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.

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.

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 highdose 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-␥, tumor necrosis factor-␣, 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. (Blood. 2010; 115:1512-1518)

NPM1 mutations may reveal acute myeloid leukemia in cases otherwise morphologically diagnosed as myelodysplastic syndromes or myelodysplastic/myeloproliferative neoplasms

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.

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.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is increasingly used in the treatment of chronic lymphocytic leukemia (CLL). Moreover, very promising results have been reported in other B-cell malignancies, including primary central nervous system lymphoma (PCNSL). Although well-tolerated in the majority of patients, ibrutinib demonstrates in some cases troublesome toxicities, including invasive fungal infections (IFIs). In the present review, we summarize clinical manifestations of IFIs in patients treated with ibrutinib, generally characterized by an early onset, mild clinical manifestations, asymptomatic/low symptomatic pulmonary localization and high incidence of central nervous system (CNS) involvement. IFI risk appears particularly increased in patients receiving ibrutinib associated with other immune modulator agents, especially with steroids or immune-chemotherapy. Moreover, the immunomodulatory effect of ibrutinib is described, pointing the attention on the involvement of specific molecules targeted by ibrutinib in innate and adaptive response to fungal infection. Overall, the findings indicate the ibrutinib may rapidly impair innate immune cell functions, while concomitantly restoring an effective protective potential of adaptive immune compartment. A correct awareness, especially when other predisposing factors are present, is warranted about the potential risk of IFIs in ibrutinibtreated patients.

BackgroundInvasive 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 FindingsBy 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.ConclusionsMucorales-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.