Alessandra Battaglia

Erythropoietin exerts anti‐apoptotic effects on rat microglial cells in vitro

Erythropoietin (EPO), a renal cytokine regulating haematopoiesis, is also produced by different cell types within the central nervous system, where it acts via the activation of specific receptors. Current evidence shows that EPO exerts neurotrophic and neuroprotective activities in different in vivo and in vitro models of brain damage. In the present study we investigated the effects of EPO on primary cultures of rat cortical microglia and astrocytes. We found that: (i) EPO exerted a marked stimulatory effect on microglial cell viability, assessed through the MTS assay, whereas astrocytes were almost unaffected; (ii) the cytokine increased microglial cell population size in a concentration‐dependent manner; however, as microglia cultures undergo spontaneous apoptosis after separation from astrocytes, the apparent effect on cell proliferation could be attributed to EPO antagonism of normal apoptosis; (iii) subsequent flow cytometry analysis on microglial cells demonstrated both the trophic role of factor(s) released by astrocytes in mixed cultures, and the putative anti‐apoptotic action of EPO; (iv) the latter was further confirmed through the assessment of gene expression of anti‐ and pro‐apoptotic factors, which showed that EPO is able to shift the Bcl : Bax ratio towards a net anti‐apoptotic effect; (v) EPO did not affect the pro‐inflammatory function of microglial cells.

Circulating and thymic CD4+ CD25+ T regulatory cells in myasthenia gravis: effect of immunosuppressive treatment

Accumulating evidence indicates an immunosuppressive role of the thymus‐derived CD4+ T‐cell population constitutively expressing high level of CD25, T regulatory (Treg) cells, in autoimmune diseases. Here we show that the number of Treg cells in the blood is significantly lower in untreated myasthenia gravis patients than in age‐matched healthy subjects, whereas it is normal or elevated in patients on immunosuppressive therapy (prednisone frequently associated with azathioprine). Therapeutic thymectomy (Tx) for either the thymoma or non‐neoplastic thymic alterations that are often associated with myasthenia gravis provided unique material for studying intrathymic Treg cells and correlating them with their peripheral counterparts. We observed that Tx prevents the increase of Treg cells in the circulation that follows immunosuppressive therapy (particularly evident if the thymus is not neoplastic), indicating that the thymus contributes to Treg‐cell normalization. However, thymic Treg cells are not modulated by immunosuppressive therapy and even in thymectomized patients Treg‐cell numbers in the blood eventually recover. The present findings suggest that a deficiency in Treg cells favours the development of myasthenia gravis and that their normalization is an important clinical benefit of immunosuppressive therapy. Treg normalization appears to be largely thymus independent and possibly reflects the reported capacity of corticosteroids to promote Treg‐cell development.

Prognostic relevance of the expression of Tdt and CD7 in 335 cases of acute myeloid leukemia

We have analyzed the expression of Tdt and CD7 in 335 cases of unequivocal acute myeloid leukemia (AML). Tdt was expressed in 80 (25%) of 321 evaluable cases. Twenty-six of 77 (34%) Tdt+ patients assessable for response, entered complete remission (CR) vs 121 of 209 (58%) Tdt− cases (P < 0.001). cd7 was expressed in 102 of 332 (30%) evaluable cases; 37 of 93 assessable (40%) cd7+ patients attained a CR as compared to 114/204 (56%) CD7− (P = 0.013). Duration of survival was significantly shorter for patients with CD7+ or Tdt+ AML (P = 0.006 and 0.001, respectively). In a multivariate analysis, Tdt was found to significantly adverse achievement of CR (P = 0.018), while CD7 affected duration of CR (P = 0.037). Overall the expression of either Tdt or CD7 correlated with a relatively high expression of CD34 (P < 0.001), gp-170 (P = 0.003), lymphoid antigens (LyAg) (P < 0.001), t(9;22) or anomalies of chromosome 5/7 (P < 0.001). finally, we pooled the patients into four phenotypic classes, according to the presence of tdt, cd7 or both: [tdt−CD7−], [Tdt+CD7−], [Tdt−CD7+] and [Tdt+CD7+]. The category [Tdt+CD7+] was characterized by a more unfavorable outcome as suggested by a lower rate of CR (P < 0.001) and a shorter duration of survival as compared to cases [tdt−CD7−], [Tdt+CD7−] and [Tdt−CD7+] (P = 0.002). This figure is consistent with the frequent convergence in the subset [Tdt+CD7+] of GP-170 positivity (P = 0.003), translocation t(9;22), anomalies of chromosome 5 and/or 7 (P < 0.001) and signs of lineage infidelity (deviant expression of lymphoid antigens) (P < 0.001). we conclude that the expression of tdt or cd7 is associated with an unfavorable outcome and that the combination of both defines a clinical subset with a poorer prognosis due to the significantly higher association with mdr phenotype, and ‘poor prognostic’ chromosomal abnormalities.

Neuropilin-1 expression identifies a subset of regulatory T cells in human lymph nodes that is modulated by preoperative chemoradiation therapy in cervical cancer

We examined the phenotype and function of CD4+ T cells expressing the semaphorin III receptor neuropilin‐1 (Nrp1) in human lymph nodes and peripheral blood. In lymph nodes, Nrp1 identified a small regulatory CD4+ CD25high T‐cell subpopulation (Nrp1+ Treg) that expressed higher levels of Forkhead box P3 (Foxp3) message and protein than Nrp1− Treg, and various molecular markers of activated Treg, i.e. CD45RO, human leucocyte antigen (HLA)‐DR and glucocorticoid‐induced tumour necrosis factor receptor (GITR). Similarly to conventional Treg, Nrp1+ Treg proliferated poorly in vitro, and exerted contact‐dependent in vitro suppression of T‐cell proliferation and cytokine secretion. However, Nrp1+ Treg were more efficient than Nrp1− Treg at inducing suppression. Nrp1 was also expressed on a small subpopulation of CD25int and CD25− CD4+ T cells that expressed more Foxp3, CD45RO, HLA‐DR and GITR than their Nrp1− counterparts. In contrast, in peripheral blood Nrp1 identified a minor CD4+ T‐cell subset that did not display the phenotypic features of Treg lacking Foxp3 expression and marginally expressing CD25. Hence, the function of Nrp1+ CD4+ T cells seemingly depends on their anatomical location. In a previous report, we proposed that Treg may curb the anti‐tumour T‐cell response in cervical cancer. We show here that Treg and Nrp1+ Treg levels dropped in the tumour‐draining lymph nodes of patients with cervical cancer following preoperative chemoradiotherapy in a direct relationship with the reduction of tumour mass, suggesting that suppressor cell elimination facilitated the generation of T cells mediating the destruction of the neoplastic cells left behind after cytotoxic therapy.

Paraneoplastic diseases associated with thymoma.

BackgroundThymoma is frequently associated with paraneoplastic diseases (PDs), most commonly with myasthenia gravis (MG). This association is thought to depend on thymoma’s capacity to produce and export T lymphocytes.Objective(1) To determine the frequency and characteristics of thymoma-associated PDs other than MG; (2) to evaluate T cell maturation in thymomas with and without PDs.MethodsWe studied 260 patients with thymoma (associated with MG in 228). The occurrence of PDs was monitored together with the tumor outcome. Phenotypic characterization of thymocyte subsets in 14 thymoma samples (7 with and 7 without MG) was performed by FACS.ResultsA total of 47 PDs was diagnosed in 41/260 patients (15.8 %). Neurological PDs included neuromyotonia, limbic encephalitis, polymyositis, subacute hearing loss, psychosis and sleep disorders. A broad spectrum of nonneurological PDs were observed, among these, hematological and cutaneous diseases prevailed. Like MG, these disorders occurred either in the presence of the thymoma or at different times after thymomectomy; their onset often heralded a tumor recurrence. In thymomas from MG subjects, we found an increased proportion of fully mature CD4 single positive (SP) thymocytes and a reduced frequency of CD4SPCD25+ cells; the latter finding may reflect a deficient generation of T regulatory cells, a reduced intratumorous activation of T cells, or both.ConclusionsWe confirm the strong association of thymoma with PDs. These disorders often occurred in MG patients and their course in relation to thymoma was similar to that of MG. In accordance with previous observations, we found some alterations in the intratumorous production of mature CD4+ T cells that could be involved in the pathogenesis of paraneoplastic autoimmunity.

CD34+/CD105+ cells are enriched in primitive circulating progenitors residing in the G0 phase of the cell cycle and contain all bone marrow and cord blood CD34+/CD38low/− precursors

A subset of circulating CD34+ cells was found to express CD105 antigen. Sorting experiments showed that most granulocyte–macrophage colony‐forming units (GM‐CFU) and burst‐forming units — erythroid (BFU‐E) were retained in the CD34+/CD105− fraction, whereas rare GM‐CFU/BFU‐E were generated from CD34+/CD105+ cells. Megakaryocytic aggregates were entirely retained in the CD34+/CD105+ fraction. Neutralizing doses of an anti‐TGF‐β1 antibody demonstrated CD34+/CD105+ cells capable of colony‐forming activity without any significant effect on CD34+/CD105− cells. Cloning of secondary colonies revealed that CD34+/CD105+ cells had a significantly higher secondary cloning efficiency than CD34+/CD105− cells. CD34+/CD105+ cells had a significantly higher long‐term culture‐initiating cell (LTC‐IC) frequency than CD34+/CD105− cells. Kinetic analysis showed that 75% of CD34+/CD105+ cells consisted of DNA 2n G0Ki‐67− cells whereas 82% of CD34+/CD105− were DNA 2n G1Ki‐67+ cells, and this latter subset showed a RNA content consistently higher than CD34+/CD105+ cells. CD34+/CD105+ progenitors were CD25+, whereas CD34+/CD105− contained a small CD25+ subset. Three‐colour analysis of bone marrow and cord blood CD34+ cells demonstrated that all the CD34+/CD38low/− primitive precursors were contained in CD34+/CD105+ cells. Extensive characterization of these CD105+ precursors indicated that they have biological properties associated with primitive haematopoietic precursors.

Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry

Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of “Treg markers”. Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry.

Collagen-specific T-cell repertoire in blood and synovial fluid varies with disease activity in early rheumatoid arthritis

IntroductionType II collagen is a DR4/DR1 restricted target of self-reactive T cells that sustain rheumatoid arthritis. The aim of the present study was to analyze the T-cell receptor repertoire at the onset of and at different phases in rheumatoid arthritis.MethodsWe used the CDR3 BV-BJ spectratyping to study the response to human collagen peptide 261–273 in 12 patients with DR4+ rheumatoid arthritis (six at the onset of disease and six during the course of disease) and in five healthy DR4+ relatives.ResultsThe collagen-specific T-cell repertoire is quite restricted at the onset of disease, involving approximately 10 rearrangements. Within the studied collagen-specific rearrangements, nearly 75% is shared among patients. Although the size of the repertoire used by control individuals is comparable to that of patients, it is characterized by different T-cell receptors. Part of the antigen-specific T-cell repertoire is spontaneously enriched in synovial fluid. The specific T-cell repertoire in the periphery was modulated by therapy and decreased with the remission of the disease. Failure of immunoscopy to detect this repertoire was not due to suppression of collagen-driven proliferation in vitro by CD4+ CD25+ T cells. Clinical relapse of the disease was associated with the appearance of the original collagen-specific T cells.ConclusionsThe collagen-specific T-cell receptor repertoire in peripheral blood and synovial fluid is restricted to a limited number of rearrangements in rheumatoid arthritis. The majority of the repertoire is shared between patients with early rheumatoid arthritis and it is modulated by therapy.

Lymphocyte populations in human lymph nodes. Alterations in CD4+ CD25+ T regulatory cell phenotype and T‐cell receptor Vβ repertoire

Here we provide a description of lymphocyte populations in human lymph nodes (LN) with a special emphasis on the CD4+ lymphocyte population constitutively expressing CD25 at a high level and endowed with immunoregulatory properties [T regulatory (Treg) cells]. Lymph nodes were analysed by multicolour flow cytometry in parallel with correspondent peripheral blood (PB). Immunomagnetically purified Treg cells were tested for anergy and suppressive activity in a CD3/T‐cell receptor (TCR)‐driven proliferation assay. Compared to PB, there was a reduced T/B lymphocyte ratio in LN. Both LN and PB contained a similar proportion of CD4+ lymphocytes but, conversely, CD8+ lymphocytes were less represented in PB, with a consequent increase in the ratio of CD4+/CD8+ natural killer cells were < 2% (PB range 6–22%). No significant differences existed in the frequency of the other lymphocyte subpopulations examined (naïve‐type CD4+ and CD8+ lymphocytes, activated B and CD4+ lymphocytes, and effector‐type CD8+ lymphocytes). LN and PB contained similar percentages of CD4+ lymphocytes constitutively expressing intermediate or high levels of CD25. CD4+ CD25++ cells constitutively coexpressed high levels of CD152 and were therefore identified as Treg cells. Treg cells in LN and PB differed in terms of CD45RB, HLA‐DR, CD45RO, and CD62L expression. Also the TCRVβ repertoire diverged between Treg cells from LN and PB. Similar to Treg cells from PB, Treg cells from LN were anergic and efficiently inhibited other CD4+ and CD8+ lymphocyte proliferation. This study extends the information on the diversities in lymphocyte composition between human LN and PB, and reports for the first time a description of the phenotypic and functional characteristics of Treg cells in human LN, highlighting the importance of the LN microenvironment in shaping the surface phenotype of Treg cells.

P-glycoprotein and terminal transferase expression identify prognostic subsets within cytogenetic risk classes in acute myeloid leukemia

Clinical and biological features were assessed in 204 consecutive de novo adult acute myeloid leukemia (AML) patients who received intensive chemotherapy regimens. Multiparameter flow cytometric assays both of the multidrug resistance (MDR-1)-associated P-glycoprotein (PGP) using the UIC2 monoclonal antibody (MoAb), and of terminal transferase (TdT) were performed. Cytogenetic findings were obtained from 196 patients with high resolution banding. At onset, UIC2 and TdT positivities were detected in 58.5% and 24% of cases, respectively. There were strict correlations either between UIC2 negativity and FAB M3 or between TdT and FAB M0-M1 (P = 0.001 and < 0.0001, respectively). On the other hand, age was significantly associated with cytogenetic risk classes (P < 0.0001). CD34 positivity was highly correlated with TdT expression (P < 0.0001). Moreover, CD7 and CD11b were significantly represented in UIC2+ subset (P < 0.0001). Rhodamine 123 (Rh 123) efflux was significantly higher in 75 UIC2 positive patients compared to 65 UIC2 negative ones (P < 0.001). As regards to cytogenetics, TdT positivity was strongly related either to t(9;22) or single/associated anomalies of chromosome 7; on the other hand, most or all cases with t(8;21) or t(15;17) were UIC2 or TdT negative, respectively. The rate of first complete remission (CR) differed both between UIC2+ and UIC2- cases and between TdT+ and TdT- ones (40% versus 72%, P < 0.001; and 36% versus 61%, P = 0.001, respectively). The survival rates (Kaplan-Meier method) were significantly shorter either in UIC2+ or in TdT+ patients (P = 0.005 and = 0.011, respectively). UIC2 and TdT negative cases showed longer remission duration (P = 0.03 and = 0.22, respectively). The additional effect of UIC2 and TdT on prognosis allowed us to identify two subsets of patients, the first [UIC2- TdT-] at better and the second [UIC2+ TdT+] at worse clinical outcome compared to single UIC2 and TdT cases, concerning CR (P < 0.001), survival (P < 0.0001) and CR duration (P = 0.007). The combinations [UIC2+ TdT-] and [UIC2- TdT+] showed an intermediate clinical course. A strong difference was found between poor risk and intermediate/favorable risk cytogenetic classes with regard to CR rate (P < 0.0001), overall survival and CR duration (P < 0.001). Nevertheless, within the poor risk class, UIC2 positivity was able to identify patients at worst prognosis with regard to CR (P = 0.005), survival (P = 0.02) and CR duration (P = 0.015). On the other hand, UIC2 and TdT negativity allowed us to distinguish patients with longer survival (P = 0.012 and = 0.04, respectively) and CR duration (P = 0.04 and = 0.025, respectively) within the intermediate/favorable risk class. The independent prognostic value of UIC2, TdT and cytogenetic risk classes was confirmed in multivariate analysis. These results suggest that PGP and TdT expressions, together with cytogenetic findings, may represent a basic predictor of chemotherapeutic failure in AML.