation was observed between PD-L1 expression on melanoma cells and OS (p = 0.05). Correlation of advanced tumor stage with increased A2/Melan-A-multimer(+) PD-1(+) T cells in the peripheral blood suggest that blocking of PD-1 could have therapeutic potential in advanced stage melanoma.

Allorestricted T lymphocytes with a high avidity T-cell receptor towards NY-ESO-1 have potent anti-tumor activity.

The cancer-testis antigen NY-ESO-1 has been targeted as a tumor-associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T-cell-based therapy is the induction of T cells capable of recognizing the NY-ESO-1-expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY-ESO-1(157-165) epitope known to be naturally presented with HLA-A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY-ESO-1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA-A2-positive, NY-ESO-1-expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY-ESO-1-specific T lymphocytes displayed TCRs with the highest avidity and best anti-tumor recognition activity. TCRs derived from allorestricted, NY-ESO-1-specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of NY-ESO-1-expressing hematological malignancies and solid tumors.

Decreased numbers of regulatory T cells suggest impaired immune tolerance in children with tourette syndrome: a preliminary study.

BACKGROUND: Post-streptococcal autoimmune inflammation of basal ganglia was suggested to be an etiological factor in some cases of Tourette syndrome (TS). Since regulatory T (T reg) cells play a major role in preventing autoimmunity, we hypothesized that a defect in T reg cells may be present in children with TS. We also postulated that group A beta hemolytic streptococcal infections could promote autoimmune responses by releasing exotoxins (streptococcal pyrogenic exotoxins [SPE]). METHODS: We analyzed peripheral blood of TS patients and healthy age-matched control subjects by fluorescence-activated cell sorting (FACS) on multiple occasions and determined the numbers of CD4(+)CD25(+)CD69(-) T reg cells. Further, we quantified the number of CD4(+) and CD8(+) lymphocytes with regard to Vbeta chains to which SPEs are known to bind. RESULTS: A significant decrease in T reg cells was observed in patients with moderate to severe TS symptoms compared with healthy age-matched control children. A decrease in T reg cell number was also noted during symptom exacerbations in five out of six patients. Further, we found a significant decrease in numbers of CD8(+)Vbeta18(+) T cells in moderate to severe TS patients. CONCLUSIONS: These data support our hypothesis that at least some TS patients may have a decreased capacity to inhibit autoreactive lymphocytes through a deficit in T reg cells. Interactions of host T cell immunity and microbial factors may also contribute to the pathogenesis of TS.

Increased serum levels of interleukin-12 and tumor necrosis factor-alpha in Tourette's syndrome.

BACKGROUND: The hypothesis that common infections can modulate the onset and course of tic disorders and early-onset obsessive-compulsive disorder (OCD) in pediatric populations is longstanding. To date, most investigations have focused on the hypothesis of molecular mimicry and humoral immune responses. This study was carried out to investigate whether cytokines associated with the innate immune response or T cell activation were altered under baseline conditions and during periods of symptom exacerbation. METHODS: Forty-six patients with Tourette's syndrome and/or early-onset OCD, aged 7-17 years, and 31 age-matched control subjects participated in a prospective longitudinal study. Ratings of clinical severity and serum were collected at regular intervals, and serum concentrations of 10 cytokines were measured repeatedly. RESULTS: Interleukin-12 and tumor necrosis factor alpha concentrations at baseline were elevated in patients compared with control subjects. Both of these markers were further increased during periods of symptom exacerbation. CONCLUSIONS: These findings suggest that symptom exacerbations are associated with an inflammatory process propagated by systemic and local cytokine synthesis that might involve the central nervous system. We conclude that, in the future, longitudinal studies of children with neuropsychiatric disorders should examine the involvement of innate and T cell immunity.

CTLs directed against HER2 specifically cross-react with HER3 and HER4.

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated Ag by T cell-based immunotherapeutical strategies such as cancer vaccines and adoptive T cell transfer. The prerequisite for a successful T cell-based therapy is the induction of T cells capable of recognizing the HER2-expressing tumor cells. In this study, we generated human cytotoxic T cell clones directed against the HER2(369-377) epitope known to be naturally presented with HLA-A*0201. Those HER2-reactive CTLs, which were also tumor lytic, exhibited a similar lysis pattern dividing the targets in lysable and nonlysable tumor cells. Several HER2-expressing tumor cells became susceptible to CTL-mediated lysis after IFN-gamma treatment and, in parallel, up-regulated molecules of the Ag-presenting machinery, indicating that the tumor itself also contributes to the success of CTL-mediated killing. Some of the HER2(369-377)-reactive T cells specifically cross-reacted with the corresponding peptides derived from the family members HER3 and/or HER4 due to a high sequence homology. The epitopes HER3(356-364) and HER4(361-369) were endogenously processed and contributed to the susceptibility of cell lysis by HER cross-reacting CTLs. The principle of "double" or "triple targeting" the HER Ags by cross-reacting T cells will impact the further development of T cell-based therapies.

Visualization of antigen-specific human cytotoxic T lymphocytes labeled with superparamagnetic iron-oxide particles.

New technologies are needed to characterize the migration and survival of antigen-specific T cells in vivo. In this study, we developed a novel technique for the labeling of human cytotoxic T lymphocytes with superparamagnetic iron-oxide particles and the subsequent depiction with a conventional 1.5-T magnetic resonance scanner. Antigen-specific CD8(+) T lymphocytes were labeled with ferucarbotran by lipofection. The uptake of ferucarbotran was confirmed by immunofluorescence microscopy using a dextran-specific antibody, and the intracellular enrichment of iron was measured by atomic absorption spectrometry. The imaging of T cells was performed by magnetic resonance on day 0, 2, 7 and 14 after the labeling procedure. On day 0 and 2 post labeling, a pronounced shortening of T2*-relaxation times was observed, which diminished after 7 days and was not detectable anymore after 14 days, probably due to the retained mitotic activity of the labeled T cells. Of importance, the antigen-specific cytolytic activity of the T cells was preserved following ferucarbotran labeling. Efficient ferucarbotran labeling of functionally active T lymphocytes and their detection by magnetic resonance imaging allows the in vivo monitoring of T cells and, subsequently, will impact the further development of T cell-based therapies.

IL-2 and IL-4 polymorphisms as candidate genes in schizophrenia.

An immune process, characterized by a relative predominance of the T helper-2 (Th2) system and possibly induced by a viral infection,may be involved in the pathophysiology of schizophrenia. In this context, functional polymorphisms in the Interleukin-2 (IL-2) and Interleukin-4 (IL-4) genes appear to be principal candidates for genetic schizophrenia research. Further evidence for these candidate genes comes from several linkage analyses, pointing to susceptibility gene loci on chromosomes 4q and 5q, where the genes coding for IL- 2 and IL-4 are located. We carried out a case-control study including 230 schizophrenic patients and 251 healthy persons, investigating the IL-2 -330 T/G single nucleotide polymorphism (SNP) and the IL-4 -590 C/T SNP. A significant association of the IL-2 -330 TT genotype and of the IL-4 -590 CC genotype with schizophrenia could be identified. Our findings may partly account for the relative predominance of the Th2 system in schizophrenia, although they cannot directly explain this immunological imbalance, but may be related to an altered antiviral immune response in patients with schizophrenia.

ICAM G241A polymorphism and soluble ICAM-1 serum levels: evidence for an active immune process in schizophrenia.

OBJECTIVES: We have previously reported reduced serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) in schizophrenic patients. A single-nucleotide polymorphism (SNP) of the ICAM-1 gene was described at position 241. The G-->A SNP results in a nonsynonymous amino acid exchange of the ICAM-1 protein, and the A allele was shown to be also associated with several immunological disorders like rheumatoid arthritis. METHODS: We investigated 70 schizophrenic patients and 128 unrelated healthy control persons regarding the relationship between the serum levels of sICAM-1 and the ICAM-1 G214A polymorphism. RESULTS: We were able to replicate our previous finding of reduced sICAM-1 levels in schizophrenia. Healthy control persons carrying the polymorphic A allele showed markedly lower sICAM-1 serum levels than carriers of the homozygous GG wild type (p < 0.004). In contrast, no significant difference in the sICAM-1 serum levels were seen regarding the G241A genotype distribution in schizophrenic patients. CONCLUSION: We hypothesize that the biochemical effect of the G241A SNP is masked in schizophrenic patients, indicating a disease-related mechanism leading to reduced levels of sICAM-1 in schizophrenia.

No association between the G308A polymorphism of the tumor necrosis factor-alpha gene and schizophrenia.

Several linkage analyses in schizophrenia research point to a locus on chromosome 6p22, where the gene coding for tumor necrosis factor-alpha (TNF-alpha) is located. A marked influence of antipsychotic medication on TNF-alpha has been described. As the involvement of an immune process in the pathophysiology of schizophrenia has been discussed, a functional TNF-alpha polymorphism appears to be a candidate in genetic schizophrenia research. The G308A polymorphism of the TNF-alpha gene was described to be associated with increased TNF-alpha production. Boin and colleagues have already described a significant association between the polymorphic allele and schizophrenia, investigating 84 schizophrenic patients (21 % polymorphic allele) and 138 healthy volunteers (11 % polymorphic allele), recruited in Northern Italy. We carried out a replication study including 157 schizophrenic patients and 186 healthy persons, who were recruited in Southern Germany. Psychopathology was additionally monitored by PANSS. We were not able to replicate the findings of Boin et al., as we did not find any difference in allele frequency or genotype distribution between our schizophrenic patients (13.7 % polymorphic allele) and healthy controls (16.9 % polymorphic allele). Moreover, we did not find any association between genotype and psychopathology, as measured by PANSS. The different results between these two studies may be due to ethnic differences.