Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) has been known to be a strong tolerance-inducing inhibitory receptor on T-cell surface. Systemic blocking of CTLA4 function with blocking antibodies has been regarded as an attractive strategy to enhance antitumor immunity. However, this strategy accompanies systemic autoimmune side effects that are sometimes problematic. Therefore, we developed a novel CTLA4 mutant that could be expressed in tumor antigen-specific T cells to enhance antitumor effect without systemic autoimmunity. This mutant, named CTLA4-CD28 chimera, consists of extracellular and transmembrane domains of CTLA4, linked with cytoplasmic CD28 domain. Overexpression of CTLA4-CD28 chimera in T cells delivered stimulatory signals rather than inhibitory signals of CTLA4 and significantly enhanced T-cell reactivity. Although this effect was observed in both CD4 and CD8 T cells, the effect on CD4 T cells was predominant. CTLA4-CD28 chimera gene IntroductionCytotoxic T lymphocyte-associated antigen 4 (CTLA4) is an inhibitory receptor expressed on T-cell surface that is induced by T-cell activation to negatively regulate excessive activation of T cells. 1 CTLA4-deficient mice develop lethal lymphoproliferative diseases within 3 to 4 weeks of birth. 2 CTLA4 deficiency or anti-CTLA4 antibody treatment ameliorated induction of peripheral T-cell tolerance 3 and exacerbated autoimmune diseases 4,5 in mouse models. Therefore, CTLA4 is thought to play a critical role in maintaining peripheral T-cell tolerance in vivo to prevent autoimmunity. Because tumor cells also were known to induce T-cell tolerance to tumor antigens to resist to antitumor immunity, 6 CTLA4 blockade was believed to be able to break tumor-mediated T-cell tolerance, resulting in enhanced antitumor immunity. Indeed, systemic administration of anti-CTLA4 blocking antibody in preclinical murine tumor models led to efficient regression of several syngenic tumors. [7][8][9] Thus, blocking CTLA4 in human malignancies has been regarded as a promising therapeutic target of antitumor immunotherapy.Therapeutic efficacy of 2 fully human anti-CTLA4 antibodies is currently being tested for several malignancies in clinical trials. 10 For metastatic melanoma, anti-CTLA4 monotherapy or combination therapy with other agents shows an overall response rate of 5% to 15%. A recent study indicated that combination therapy of anti-CTLA4 plus dacarbazine, a chemotherapeutic agent, could improve the overall survival compared with dacarbazine monotherapy in a phase 3 randomized clinical trial. 11 However, a significant proportion of patients who received anti-CTLA4 therapy developed grade III or IV autoimmune side effects, such as enterocolitis, skin rash, hypophysitis, hepatitis, and uveitis. 12 Although most of these side effects could be managed by steroid treatment, several patients experienced the serious conditions such as intestinal perforation. 13 The more intriguing observation was that the degree of autoimmunity was correlated with treatment efficacy. 12 T...

Induction of T-cell clonal anergy involves serial activation of transcription factors, including NFAT and Egr2/3. However, downstream effector mechanisms of these transcription factors are not fully understood yet. Here we identify Ndrg1 as an anergy factor induced by Egr2. Ndrg1 is upregulated by anergic signalling and maintained at high levels in resting anergic T cells. Overexpression of Ndrg1 mimics the anergic state and knockout of the gene prevents anergy induction. Interestingly, Ndrg1 is phosphorylated and degraded by CD28 signalling in a proteasome-dependent manner, explaining the costimulation dependence of anergy prevention. Similarly, IL-2 treatment of anergic T cells, under conditions that lead to the reversal of anergy, also induces Ndrg1 phosphorylation and degradation. Finally, older Ndrg1-deficient mice show T-cell hyperresponsiveness and Ndrg1-deficient T cells aggravate inducible autoimmune inflammation. Thus, Ndrg1 contributes to the maintenance of clonal anergy and inhibition of T-cell-mediated inflammation.

Background and PurposeThe detection of aquaporin 4-IgG (AQP4-IgG) is now a critical diagnostic criterion for neuromyelitis optica spectrum disorder (NMOSD). To evaluate the serostatus of NMOSD patients based on the 2015 new diagnostic criteria using a new in-house cell-based assay (CBA).MethodsWe generated a stable cell line using internal ribosome entry site-containing bicistronic vectors, which allow the simultaneous expression of two proteins (AQP4 and green fluorescent protein) separately from the same RNA transcript. We performed in-house CBA using serum from 386 patients: 178 NMOSD patients diagnosed according to the new diagnostic criteria without AQP4-IgG, 63 high risk NMOSD patients presenting 1 of the 6 core clinical characteristics of NMOSD but not fulfilling dissemination in space, and 145 patients with other neurological diseases, including 66 with multiple sclerosis. The serostatus of 111 definite and high risk NMOSD patients were also tested using a commercial CBA kit with identical serum to evaluate the correlation between the 2 methods. All assays were performed by two independent and blinded investigators.ResultsOur in-house assay yielded a specificity of 100% and sensitivities of 80% (142 of 178) and 76% (48 of 63) when detecting definite- and high risk NMOSD patients, respectively. The comparison with the commercial CBA kit revealed a correlation for 102 of the 111 patients: no correlation was present in 7 patients who were seronegative using the commercial method but seropositive using the in-house method, and in 2 patients who were seropositive using the commercial method but seronegative using the in-house method.ConclusionsThese results demonstrate that our in-house CBA is a highly specific and sensitive method for detecting AQP4-IgG in NMOSD patients.