Regulatory T cells (Tregs

IntroductionRetroviral T-cell receptor (TCR) gene transfer is an attractive strategy by which large numbers of antigen-specific T cells can be generated for adoptive transfer. [1][2][3][4] One of the advantages of this technique is that it can be used to circumvent possible impairment of autologous T-cell responses against tumor associated antigens as it bypasses central tolerance. 5 In addition, the introduced TCR specificity can be targeted against poorly immunogenic antigens and high affinity TCR can be selected for transfer. 6 This method has recently seen success in the first clinical trial of TCR gene therapy, where MART1 specific T cells, generated by retroviral gene transfer, were adoptively transferred into patients with metastatic melanoma. The engineered T cells engrafted in 15 of 17 patients, 2 of whom demonstrated long term tumor regression. 7 T cells engineered to express CEA-specific TCR also induced a decrease in CEA levels in 3 patients with metastatic colorectal cancer, and regression of tumor metastases in 1 patient, but were associated with a severe colitis in all 3 patients. 8 The most promising results were seen after adoptive transfer of TCR transduced T cells specific for the NY-ESO-1antigen which resulted in clinical responses in synovial cell sarcoma and in melanoma. 9 As the density of TCR on the surface of cells affects their functional avidity, inefficient TCR expression may impair the success of TCR gene therapy. [10][11][12][13][14] Several different strategies have been developed to increase the expression of introduced ␣ and ␤ chains by reducing the mispairing with endogenous TCR chains. The strategies include replacing the human TCR constant domain with murine sequences, the introduction of an additional disulphide bond into the constant regions, and the production of hybrid molecules consisting of the extracellular portion of TCR chains fused to the intracellular CD3 domain. [15][16][17][18] TCR ␣ and ␤ chains form a complex with 4 invariant CD3 chains: ␥,␦,⑀ and . This complex formation is required in order for the TCR to be expressed on the cell surface, and for signal transduction on antigen recognition. TCR introduced by retroviral gene transfer are likely to be in competition with endogenous TCR molecules for CD3 chains. We used a murine model system to explore whether the co-transfer of TCR genes together with the genes encoding the ␥,␦,⑀ and chains of the CD3 complex can augment TCR expression. Two different TCR, with specificity for Wilms' Tumor antigen 1 (WT1) and influenza nucleoprotein, were used to demonstrate that CD3 is rate limiting for the expression of introduced TCR in gene modified T cells. Co-transduction of CD3 and TCR genes resulted in up to 16-20 fold increase in TCR surface expression and tetramer binding compared with transduction of TCR genes alone. The increase in TCR expression was associated with increased T-cell avidity leading to improved recognition of low concentration of peptide antigen. In vivo TCRϩCD3 co-transduced T cells eradicate tumors fa...

Epstein Barr virus (EBV) is one of the most ubiquitous human pathogens in the world, persistently infecting more than 90% of the adult human population. It drives some of the strongest human CD8 + T cell responses, which can be observed during symptomatic primary infection known as infectious mononucleosis (IM). Despite high viral loads and prolonged CD8 + T cell stimulation during IM, EBV enters latency and is under lifelong immune control in most individuals that experience this disease. We investigated whether changes in T cell function, as frequently characterized by PD-1 up-regulation, occur during IM due to the prolonged exposure to high antigen levels. We readily detected the expansion of PD-1 positive CD8 + T cells together with high frequencies of Tim-3, 2B4, and KLRG1 expression during IM and in mice with reconstituted human immune system components (huNSG mice) that had been infected with a high dose of EBV. These PD-1 positive CD8 + T cells, however, retained proliferation, cytokine production, and cytotoxic abilities. Multiple subsets of CD8 + T cells expanded during EBV infection, including PD-1 + Tim-3 + KLRG1 + cells that express CXCR5 and TCF-1 germinal center homing and memory markers, and may also contain BATF3. Moreover, blocking the PD-1 axis compromised EBV specific immune control and resulted in virus-associated lymphomagenesis. Finally, PD-1 + , Tim-3 + , and KLRG1 + CD8 + T cell expansion coincided with declining viral loads during low dose EBV infection. These findings suggest that EBV infection primes PD-1 positive CD8 + T cell populations that rely on this receptor axis for the efficient immune control of this ubiquitous human tumor virus.