Mechanical behaviour of pressed and sintered CP Ti and Ti-6Al-7Nb alloy obtained from master alloy addition powder

The Ti-6Al-7Nb alloy was obtained using the blending elemental approach with a master alloy and elemental titanium powders. Both the elemental titanium and the Ti-6Al-7Nb powders were characterised using X-ray diffraction, differential thermal analysis and dilatometry. The powders were processed using the conventional powder metallurgy route that includes uniaxial pressing and sintering. The trend of the relative density with the sintering temperature and the microstructural evolution of the materials sintered at different temperatures were analysed using scanning electron microscopy and X-ray diffraction. A minimum sintering temperature of 1200 °C has to be used to ensure the homogenisation of the alloying elements and to obtain a pore structure composed of spherical pores. The sintered samples achieve relative density values that are typical for powder metallurgy titanium and no intermetallic phases were detected. Mechanical properties comparable to those specified for wrought Ti-6Al-7Nb medical devices are normally obtained. Therefore, the produced materials are promising candidates for load bearing applications as implant materials.The authors want to acknowledge the financial support from the Spanish Ministry of Science through the R&D Projects MAT2009-14448-C02-02 and MAT2009-14547-C02-02, and from the Regional Government of Madrid through the ESTRUMAT (S2009/MAT-1585) projectPublicad

Thymus-Derived Regulatory T Cells Are Positively Selected on Natural Self-Antigen through Cognate Interactions of High Functional Avidity

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here, we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen, while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner, while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of T cell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells, a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack

A new safeguard eliminates T cell receptor gene-modified auto-reactive T cells after adoptive therapy

Der adoptive Transfer von TZR-modifizierten T Zellen ist mit potentiellen Risiken verbunden. Autoimmunreaktionen können auftreten, wenn Tumor-assoziierte Antigene auf normalem Gewebe erkannt werden, Fehlpaarung der TZR-Ketten zur Bildung eines autoreaktiven Rezeptors führen oder ein sonst anerger auto-reaktiver endogener Rezeptor aktiviert wird. Auch besteht das Risiko der malignen Transformation der Zelle durch Insertionsmutagenese. Daher ist es notwendig, die transferierten T Zellen im Fall schwerer Nebenwirkungen eliminieren zu können. Derzeit verfügbare Sicherheitsmechanismen sind für die Therapie mit TZR-modifizierten T Zellen ungeeignet. In dieser Arbeit wurde ein neuer Sicherheitsansatz entwickelt, der auf einem TZR-intrinsischen Depletionsmechanismus beruht und TZR-veränderte T Zellen eliminieren kann. Durch Einfügen eines myc-tags in murine (OT-I, P14) und humane (gp100) TZRs konnten TZR-exprimierende T Zellen in vitro und in vivo mittels eines myc-spezifischen Antikörpers depletiert werden. Die T Zellen behielten vergleichbare Funktionalität hinsichtlich Antigenerkennung und Zytokinsekretion wie Zellen, die den Wild-Typ Rezeptor exprimierten. Die Depletion adoptiv transferierter T Zellen verhinderte lethalen Diabetes in einem Mausversuch. Im verwendeten Modell wurden Splenozyten, die einen myc-getagten OT-I TZR exprimierten, in RIP-mOVA Mäuse injiziert, welche in den Inselzellen des Pankreas das OT-I-spezifische Antigen Ovalbumin exprimieren. Zerstörung der Inselzellen durch die T-Zellen induzierte lethalen Diabetes in unbehandelten Mäusen. Tiere, denen ein myc-spezifischer Antikörper verabreicht wurde, zeigten keine Symptome. Dieser neuartige Sicherheitsmechanismus erlaubt es, adoptive T Zelltherapie abzubrechen, falls schwere Nebenwirkungen auftreten. Im Gegensatz zu früheren Strategien muss kein zusätzliches Sicherheitsgen eingebaut werden und die Sicherheit des Ansatzes wird durch Verlust oder Herunterregulierung des Transgens nicht beeinflusst.Adoptive transfer of TCR gene-modified T lymphocytes into patients is associated with potential risk factors. First, auto-immunity may occur if a tumor-associated antigen is targeted on normal tissue, if TCR chain mispairing leads to the formation of an auto-reactive receptor or if an otherwise anergic endogenous receptor specific for an auto-antigen becomes activated. Second, retroviral integration could lead to malignant transformation of the T cell. Therefore, it is essential to have the possibility to deplete the transferred T cells in vivo in case of severe side effects. The available safety modalities comprise disadvantages rendering them less feasible for the application in therapy with TCR gene-modified T cells. In this study, a safeguard based on a TCR-intrinsic depletion mechanism has been developed that eliminates auto-reactive TCR-redirected T cells. By introducing a myc-tag into the murine (OT-I, P14) or human (gp100) TCRs it was possible to deplete TCR-expressing T cells in vitro and in vivo with a myc-specific antibody. The T cells maintained equal function compared to cells expressing the wild-type receptor as shown by antigen binding and cytokine secretion. Importantly, the in vivo depletion of adoptively transferred T cells prevented disease in an auto-immune mouse model. Here, splenocytes transduced with a myc-tagged OT-I TCR were injected into RIP-mOVA mice expressing the OT-I-specific antigen ovalbumin in the pancreatic beta-cells. Destruction of these cells by the adoptively transferred T cells led to severe diabetes in untreated mice. Animals receiving a myc-specific antibody after T cell transfer showed no increase in blood glucose levels. The developed safeguard allows termination of adoptive therapy in case of severe side-effects. The strategy is superior to previous ones as it relies on a TCR-intrinsic mechanism which does not require introduction of an additional gene and safety is not hampered by loss or low expression of the transgene

CAR T Cells with Enhanced Sensitivity to B Cell Maturation Antigen for the Targeting of B Cell Non-Hodgkin's Lymphoma and Multiple Myeloma

Autologous T cells genetically modified with a chimeric antigen receptor (CAR) redirected at CD19 have potent activity in the treatment of B cell leukemia and B cell non-Hodgkin's lymphoma (B-NHL). Immunotherapies to treat multiple myeloma (MM) targeted the B cell maturation antigen (BCMA), which is expressed in most cases of MM. We developed a humanized CAR with specificity for BCMA based on our previously generated anti-BCMA monoclonal antibody. The targeting single chain variable fragment (scFv) domain exhibited a binding affinity in the low nanomolar range, conferring T cells with high functional avidity. Redirecting T cells by this CAR allowed us to explore BCMA as an alternative target for mature B-NHLs. We validated BCMA expression in diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia. BCMA CAR T cells triggered target cell lysis with an activation threshold in the range of 100 BCMA molecules, which allowed for an efficient eradication of B-NHL cells in vitro and in vivo. Our data corroborate BCMA is a suitable target in B cell tumors beyond MM, providing a novel therapeutic option for patients where BCMA is expressed at low abundance or where anti-CD19 immunotherapies have failed due to antigen loss

Epitope mapping identifies a cryptic epitope from +3 ARF of E7co as TCR target.

<p>(a–b) The +2 and +3 ARFs of E7co translate for cryptic peptide sequences (bold italic) not encoded by the +2 and +3 ARFs of E7wt. (c) T cells transduced with TCRs B21, B23, S16 and S51 were cocultured with K562-B*27:05 cells pulsed with candidate epitopes from the +1 ORF and the +3 ARF of E7co and a selection of control peptides (Ctrl.). Untransduced T cells were used as negative control. Reactivity was asessed by IFNγ ELISA. Results are shown as mean +/− SEM of duplicates.</p

Redesign of +3 ARF prevents E7co from recognition by TCR-transduced T cells.

<p>(a–c) Exchanging wobble nucleotides (bold) of the primary +1 ORF leads to (a) introduction of stop codons or (b–c) amino acid exchanges (1mut, 2mut) at anchor residue positions only in the +3 ARF (bold amino acids) without changing the polypeptide sequence from +1 ORF translation. (d) TCR-tranduced T cells (B21, B23, S16 and S51) were cocultured with K562-B*27:05 target cells that express one of the mutant microgene constructs (1–30 nt) of E7co. Untransduced T cells were used as a negative control. Reactivity was assessed by IFNγ ELISA. Results are shown as mean +/− SEM of duplicates.</p

Epitope binding prediction to HLA-B*27:05.

<p>Epitope binding prediction to HLA-B*27:05.</p

TCRs are specific for HLA-B*27:05 and E7co but not E7wt.

<p>TCR-transduced T cells were tested for HLA restriction and antigen specificity by coculture with different target cells to measure IFNγ release by ELISA. Untransduced (ut) T cells were used as negative control. Results are shown as mean +/− SEM of duplicates. (a) Restriction mapping of four different TCRs (B21, B23, S16 and S51) was perfomed using K562 target cells carrying E7co and one of the six cognate MHC class I molecules of the original donor. (b) HLA-B*27:05-engineered target cell lines of different origin (CaSki, HT-3 and K562) were tested for recognition by TCR-transduced T cells.</p

Epitope mapping with truncated E7co microgene fragments.

<p>(a) Scheme of truncated E7co microgenes (grey bars) and full-length E7wt (white bar) that were stably expressed in K562-HLA-B*27:05 target cells through MP71 retrovirus transduction. Microgenes were coupled to mCherry expression marker via an IRES element to confirm transgene expression. (b) TCR-transduced T cells (B21, B23, S16 and S51) were cocultured with microgene-expressing target cells and supernatant was tested for IFNγ by ELISA. Untransduced T cells were used as negative control. Results are shown as mean +/− SEM of duplicates. n.d., not detectable.</p