Chimeric receptors that include CD28 signaling in series with TCRζ in the same receptor have been demonstrated to activate prestimulated human primary T cells more efficiently than a receptor providing TCRζ signaling alone. We examined whether this type of receptor can also activate resting human primary T cells, and whether molecules other than CD28 could be included in a single chimeric receptor in series with TCRζ to mediate the activation of resting human primary T cells. Human CD33-specific chimeric receptors were generated with CD28, inducible costimulator, CD134, or CD137 signaling regions in series with TCRζ signaling region and transfected by electroporation into resting human primary T cells. Their ability to mediate Ag-specific activation was analyzed in comparison with a receptor providing TCRζ signaling alone. Inclusion of any of the costimulatory signaling regions in series with TCRζ enhanced the level of specific Ag-induced IL-2, IFN-γ, TNF-α, and GM-CSF cytokine production and enabled resting primary T cells to survive and proliferate in response to Ag in the absence of any exogenous factors. Inclusion of CD28, inducible costimulator, or CD134 enhanced TCRζ-mediated, Ag-specific target cell lysis. Chimeric receptors providing B7 and TNFR family costimulatory signals in series with TCRζ in the same receptor can confer self-sufficient clonal expansion and enhanced effector function to resting human T cells. This type of chimeric receptor may now be used to discover the most potent combination of costimulatory signals that will improve current immunotherapeutic strategies.

The enzyme telomerase is essential for maintaining the replicative capacity of memory T cells. Although CD28 costimulatory signals can up-regulate telomerase activity, human CD8+ T cells lose CD28 expression after repeated activation. Nevertheless, telomerase is still inducible in CD8+CD28− T cells. To identify alternative costimulatory pathways that may be involved, we introduced chimeric receptors containing the signaling domains of CD28, CD27, CD137, CD134, and ICOS in series with the CD3 zeta (ζ) chain into primary human CD8+ T cells. Although CD3 ζ-chain signals alone were ineffective, triggering of all the other constructs induced proliferation and telomerase activity. However, not all CD8+CD28− T cells could up-regulate this enzyme. The further fractionation of CD8+CD28− T cells into CD8+CD28− CD27+ and CD8+CD28−CD27− subsets showed that the latter had significantly shorter telomeres and extremely poor telomerase activity. The restoration of CD28 signaling in CD8+CD28−CD27− T cells could not reverse the low telomerase activity that was not due to decreased expression of human telomerase reverse transcriptase, the enzyme catalytic subunit. Instead, the defect was associated with decreased phosphorylation of the kinase Akt, that phosphorylates human telomerase reverse transcriptase to induce telomerase activity. Furthermore, the defective Akt phosphorylation in these cells was specific for the Ser473 but not the Thr308 phosphorylation site of this molecule. Telomerase down-regulation in highly differentiated CD8+CD28−CD27− T cells marks their inexorable progress toward a replicative end stage after activation. This limits the ability of memory CD8+ T cells to be maintained by continuous proliferation in vivo.

Persistent infection with hepatitis B virus (HBV) remains a major global health threat, currently aff ecting over 350 million individuals worldwide and causing more than 1 million deaths annually. Immune clearance is thought to be mediated primarily through a strong virus-specifi c CD8 T cell response ( 1 ); marked quantitative and qualitative defects in this response have been described in patients with chronic HBV infection (CHB) ( 2 -4 ). Patients with uncontrolled infection are distinguished from healthy HBV carriers by the presence of a large lymphocytic infi ltrate in their livers, containing a high proportion of non -antigen-specifi c CD8 T cells ( 3,5 ). Little is known about the characteristics of this generalized CD8 T cell population and its potential contribution to the failure of viral control and liver immunopathogenesis.In this study, we defi ne the functional profi le of non -HBV-specifi c CD8 T cells in patients with CHB, and investigate aberrant expression of the proximal TCR-associated signaling molecules CD3 and CD28 as putative mechanisms contributing to the defects seen.In other diseases of chronic infl ammation and high-load antigenic persistence analogous to the situation in CHB, selective defects in global CD8 function have been associated with downregulation of CD3 ± CD28. These include autoimmune disorders ( 6, 7 ), malignancy ( 8 ), and chronic viral ( 9, 10 ) and bacterial ( 11 ) The infl amed liver in chronic hepatitis B virus (HBV) infection (CHB) is characterized by a large infl ux of non -virus-specifi c CD8 T cells. Little is known about the functional capacity of these lymphocytes, which could provide insights into mechanisms of failure of viral control and liver damage in this setting. We compared the effector function of total circulating and intrahepatic CD8 T cells in CHB patients and healthy donors. We demonstrated that CD8 T cells from CHB patients, regardless of their antigen specifi city, were impaired in their ability to produce interleukin-2 and proliferate upon TCR-dependent stimulation. In contrast, these CD8 T cells had preserved production of the proinfl ammatory cytokines interferon-␥ and tumor necrosis factor-␣ . This aberrant functional profi le was partially attributable to down-regulation of the proximal T cell receptor signaling molecule CD3 , and could be corrected in vitro by transfection of CD3 or replenishment of the amino acid arginine required for its expression. We provide evidence for depletion of arginine in the infl amed hepatic microenvironment as a potential mechanism for these defects in global CD8 T cell signaling and function. These data imply that polarized CD8 T cells within the HBV-infected liver may impede proliferative antiviral effector function, while contributing to the proinfl ammatory cytokine environment.

SummaryFOXP3 has been identified as a key regulator of immune homeostasis. Mutations within the FOXP3 gene result in dysregulated CD4 + T-cell function and elevated cytokine production, leading to lymphoproliferative disease. FOXP3 expression in CD4 + T cells is primarily detected with the CD4 + CD25 + regulatory T-cell population. In humans the protein is detected as a doublet following immunoblot analysis. The lower band of the doublet has been identified as a splice isoform lacking a region corresponding to exon 2. The aim of this study was to investigate whether the splice variant form lacking exon 2 and a new novel splice variant lacking both exons 2 and 7, were functional inhibitors of CD4 + T-cell activation. The data generated showed that full-length FOXP3 and both splice variant forms of the protein were functional repressors of CD4 + T-cell activation.

The online version of this article has a Supplementary Appendix. BackgroundCytokine-induced killer cells are ex vivo-expanded cells with potent antitumor activity. The infusion of cytokine-induced killer cells in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses have been observed. To improve their effector functions against acute myeloid leukemia, we genetically modified cytokine-induced killer cells with chimeric receptors specific for the CD33 myeloid antigen. Design and MethodsSFG-retroviral vectors coding for anti-CD33-ζ and anti-CD33-CD28-OX40-ζ chimeric receptors were used to transduce cytokine-induced killer cells. Transduced cells were characterized in vitro for their ability to lyse leukemic targets (4-hour 51 chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate ( 3 H-thymidine-incorporation assay) and to secrete cytokines (flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34 + hematopoietic progenitor cells was evaluated by analyzing the colony-forming unit capacity after co-incubation. ResultsCytokine-induced killer cells were efficiently transduced with the anti-CD33 chimeric receptors, maintaining their native phenotype and functions and acquiring potent cytotoxicity (up to 80% lysis after 4-hour incubation) against different acute myeloid leukemia targets, as also confirmed in long-term killing experiments. Moreover, introduction of the anti-CD33 chimeric receptors was accompanied by prominent CD33-specific proliferative activity, with the release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of the anti-leukemic activity of cytokine-induced killer cells. Importantly, even though the cytokine-induced killer cells transduced with anti-CD33 chimeric receptors showed toxicity against normal hematopoietic CD34 + progenitor cells, residual clonogenic activity was preserved. ConclusionsOur results indicate that anti-CD33 chimeric receptors strongly enhance anti-leukemic cytokine-induced killer cell functions, suggesting that cytokine-induced killer cells transduced with these molecules might represent a promising optimized tool for acute myeloid leukemia immunotherapy.Key words: chimeric T-cell receptor, acute myeloid leukemia, CD33, cell therapy, gene therapy. Haematologica 2010;95(12):2144-2152. doi:10.3324/haematol.2010 This is an open-access paper. © F e r r a t a S t o r t i F o u n d a t i o n

These data suggest that platelet-derived growth factor plays a key role in the development of intimal lesions at sites of acute vascular injury in the nonhuman primate.

Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).

Summary A humanised IgGl/k version of A33 (hA33) has been constructed and expressed with yields up to 700 mg 1' in mouse myeloma NS0 cells in suspension culture. The equilibrium dissociation constant of hA33 (KD= 1.3 al., 1990, 1994). A phase I/II study has been conducted (Welt et al., 1994) with this murine antibody, in which some tumour responses were observed at the maximum tolerated dose (75 mCi m-2). The major limiting toxicity was haematological, as observed in almost all therapy studies with radioimmunoconjugates. All patients treated developed a human anti-mouse antibody (HAMA) response after one administration, and this led to very rapid clearance of the conjugate upon retreatment, consistent with all previous results with rodent antibodies. These data suggest that A33 is a promising antibody for successful radioimmunotherapy of colon cancer, and the purpose of this study has been to design and develop a second generation reagent based upon it. The key to the development of successful radioimmunotherapy will be the identification of reagents capable of delivering a killing dose to tumour cells without unacceptable toxicity to normal tissues. To this end we are evaluating several alternative radioimmunotherapeutic strategies including the use of isotopes which require internalisation into the cell for cytotoxicity, such as 125I (which are less toxic to normal tissues), and engineering the antibody for the optimal delivery of highly cytotoxic agents such as 90Y.The radioisotope 9'Y has been used in several radioimmunotherapy studies and is an attractive isotope for this purpose owing to its appropriate physical properties. As a pure high-energy P-emitter 9Y has advantages over the more