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Posttransplantation primary cutaneous T-cell lymphomas (PT-CTCL) are a rare complication of sustained immunosuppression in the posttransplant setting. When present, PT-CTCLs are typically EBV- and exhibit features of mycosis fungoides/Sézary syndrome or CD30+ lymphoproliferative disorders. We present a case of a 75-year-old individual who developed skin lesions 30 years after liver transplantation. Pathologic evaluation of the skin biopsy revealed involvement by a clonal, EBV+ T-cell population of gamma/delta lineage with no evidence of systemic disease. Comprehensive genomic profiling was performed, confirming focal one-copy loss of 6q23.3, altogether consistent with the extremely rare and unusual diagnosis of primary cutaneous EBV+ extranodal NK/T-cell lymphoma of gamma/delta T-cell lineage in the posttransplantation setting., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: This study investigated the effectiveness of algorithmic testing in hematopathology at the Brigham and Women's Hospital and Dana-Farber Cancer Institute (DFCI). The algorithm was predicated on test selection after an initial pathologic evaluation to maximize cost-effective testing, especially for expensive molecular and cytogenetic assays., Materials and Methods: Standard ordering protocols (SOPs) for 17 disease categories were developed and encoded in a decision support application. Six months of retrospective data from application beta testing was obtained and compared with actual testing practices during that timeframe. In addition, 2 years of prospective data were also obtained from patients at one community satellite site., Results: A total of 460 retrospective cases (before introduction of algorithmic testing) and 109 prospective cases (following introduction) were analyzed. In the retrospective data, 61.7% of tests (509 of 825) were concordant with the SOPs while 38.3% (316 of 825) were overordered and 30.8% (227 of 736) of SOP-recommended tests were omitted. In the prospective data, 98.8% of testing was concordant (244 of 247 total tests) with only 1.2% overordered tests (3 of 247) and 7.6% omitted tests (20 of 264 SOP-recommended tests; overall P < .001). The cost of overordered tests before implementing SOP indicates a potential annualized saving of $1,347,520 in US dollars (USD) in overordered testing at Brigham and Women's Hospital/DFCI. Only two of 316 overordered tests (0.6%) returned any additional information, both for extremely rare clinical circumstances., Conclusion: Implementation of SOPs dramatically improved test ordering practices, with a just right number of ancillary tests that minimizes cost and has no significant impact on acquiring key informative test results.

In this study, we performed a comprehensive molecular analysis of paired skin and peripheral blood/bone marrow (BM) samples from 17 patients with cutaneous myeloid or cutaneous histiocytic-dendritic neoplasms. The cutaneous manifestations included 10 patients with cutaneous acute myeloid leukemia (c-AML), 2 patients with full or partial Langerhans cell differentiation, 2 patients with blastic plasmacytoid dendritic cell neoplasms (BPDCN), 1 patient with both Langerhans cell differentiation and BPDCN, and 2 patients with full or partial indeterminate dendritic cell differentiation. Seven of the 10 c-AML patients (70%) exhibited concurrent or subsequent marrow involvement by acute myeloid leukemia, with all 7 cases (100%) demonstrating shared clonal mutations in both the skin and BM. However, clonal relatedness was documented in one additional case that never had any BM involvement. Nevertheless, NPM1 mutations were identified in 7 of the 10 (70%) of these c-AML cases while one had KMT2A rearrangement and one showed inv(16). All 3 patients (100%) with Langerhans cell neoplasms, 2 patients with BPDCN (100%), and one of the 2 patients (50%) with other cutaneous dendritic cell neoplasms also demonstrated shared mutations between the skin and concurrent or subsequent myeloid neoplasms. Both BM and c-AML shared identical founding drivers, with a predominance of NPM1, DNMT3A, and translocations associated with monocytic differentiation, with common cutaneous-only mutations involving genes in the signal transduction and epigenetic pathways. Cutaneous histiocytic-dendritic neoplasms shared founding drivers in ASXL1, TET2, and/or SRSF2. However, in the Langerhans cell histiocytosis or histiocytic sarcoma cases, there exist recurrent secondary RAS pathway hits, whereas cutaneous BPDCN cases exhibit copy number or structural variants. These results enrich and broaden our understanding of clonally related cutaneous manifestations of myeloid neoplasms and further illuminate the highly diverse spectrum of morphologic and immunophenotypic features they exhibit., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

TP53 mutation predicts adverse prognosis in many cancers, including myeloid neoplasms, but the mechanisms by which specific mutations impact disease biology, and whether they differ between disease categories, remain unknown. We analyzed TP53 mutations in four myeloid neoplasm subtypes (MDS, AML, AML with myelodysplasia-related changes (AML-MRC), and therapy-related acute myeloid leukemia (tAML)), and identified differences in mutation types, spectrum, and hotspots between disease categories and compared to solid tumors. Missense mutations in the DNA-binding domain were most common across all categories, whereas inactivating mutations and mutations outside the DNA binding domain were more common in AML-MRC compared to MDS. TP53 mutations in MDS were more likely to retain transcriptional activity, and co-mutation profiles were distinct between disease categories and mutation types. Our findings suggest that mutated TP53 contributes to initiation and progression of neoplasia via distinct mechanisms, and support the utility of specific identification of TP53 mutations in myeloid malignancies., Competing Interests: Disclosure of Conflicts of Interest The authors declare no competing financial interests.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDC). BPDCN occurs at least three times more frequently in men than in women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2 , an X chromosome gene encoding a splicing factor, are enriched in BPDCN, and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo , BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation. SIGNIFICANCE: Sex bias in cancer is well recognized, but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway. This article is highlighted in the In This Issue feature, p. 275 ., (©2021 The Authors; Published by the American Association for Cancer Research.)

A hallmark of acute myeloid leukemia (AML) is the inability of self-renewing malignant cells to mature into a non-dividing terminally differentiated state. This differentiation block has been linked to dysregulation of multiple cellular processes, including transcriptional, chromatin, and metabolic regulation. The transcription factor HOXA9 and the histone demethylase LSD1 are examples of such regulators that promote differentiation blockade in AML. To identify metabolic targets that interact with LSD1 inhibition to promote myeloid maturation, we screened a small molecule library to identify druggable substrates. We found that differentiation caused by LSD1 inhibition is enhanced by combined perturbation of purine nucleotide salvage and de novo lipogenesis pathways, and identified multiple lines of evidence to support the specificity of these pathways and suggest a potential basis of how perturbation of these pathways may interact synergistically to promote myeloid differentiation. In sum, these findings suggest potential drug combination strategies in the treatment of AML., Competing Interests: Y.S. is a co-founder and equity holder of Constellation Pharmaceuticals, Athelas Therapeutics and K36 Therapeutics, a consultant for Active Motif, and an equity holder of Imago Biosciences. I.S.H. is a consultant for ONO Pharma USA. D.B.S. is a co-founder and an equity holder of Clear Creek Bio. Y.S., B.M.Z, J.M., and A.D. are developers of the LSD1 inhibitor combinations described in this report and Boston Children's Hospital has pursued a patent application of this technology. All other authors declare no competing financial interests., (© 2021 The Author(s).)

Prediction of long-term outcomes from short-term measurements remains a fundamental challenge. Quantitative assessment of signaling dynamics, and the resulting transcriptomic and proteomic responses, has yielded fundamental insights into cellular outcomes. However, the utility of these measurements is limited by their short timescale (hours to days), while the consequences of these events frequently unfold over longer timescales. Here, we discuss the predictive power of static and dynamic measurements, drawing examples from fields that have harnessed the predictive capabilities of such measurements. We then explore potential approaches to close this timescale gap using complementary measurements and computational approaches, focusing on the example of dynamic measurements of signaling factors and their impacts on cellular outcomes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

T-cell/histiocyte-rich large B-cell lymphoma (TCRLBCL) is an aggressive variant of diffuse large B-cell lymphoma (DLBCL) characterized by rare malignant B cells within a robust but ineffective immune cell infiltrate. The mechanistic basis of immune escape in TCRLBCL is poorly defined and not targeted therapeutically. We performed a genetic and quantitative spatial analysis of the PD-1/PD-L1 pathway in a multi-institutional cohort of TCRLBCLs and found that malignant B cells harbored PD-L1/PD-L2 copy gain or amplification in 64% of cases, which was associated with increased PD-L1 expression (P = .0111). By directed and unsupervised spatial analyses of multiparametric cell phenotypic data within the tumor microenvironment, we found that TCRLBCL is characterized by tumor-immune "neighborhoods" in which malignant B cells are surrounded by exceptionally high numbers of PD-L1-expressing TAMs and PD-1+ T cells. Furthermore, unbiased clustering of spatially resolved immune signatures distinguished TCRLBCL from related subtypes of B-cell lymphoma, including classic Hodgkin lymphoma (cHL) and DLBCL-NOS. Finally, we observed clinical responses to PD-1 blockade in 3 of 5 patients with relapsed/refractory TCRLBCL who were enrolled in clinical trials for refractory hematologic malignancies (NCT03316573; NCT01953692), including 2 complete responses and 1 partial response. Taken together, these data implicate PD-1 signaling as an immune escape pathway in TCRLBCL and also support the potential utility of spatially resolved immune signatures to aid the diagnostic classification and immunotherapeutic prioritization of diverse tumor types., (© 2021 by The American Society of Hematology.)

Loss of the Y chromosome (LOY) is one of the most common somatic genomic alterations in hematopoietic cells in men. However, due to the high prevalence of LOY as the sole cytogenetic finding in the healthy older population, differentiating isolated LOY associated with clonal hematologic processes from aging-associated mosaicism can be difficult in the absence of definitive morphological features of disease. In the past, various investigators have proposed that a high percentage of metaphases with LOY is more likely to represent expansion of a clonal myeloid disease-associated population. It is unknown whether the proportion of metaphases with LOY is associated with the incidence of myeloid neoplasia-associated genomic alterations. To address this question, we identified marrow samples with LOY as isolated cytogenetic finding and used targeted next generation sequencing-based molecular analysis to identify common myeloid neoplasia-associated somatic mutations. Among 73 patients with median age of 75 years (range 29-90), the percentage of metaphases with LOY was <25% in 23 patients, 25-49% in 10, 50-74% in 8 and ≥75% in 32. A threshold of ≥75% LOY was significantly associated with morphologic diagnosis of myeloid neoplasm (p = 0.004). Further, ≥75% LOY was associated with a higher lifetime incidence of diagnosis of myelodysplastic syndromes (MDS; p < 0.0001), and in multivariate analysis ≥75% LOY was a statistically significant independent predictor of myeloid neoplasia [OR 6.17; 95% CI = 2.15-17.68; p = 0.0007]. Higher LOY percentage (≥75%) was associated with greater likelihood of having somatic mutations (p = 0.0009) and a higher number of these mutations (p = 0.0002). Our findings indicate that ≥75% LOY in marrow is associated with increased likelihood of molecular alterations in genes commonly seen in myeloid neoplasia and with morphologic features of MDS. These observations suggest that ≥75% LOY in bone marrow should be considered an MDS-associated cytogenetic aberration.

Inhibitory signals through the PD-1 pathway regulate T cell activation, T cell tolerance, and T cell exhaustion. Studies of PD-1 function have focused primarily on effector T cells. Far less is known about PD-1 function in regulatory T (T reg) cells. To study the role of PD-1 in T reg cells, we generated mice that selectively lack PD-1 in T reg cells. PD-1-deficient T reg cells exhibit an activated phenotype and enhanced immunosuppressive function. The in vivo significance of the potent suppressive capacity of PD-1-deficient T reg cells is illustrated by ameliorated experimental autoimmune encephalomyelitis (EAE) and protection from diabetes in nonobese diabetic (NOD) mice lacking PD-1 selectively in T reg cells. We identified reduced signaling through the PI3K-AKT pathway as a mechanism underlying the enhanced suppressive capacity of PD-1-deficient T reg cells. Our findings demonstrate that cell-intrinsic PD-1 restraint of T reg cells is a significant mechanism by which PD-1 inhibitory signals regulate T cell tolerance and autoimmunity., Competing Interests: Disclosures: P.T. Sage reported a patent to PCT/US16/57031 pending. L.M. Francisco reported "other" from Evelo Biosciences and GlaxoSmithKline outside the submitted work; in addition, L.M. Francisco had a patent to US Patent 10,577,586 pending, a patent to WO2017066561A2 pending, and a patent to US20150299322A1 with royalties paid; and is currently an employee of Evelo Biosciences. V.R. Juneja reported a patent number 20180303922 issued. B.R. Blazar reported grants from the National Institutes of Health and personal fees from BlueRock Therapeutics, Magenta Therapeutics, Kadmon Pharmaceuticals, Incyte Corp, Obsidian Therapeutics, Regeneron Pharmaceuticals, Regimmune, Dr. Reddy, and Equillium Inc. outside the submitted work. D.A.A. Vignali reported personal fees from Almirall outside the submitted work and reported, "cofounder and stock holder - Novasenta and Tizona; stock holder - Oncorus and Werewolf; patents licensed and royalties - Astellas, BMS; scientific advisory board member - Tizona, Werewolf and F-Star; consultant - Astellas, BMS, Almirall; research funding - BMS, Astellas and Novasenta." G.J. Freeman reported a patent with royalties paid (Roche); a patent with royalties paid (Merck MSD); a patent with royalties paid (Bristol-Myers Squibb); a patent with royalties paid (Merck KGA); a patent with royalties paid (Boehringer-Ingelheim); a patent with royalties paid (AstraZeneca); a patent with royalties paid (Dako); a patent with royalties paid (Leica); a patent with royalties paid (Novartis); and a patent with royalties paid (Mayo Clinic). G.J. Freeman has served on advisory boards for Roche, Bristol-Myers-Squibb, Xios, Origimed, Triursus, iTeos, NextPoint, IgM, Jubilant and GV20, and has equity in Nextpoint, Triursus, Xios, iTeos, IgM, and GV20. A.H. Sharpe reported personal fees from Surface Oncology, Sqz Biotech, Selecta, Elstar, and Elpiscience; "other" from Monopteros; grants from Novartis, Merck, Roche, Ipsen, UCB, and Quark Ventures outside the submitted work; and is on the scientific advisory boards for the Massachusetts General Cancer Center, Program in Cellular and Molecular Medicine at Boston Children's Hospital and the Human Oncology and Pathogenesis Program at Memorial Sloan Kettering Cancer Center and Scientific Editor for the Journal of Experimental Medicine. A.H. Sharpe reported the following patents: 7,432,059, with royalties paid (Roche, Merck, Bristol-Myers-Squibb, EMD-Serono, Boehringer-Ingelheim, AstraZeneca, Leica, Mayo Clinic, Dako and Novartis); 7,722,868, with royalties paid (Roche, Merck, Bristol-Myers-Squibb, EMD-Serono, Boehringer-Ingelheim, AstraZeneca, Leica, Mayo Clinic, Dako and Novartis); 8,652,465, licensed (Roche), 9,457,080, licensed (Roche); 9,683,048, licensed (Novartis); 9,815,898, licensed (Novartis); 9,845,356, licensed (Novartis); 10,202,454, licensed (Novartis); 10,457,733, licensed (Novartis); 9,580,684, issued; 9,988,452, issued; 10,370,446, issued. No other disclosures were reported., (© 2020 Tan et al.)

Follicular regulatory T (T FR ) cells have specialized roles in modulating follicular helper T (T FH ) cell activation of B cells. However, the precise role of T FR cells in controlling antibody responses to foreign antigens and autoantigens in vivo is still unclear due to a lack of specific tools. A T FR cell-deleter mouse was developed that selectively deletes T FR cells, facilitating temporal studies. T FR cells were found to regulate early, but not late, germinal center (GC) responses to control antigen-specific antibody and B cell memory. Deletion of T FR cells also resulted in increased self-reactive immunoglobulin (Ig) G and IgE. The increased IgE levels led us to interrogate the role of T FR cells in house dust mite models. T FR cells were found to control T FH 13 cell-induced IgE. In vivo, loss of T FR cells increased house-dust-mite-specific IgE and lung inflammation. Thus, T FR cells control IgG and IgE responses to vaccines, allergens and autoantigens, and exert critical immunoregulatory functions before GC formation.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an uncommon hematologic malignancy with poor outcomes. Existing data on the clinical behavior of BPDCN are limited because reported outcomes are from small retrospective series, and standardized treatment guidelines are lacking. The interleukin-3 cytotoxin conjugate tagraxofusp was recently tested in phase 1/2 trials that led to US Food and Drug Administration approval, the first ever for BPDCN. However, because there was no matched internal comparator in this or any clinical study to date, results of BPDCN trials testing new drugs are difficult to compare with alternative therapies. We therefore sought to define the clinical characteristics and outcomes of a group of patients with BPDCN treated at 3 US cancer centers in the modern era but before tagraxofusp was available. In 59 studied patients with BPDCN, the median overall survival from diagnosis was 24 months, and outcomes were similar in patients with "skin only" or with systemic disease at presentation. Intensive first-line therapy and "lymphoid-type" chemotherapy regimens were associated with better outcomes. Only 55% of patients received intensive chemotherapy, and 42% of patients underwent stem cell transplantation. Clinical characteristics at diagnosis associated with poorer outcomes included age >60 years, abnormal karyotype, and terminal deoxynucleotidyltransferase (TdT) negativity in the BPDCN cells. We also identified disease responses to pralatrexate and enasidenib in some patients. This study highlights poor outcomes for patients with BPDCN in the modern era and the need for new treatments. Outcomes from ongoing clinical trials for BPDCN can be evaluated relative to this contemporary cohort., (© 2019 by The American Society of Hematology.)

Combination anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) and anti-programmed cell death protein 1 (PD-1) therapy promotes antitumor immunity and provides superior benefit to patients with advanced-stage melanoma compared with either therapy alone. T cell immunity requires recognition of antigens in the context of major histocompatibility complex (MHC) class I and class II proteins by CD8 + and CD4 + T cells, respectively. We examined MHC class I and class II protein expression on tumor cells from previously untreated melanoma patients and correlated the results with transcriptional and genomic analyses and with clinical response to anti-CTLA-4, anti-PD-1, or combination therapy. Most (>50% of cells) or complete loss of melanoma MHC class I membrane expression was observed in 78 of 181 cases (43%), was associated with transcriptional repression of HLA-A , HLA-B , HLA-C , and B2M , and predicted primary resistance to anti-CTLA-4, but not anti-PD-1, therapy. Melanoma MHC class II membrane expression on >1% cells was observed in 55 of 181 cases (30%), was associated with interferon-γ (IFN-γ) and IFN-γ-mediated gene signatures, and predicted response to anti-PD-1, but not anti-CTLA-4, therapy. We conclude that primary response to anti-CTLA-4 requires robust melanoma MHC class I expression. In contrast, primary response to anti-PD-1 is associated with preexisting IFN-γ-mediated immune activation that includes tumor-specific MHC class II expression and components of innate immunity when MHC class I is compromised. The benefits of combined checkpoint blockade may be attributable, in part, to distinct requirements for melanoma-specific antigen presentation to initiate antitumor immunity., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The contribution of the kidney-draining lymph node (KLN) to the pathogenesis of ischemia-reperfusion injury (IRI) of the kidney and its subsequent recovery has not been explored in depth. In addition, the mechanism by which repetitive IRI contributes to renal fibrosis remains poorly understood. Herein, we have found that IRI of the kidney is associated with expansion of high endothelial venules (HEVs) and activation of fibroblastic reticular cells (FRCs) in the KLN, as demonstrated by significant expansion in the extracellular matrix. The lymphotoxin α signaling pathway mediates activation of FRCs, and chronic treatment with lymphotoxin β receptor-immunoglobulin fusion protein (LTβr-Ig) resulted in marked alteration of the KLN as well as augmentation of renal fibrosis. Depletion of FRCs reduced T cell activation in the KLN and ameliorated renal injury in acute IRI. Repetitive renal IRI was associated with senescence of FRCs, fibrosis of the KLN, and renal scarring, which were ameliorated by FRC administration. Therefore, our study emphasizes the critical role of FRCs in both the initiation and repair phases of injury following IRI of the kidney.

Background: Histiocytic sarcoma (HS) is a rare malignant neoplasm showing morphologic and immunophenotypic features of histiocytes. Molecular characteristics of HS and fine-needle aspiration (FNA) criteria for its diagnosis have not been established., Methods: A case series of HS in 8 FNA samples from 6 patients was reviewed along with histopathologic and clinical data. Immunohistochemistry was performed on cell blocks (3 cases), core biopsies (5 cases), and surgical specimens (4 cases). Targeted-exome next-generation sequencing (NGS) was performed on surgical resection specimens in 4 cases., Results: Four patients had a known history of hematolymphoid malignancy. Cytomorphologic features included variably cellular smears composed of large epithelioid cells with reniform nuclei and abundant vacuolated cytoplasm, in an inflammatory background, with occasional cytophagocytosis and lymphoglandular bodies. Marked pleomorphism, multinucleated monster cells, and binucleated histiocytoid cells with partially overlapping, eccentrically placed nuclei resembling Pac-Man were common. Most cases expressed histiocytic markers CD68 (6 of 7 cases), CD163 (5 of 5 cases), and PU.1 (3 of 4 cases). In 3 cases, NGS analysis revealed alterations in lysine methyltransferase 2D (KMT2D)/mixed-lineage leukemia 2 (MLL2), a gene involved in chromatin regulation and previously implicated in the pathogenesis of follicular lymphoma., Conclusions: Although diagnosing HS with FNA alone is extremely challenging, the presence of pleomorphic and epithelioid large cells with binucleation and/or multinucleation in an inflammatory background should prompt the diagnosis of HS with judicious use of confirmatory histiocytic lineage markers. The detection of recurrent KMT2D/MLL2 alterations implicates epigenetic regulation in the pathogenesis of HS and supports the notion of transdifferentiation from a genetically similar but phenotypically distinct tumor of a different lineage. Cancer Cytopathol 2017;125:604-14. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Aims: Pure erythroid leukaemia (PEL) is an extremely rare and aggressive subtype of acute myeloid leukaemia defined by the World Health Organization (WHO) as a neoplastic proliferation of immature cells committed exclusively to the erythroid lineage, comprising >80% of bone marrow cells and not meeting the criteria of other well-defined myeloid neoplasms. The aim of this study was to describe the clinicopathological features of acute leukaemias with a pure erythroid phenotype (ALPEP) irrespective of their WHO classification and to determine if ALPEP represents a distinct clinicopathological entity., Methods and Results: We identified seven cases of ALPEP, in which immature cells fulfilled WHO morphological and immunophenotypical criteria for PEL. All patients except one were male, with a median age of 60 years. Three cases represented de novo PEL, three were therapy-related myeloid neoplasms and one was a blast phase of a myeloproliferative neoplasm. Extensive tumour necrosis was present in five cases (71%). Five cases with available modal karyotypes all demonstrated a complex karyotype involving the TP53 gene locus, with three cases (60%) also showing a monosomy 5 or deletion 5q and additional material on chromosome 19q13. All patients died of their disease, with a mean overall survival of 189 and 64.7 days in cases without and with necrosis on the initial biopsy, respectively., Conclusions: We describe previously unreported but relatively common findings of extensive tumour necrosis and recurring cytogenetic abnormalities in ALPEP. Our findings suggest strongly that ALPEP represents a distinct clinicopathological entity regardless of its WHO classification., (© 2017 John Wiley & Sons Ltd.)

Naive T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naive CD4(+) T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one-carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one-carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one-carbon metabolism that is critical for T cell activation and survival., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1-/- donors. PD-L1-deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1-/- donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell-mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.

The recognition that the immune system can identify and destroy tumor cells has driven a paradigm shift in our understanding of human cancer. Therapies designed to enhance this capacity, including cancer vaccines and coinhibitory receptor blockade, have demonstrated clinical efficacy in treating tumors refractory to conventional therapy. In this review, we discuss how the analysis of the immune microenvironment in primary tissue biopsy samples can be used to stratify patients according to clinical outcome, identify patients likely to benefit from specific immunotherapies, and tailor combination immunotherapy to individual patients and tumor types. As immunotherapy gains in complexity and is used in combination with agents that target oncogenic, intracellular signaling pathways, diagnostic pathologists will play an increasingly important part in identifying and quantifying cellular and molecular biomarkers in tissue samples that reflect the nature and magnitude of the antitumor immune response.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of T cell responses. Germline Ctla4 deficiency is lethal, making investigation of the function of CTLA-4 on mature T cells challenging. To elucidate the function of CTLA-4 on mature T cells, we have conditionally ablated Ctla4 in adult mice. We show that, in contrast to germline knockout mice, deletion of Ctla4 during adulthood does not precipitate systemic autoimmunity, but surprisingly confers protection from experimental autoimmune encephalomyelitis (EAE) and does not lead to increased resistance to MC38 tumors. Deletion of Ctla4 during adulthood was accompanied by activation and expansion of both conventional CD4(+)Foxp3(-) (T conv) and regulatory Foxp3(+) (T reg cells) T cell subsets; however, deletion of CTLA-4 on T reg cells was necessary and sufficient for protection from EAE. CTLA-4 deleted T reg cells remained functionally suppressive. Deletion of Ctla4 on T reg cells alone or on all adult T cells led to major changes in the Ctla4 sufficient T conv cell compartment, including up-regulation of immunoinhibitory molecules IL-10, LAG-3 and PD-1, thereby providing a compensatory immunosuppressive mechanism. Collectively, our findings point to a profound role for CTLA-4 on T reg cells in limiting their peripheral expansion and activation, thereby regulating the phenotype and function of T conv cells., (© 2015 Paterson et al.)

A man in his 40s presented with 1 month of worsening confusion, fatigue, and headache. Results from laboratory analyses were notable for a complete white blood cell count of 17 000/μL (31% blast cells), a platelet count of 76 000/μL, and a hemoglobin level of 16.6 g/dL. Imaging studies revealed a large mixed-attenuation subdural collection in the right frontal region with prominent mass effect. The patient underwent an emergency neurosurgical procedure. The differential diagnosis, pathologic findings, and diagnosis are discussed.

Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5'-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5'-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell-nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease.

The receptor CTLA-4 has been implicated in controlling B cell responses, but the mechanisms by which CTLA-4 regulates antibody production are not known. Here we showed deletion of CTLA-4 in adult mice increased Tfh and Tfr cell numbers and augmented B cell responses. In the effector phase, loss of CTLA-4 on Tfh cells resulted in heightened B cell responses, whereas loss of CTLA-4 on Tfr cells resulted in defective suppression of antigen-specific antibody responses. We also found that non-Tfr Treg cells could suppress B cell responses through CTLA-4 and that Treg and/or Tfr cells might downregulate B7-2 on B cells outside germinal centers as a means of suppression. Within the germinal center, however, Tfr cells potently suppress B cells through CTLA-4, but with a mechanism independent of altering B7-1 or B7-2. Thus, we identify multifaceted regulatory roles for CTLA-4 in Tfh, Tfr, and Treg cells, which together control humoral immunity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis, whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism, leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Type B T cells recognize a peptide-MHC conformer generated in recycling endosomes and eliminated by H2-DM in late endosomes; as a result, they recognize exogenous peptide, but fail to respond to the identical epitope generated from the native protein. To investigate the behavior of these cells in vivo, we generated mice transgenic for a type B TCR recognizing the 48-62 epitope of hen egg white lysozyme (HEL) presented by I-A(k). Type B T cells responded only to peptide ex vivo, but responded in vivo to immunization with either protein or peptide in the presence of Freund's adjuvant or LPS. Presentation of the type B conformer was MyD88-independent, evident within 24 h after HEL immunization, and restricted to the CD11b/c(+) APC subset. Immunization with listeriolysin O, a potent inducer of cell death, also primed type B T cells in vivo, and transfer of HEL-bearing allogeneic dendritic cells activated type B T cells. We conclude that a number of conditions in vivo, some of which induce inflammation and cell death, lead to peptide presentation through mechanisms distinct from the classical pathways involving H-2DM molecules.

The peptide spanning residues 48-62 of hen egg white lysozyme presented by I-A(k) molecules gives rise to two T cell populations, types A and B, that recognize distinct conformers of the complex generated in late and recycling endosomes. The class II-like accessory molecule H2-DM functions as a conformational editor, eliminating the type B conformer in late endosomes. Here, we show that the conformation of the complex, and its susceptibility to editing by H2-DM, are determined by peptide amino-terminal flanking residues. Elimination of these residues abolished editing, permitting formation of the type B conformer in late endosomes. Substitutions at P(-2) affected the stability of the type B conformer, preventing its formation and/or editing, without hindering peptide binding or formation of the type A conformer of the complex. We conclude that interactions involving amino-terminal flanking residues stabilize peptide-MHC conformers and confer resistance to editing by H2-DM, influencing the nature of the T cell repertoire.

The identification and quantitation of peptides selected by class II MHC molecules during natural processing of proteins is of key importance in understanding the repertoire and distribution of T cells. The examination of peptides selected by class II MHC molecules has depended greatly on mass spectrometry, a powerful technique that identifies and sequences peptides in complex mixtures with great sensitivity and precision. Such analysis has resulted in the identification of several factors, including the repertoire of peptides selected by MHC molecules during natural processing of proteins, motifs important for selection of processed peptides, conformational isomers of peptide-MHC complexes, and post-translational changes to the peptides.

The relative plasticity of peptide binding to class II major histocompatibility complex (MHC) molecules permits formation of multiple conformational isomers by the same peptide and MHC molecule; such conformers are specifically recognized by distinct subsets of T cells. Here, we review current knowledge and recent advances in our understanding of peptide-class II MHC conformational isomerism and the mechanisms that generate distinct MHC-peptide conformers. We focus on our studies of two T-cell subsets, type A and B, which recognize distinct conformers of the dominant epitope of hen egg white lysozyme presented by I-A(k). These conformers form via different pathways and in distinct intracellular vesicles: the type A conformer forms in late endosomes upon processing of native protein, while the more flexible type B conformer forms in early endosomes and at the cell surface. In this process, H2-DM acts as a conformational editor, eliminating the type B conformer in late endosomes. Type B T cells constitute a significant component of the naïve T-cell repertoire; furthermore, self-reactive type B T cells escape negative selection and are present in abundance in the periphery. Ongoing studies should elucidate the role of type B T cells in immunity to pathogens and in autoimmune pathology.

The peptide spanning residues 48-61 of hen egg white lysozyme (HEL) presented by I-A(k) gives rise to two T cell populations, referred to as type A and B, that distinguish the complex generated intracellularly upon processing of HEL from that formed with exogenous peptide. Here, we ascribe this difference to recognition of distinct conformers of the complex and show that formation of the two complexes results from antigen processing in different intracellular compartments and is dependent upon H2-DM. While the type A complex preferentially formed in a lysosome-like late vesicle, the type B complex failed to form in this compartment; this distinction was abolished in antigen-presenting cells lacking DM. Experiments in vitro indicated that H2-DM acts directly on the complex to eliminate the type B conformation. We conclude that different antigen-processing pathways generate distinct MHC-peptide conformers, priming T cells with distinct specificity that may play unique roles in immunity.

We quantitated the amounts of peptides from hen egg-white lysozyme presented by I-A(k) molecules in APC lines. The large chemical gradient of presentation of the four hen egg-white lysozyme epitopes observed in cell lines expressing HLA-DM or H-2DM (referred to in this study as DM) was significantly diminished in the T2.A(k) line lacking DM. Differences in levels of presentation between wild-type and DM-deficient APC were observed for all four epitopes, but differences were most evident for the highest affinity epitope. As a result of these quantitative differences in display, presentation of all four epitopes to T cells was impaired in the line lacking DM. The binding affinity of the pool of naturally processed peptides from DM-expressing lines was higher than that from the DM-deficient line. Thus, using a direct biochemical approach in APC, we demonstrate that DM influences the selection of peptides bound to MHC class II by favoring high affinity peptides.

Type B T cells recognize peptide provided exogenously but are ignorant of the same epitope derived from intracellular processing. In this study, we demonstrate the existence of type B T cells to an abundant autologous peptide derived from processing of the I-A(k) beta-chain. T cell hybridomas raised against this peptide fail to recognize syngeneic APC despite abundant presentation of the naturally processed epitope but react in a dose-dependent manner to exogenous peptide. Moreover, these hybridomas respond to Abeta(k) peptide extracted from the surface of I-A(k)-expressing APC. This peptide was isolated from B cell lines where it was found in high abundance; it was also present in lines lacking HLA-DM, but in considerably lower amounts. Therefore, type B T cells exist in the naive repertoire to abundant autologous peptides. We discuss the implications of these findings to the potential biological role of type B T cells in immune responses and autoimmune pathology.