Abstract Background: Recent research has verified that blood group or Rh factor can influence susceptibility to various cardiovascular, neoplastic and infectious diseases including COVID-19. While a number of studies have looked at correlations between blood group and various rheumatological diseases, findings have been inconsistent, often because many of these studies suffered from small sample size issues. In order to better understand the potential relationships between blood group/Rh factor and rheumatological diseases, we performed a large-scale self-report pilot study of blood type distributions in five autoimmune diseases.Methods: Five autoimmune diseases were included in the study: systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and ankylosing spondylitis. We also included a control group in which participants did not have any autoimmune diseases. The participants were recruited through social media and organizations such as the Lupus Foundation and the National Psoriasis Foundation. Respondents who met the inclusion criteria were asked only two questions by anonymous survey: blood type and country of birth.Results: Each autoimmune disorder group included between 570 and 951 US participants. While there was little difference in blood type distribution patterns among the five diseases, unexpectedly, all five disease groups showed a consistent pattern where Rh negative was almost twice as high as US population norms. A post-hoc non-autoimmune control group was added in order to determine if this anomalous finding was an artifact of the study design. The control group displayed a similar unexpected increase in the Rh-negative blood type prevalence, suggesting that the very high Rh-negative frequency among the tested disease groups was likely to be an artifact of the study design. Conclusions: Overall, our preliminary study results show no meaningful differences between the disease groups and the post-hoc control group, suggesting that neither ABO type nor Rh factor affects susceptibility to the development of any of the five studied autoimmune diseases. Nevertheless, the unexpected observed difference in Rh factor distribution between the studied groups/control group and the corresponding US population norms has important implications for any research study using self-selected subjects. Our results suggest that such studies may be subject to unanticipated biases, requiring meticulous controls to confirm impartiality and exclude any artifacts of the study design.

gamma delta T cells in human solid tumors remain poorly defined. Here, we describe molecular and functional analyses of T-cell receptors (TCR) from tumor-infiltrating gamma delta T lymphocytes (gamma delta TIL) that were in direct contact with tumor cells in breast cancer lesions from archival material. We observed that the majority of gamma delta TILs harbored a proinflammatory phenotype and only a minority associated with the expression of IL17. We characterized TCR gamma or TCR delta chains of gamma delta TILs and observed a higher proportion of V delta 2(+) T cells compared with other tumor types. By reconstructing matched V delta 2(-) TCR gamma and TCR delta pairs derived from single-cell sequencing, our data suggest that gd TILs could be active against breast cancer and other tumor types. The reactivity pattern against tumor cells depended on both the TCR gamma and TCR delta chains and was independent of additional costimulation through other innate immune receptors. We conclude that gamma delta TILs can mediate tumor reactivity through their individual gamma delta TCR pairs and that engineered T cells expressing TCR gamma and delta chains derived from gamma delta TILs display potent antitumor reactivity against different cancer cell types and, thus, may be a valuable tool for engineering immune cells for adoptive cell therapies.

To the Editor––Zeng et al [1] describe discouraging effects of convalescent plasma therapy on survival in patients with coronavirus disease 2019 (COVID-19). This contrasts with quite opposite results of a pilot study [2] published earlier. The fundamental difference between these 2 studies is that the presence of virus-neutralizing antibodies (nAbs) in convalescent plasma was not assessed by Zeng et al [1], whereas convalescent plasma in the earlier and very encouraging study [2] was selected to contain substantial amounts of nAbs. These findings highlight the importance of assessing the presence of nAbs in convalescent plasma used therapeutically in patients with COVID-19. Zeng et al [1] also describe a 30-yearold severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)–positive woman with bilateral pneumonia, in whom a sepsislike syndrome progressively developed. This progression was not affected by 2 convalescent plasma transfusions (400 and 200 mL, with unknown amounts of nAbs, administered 2 days apart), ultimately resulting in a fatal outcome. Indeed, in many patients with COVID-19, sepsislike syndromes develop, in which therapeutic plasma exchange (TPE) may significantly reduce the levels of key proinflammatory cytokines and permeability factors [3, 4] elevated owing to inappropriate inflammatory responses [5]. TPE is an effective treatment in many clinical situations, and a typical single TPE treatment takes about 2 hours and replaces 1–1.5 blood volumes; the plasma is discarded and replaced by an isotonic solution of approximately 5% human albumin, which typically does not induce any detectable hypersensitivity or toxic reactions. TPE may be complemented by replacing 400 mL of the isotonic human albumin solution with 400 mL of ABOmatched convalescent plasma [1, 2] containing high titers of anti-SARS-CoV-2 nAbs. Significantly higher nAb titers are present in elderly and middle-aged patients than in young recovered patients [6]. Many hospitals probably cannot perform the SARS-CoV-2-neutralization assay, which requires a high-containment (biosafety level 3 or 4) facility. Therefore, plasma with high titers of antibodies binding to the S1 receptor binding domain, S1-N-terminal domain (NTD) and S2 should be selected. Testing with enzyme-linked immunosorbent assay (or another suitable assay) can replace the virus neutralization assay, because practically all known nAbs bind to 1 of these 3 SARS-CoV-2 regions and interfere with binding to angiotensin-converting enzyme 2 or with S2-mediated membrane fusion [7]. Alternatively, nAbs-containing allogeneic plasma could be replaced by cross-neutralizing SARS-CoV receptor binding domain–specific (human or “humanized”) antibodies [8] or SARSCoV-2–specific monoclonal nAbs [9].

The pandemic spread of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires very urgently to identify effective therapies and prophylaxis. This appears perhaps even more imperative, after remdesivir, seemingly the most promising antiviral drug with very potent in vitro activity against SARS-CoV-2, was tested in adults with severe COVID-19 in a randomized, double-blind, placebo-controlled, multi-centre trial with somewhat disappointing results [1] (quote): “In this study of adult patients admitted to hospital for severe COVID-19, remdesivir was not associated with statistically significant clinical benefits.”

Human T-cell lymphotropic virus (HTLV) belongs to a larger group of primate T-cell lymphotropic viruses (PTLVs) within the family Retroviridae. It is estimated that 10 to 20 million people worldwide may be infected with HTLV-1. Although most of them are asymptomatic, around 5% of infected individuals may develop either HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) or Adult T-cell Leukaemia/Lymphoma (ATLL). Public Health authorities in many countries have implemented routine blood-donor tests for HTLV-specific antibodies; but this is not the case for Germany since the reported prevalence is very low (7/100,000). With the aim to evaluate retrospectively the presence of HTLV-1 among oncology patients in this country, samples stored at the Universitätsklinikum Freiburg, were analyzed. For this purpose, two different nested-PCR (n-PCR) protocols have been modified and set up for HTLV-1 detection. One positive case was detected by n-PCR among 406 samples (0,25%) in a period of 5 years (2008-2012) corresponding to a T-Cell Lymphoma. Despite the low prevalence, this virus is circulating in Germany, probably due to the increasing numbers of immigrants in these last years. Physicians should consider HTLV-1 infection and suspect it taking in account the ethnic and relation to endemic regions regardless the patient's residence.

HTLV-1 diagnosis using PCR is based on the amplification of the viral DNA sequences. This is usually conducted using a nested PCR which involves two rounds of DNA amplification reactions targeting the viral polymerase gene. In this case, we set up the conditions to analyze a pool of 10 samples in a single n-PCR detecting the presence of HTLV-1.

In the wake of the sudden passing of Professor Paul Fisch, colleagues, collaborators and friends shared their thoughts on Paul's significant contributions to γδ T cell research and the scientific community at large. In 1968, shortly after the Russian invasion and occupation of Czechoslovakia, Dr Arthur Fisch, an Auschwitz survivor, relocated with his wife and 8-year-old Paul from Bratislava to Bad Neustadt an der Saale (West Germany). Paul studied medicine with an internship in Neurology in Cologne. After residency in Internal Medicine in Würzburg, Paul joined Paul Sondel's group at the Carbone Cancer Center at the University of Wisconsin in Madison, as a Leukemia Society of America Fellow. He was then awarded a German Research Foundation scholarship to work with Terry Rabbits at the Laboratory of Molecular Biology in Cambridge, UK. Paul returned to Germany as a group leader and Leukemia Society Fellow in the Department of Hematology and Oncology at the University of Freiburg, completing a Habilitation degree in Immunology under Thomas Boehm. Paul was then awarded the prestigious Heisenberg scholarship, joining Hans-Georg Rammensee's department at the University of Tübingen, where he completed a Habilitation in Immunology and Molecular Genetics. There he also received the Artur-Pappenheim-Award from the German Society of Hematology and Medical Oncology. In 1998, Paul returned to Freiburg as Professor of Molecular Pathology at the Institute for Clinical Pathology. In 2016, on his return from a scientific trip to Buenos Aires, Paul suffered a fall causing two vertebral fractures. Subsequent medical intervention and further complications thereof ultimately led to his death on 26 November 2018. Paul was a true pioneer of γδ T cell research, interested in multiple facets of the cells’ biology. Drawing scientific insight from meticulous investigation of human clinical data and cell culture systems, Paul made profound contributions to our understanding of γδ T cells, and to cancer immunology more broadly. After the wholly unanticipated discovery of the TCRγ chain1 and the subsequent discovery of γδ T cells,2, 3 some immunologists doubted that γδ T cell antigen recognition would differ from that of αβ T cells. However, word emerged from the Sondel lab in Wisconsin that – incontrovertibly – peripheral blood γδ T cells kill haematological malignancies in the absence of known MHC I or MHC II elements. The young clinician scientist Paul Fisch spearheaded these studies, bringing his findings to the 1990 International Workshop on the ‘Specificity and Function of γδ T cells’ at Schloss Elmau in Bavaria, Germany. Paul's time in Madison was fundamental to both his professional and personal life. Not only Paul's scientific discoveries,4-6 but also his friendships from those years would carry on for the rest of his life (Figure 1). In his seminal paper on γδ T cell specificity, Paul was one of the first to describe the molecular mimicry that allows γδ T cells to be shaped by infections but also to recognize tumour cells.5 In addition, Paul showed that γδ T cells can be subdivided into distinct subsets according to their cytotoxic responses,6 and that while inhibitory receptors typically found on NK cells likewise control γδ T cell cytotoxicity against Daudi lymphoma targets,7 γδ T cells are functionally distinct from NK cells.4, 6 These important discoveries set the landscape for a wave of studies about the role of NK receptors on γδ T cells, and their innate immune responses in tumour immunity,8, 9 which remains a very active research field.10, 11 With these early studies, Paul moved a large and important stone into place on the pathway to recognizing γδ T cell biology as unique and clinically profound. Indeed, Paul's work has proven prescient, as reflected in the wealth of strategies under development to harvest this potential. Biotech company founders in the γδ T cell space agree that Paul's discoveries provided scientific underpinnings that have enabled progress towards clinical trials. Beyond his early work, Paul continued to make crucial contributions as a sharp, fair reviewer and critical discussion partner for these companies. Naturally, Paul's research also continued in this vein, with recent papers on enhancement of tumour cell killing via manipulation of the γδ T cell antigen receptor 12, 13 and analysis of γδ T cells infiltrating triple-negative breast cancers.14 Paul not only thought about science, he also loved performing experiments! His keen interest in how natural phosphoantigens activate γδ T cells led Paul to bring a huge dry pellet of Daudi cells to Jean-Jacques Fournié in Toulouse for chromatographic separation. Careful analyses revealed only trace amounts of these very labile metabolites, unfortunately an unpublishable result. Undeterred, the duo screened a collection of ~2000 natural product extracts that Jean-Jacques had assembled during family holidays. Paul consistently observed activating compounds in these samples, pointing to ubiquitous stimuli for these unconventional T lymphocytes. More recently, while studying the diversity of γδ T cell expansions in immunodeficient patients and analysing the underlying mechanisms, Paul substantially improved the TCRγ and δ spectratyping method through intense laboratory experimentation.15 Paul was a central figure in the γδ T cell arena, highly respected for his honesty, work ethic and outstanding commitment to research. Paul's eminent role was honoured with his appointment as a chairman for the 2010 γδ T cell meeting in Kiel, Germany. Then, when the next organizers were unable to host the following conference, Paul stepped up and, together with Wolfgang Schamel, rescued the 2012 γδ T cell meeting on short notice. Paul wrote a conference grant application to the German Research Foundation that, hastily signed on the hood of Paul's car under a street lamp on a dark and drizzling Sunday evening, was thankfully successful!16 Paul was also important to the γδ T cell community on a personal level. Paul warmly welcomed researchers from other fields and befriended newcomers at γδ T cell conferences. Deeply committed to family, Paul would sometimes bring one of his children to γδ T cell meetings, and could carry on discussions about the unexpected characteristics and multifunctionality of γδ T cells while picking out colourful doughnuts! Not without some measure of unforeseen disarray, Paul was a generous and reliable collaborator, and helped junior and senior researchers alike gain a step up into the γδ T cell world by freely sharing his expertise and support. Paul inspired a new generation of scientists to pursue greater knowledge of γδ T cells. Many scientists cloning γδ T cells use Paul's protocol,4 often accompanied by his personal guidance. Paul also collaborated with clinicians outside of the γδ T cell field, studying patients with inherited immunodeficiencies and analysing the consequences of hypomorphic mutations in genes critical for VDJ recombination.17 This intriguing work was the beginning of several interesting discoveries in this area spanning over a decade, the most recent published in Blood in 2016.18 Paul's additional non-γδ T cell studies included thymoma-associated immunodeficiencies19 and hematopoietic cell transplantations.20 Paul was a quiet and humble person, with a demeanour that belied his vast intelligence. In his calm, friendly and reassuring way, he treated his conversation partners as equals, thereby instilling confidence in those who sought him out. It was easy to speculate and hypothesize with Paul, as he was an excellent listener, was very well read and his comments were thoughtful, pragmatic and to the point. Paul stood by his ideas, which collaborators and competitors alike sometimes found challenging, but they would always recognize the benefits of his input nonetheless. Paul's way of thinking might affectionately be described as a sort of ‘creative chaos’, as might his appearance, but his mind was sharp and analytical. Paul was a loyal friend, and an encouraging and generous mentor. He was quirky, yet warm and open not only with friends, but also with collaborators and students; discussions often went beyond science, to include family, philosophy or politics, sometimes over döner kebab or cheesecake and fine coffee. Those who were close to him appreciated Paul's special sense of humour, humanity and kindness. In his final year, Paul impressed all those around him with his energy and scientific drive, despite his failing health. Paul's unstoppable vision was to develop the use of γδ T cells for anticancer immunotherapy. As such, he mustered his remaining strength, will and passion to rally scientists to form a German consortium to analyse the role of γδ T cells in cancer, and submitted a funding proposal to the Deutsche Krebshilfe. When awarded an invitation to proceed, Paul fought his ill health to prepare for the defence in viva voce. With his iron will to advance science, Paul disregarded his deteriorating health, travelling to Bonn to defend his dream. On this unforgettable day, those with him shared moments of excitement, laughter, anger, eagerness and empathy. Despite his weakened state, Paul was following his passion and was therefore happy. He passed away a few days later, never having learned the outcome. The news of Paul's death came as a shock to all who knew him. Paul was a rare combination of kindness of heart and keenness of mind, and we feel very lucky to have had him as a friend and colleague. He brought us an abundance of beauty, inspiration, originality, authenticity, kindness and love. We sincerely hope Paul's wife Anja, daughter Nora and sons David and Aron will find some comfort and solace in knowing what an indelible impression Paul left on so many of us. He did not need a long life for us to measure. It was, rather, we who needed his life to be longer, and though he is gone, his unconditional love for science will continue to inspire us. This piece was a truly collaborative effort based on heartfelt contributions from Ilan Bank, Willi Born, Eric Champagne, Matthias Eberl, Stephan Ehl, Jean-Jacques Fournié, Adrian Hayday, José Villacorta Hidalgo, Jürgen Kuball, Lawrence Lamb Jr, Susana Minguet, Hans-Heinrich Oberg, Immo Prinz, Hans-Georg Rammensee, Wolfgang Schamel, Karin Schilbach, Paul Sondel and Daniela Wesch. Our sincere thanks go to the Fisch family for confirming the accuracy of dates and places, and for providing Paul's photo (Figure 2). The authors declare no conflict of interest.

HTLV-1 diagnosis using PCR is based on the amplification of the viral DNA sequences. This is usually conducted using a nested PCR which involves two rounds of DNA amplification reactions targeting the viral polymerase gene. In this case, we also add in the second round primers to amplify the human actin gene.

Background: Therapeutic plasma exchange has been tried as a treatment approach for systemic sclerosis since 1978 based on the rationale that some circulating factor is involved in disease pathogenesis, for example, autoantibodies or immune complexes, and that removing the potential pathogenic factors could lead to symptom improvement. Based on our impression that clinicians and researchers are largely unaware that a large volume of research has been published about the use of therapeutic plasma exchange as a treatment for systemic sclerosis, we conducted a comprehensive review and analysis of all published research on this topic. Results: We identified 46 relevant articles that met our search criteria, involving a total of 572 patients. Of these, 19 were case studies; the rest ranged from small observational studies to prospective randomized clinical trials. In all but two studies, most patients receiving therapeutic plasma exchange showed improvements in both clinical symptoms and laboratory markers, including significant improvement in Raynaud's symptoms and healing of digital ulceration after three to four weekly treatments. The beneficial effects from even a short course of therapeutic plasma exchange treatments were long-lasting, typically 6 months or longer. Therapeutic plasma exchange was very well tolerated. Adverse events were rare and, in almost all cases, mild and transitory. Conclusion: These results suggest that long-term therapeutic plasma exchange may offer a low-risk way to control and in some cases reverse systemic sclerosis symptoms. The mechanism for the clinical improvements seen from therapeutic plasma exchange in systemic sclerosis patients is unclear. Therefore, additional studies of therapeutic plasma exchange effects in systemic sclerosis appear to be highly desirable.

BRAF is a serine/threonine-protein kinase in the MAPK/ERK signaling pathway, which is often activated by somatic point mutations in various cancers (Davies et al., 2002Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8270) Google Scholar). About 40–60% of all malignant melanomas (MMs) carry BRAF mutations and ∼90% of these are the V600E type with an increased kinase activity (Chapman et al., 2011Chapman P.B. Hauschild A. Robert C. Haanen J.B. Ascierto P. Larkin J. et al.Improved survival with vemurafenib in melanoma with BRAF V600E mutation.N Engl J Med. 2011; 364: 2507-2516Crossref PubMed Scopus (6112) Google Scholar, Davies et al., 2002Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8270) Google Scholar). Because BRAF mutations are more frequent in benign nevi than in MMs (Pollock et al., 2003Pollock P.M. Harper U.L. Hansen K.S. Yudt L.M. Stark M. Robbins C.M. et al.High frequency of BRAF mutations in nevi.Nat Genet. 2003; 33: 19-20Crossref PubMed Scopus (1358) Google Scholar, Poynter et al., 2006Poynter J.N. Elder J.T. Fullen D.R. Nair R.P. Soengas M.S. Johnson T.M. et al.BRAF and NRAS mutations in melanoma and melanocytic nevi.Melanoma Res. 2006; 16: 267-273Crossref PubMed Scopus (195) Google Scholar, Shain et al., 2015Shain A.H. Yeh I. Kovalyshyn I. Sriharan A. Talevich E. Gagnon A. et al.The genetic evolution of melanoma from precursor lesions.N Engl J Med. 2015; 373: 1926-1936Crossref PubMed Scopus (621) Google Scholar, Shain and Bastian, 2016Shain A.H. Bastian B.C. From melanocytes to melanomas.Nat Rev Cancer. 2016; 16: 345-358Crossref PubMed Scopus (446) Google Scholar), they could be an initiating event in melanocytic proliferation (Patton et al., 2005Patton E.E. Widlund H.R. Kutok J.L. Kopani K.R. Amatruda J.F. Murphey R.D. et al.BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.Curr Biol. 2005; 15: 249-254Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, Poynter et al., 2006Poynter J.N. Elder J.T. Fullen D.R. Nair R.P. Soengas M.S. Johnson T.M. et al.BRAF and NRAS mutations in melanoma and melanocytic nevi.Melanoma Res. 2006; 16: 267-273Crossref PubMed Scopus (195) Google Scholar, Uribe et al., 2003Uribe P. Wistuba II, González S. BRAF mutation: a frequent event in benign, atypical, and malignant melanocytic lesions of the skin.Am J Dermatopathol. 2003; 25: 365-370Crossref PubMed Scopus (129) Google Scholar). To corroborate this possibility, we analyzed the BRAF V600E mutation rates in benign nevi as well as in lesions with histopathological characteristics of both benign nevi and MMs, designated as melanocytic tumors of uncertain malignant potential (MELTUMPs) (Barnhill et al., 2010Barnhill R.L. Cerroni L. Cook M. Elder D.E. Kerl H. LeBoit P.E. et al.State of the art, nomenclature, and points of consensus and controversy concerning benign melanocytic lesions: outcome of an international workshop.Adv Anat Pathol. 2010; 17: 73-90Crossref PubMed Scopus (57) Google Scholar, Elder and Xu, 2004Elder D.E. Xu X. The approach to the patient with a difficult melanocytic lesion.Pathology (Phila). 2004; 36: 428-434Scopus (108) Google Scholar). The histopathological definition of each lesion was established by at least two experienced dermatopathologists. This study of formalin-fixed, paraffin-embedded tissues from 45 MELTUMPs and 40 benign nevi (34 acquired melanocytic nevi, 3 congenital nevi, and 3 Clark’s nevi; Supplementary Table S1 online) was approved by the ethics committee of the University of Freiburg Medical Center (ethics vote 324/09). Under German law, written consent from the patients was not required because the material used had been collected for diagnostic and therapeutic purposes in the years 2006–2011 in the archives of the Institute for Surgical Pathology, University Hospital Freiburg and used for this study in pseudonymized form. All 85 samples were stained with the anti-BRAF V600E (clone VE1) monoclonal antibody, followed by the DakoREAL Detection System (Dako, Glostrup, Denmark) (Figure 1) and analyzed on a modified immunoreactive score (IRS) of the one described by Remmele and Stegner, 1987Remmele W. Stegner H.E. Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue.Pathol. 1987; 8: 138-140Google Scholar (Figure 1 and Supplementary Table S2 online). Additionally, the BRAF V600E allele-specific PCR (Arcaini et al., 2012Arcaini L. Zibellini S. Boveri E. Riboni R. Rattotti S. Varettoni M. et al.The BRAF V600E mutation in hairy cell leukemia and other mature B-cell neoplasms.Blood. 2012; 119: 188-191Crossref PubMed Scopus (143) Google Scholar) was performed in 31 MELTUMPs and 24 benign nevi samples (Supplementary Materials and Methods online and Supplementary Figure S1 online) and the PCR fragments were analyzed using a Genetic Analyzer 3130xl (Applied Biosystems, Carlsbad, CA) (Figure 2a–2c ).Figure 2Detection of the BRAF V600E mutation frequency by IHC or AS-PCR. (a–c) The lesions of interest in the IHC stains are marked by black arrows. (a) A MELTUMP that was positive for BRAF V600E in IHC (IRS n = 4), but not in the AS-PCR (scale bar = 25 μm). (b) Compound congenital nevus that was positive for the BRAF V600E mutation in IHC (IRS n = 12) and AS-PCR (scale bar = 50 μm). (c) MELTUMP that tested positive for the wild-type BRAF in IHC (IRS n = 0) and AS-PCR (scale bar = 100 μm). (d) Depiction of the final mutation status determined through one or both of our applied methods (AS-PCR and IHC) for all lesions. AS, allele-specific; bp, base pairs; IHC, immunohistochemistry; IRS, immunoreactive score; MUT, BRAF V600E mutation–specific primer; MELTUMP, melanocytic tumor of uncertain malignant potential; RFU, relative fluorescence unit; V600E, BRAF V600E; WT, wild-type BRAF.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The immunohistochemistry with the VE1 monoclonal antibody produced at least one interpretable stain for all 40 nevi and for 43 of 45 MELTUMPs and the BRAF V600E mutations were detected in 70.0% (28 of 40) of nevi and 51.2% (22 of 43) of MELTUMPs. The allele-specific PCR produced a valid result for all 55 tested lesions, with 62.5% (15 of 24) of the nevi and 51.6% (16 of 31) of MELTUMPs being positive for the BRAF V600E mutation. The overall mutation status of each lesion was defined as positive if at least one method detected the BRAF V600E mutation (Figure 2d). The overall BRAF V600E mutation rate in nevi was 70.0% (28 of 40) and higher than that in MELTUMPs—51.1% (23 of 45), but this difference (P = 0.083) (two-sided Fisher's exact test) was not statistically significant. The male-to-female ratio for all 85 lesions was 42:43, and the mean patient’s age at the time of tissue excision was 45.4 years. Melanocytic lesions located on the leg were significantly more frequently associated with the wild-type BRAF (P = 0.00138), whereas trunk lesions were significantly more likely to be BRAF V600E–mutated (P = 0.00677). MELTUMPs located on the trunk were significantly more likely to possess the BRAF V600E mutation than MELTUMPs located elsewhere (P = 0.0419). The age difference between BRAF V600E–positive MELTUMP patients (46.3 years) and MELTUMP patients with wild-type BRAF (52 years) was not statistically significant (P = 0.241). However, the mutated V600E allele was significantly more likely (P = 0.00502) to be present in men’s nevi than in women’s nevi, and significantly less likely (P = 0.00299) in nevi on the leg than in nevi elsewhere. Our results showing that the BRAF V600E mutation rate in MELTUMPs (51.1%) is lower than that of benign nevi (70%) are consistent with previously published data (Pollock et al., 2003Pollock P.M. Harper U.L. Hansen K.S. Yudt L.M. Stark M. Robbins C.M. et al.High frequency of BRAF mutations in nevi.Nat Genet. 2003; 33: 19-20Crossref PubMed Scopus (1358) Google Scholar, Poynter et al., 2006Poynter J.N. Elder J.T. Fullen D.R. Nair R.P. Soengas M.S. Johnson T.M. et al.BRAF and NRAS mutations in melanoma and melanocytic nevi.Melanoma Res. 2006; 16: 267-273Crossref PubMed Scopus (195) Google Scholar, Shain et al., 2015Shain A.H. Yeh I. Kovalyshyn I. Sriharan A. Talevich E. Gagnon A. et al.The genetic evolution of melanoma from precursor lesions.N Engl J Med. 2015; 373: 1926-1936Crossref PubMed Scopus (621) Google Scholar, Shain and Bastian, 2016Shain A.H. Bastian B.C. From melanocytes to melanomas.Nat Rev Cancer. 2016; 16: 345-358Crossref PubMed Scopus (446) Google Scholar, Uribe et al., 2003Uribe P. Wistuba II, González S. BRAF mutation: a frequent event in benign, atypical, and malignant melanocytic lesions of the skin.Am J Dermatopathol. 2003; 25: 365-370Crossref PubMed Scopus (129) Google Scholar) and compatible with the idea that the BRAF mutation might be an early step in melanocytic transformation (Uribe et al., 2003Uribe P. Wistuba II, González S. BRAF mutation: a frequent event in benign, atypical, and malignant melanocytic lesions of the skin.Am J Dermatopathol. 2003; 25: 365-370Crossref PubMed Scopus (129) Google Scholar). In fact, Shain et al., 2015Shain A.H. Yeh I. Kovalyshyn I. Sriharan A. Talevich E. Gagnon A. et al.The genetic evolution of melanoma from precursor lesions.N Engl J Med. 2015; 373: 1926-1936Crossref PubMed Scopus (621) Google Scholar concluded that the intermediate lesions are a biologically distinct group with unique genetic markers—such as frequent TERT promoter mutations, NRAS or the BRAF V600K mutations rather than the BRAF V600E—that differentiate them from benign nevi and MMs (Shain et al., 2015Shain A.H. Yeh I. Kovalyshyn I. Sriharan A. Talevich E. Gagnon A. et al.The genetic evolution of melanoma from precursor lesions.N Engl J Med. 2015; 373: 1926-1936Crossref PubMed Scopus (621) Google Scholar, Shain and Bastian, 2016Shain A.H. Bastian B.C. From melanocytes to melanomas.Nat Rev Cancer. 2016; 16: 345-358Crossref PubMed Scopus (446) Google Scholar). Consistent with these findings, in a zebrafish model, the BRAF V600E mutation led to benign melanocytic proliferation, requiring an additional p53 mutation for melanoma development (Patton et al., 2005Patton E.E. Widlund H.R. Kutok J.L. Kopani K.R. Amatruda J.F. Murphey R.D. et al.BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.Curr Biol. 2005; 15: 249-254Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). BRAF V600E–mutated lesions were significantly more frequently found on the trunk, a location often associated with intermitted sun exposure, than elsewhere. This observation was also made by others in MMs (Liu et al., 2006Liu W. Kelly J.W. Trivett M. Murray W.K. Dowling J.P. Wolfe R. et al.Distinct clinical and pathological features are associated with the BRAFT1799A(V600E) mutation in primary melanoma.J Invest Dermatol. 2006; 127: 900-905Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, Long et al., 2011Long G.V. Menzies A.M. Nagrial A.M. Haydu L.E. Hamilton A.L. Mann G.J. et al.Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma.J Clin Oncol. 2011; 29: 1239-1246Crossref PubMed Scopus (825) Google Scholar), as well as in benign nevi (Karram et al., 2013Karram S. Novy M. Saroufim M. Loya A. Taraif S. Houreih M.A. et al.Predictors of BRAF mutation in melanocytic nevi: analysis across regions with different UV radiation exposure.Am J Dermatopathol. 2013; 35: 412-418Crossref PubMed Scopus (24) Google Scholar). In agreement with Karram et al., 2013Karram S. Novy M. Saroufim M. Loya A. Taraif S. Houreih M.A. et al.Predictors of BRAF mutation in melanocytic nevi: analysis across regions with different UV radiation exposure.Am J Dermatopathol. 2013; 35: 412-418Crossref PubMed Scopus (24) Google Scholar, the BRAF V600E–mutated melanocytic lesions were significantly less frequently located on the leg. Contrastingly, either no association between the BRAF V600E status and location on the extremities or positive correlations were reported in MMs (Liu et al., 2006Liu W. Kelly J.W. Trivett M. Murray W.K. Dowling J.P. Wolfe R. et al.Distinct clinical and pathological features are associated with the BRAFT1799A(V600E) mutation in primary melanoma.J Invest Dermatol. 2006; 127: 900-905Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, Long et al., 2011Long G.V. Menzies A.M. Nagrial A.M. Haydu L.E. Hamilton A.L. Mann G.J. et al.Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma.J Clin Oncol. 2011; 29: 1239-1246Crossref PubMed Scopus (825) Google Scholar). Although the age differences among all our defined patient groups were statistically insignificant, at the time of diagnosis, patients with BRAF V600E–mutated lesions appeared to be slightly younger than patients with BRAF wild-type lesions similar to previous MM studies (Liu et al., 2006Liu W. Kelly J.W. Trivett M. Murray W.K. Dowling J.P. Wolfe R. et al.Distinct clinical and pathological features are associated with the BRAFT1799A(V600E) mutation in primary melanoma.J Invest Dermatol. 2006; 127: 900-905Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, Long et al., 2011Long G.V. Menzies A.M. Nagrial A.M. Haydu L.E. Hamilton A.L. Mann G.J. et al.Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma.J Clin Oncol. 2011; 29: 1239-1246Crossref PubMed Scopus (825) Google Scholar). Thus, it appears that the BRAF V600E mutation might inversely reflect the degree of sun-induced skin damage (Landi et al., 2006Landi M.T. Bauer J. Pfeiffer R.M. Elder D.E. Hulley B. Minghetti P. et al.MC1R germline variants confer risk for BRAF-mutant melanoma.Science. 2006; 313: 521-522Crossref PubMed Scopus (269) Google Scholar, Liu et al., 2006Liu W. Kelly J.W. Trivett M. Murray W.K. Dowling J.P. Wolfe R. et al.Distinct clinical and pathological features are associated with the BRAFT1799A(V600E) mutation in primary melanoma.J Invest Dermatol. 2006; 127: 900-905Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) that is likely to be higher in older people (Schäfer et al., 2006Schäfer T. Merkl J. Klemm E. Wichmann H.-E. Ring J. The epidemiology of nevi and signs of skin aging in the adult general population: results of the KORA-survey 2000.J Invest Dermatol. 2006; 126: 1490-1496Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In the nevi subgroup, men had significantly more BRAF V600E–mutated nevi than women. This sex-dependent difference has not been noted in previous studies of MMs or benign nevi (Karram et al., 2013Karram S. Novy M. Saroufim M. Loya A. Taraif S. Houreih M.A. et al.Predictors of BRAF mutation in melanocytic nevi: analysis across regions with different UV radiation exposure.Am J Dermatopathol. 2013; 35: 412-418Crossref PubMed Scopus (24) Google Scholar, Uribe et al., 2003Uribe P. Wistuba II, González S. BRAF mutation: a frequent event in benign, atypical, and malignant melanocytic lesions of the skin.Am J Dermatopathol. 2003; 25: 365-370Crossref PubMed Scopus (129) Google Scholar). In conclusion, BRAF-mutated melanocytic lesions, especially MELTUMPs, are significantly more commonly located on the trunk and tend to occur at a younger age. MELTUMPs have a lower BRAF mutation frequency than benign nevi, which is compatible with genetic concepts where the BRAF mutation might be an event leading to cell proliferation of melanocytes, but not to their malignant transformation. Andreas von Deimling, is the inventor of the VE1 monoclonal antibody, which is commercially available. The remaining authors state no conflict of interest. We thank Marie Follo, PhD, for reading the manuscript. Jose Villacorta Hidalgo was supported by a PhD scholarship from Deutscher Akademischer Austauschdienst. The laboratory of PF is supported by a grant from Deutsche Forschungsgemeinschaft (SFB 1160-Z1). Download .pdf (.07 MB) Help with pdf files Supplementary Data

The success of active immunotherapies in the prevention of many infectious diseases over the course of over 200 years has lead scientists to wonder if the same principles could be applied to cancer. Antigen-specific active immunotherapies for the treatment of cancer have been researched for over two decades, however, the overwhelming majority of these studies have failed to stimulate robust clinical responses. It is clear that current active immunotherapy research should incorporate methods to increase the immunostimulatory capacity of these therapies. To directly address this need, we propose the addition of the immunostimulatory heat shock proteins (HSPs) to active immunotherapeutic strategies to augment their efficacy. Heat shock proteins are a family of highly conserved intracellular chaperone proteins, and are the most abundant family proteins inside cells. This ubiquity, and their robust immunostimulatory capacity, points to their importance in regulation of intracellular processes and, therefore, indicators of loss of cellular integrity if found extracellularly. Thus, we emphasize the importance of taking into consideration the location of vaccine-derived HSP/tumor-antigen complexes when designing active immunotheraputic strategies.

Natural killer (NK) cell subpopulations from 8 HLA-matched but killer cell immunoglobulin-like receptor (KIR)/HLA-ligand-mismatched patient-donor pairs were analyzed in the course of allogeneic hematopoietic stem cell transplantation (HCT). The patients' post-transplantation NKG2A-/LIR-1- NK cells, which expressed only inhibitory KIRs for which the patient had no HLA class I ligands, showed higher cytotoxic capacity than the NKG2A-/LIR-1- NK cells lacking any inhibitory KIRs that remained tolerant throughout the course of HCT. The NKG2A+ NK cell subpopulations displayed the highest levels of cytotoxic activation, which appeared to be significantly enhanced in comparison with that in allogeneic graft's donors. LIR-1- NK cells were much more frequent after HCT than LIR-1+ NK cells and LIR-1 expression on NKG2A+ or NKG2A- NK cells was associated with significantly lower cytotoxic activities. Thus NKG2A-/LIR-1- NK cells expressing only HLA-mismatched KIRs show a partial break in tolerance in the first year following HCT. The failure to exclude LIR-1+ cells within the NKG2A- NK cell subset in previous studies could explain the earlier conflicting results. Thus systemic immune activation in patients following HCT augments the GvL effect through both increasing overall NK cell activities and partially breaking tolerance of unlicensed NK cells.

Deeper understanding of γδ Τ cell increases in various clinical situations requires the assessment of TCRγ and δ variable (V) region gene expression and junctional diversity. Here we describe an improved TCRγ and δ spectratyping method used to study the γδ T-cell expansions in two patients with thymoma and immunodeficiency. One of these patients also suffered from chronic CMV infection and pure red cell aplasia and the other from chronic visceral leishmaniasis and myasthenia gravis. Analyses of the junctional diversity of the TCRγ and δ chains, flow cytometry with a panel of non-commercially available anti-TCRγδ V region monoclonal antibodies and functional studies were performed. The results clearly distinguished an expansion of oligoclonal, most likely antigen-driven, cytotoxic γδ T cells in the first patient from a naive pattern of polyclonal γδ T-cell proliferation in the second. These findings demonstrate the diversity of γδ T-cell expansions in immunodeficient patients and highlight the value of spectratyping as a tool for their characterization and understanding of the underlying mechanisms.

The mechanisms underlying thymoma-associated immunodeficiency are largely unknown, and the significance of increased blood γδ Τ cells often remains elusive. In this study we address these questions based on an index patient with thymoma, chronic visceral leishmaniasis, myasthenia gravis, and a marked increase of rare γδ T cell subsets in the peripheral blood. This patient showed cutaneous anergy, even though he had normal numbers of peripheral blood total lymphocytes as well as CD4+ and CD8+ T cells. Despite his chronic infection, analyses of immunophenotypes and spectratyping of his lymphocytes revealed an unusual accumulation of naive γδ and αβ T cells, suggesting a generalized T cell activation defect. Functional studies in vitro demonstrated substantially diminished IL-2 and IFN-γ production following TCR stimulation of his “untouched” naive CD4+ T cells. Biochemical analysis revealed that his γδ and αβ T cells carried an altered TCR complex with reduced amounts of the ζ-chain (CD247). No mutations were found in the CD247 gene that encodes the homodimeric ζ protein. The diminished presence of CD247 and increased numbers of γδ T cells were also observed in thymocyte populations obtained from three other thymoma patients. Thus, our findings describe a novel type of a clinically relevant acquired T cell immunodeficiency in thymoma patients that is distinct from Good’s syndrome. Its characteristics are an accumulation of CD247-deficient, hyporresponsive naive γδ and αβ T cells and an increased susceptibility to infections.

Breast cancer is the leading cause of cancer death in women and the second most common cancer worldwide after lung cancer. The remarkable heterogeneity of breast cancers influences numerous diagnostic, therapeutic, and prognostic factors. Triple-negative breast carcinomas (TNBCs) lack expression of HER2 and the estrogen and progesterone receptors and often contain lymphocytic infiltrates. Most of TNBCs are invasive ductal carcinomas (IDCs) with poor prognosis, whereas prognostically more favorable subtypes such as medullary breast carcinomas (MBCs) are somewhat less frequent. Infiltrating T-cells have been associated with an improved clinical outcome in TNBCs. The prognostic role of γδ T-cells within CD3(+) tumor-infiltrating T lymphocytes remains unclear. We analyzed 26 TNBCs, 14 IDCs, and 12 MBCs, using immunohistochemistry for the quantity and patterns of γδ T-cell infiltrates within the tumor microenvironment. In both types of TNBCs, we found higher numbers of γδ T-cells in comparison with normal breast tissues and fibroadenomas. The numbers of infiltrating γδ T-cells were higher in MBCs than in IDCs. γδ T-cells in MBCs were frequently located in direct contact with tumor cells, within the tumor and at its invasive border. In contrast, most γδ T-cells in IDCs were found in clusters within the tumor stroma. These findings could be associated with the fact that the patient's prognosis in MBCs is better than that in IDCs. Further studies to characterize these γδ T-cells at the molecular and functional level are in progress.

Whether vaccination against a virus can protect against more virulent coinfection with the virus and additional pathogen(s) remains poorly characterized. Overlapping endemicity of human immunodeficiency virus (HIV) and malaria suggests that HIV/malaria coinfection frequently complicates acute and chronic HIV infection. Here we showed that vaccination of macaques with recombinant Listeria ΔactA prfA* expressing simian/human immunodeficiency virus (SHIV) gag and env elicited Gag- and Env-specific T-cell responses, and protected against life-threatening SHIV-related malaria after SHIV/Plasmodium fragile coinfection. SHIV antigen immunization reduced peak viremia, resisted SHIV/malaria-induced lymphoid destruction, and blunted coinfection-accelerated decline of CD4(+) T-cell counts after SHIV/malaria coinfection. SHIV antigen immunization also weakened coinfection-driven overreactive proinflammatory interferon-γ (IFNγ) responses and led to developing T helper cell 17/22 (Th17/Th22) responses after SHIV/malaria coinfection. The findings suggest that vaccination against AIDS virus can alter patterns of immune responses to the SHIV/malaria coinfection and protect against life-threatening SHIV-related malaria.

OBJECTIVE:To assess the effects of timing and schedule of zoledronic acid (ZA) administration on bone mineral density (BMD) in patients beginning androgen deprivation therapy (ADT) for the treatment of recurrent prostate cancer. PATIENTS AND METHODS:In this randomized, 3-arm trial, we evaluated changes in BMD after 3 different ZA administration schedules in men with recurrent prostate cancer who were beginning ADT. Forty-four patients were enrolled and randomized to receive a single dose of ZA given 1 week before beginning ADT (arm 1), a single dose of ZA given 6 months after beginning ADT (arm 2), or monthly administration of ZA starting 6 months after beginning ADT, for a total of 6 doses (arm 3). RESULTS:Patients who received ZA before ADT had a significant improvement in BMD at the total proximal femur and trochanter after 6 months compared with the other groups. In addition, only patients in the arm that received multiple doses improved lumbar spine BMD while on ADT, with these findings persisting to 24 months. However, this group also experienced more grade 1 adverse events. CONCLUSIONS:Analysis of these data suggests that ZA administration before initiation of ADT was superior to treatment 6 months after starting ADT in maintaining BMD. In addition, monthly ZA administration can increase BMD above baseline but is associated with more adverse events. Further study is needed to examine whether the timing and frequency of ZA therapy in patients on ADT can reduce fracture risk.

Prior to the advent of VEGF-targeted therapies, renal cell carcinoma (RCC) was among the few solid tumors shown to respond to cytokine-based therapies such as interleukin-2 (IL-2) and interferon alpha. Previous work has shown that aminobisphosphonates, including zoledronic acid (ZA), are capable of activating human Vγ9 Vδ2 T cells in vitro, and these cells can be further expanded with IL-2. Moreover, these Vγ9 Vδ2 T cells have cytolytic activity in vitro to multiple human tumor cell lines. In the current report, we have conducted a pilot trial in patients with metastatic RCC, evaluating different doses of ZA in combination with low-dose IL-2 to determine whether combining these agents can promote in vivo proliferation of Vγ9 Vδ2 T cells and elicit an antitumor response. In 12 patients evaluated, no objective clinical responses were observed by RECIST criteria; however, two patients experienced prolonged stable disease. A modest increase in Vγ9 Vδ2 T-cell frequency could be detected by Day 8 of therapy in four of the nine patients who received at least one cycle of therapy, but not to the magnitude anticipated from preclinical models. Repeated administration of IL-2 and ZA resulted in both a diminished in vivo percentage of Vγ9 Vδ2 T cells as well as impaired expansion in vitro after the first cycle of therapy. These results suggest that repeated administration of IL-2 and ZA, at the doses and schedules used in this trial, may actually inhibit the proliferative capacity of Vγ9 Vδ2 T cell in patients with metastatic RCC.

Despite a long-lasting global effort, the Holy Grail quest for a protective vaccine, able to confer prevention to HIV infection, did not reach the hoped for results, nor seems able to do so in the near future. Since mucosal surfaces of the host serve as the main entry point for HIV, it seems now logical to switch from a systemic to a localized view of events, in order to reveal critical steps useful in designing new and different vaccination strategies. In this context, the recent description of the very early phases of infection, from the eclipse to the viremia peak phase, seems to define a point-of-no-return threshold after which the main HIV infection steps, i.e. the massive destruction of the CD4+CCR5+ cell pool, the destruction of the mucosal physical barrier, and the establishment of reservoir sanctuaries, have already been accomplished. Nevertheless, the underlying mechanisms, the timing, and the consequences of evasion mechanisms exploited by HIV are still under scrutiny. Innate immunity, as part of a rapid lymphoid stress surveillance system, is known to play a central role in host responses to many infectious agents. In particular, Vγ9Vδ2 T-cells are able to quickly respond to danger signals without the need for classical major histocompatibility complex presentation, and may act as a bridge between innate and acquired arms of immune response, being able to kill infected/transformed cells, release antimicrobial soluble factors, and increase the deployment of other innate and acquired responses. Many experimental evidences suggest a direct role of circulating Vγ9Vδ2 T-cells during HIV disease. They may exert a direct anti-HIV role by secreting chemokines competing for HIV entry coreceptors as well as other soluble antiviral factors, and by killing infected cells by cytotoxic natural killer-like mechanisms. Moreover, they were found progressively depleted and anergic in advanced stages of HIV disease, this effect being directly linked to uncontrolled HIV replication. Scarce evidences are available on the involvement of mucosal gamma/delta T-cells during the early phases of HIV infection. In particular, the relative cause/effect links between HIV infection, destruction of the mucosal physical barrier, nonspecific activation of the immune system, and mucosal innate cell activation and effector functions, are still not completely defined. In order to attain an effective manipulation of innate immune cells, aiming at the induction of an effective adaptive immunity against HIV, any information on the role of mucosal antiviral factors and innate immune cells will be very important. The aim of this review is to summarize the information on the role of gamma/delta T-cells during HIV infection, from the general circulating population to mucosal sites, in order to better describe areas deserving increased attention. In particular, strategies enhancing gamma/delta T-cell functions may open the possibility to formulate new immunotherapeutic regimens, which could impact the improvement of immune control of HIV disease.

Most vaccine strategies are designed to elicit adaptive immune responses to a variety of microbial or tumor-associated antigens. These immune responses are predominantly mediated by αβ T cells, B cells and antibodies. Nevertheless, approximately 1-5% of human peripheral blood lymphocytes possess the surface gd T-cell receptor, predominantly expressing the Vγ9Vδ2 variable segments.