ABSTRACT
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.
Subject(s)
Enzyme-Linked Immunospot Assay , Humans , Enzyme-Linked Immunospot Assay/methods , Male , Female , Middle Aged , Aged , Adult , Prospective Studies , Aspergillosis/diagnosis , Aspergillosis/immunology , Interleukin-10/immunology , Hematologic Neoplasms/immunology , Hematologic Neoplasms/diagnosis , Sensitivity and Specificity , T-Lymphocytes/immunologyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Monoclonal Gammopathy of Undetermined Significance , Multiple Myeloma , Paraproteinemias , Smoldering Multiple Myeloma , Disease Progression , Humans , Monoclonal Gammopathy of Undetermined Significance/pathology , Multiple Myeloma/pathology , Multiple Myeloma/therapy , Paraproteinemias/therapy , T-Lymphocytes/pathologyABSTRACT
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.
Subject(s)
Aspergillosis , Invasive Fungal Infections , Leukemia, Lymphocytic, Chronic, B-Cell , Agammaglobulinaemia Tyrosine Kinase/metabolism , Antifungal Agents/pharmacology , Antifungal Agents/therapeutic use , Aspergillosis/drug therapy , Aspergillosis/metabolism , Blood Platelets/metabolism , Humans , Invasive Fungal Infections/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Protein Kinase Inhibitors/therapeutic useABSTRACT
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: â¢BTK inhibition affects a productive immune response of CLL-associated macrophages (NLC) during Aspergillus fumigatus infection.â¢Reduction of TNF-α secretion and phagocytosis are detected in monocytes isolated from CLL patients during ibrutinib therapy.
Subject(s)
Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Antineoplastic Agents/therapeutic use , Aspergillosis/immunology , Aspergillus fumigatus/physiology , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Macrophages/immunology , Piperidines/therapeutic use , Adenine/pharmacology , Adenine/therapeutic use , Antineoplastic Agents/pharmacology , Humans , Immunity, Innate , Immunomodulation , Macrophages/microbiology , Phagocytosis , Piperidines/pharmacology , Signal Transduction , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolismSubject(s)
Anemia, Aplastic/epidemiology , Latent Tuberculosis/epidemiology , Leukemia, Myeloid, Acute/epidemiology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/epidemiology , Adolescent , Adult , Aged , Aged, 80 and over , Anemia, Aplastic/drug therapy , Antitubercular Agents/therapeutic use , BCG Vaccine , Combined Modality Therapy , Comorbidity , Febrile Neutropenia/epidemiology , Hematopoietic Stem Cell Transplantation , Humans , Interferon-gamma Release Tests , Isoniazid/therapeutic use , Latent Tuberculosis/diagnostic imaging , Latent Tuberculosis/drug therapy , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/therapy , Middle Aged , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Retrospective Studies , Young AdultABSTRACT
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.
Subject(s)
Biosimilar Pharmaceuticals/pharmacology , Filgrastim/pharmacology , Hematopoietic Stem Cell Mobilization/methods , Leukemia, Myeloid/therapy , Peripheral Blood Stem Cell Transplantation/methods , Peripheral Blood Stem Cells/drug effects , Acute Disease , Adult , Female , Hematologic Agents/pharmacology , Humans , Leukemia, Myeloid/pathology , Male , Middle Aged , Retrospective Studies , Transplantation, Autologous , Treatment Outcome , Young AdultABSTRACT
Although the emergence of bone marrow (BM)-resident p190BCR-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 p190BCR-ABL-specific T cells in vitro by stimulation with dendritic cells pulsed with p190BCR-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 p190BCR-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 p190BCR-ABL-specific T cells in the BM. Our results show that p190BCR-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.
Subject(s)
Adoptive Transfer/methods , Fusion Proteins, bcr-abl/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Protein Kinase Inhibitors/therapeutic use , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantation , Adult , Cells, Cultured , Female , Humans , Male , Middle Aged , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunologyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Immunocompromised Host , Leukemia, Myeloid, Acute/immunology , Mucorales/immunology , Mucormycosis/immunology , Th2 Cells/immunology , Adolescent , Adult , Aged , Female , Humans , Leukemia, Myeloid, Acute/microbiology , Male , Middle Aged , Mucormycosis/diagnostic imaging , Mucormycosis/microbiology , Radiography , Th2 Cells/microbiology , Young AdultABSTRACT
Chronic/recurrent behaviour may be encountered in some distinct atypical or malignant lymphoproliferations, while recurrences are not generally observed in reactive/benign lymphadenopathies. We retrospectively analysed a consecutive series of 486 human immunodeficiency virus-negative adults, who underwent lymphadenectomy. Neoplastic and benign/reactive histopathological pictures were documented in 299 (61·5%) and 187 (38·5%) cases, respectively. Of note, seven of the 111 (6·3%) patients with benign lymphadenopathy without well-defined aetiology, showed chronic/recurrent behaviour, without constitutional symptoms. Enlarged lymph nodes were round in shape and hypoechoic, mimicking lymphoma. Reactive follicular hyperplasia and paracortical expansion were observed. Human herpesvirus (HHV)-6B positive staining in follicular dendritic cells (FDCs) was documented in all seven patients. Serological, molecular and immunological examinations suggested HHV-6B reactivation. Among the remaining 104 cases with reactive lymphoid hyperplasia in the absence of well-known aetiology and without recurrences, positivity for HHV-6B on FDCs was found in three cases, whereas in seven further patients, a scanty positivity was documented in rare, scattered cells in inter-follicular regions. Immunohistochemistry for HHV-6A and HHV-6B was invariably negative on 134 lymph nodes, with either benign pictures with known aetiology or malignant lymphoproliferative disorders, tested as further controls. Future studies are warranted to investigate a potential association between HHV-6B reactivation and chronic/recurrent benign lymphadenopathy.
Subject(s)
Herpesvirus 6, Human/physiology , Lymphatic Diseases/virology , Roseolovirus Infections/complications , Adult , Aged , Chronic Disease , Dendritic Cells/pathology , Female , Humans , Hyperplasia/virology , Immunohistochemistry , Lymph Node Excision , Lymph Nodes/pathology , Male , Middle Aged , Neoplasms/virology , Recurrence , Retrospective Studies , Virus ActivationABSTRACT
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.
Subject(s)
Bone Marrow/immunology , CD8-Positive T-Lymphocytes/immunology , Fungemia/immunology , Fungi/immunology , Adult , Aged , Blood/immunology , Cohort Studies , Female , Humans , Interferon-gamma/metabolism , Male , Middle AgedSubject(s)
Antineoplastic Agents/pharmacology , Lymphoma, Primary Effusion/drug therapy , Neoplasm Proteins/antagonists & inhibitors , RNA, Small Interfering/pharmacology , Repressor Proteins/antagonists & inhibitors , Cell Line, Tumor , Female , Humans , Liposomes , Lymphoma, Primary Effusion/genetics , Lymphoma, Primary Effusion/metabolism , Male , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Positive Regulatory Domain I-Binding Factor 1 , RNA, Small Interfering/genetics , Repressor Proteins/biosynthesisSubject(s)
Anemia, Aplastic/diagnosis , Anemia, Aplastic/epidemiology , Leukemia/diagnosis , Leukemia/epidemiology , Respiratory Syncytial Virus Infections/epidemiology , Respirovirus Infections/epidemiology , Acute Disease , Adolescent , Adult , Aged , Aged, 80 and over , Anemia, Aplastic/complications , Anemia, Aplastic/therapy , Female , Humans , Leukemia/complications , Leukemia/therapy , Male , Middle Aged , Parainfluenza Virus 3, Human/physiology , Prognosis , Respiratory Syncytial Virus Infections/complications , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Viruses/physiology , Respiratory Tract Infections/complications , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/epidemiology , Respirovirus Infections/complications , Respirovirus Infections/diagnosis , Retrospective Studies , Treatment Outcome , Young AdultSubject(s)
Leukemia, Myeloid, Acute/genetics , Mutation , Myelodysplastic Syndromes/genetics , Myeloproliferative Disorders/genetics , Nuclear Proteins/genetics , Aged , Biopsy , Bone Marrow/pathology , Cell Transformation, Neoplastic/genetics , DNA Mutational Analysis , Female , Humans , Leukemia, Myeloid, Acute/diagnosis , Male , Middle Aged , Myelodysplastic Syndromes/diagnosis , Myeloproliferative Disorders/diagnosis , NucleophosminSubject(s)
Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , T-Lymphocytes, Cytotoxic/immunology , Aged , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Benzamides/adverse effects , Benzamides/therapeutic use , Female , Humans , Imatinib Mesylate , Immunologic Memory , Immunophenotyping , Piperazines/adverse effects , Piperazines/therapeutic use , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/adverse effects , Pyrimidines/therapeutic use , Remission Induction , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
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, ß1-3glucan, 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.