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Similarly to Epstein-Barr virus (EBV), the human herpesvirus-8 (HHV-8) is a γ-herpesvirus, recently recognized to be associated with the occurrence of rare B cell lymphomas and atypical lymphoproliferations, especially in the human immunodeficiency virus (HIV) infected subjects. Moreover, the human herpesvirus-6 (HHV-6), a β-herpesvirus, has been shown to be implicated in some non-malignant lymph node proliferations, such as the Rosai Dorfman disease, and in a proportion of Hodgkin's lymphoma cases. HHV-6 has a wide cellular tropism and it might play a role in the pathogenesis of a wide variety of human diseases, but given its ubiquity, disease associations are difficult to prove and its role in hematological malignancies is still controversial. The involvement of another β-herpesvirus, the human cytomegalovirus (HCMV), has not yet been proven in human cancer, even though recent findings have suggested its potential role in the development of CD4+ large granular lymphocyte (LGL) lymphocytosis. Here, we review the current knowledge on the pathogenetic role of HHV-8 and human β-herpesviruses in human lymphoproliferative disorders. [ABSTRACT FROM AUTHOR]

Objective: We investigated the performance of enzyme linked immunospot (ELISpot) assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematologic malignancies., Methods: We prospectively enrolled two cohorts of patients undergoing intensive myelosuppressive or immunosuppressive treatments at high risk for IA. ELISpot was performed to detect Aspergillus-specific T cells producing Interleukin-10., Results: In the discovery cohort, a derived cut-off of 40 spot forming cells (SFCs)/106 PBMCs has shown to correctly classify IA cases with a sensitivity and specificity of 89.5% and 88.6%, respectively. This cut-off is lowered to 25 SFC when considering the subset of possible IA patients, with sensitivity and specificity of 76% and 93%, respectively. The application of the 40 SFCs cut-off to the validation cohort resulted in a positivity rate of 83.3% in proven/probable cases and a negativity rate of 92.5% in possible/non-IA cases. Adopting the 25 SCFs cut-off, the assay resulted positive in 83.3% of proven/probable cases while it resulted negative in 66.7% of possible/non-IA cases., Conclusions: ELISpot shows promises in the diagnosis of IA and the possibility to use two distinct cut-offs with similar diagnostic performances according to patients' different pre-test probability of infection can widen its use in patients at risk., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: M. Luppi, L. Potenza and P. Barozzi have applied for an Italian patent regarding clinical applications of the ELISpot assay for the diagnosis of Aspergillus infection [PCT: WO2008/075395A3, IT2007/000867]. M. Luppi, L. Potenza, D. Vallerini, P. Barozzi and F. Forghieri have applied for an Italian and US patent regarding clinical applications of the ELISpot assay for the diagnosis of Mucorales infection [PCT: WO2012073160 A1, IT MI20102224, US 2013/0260395]. This does not alter the authors’ adherence to all Journal policies. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Other authors of this manuscript have no conflicts of interest related to the study to disclose., (Copyright: © 2024 Bettelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Multiple Myeloma (MM) typically originates from underlying precursor conditions, known as Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Validated risk factors, related to the main features of the clonal plasma cells, are employed in the current prognostic models to assess long-term probabilities of progression to MM. In addition, new prognostic immunologic parameters, measuring protective MM-specific T-cell responses, could help to identify patients with shorter time-to-progression. In this report, we described a novel Multi-antigenic Myeloma-specific (MaMs) T-cell assay, based on ELISpot technology, providing simultaneous evaluation of T-cell responses towards ten different MM-associated antigens. When performed during long-term follow-up (mean 28 months) of 33 patients with either MGUS or SMM, such deca-antigenic myeloma-specific immunoassay allowed to significantly distinguish between stable vs. progressive disease ( p < 0.001), independently from the Mayo Clinic risk category. Here, we report the first clinical experience showing that a wide (multi-antigen), standardized (irrespective to patients' HLA), MM-specific T-cell assay may routinely be applied, as a promising prognostic tool, during the follow-up of MGUS/SMM patients. Larger studies are needed to improve the antigenic panel and further explore the prognostic value of MaMs test in the risk assessment of patients with monoclonal gammopathies.

Multiple Myeloma (MM) is a malignant growth of clonal plasma cells, typically arising from asymptomatic precursor conditions, namely monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Profound immunological dysfunctions and cytokine deregulation are known to characterize the evolution of the disease, allowing immune escape and proliferation of neoplastic plasma cells. In the past decades, several studies have shown that the immune system can recognize MGUS and MM clonal cells, suggesting that anti-myeloma T cell immunity could be harnessed for therapeutic purposes. In line with this notion, chimeric antigen receptor T cell (CAR-T) therapy is emerging as a novel treatment in MM, especially in the relapsed/refractory disease setting. In this review, we focus on the pivotal contribution of T cell impairment in the immunopathogenesis of plasma cell dyscrasias and, in particular, in the disease progression from MGUS to SMM and MM, highlighting the potentials of T cell-based immunotherapeutic approaches in these settings.

In recent years, the introduction of new drugs targeting Bruton's tyrosine kinase (BTK) has allowed dramatic improvement in the prognosis of patients with chronic lymphocytic leukemia (CLL) and other B-cell neoplasms. Although these small molecules were initially considered less immunosuppressive than chemoimmunotherapy, an increasing number of reports have described the occurrence of unexpected opportunistic fungal infections, in particular invasive aspergillosis (IA). BTK represents a crucial molecule in several signaling pathways depending on different immune receptors. Based on a variety of specific off-target effects on innate immunity, namely on neutrophils, monocytes, pulmonary macrophages, and nurse-like cells, ibrutinib has been proposed as a new host factor for the definition of probable invasive pulmonary mold disease. The role of platelets in the control of fungal growth, through granule-dependent mechanisms, was described in vitro almost two decades ago and is, so far, neglected by experts in the field of clinical management of IA. In the present study, we confirm the antifungal role of platelets, and we show, for the first time, that the exposure to BTK inhibitors impairs several immune functions of platelets in response to Aspergillus fumigatus , i.e., the ability to adhere to conidia, activation (as indicated by reduced expression of P-selectin), and direct killing activity. In conclusion, our experimental data suggest that antiplatelet effects of BTK inhibitors may contribute to an increased risk for IA in CLL patients.

Infections represent a cause of morbidity and mortality in patients affected by chronic lymphocytic leukemia (CLL). Introduction of new drugs in CLL clinical practice has showed impressive efficacy, in particular those targeting BTK. Among the consistent clinical data, an increasing number of reports describing the occurrence of unexpected opportunistic fungal infections has been reported during treatment with ibrutinib in the first 6 months of treatment. The reason underlying manifestations of invasive fungal infections in patients treated with ibrutinib is still under investigation. Our study aimed to understand the impact of BTK inhibition on immune response to fungal infection mediated by macrophages and CD14+ monocytic population obtained from CLL patients. Exposure to ibrutinib and acalabrutinib reduced signaling pathways activated by Aspergillus fumigatus determining an exacerbation of an immunosuppressive signature, a reduction of phagocytosis and a significant deficit in the secretion of inflammatory cytokines either in macrophages and monocytes isolated from CLL patients and healthy donors. These effects lead to a failure in completely counteracting conidia germination. In addition we investigated the biological effects of ibrutinib on monocyte counterpart in patients who were undergoing therapy. A significant impairment in cytokine secretion and a deficit of phagocytosis in circulating monocytes were detected after 3 months of treatment. Thus, our results uncover modifications in the innate response in CLL patients induced by ibrutinib that may impair the immunological response to fungal infection., Keypoints: •BTK inhibition affects a productive immune response of CLL-associated macrophages (NLC) during Aspergillus fumigatus infection.•Reduction of TNF-α secretion and phagocytosis are detected in monocytes isolated from CLL patients during ibrutinib therapy., (Copyright © 2020 Fiorcari, Maffei, Vallerini, Scarfò, Barozzi, Maccaferri, Potenza, Ghia, Luppi and Marasca.)

Nucleophosmin(NPM1)-mutated protein, a leukemia-specific antigen, represents an ideal target for AML immunotherapy. We investigated the dynamics of NPM1-mutated-specific T cells on PB and BM samples, collected from 31 adult NPM1 -mutated AML patients throughout the disease course, and stimulated with mixtures of 18 short and long peptides (9-18mers), deriving from the complete C-terminal of the NPM1-mutated protein. Two 9-mer peptides, namely LAVEEVSLR and AVEEVSLRK (13.9-14.9), were identified as the most immunogenic epitopes. IFNγ-producing NPM1-mutated-specific T cells were observed by ELISPOT assay after stimulation with peptides 13.9-14.9 in 43/85 (50.6%) PB and 34/80 (42.5%) BM samples. An inverse correlation between MRD kinetics and anti-leukemic specific T cells was observed. Cytokine Secretion Assays allowed to predominantly and respectively identify Effector Memory and Central Memory T cells among IFNγ-producing and IL2-producing T cells. Moreover, NPM1-mutated-specific CTLs against primary leukemic blasts or PHA-blasts pulsed with different peptide pools could be expanded ex vivo from NPM1 -mutated AML patients or primed in healthy donors. We describe the spontaneous appearance and persistence of NPM1 -mutated-specific T cells, which may contribute to the maintenance of long-lasting remissions. Future studies are warranted to investigate the potential role of both autologous and allogeneic adoptive immunotherapy in NPM1 -mutated AML patients., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Although the emergence of bone marrow (BM)-resident p190 BCR-ABL-specific T lymphocytes has been correlated with hematologic and cytogenetic remissions in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) undergoing maintenance tyrosine-kinase inhibitor treatment, little is known about the possibility of culturing these cells ex vivo and using them in T-cell therapy strategies. We investigated the feasibility of expanding/priming p190 BCR-ABL-specific T cells in vitro by stimulation with dendritic cells pulsed with p190 BCR-ABL peptides derived from the BCR-ABL junctional region and alternative splicing, and of adoptively administering them to patients with relapsed disease. We report on the feasibility of producing clinical-grade BCR-ABL-specific cytotoxic T lymphocytes (CTLs), endowed with antileukemia activity, from Ph + ALL patients and healthy donors. We treated 3 patients with Ph + ALL with autologous or allogeneic p190 BCR-ABL-specific CTLs. No postinfusion toxicity was observed, except for a grade II skin graft-versus-host disease in the patient treated for hematologic relapse. All patients achieved a molecular or hematologic complete remission (CR) after T-cell therapy, upon emergence of p190 BCR-ABL-specific T cells in the BM. Our results show that p190 BCR-ABL-specific CTLs are capable of controlling treatment-refractory Ph + ALL in vivo, and support the development of adoptive immunotherapeutic approaches with BCR-ABL CTLs in Ph + ALL., (© 2017 by The American Society of Hematology.)

Based upon the clinical behavior of three patients, we suggest that the combination of low-dose Ara-C and all-trans retinoic acid may potentially be effective in some elderly patients, unfit for intensive chemotherapy, affected with NPM1 -mutated acute myeloid leukemia without FLT3 mutations, warranting perspective clinical studies in these selected patients.

Bone marrow has already been described as an enrichment site for several antigen-specific T lymphocytes, but the presence of mould-specific T cells has never been investigated in the bone marrow. We have previously demonstrated that mould-specific T cells emerge in the peripheral blood of patients with invasive fungal infections (IFI) but tend to become undetectable after disease resolution. In seven patients with a history of IFI, we investigated the presence of mould-specific T cells secreting different cytokines in bone marrow and peripheral blood paired samples. The results showed that the frequencies of mould-specific T cells secreting the protective cytokine IFNγ are significantly higher in bone marrow (BM) and are mainly represented by CD8+ T lymphocytes with effector phenotype. A putative disappearance of such protective BM responses after myeloablative therapy could contribute to the increased risk of IFI in hematologic patients., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Invasive mucormycosis (IM) is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients., Methods and Findings: By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB) samples has been investigated at three time points during high-dose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3%) of 204 patients, accounting for 32 (5.3%) of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD), showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD): 2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD., Conclusions: Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1-3glucan, β1-3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with IA.

Mucorales-specific T cells were investigated in 28 hematologic patients during the course of their treatment. Three developed proven invasive mucormycosis (IM), 17 had infections of known origin but other than IM, and 8 never had fever during the period of observation. Mucorales-specific T cells could be detected only in patients with IM, both at diagnosis and throughout the entire course of the IM, but neither before nor for long after resolution of the infection. Such T cells predominantly produced IL-4, IFN-γ, IL-10, and to a lesser extent IL-17 and belonged to either CD4(+) or CD8(+) subsets. The specific T cells that produced IFN-γ were able to directly induce damage to Mucorales hyphae. None of the 25 patients without IM had Mucorales-specific T cells. Specific T cells contribute to human immune responses against fungi of the order Mucorales and could be evaluated as a surrogate diagnostic marker of IM.

Clinical charts from 63 consecutive highly immunocompromised haematologic patients presenting with pulmonary nodular lesions on CT scan, classified as either probable or possible invasive fungal disease (IFD) according to the revised EORTC/MSG classification, were retrospectively studied. Histopathological analysis of lung tissues, available for 23 patients, demonstrated proven IFD in 17 cases (14 invasive aspergillosis and 3 invasive zygomycosis), diffuse alveolar damage in one and organising pneumonia (OP) in five cases. In the OP cases, three of which have been defined as probable IFD according to EORTC/MSG classification, extensive immunohistochemical, molecular and immunological analyses for fungi were negative. Our case descriptions extend the notion that OP may be encountered as a distinct histopathological entity in pulmonary nodular lesions in patients with leukaemia with probable/possible IFD.

Imatinib mesylate has been demonstrated to allow the emergence of T cells directed against chronic myeloid leukemia cells. A total of 10 Philadelphia chromosome-positive acute lymphoblastic leukemia patients receiving high-dose imatinib mesylate maintenance underwent long-term immunological monitoring (range, 2-65 months) of (p190)BCR-ABL-specific T cells in the bone marrow and peripheral blood. (p190)BCR-ABL-specific T lymphocytes were detected in all patients, more frequently in bone marrow than in peripheral blood samples (67% vs 25%, P < .01) and resulted significantly associated with lower minimal residual disease values (P < .001), whereas absent at leukemia relapse. Specific T cells were mainly effector memory CD8(+) and CD4(+) T cells, producing interferon-gamma, tumor necrosis factor-alpha, and interleukin-2 (median percentage of positive cells: 3.34, 3.04, and 3.58, respectively). Cytotoxic subsets able to lyse BCR-ABL-positive leukemia blasts also were detectable. Whether these autologous (p190)BCR-ABL-specific T cells may be detectable under other tyrosine-kinase inhibitors, expanded ex vivo, and exploited for immunotherapy remains to be addressed.

A child with acute myeloid leukemia presented with multiple liver lesions mimicking hepatosplenic candidiasis during the neutropenic phase following the induction chemotherapy. All the available diagnostic tools showed repeatedly negative results, including galactomannan. An enzyme-linked immunospot (ELISPOT) assay showed a high number of Aspergillus-specific T cells producing interleukin-10 [TH2(IL-10)] and a low number of Aspergillus-specific T cells producing gamma interferon [TH1(IFN-gamma)], revealing invasive aspergillosis (IA) before the confirmatory biopsy. A progressive skewing from the predominance of TH2(IL-10) to a predominance of TH1(IFN-gamma) was observed close to the complete resolution of the infection and foreshadowed the outcome. The ELISPOT assay holds promise for diagnosing pediatric IA.

In nine patients with posttransplant Kaposi sarcoma (KS) T-cell responses to human herpesvirus (HHV)-8 latent and lytic antigens, as detected by enzyme-linked-immunospot (Elispot) assay, were absent at disease onset. Virus-specific T-cell responses were detected in six renal recipients at remission after a reduction of calcineurin inhibitors (CIs), and in two HHV-8 seropositive renal recipients without KS. In two liver recipients undergoing switch from CIs to sirolimus (SRL), normalization of the T-cell repertoire and recovery of both HHV-8-specific effector and memory T lymphocytes were associated with complete KS remission. In a renal recipient undergoing SRL conversion, the early recovery of HHV-8-specific effector but not of memory T lymphocytes, was associated only with partial remission. Neither rejection nor changes in graft function were observed after SRL conversion. HHV-8-specific T-cell responses are required to achieve posttransplant KS remission, and may be restored under SRL, while maintaining effective immunosuppression.