T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1 157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1 157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes.

© F e r r a t a S t o r t i F o u n d a t i o nshown to be crucial in sustaining adult B-cell lymphopoiesis. 21 However, ablation of the FLT3/FLT3L axis alone did not result in a complete block in the generation of any hematopoietic lineage, suggesting that FLT3L might exert its crucial role in hematopoiesis through interactions with other cytokines, such as interleukin-7 (IL-7) or stem cell factor. 19,22,23 To elucidate the specific action of FLT3L on hematopoiesis in vivo, FLT3L has been administered, with the results confirming the important role of FLT3L in DC generation. 24 We have previously shown that apart from DC, FLT3L injection leads to transient expansion of a FLT3 + progenitor population with lymphoid and myeloid potential. 25 In order to evaluate the role of FLT3L in the development of different hematopoietic lineages, in this study we investigated the effects of sustained over-expression of FLT3L in a transgenic mouse model. Our study confirms the positive role of FLTL in DC development and highlights the importance of this cytokine in the survival and expansion of lymphoid and myeloid progenitors. Furthermore, our data provide evidence for an instructive role of FLT3L in hematopoietic development. Methods MiceAll mice used in this study were bred and maintained in our animal facility under pathogen-free conditions and all animal experiments were performed in accordance with institutional guidelines (permission numbers 1887 and 1888). Immunization to induce a Tdependent antibody response and FLT3L treatment of mice were carried out as previously described. 25 Cell culturesST2, OP9 and OP9 stromal cells expressing Notch ligand Deltalike 1 (OP9DL1) were maintained in IMDM supplemented with 5 x 10 -5 M β-mercaptoethanol, 1 mM glutamine, 0.03% w/v Primatone (Quest Naarden, the Netherlands), 100 U/mL penicillin, 100 μg/mL streptomycin and 5% fetal bovine serum. Co-cultures of stromal cells with sorted progenitor cells were performed as previously described 25 (and Online Supplementary Materials and Methods). Platelet countsBlood was drawn from the tail vein of mice and incubated with 1% ammonium oxalate for 10 min at room temperature. Following incubation, live cells were counted in a Neubauer hemocytometer. ImmunofluorescenceSpleens were snap-frozen and embedded in OCT-compound (Sakura, Zoetermeer, the Netherlands), and 5 μm sections were prepared. Sections were fixed in acetone for 10 min, air-dried for 60 min and subsequently stained with fluorescein isothiocyanatelabeled anti-CD90, phycoerythrin-labeled anti-IgM and allophycocyanin-labeled anti-CD11c antibodies for 30 min. Results Splenomegaly and lymphadenopathy in FLT3L transgenic miceTo investigate the effect of prolonged FLT3L overexpression, we generated mice expressing the human FLT3L gene under the control of the β-actin promoter (hereafter referred to as FLT3L-Tg mice). FLT3L levels in the blood were in the range of 500-1000 ng/mL, as assesed by enzyme-linked immunosorbent assay using an antihuman FLT3L antibody developed in our ...

MAGE-C1/CT7 and MAGE-C2/CT10 are members of the large MAGE family of cancer-testis (CT) antigens. CT antigens are promising targets for immunotherapy in cancer because their expression is restricted to cancer and germ line cells and a proportion of cancer patients presents with immune responses against CT antigens, which clearly demonstrates their immunogenicity. This study investigates the expression of MAGE-C1/CT7 and MAGE-C2/CT10 in primary and metastatic melanoma. Immunohistochemical staining of tissue microarrays that consisted of 59 primary malignant melanomas of the skin, 163 lymph node and distant melanoma metastases and 68 melanoma cell lines was performed. We found MAGE-C1/CT7 expression in 15 out of 50 (24%) primary melanomas and 15 out of 50 (24%) cell lines, whereas MAGE-C2/CT10 was detected in 17 out of 51 (33%) primary melanomas and 14 out of 68 (17%) cell lines. MAGE-C1/CT7 and MAGE-C2/CT10 were both detected in 40% of melanoma metastases. Patients with MAGE-C1/CT7 or MAGE-C2/CT10 positive primary melanoma had significantly more lymph node metastases (p = 0.005 and p<0.001, resp.). Prediction of lymph node metastasis by MAGE-C1/CT7 and MAGE-C2/CT10 was independent of tumor cell proliferation rate (Ki67 labeling index) in a multivariate analysis (p = 0.01). Our results suggest that the expression of MAGE-C1/CT7 and MAGE-C2/CT10 in primary melanoma is a potent predictor of sentinel lymph node metastasis.

Some signs of potential autoimmunity, such as the appearance of antinuclear antibodies (ANAs) become prevalent with age. In most cases, elderly people with ANAs remain healthy. Here, we investigated whether the same holds true for inbred strains of mice. Indeed, we show that most mice of the C57BL/6 (B6) strain spontaneously produced IgG ANA at 8-12 months of age, showed IgM deposition in kidneys and lymphocyte infiltrates in submandibular salivary glands. Despite all of this, the mice remained healthy. ANA production is likely CD4 + T-cell dependent, since old (40-50 weeks of age) B6 mice deficient for MHC class II do not produce IgG ANAs. BM chimeras showed that ANA production was not determined by age-related changes in radiosensitive, hematopoietic progenitor cells, and that the CD4 + T cells that promote ANA production were radioresistant.Thymectomy of B6 mice at 5 weeks of age led to premature alterations in T-cell homeostasis and ANA production, by 15 weeks of age, similar to that in old mice. Our findings suggest that a disturbed T-cell homeostasis may drive the onset of some autoimmune features.Keywords: Antinuclear antibody r Autoimmunity r Sjögren's syndrome r T-cell homeostasis See accompanying Commentary by Winkler and WaismanAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionIt is well established that the functional activity of the immune system declines with age. The fact that mortality and morbidity during annual influenza epidemics increases in the elderly population is a clear example of this [1]. Moreover, influenza vaccinations are only marginally protective for the elderly [2][3][4]. Yet another example of immune activity decline with age is the reactivation of the Varicella zoster virus in the elderly. The frequency of this reactivation increases in individuals after the fiftieth year [5] and is thought to be due to a generalized decreased memory response to Correspondence: Prof. Antonius Rolink e-mail: antonius.rolink@unibas.ch chronic infections. In addition, some malignancies are more often found among the elderly [6]. For multiple myeloma, the median age of diagnosis is 66 years and only 2% of all patients are younger than 40 years of age. The increased incidence of this plasma cell tumor and of certain other malignancies with age might suggest that, in these cases, immune surveillance is effective in young individuals, but declines with age. The decline in immune competence is thought to be due to an age-related decrease in production of both B cells and T cells. We and others have shown that in both mouse and man, B-cell production decreases with age [7][8][9]. Thus, the frequency of mouse BM derived pro-B cells, which can be grown on stromal cells in the presence of IL-7, decreases with age: from a frequency of around 1 in 50 at 2 weeks of age, whereas this frequency drops to 1 in C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 2894 Anja Nusser et al. Eur. J. Immunol. 2014. 44: 2893-2902...

Cancer/testis (CT) antigens represent prime candidates for immunotherapy in cancer patients, because their expression is restricted to cancer cells and germ cells of the testis. MAGE-C1/CT7 is a CT antigen that is highly expressed in several types of cancers. Spontaneous occurrence of CT7-specific antibodies was previously detected by SEREX screen in a melanoma patient. However, naturally occurring CT7-specific T-cell responses have thus far not been detected. Peripheral blood mononuclear cells (PBMCs) from 26 metastatic melanoma patients expressing CT7 in their tumor lesions (CT7 + ) were analyzed for CT7-specific T-cell responses using overlapping peptides. CT7-specific CD4 + T-cell responses were detected in three patients (11.5%). These CT7-specific CD4 + T-cell responses were detectable in melanoma patients' PBMCs exclusively from preexisting CD45RA − memory CD4 + T-cell pool. Additional CT7-specific memory CD4 + T-cell responses were detected in CT7 + melanoma patients after depletion of CD4 + CD25high Treg cells showing that Treg cells impact on CT7-specific CD4 + T cells in melanoma patients. CT7-specific CD4 + T-cell clones were generated and used to define minimal epitopes, restriction elements, and confirm the recognition of naturally processed antigen. Surprisingly, these clones were able to secrete perforin and exert cytotoxicity. This study shows that CT7 can induce specific cellular immunity in melanoma patients. Based on these findings, CT7 will be further explored as a potential vaccine for melanoma immunotherapy.cancer/testis antigens | T-cell response