Antonella Facchinetti

An improved method for the detection of DNA fragmentation

An application of the Southern blot technique is described which permits the detection of DNA fragmentation due to cell death by apoptosis. DNA fragments were isolated from cell suspensions and tissues, separated on agarose gel, transferred by Southern blot and hybridized with a radiolabeled total cellular DNA probe. The application of this procedure to thymus cell samples, revealed the distinct ladder pattern of DNA fragments in multiples of about 180-200 base pairs, a characteristic feature of DNA fragmentation. In comparison to conventional DNA visualization with ethidium bromide staining, the radiolabeled probe improved the detection of DNA fragments at least eight-fold. This method detects low levels of DNA fragments, as well as physiological tissue DNA fragmentation, while avoiding cell damage due to DNA radiolabeling.

Effect of IFNβ and anti-IFNβ antibodies on NK cells in multiple sclerosis patients

Abstract We analysed longitudinally the numbers of CD3–CD16+ (natural killer cells, NK) and CD3–CD57+ cells (a subset of NK) in 15 IFNβ1b- and 12 IFNβ1a-treated relapsing–remitting multiple sclerosis (RRMS) patients. IFNβ1b (Betaferon ® )-treated RRMS patients showed a rapid and marked reduction in the number of both NK subsets which started 1 month after therapy initiation, and reached highest significance after 3 months ( P =0.000); however, figures reverted to pre-treatment values following the appearance of anti-IFNβ antibodies. In IFNβ1a (Avonex™)-treated RRMS patients, the decrease in both CD3–CD16+ and CD3–CD57+ cell number was slower but more persistent; anti-IFNβ antibodies were only rarely detected in these patients, and at lower titers than in IFNβ1b-treated ones. Our findings suggest that NK cells might be one of the major immunological targets of IFNβ-based treatments.

Protection from experimental autoimmune encephalomyelitis (EAE): non-depleting anti-CD4 mAb treatment induces peripheral T-cell tolerance to MBP in PL/J mice

Following pre-treatment with a non-depleting anti-CD4 mAb (H129.19) that produces long-lasting receptor saturation, PL/J mice were fully protected from experimental auto-immune encephalomyelitis (EAE) induced by injection of myelin basic protein (MBP). These mice did not develop EAE following MBP re-challenge 5-10 weeks later when the CD4+ cells were no longer coated by the mAb and their lymph node cells were specifically unresponsive to MBP stimulation in vitro. Moreover, superantigen staphylococcal enterotoxin B (SEB) inoculation, which re-induces EAE in MBP immunized mice, failed to activate encephalitogenic T-cells in anti-CD4 + MBP treated mice, even after MBP re-challenge, indicating that tolerance in the peripheral T-cell compartment was achieved. However, MBP re-challenge 16 weeks later, but not SEB, produced an acute episode of EAE in these mice, while it failed to induce disease in a parallel group of adult thymectomized mice. These results indicate that no memory of the first priming exists at this time and that new MBP-specific T-cell precursors are peripheralized and produce EAE after MBP recognition.

A Large Number of T Lymphocytes Recognize Moloney-Murine Leukemia Virus-Induced Antigens, but a Few Mediate Long-Lasting Tumor Immunosurveillance

The CD8+ T cell response to Moloney-murine leukemia virus (M-MuLV)-induced Ags is almost entirely dominated by the exclusive expansion of lymphocytes that use preferential TCRVβ chain rearrangements. In mice lacking T cells expressing these TCRVβ, we demonstrate that alternative TCRVβ can substitute for the lack of the dominant TCRVβ in the H-2-restricted M-MuLV Ag recognition. We show that, at least for the H-2b-restricted response, the shift of TCR usage is not related to a variation of the immunodominant M-MuLV epitope recognition. After virus immunization, all the potentially M-MuLV-reactive lymphocytes are primed, but only the deletion of dominant Vβ rescues the alternative Vβ response. The mechanism of clonal T cell “immunodomination” that guides the preferential Vβ expansion is likely the result of a proliferative advantage of T cells expressing dominant Vβ, due to differences in TCR affinity and/or cosignal requirements. In this regard, a CD8 involvement is strictly required for the virus-specific cytotoxic activity of CTL expressing alternative, but not dominant, Vβ gene rearrangements. The ability of T cells expressing alternative TCRVβ rearrangements to mediate tumor protection was evaluated by a challenge with M-MuLV tumor cells. Although T cells expressing alternative Vβ chains were activated and expanded, they were not able to control tumor growth in a long-lasting manner due to their incapacity of conversion and accumulation in the T central memory pool.

In vivo and in vitro Death of Mature T Cells Induced by Separate Signals to CD4 and αβTCR

Abstract To investigate whether a clonal deletion mechanism is responsible for the mature T cell tolerance that may be induced in vivo by TCR signal to anti-CD4 (H129.19 mAb) coated cells, we analyzed the T cell repertoire in anti-CD4 mAb treated BALB/c mice by flow cytometry following TCR signals through anti-αβTCR mAb or SEB superantigen. Lymph nodes showed a strong reduction in the CD4+ /CD8+ cell ratio, and a selective clonal loss of CD4+ Vβ8+ cells 4 d following anti-αβTCR or SEB injection, respectively. Following lymph node cell activation in a short-term in vitro assay with SEB or anti-Vβ8 mAb, a selective elimination of CD4+ Vβ8+ cells was again detected, and DNA fragmentation analysis disclosed a cell death by apoptosis. These findings suggest that TCR triggering transduces an apoptotic signal into CD4+ mAb saturated cells that in turn leads to specific holes in the mature T cell repertoire.

Mechanism of Na+/K+-ATPase activation by trypsin and kallikrein

The mechanism of the Na+/K(+)-ATPase activation by trypsin (from bovine pancreas) and kallikrein (from human plasma) was investigated on enzyme preparations from different sources (beef heart and dog kidney) and at different degrees of purification (beef heart). Kallikrein was effective on both beef and dog enzymes, whereas trypsin stimulated only the beef-heart Na+/K(+)-ATPase. The extent of activation by the proteinases was inversely related to the degree of purification (maximal enzyme activation about 60 and 20% on the partially purified and the more purified enzymes, respectively). Enzyme activation was observed up to 0.5-0.6 microgram/ml of proteinase. At higher concentrations the activation decreased and was converted into inhibition at proteinase concentrations above 1.0 micrograms/ml. Na+/K(+)-ATPase stimulation was due to an increase in the Vmax of the enzyme reaction. Km for ATP remained unaffected. The activating effect was favoured by sodium and counteracted by potassium. Accordingly, Na(+)-ATPase activity was stimulated to a greater extent (up to 350%), whereas K(+)-dependent p-nitrophenylphosphatase activity proved to be insensitive to the actions of the proteinases. The Na+/K(+)-ATPase stimulation by both proteinases was antagonized by either ouabain or canrenone, two drugs that bind on the extracellular side of the Na+/K(+)-ATPase molecule. On the contrary, the enzyme inactivation observed at high proteinase concentrations was not counteracted by these two drugs. The stimulation of either Na+/K(+)- or Na(+)-ATPase activity was shown to be an irreversible effect without any significant protein degradation detectable by SDS gel electrophoresis. The results obtained suggest that proteinases exert their stimulatory effects by interacting preferentially with the E2 conformation of Na+/K(+)-ATPase at site(s) located on the extracellular moiety of the enzyme.

Functional Avidity–Driven Activation-Induced Cell Death Shapes CTL Immunodominance

Immunodominance is a complex phenomenon that relies on a mere numerical concept, while being potentially influenced at every step of the immune response. We investigated the mechanisms leading to the establishment of CTL immunodominance in a retroviral model and found that the previously defined subdominant Env-specific CD8+ T cells are endowed with an unexpectedly higher functional avidity than is the immunodominant Gag-recognizing counterpart. This high avidity, along with the Env Ag overload, results in a supraoptimal TCR engagement. The overstimulation makes Env-specific T lymphocytes more susceptible to apoptosis, thus hampering their expansion and leading to an unintentional “immune kamikazing.” Therefore, Ag-dependent, hyperactivation-induced cell death can be regarded as a novel mechanism in the establishment of the immunodominance that restrains and opposes the expansion of high-avidity T cells in favor of lower-affinity populations.

The MBP-reactive repertoire is shaped by recognition of minor histocompatibility antigens

While it is known that the degeneracy of T-cell antigen recognition is involved in many aspects of T cell-immunology, its importance in the selection of the T cell repertoire remains an aspect to be better investigated. Here we examined if an intrathymic degenerate T cell recognition mechanism shapes the myelin basic protein (MBP)-reactive repertoire inducing resistance to experimental autoimmune encephalomyelitis (EAE) in some MHC and/or minor histocompatibility antigens (MiHAs) heterozygous F1 mice bearing the H-2(s) susceptibility allele. We found a considerable degree of cross-reactivity between MBP and MiHAs encoded in various EAE resistant mouse strains: (1) MBP-specific T cells can be re-stimulated in vitro by cells expressing these MiHAs and maintain their encephalitogenic activity, and (2) lymphoid cells from parental strains that generate EAE resistant F1 hybrids can induce disease relapse when injected into EAE-susceptible hosts. The results suggest that heterozygosity, through the degeneracy of T cell antigen recognition mechanism, may provide further means to constrain the potential autoreactive repertoire.

Customizing CellSearch platform

WE read with interest the recent manuscript from Lowes et al. (1), published in Cytometry A, reporting on the developing and optimizing protocols for the characterization of circulating tumor cells (CTCs) on the CellSearch platform for two proteins of interest, CD44 and M-30. We totally agree with the authors that appropriate steps must be taken for proper optimization of user-defined protein marker assays on this system and this paper undertakes the laudable effort of defining a working method. However, we have significant issues with the overall study design and the conclusions derived from this study. A major concern regards the fact that the optimization experiments have been conducted on spiked samples only, without any further validation in ex vivo samples. To detect tumor cells in peripheral blood, a general consensus exists that an optimal immunocytochemical method has to fulfill specific criteria, including, among others, proven clinical significance. To prove the clinical significance of a new method for tumor cell detection and characterization, this should be optimized in a pilot set of patients (2). The CellSearch platform is a closed system that uses cytokeratin (CK) 8, 18, and 19 to identify tumor cells of epithelial origin. The widespread distribution of these keratins makes them useful markers for the great majority of carcinomas arising from simple epithelia. At single cell level, the cytoplasmic CK staining can be strong and evenly distributed; more often, CK expression patterns are heterogeneous, with stronglyand weakly-stained parts (3,4) asymmetrically distributed. Similarly, the expression of M30, a neo-epitope in CK18 that becomes available at a caspase cleavage event during apoptosis (4), is heterogeneous as well. We were therefore disappointed by the study design pursued by Lowes et al. who, by assuming that M30 expression in an in vitro drug-conditioned cell line (usually showing high intensity CK-staining) would equal that of CTCs coming from a patient sample, limited the integrated assay optimization steps to cell lines (added to normal donor blood) only, and evaluated the percentage of M30-positive CTCs as the only mean to determine M30 monoclonal Antibody (mAb) suitability with the CellSearch system. To support such broad conclusions about the suitability of M30 in conjunction with the CellSearch system in cancer patients, samples representing more than one kind of cancer should have been tested. To meet this criterion, when we customized M30 CTC assay, we started from cell lines spiked into whole blood samples, but afterwards we studied M30 expression on CTCs in a group of 122 patients affected by different kind of tumors, namely breast, colorectal, and renal cancer (4). Overall, the results of this study by the CellSearch platform showed that 64 (52%) cancer patients had at least one CTC, and 56 (87.5%) CTCs-positive patients had one or more M30-positive CTCs (4). These data were consistent with the results obtained by different methods, by which apoptotic CTCs were detected in breast (5), prostate (6,7), and lung (8) cancer patients; moreover an association between overall survival and presence of apoptotic CTCs has been also highlighted in CRPC (3). Furthermore, by applying the M30 mAb in conjunction with the CellSearch assay, we have recently shown that the changes in live/apoptotic CTCs predict treatment failure in a group of 53 metastatic renal cell carcinoma (mRCC) (9). A second key point concerns the choice of flow cytometry (FCM) as a reference method. Despite the author’s declaration that FCM cannot be used to compare CellSearch results, they de facto use FCM throughout the study for testing the suitability of user-defined markers. User-defined markers in conjunction with the CellSearch system have been used either to detect treatment targets in

Monitoring and Characterization of Circulating Tumor Cells (CTCs) in a Patient With EML4-ALK–Positive Non–Small Cell Lung Cancer (NSCLC)

Circulating tumor cells (CTCs) are epithelial cells that originate from primary tumor and are detectable at a low rate (typically 1 to 1000 per 7.5 mL) in the peripheral blood of patients with cancer; CTCs might settle down at distant sites to give metastasis. The presence of CTCs has been associated with worst prognosis in metastatic cancer of breast, prostate, and colon. Recent experiences demonstrated that CTC detection could help improve diagnosis and predict prognosis in patients with nonesmall cell lung cancer (NSCLC); however, few data exist in the literature on this topic in patients with EML4-ALKe positive (EML4-ALKþ) NSCLC. We report a case of a patient with EML4-ALKþ NSCLC who underwent sequential monitoring and EML4-ALK characterization of CTCs during targeted therapy. We were able to document that the levels of EML4-ALKþ CTCs reflect the response to crizotinib therapy or ongoing resistance to EML4-ALK inhibitors. We obtained for the first time the proof of principle that our quantitative assay can longitudinally monitor EML4-ALK expression in CTCs under selective pressure of a specific treatment. After demonstrating its clinical utility, in the future we will use it to predict treatment efficacy and to detect early drug resistance to ALK inhibitors.