Abrogation of SRC homology region 2 domain-containing phosphatase 1 in tumor-specific T cells improves efficacy of adoptive immunotherapy by enhancing the effector function and accumulation of short-lived effector T cells in vivo.

T cell expression of inhibitory proteins can be a critical component for the regulation of immunopathology owing to self-reactivity or potentially exuberant responses to pathogens, but it may also limit T cell responses to some malignancies, particularly if the tumor Ag being targeted is a self-protein. We found that the abrogation of Src homology region 2 domain-containing phosphatase-1 (SHP-1) in tumor-reactive CD8(+) T cells improves the therapeutic outcome of adoptive immunotherapy in a mouse model of disseminated leukemia, with benefit observed in therapy employing transfer of CD8(+) T cells alone or in the context of also providing supplemental IL-2. SHP-1(-/-) and SHP-1(+/+) effector T cells were expanded in vitro for immunotherapy. Following transfer in vivo, the SHP-1(-/-) effector T cells exhibited enhanced short-term accumulation, followed by greater contraction, and they ultimately formed similar numbers of long-lived, functional memory cells. The increased therapeutic effectiveness of SHP-1(-/-) effector cells was also observed in recipients that expressed the tumor Ag as a self-antigen in the liver, without evidence of inducing autoimmune toxicity. SHP-1(-/-) effector CD8(+) T cells expressed higher levels of eomesodermin, which correlated with enhanced lysis of tumor cells. Furthermore, reduction of SHP-1 expression in tumor-reactive effector T cells by retroviral transduction with vectors that express SHP-1-specific small interfering RNA, a translatable strategy, also exhibited enhanced antitumor activity in vivo. These studies suggest that abrogating SHP-1 in effector T cells may improve the efficacy of tumor elimination by T cell therapy without affecting the ability of the effector cells to persist and provide a long-term response.

SHP-1 in T cells limits the production of CD8 effector cells without impacting the formation of long-lived central memory cells.

During responses against viruses and malignancies, naive CD8 T lymphocytes expand to form both short-lived effector cells and a population containing cells with the potential to be long-lived and participate in memory responses (memory precursor effector cells). The strength of antigenic, costimulatory, and cytokine signals during responses impacts the magnitude and type of CD8 populations formed. In vitro studies have revealed that the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP-1) regulates signal transduction from receptors on T cells including the TCR, helping set the activation threshold, and therefore may shape responses of mature CD8 T cells in vivo. Analysis of CD8 T cells from motheaten mice, which are globally deficient in SHP-1, proved problematic due to cell-extrinsic effects of SHP-1 deficiency in non-T cells on CD8 T cells. Therefore, a conditional knockout of SHP-1 in mature single-positive T cells was developed to analyze cell-intrinsic consequences of complete and partial SHP-1 deficiency on CD8 T cell responses to acute viral infection. The results demonstrated that SHP-1 has disparate effects on subpopulations of responding cells, limiting the magnitude and quality of primary and secondary responses by reducing the number of short-lived effector cells generated without affecting the size of the memory precursor effector cell pool that leads to formation of long-term memory.

Peripheral CD8+ T cell tolerance to self-proteins is regulated proximally at the T cell receptor.

CD8(+) T cell tolerance, although essential for preventing autoimmunity, poses substantial obstacles to eliciting immune responses to tumor antigens, which are generally overexpressed normal proteins. Development of effective strategies to overcome tolerance for clinical applications would benefit from elucidation of the immunologic mechanism(s) regulating T cell tolerance to self. To examine how tolerance is maintained in vivo, we engineered dual-T cell receptor (TCR) transgenic mice in which CD8(+) T cells recognize two distinct antigens: a foreign viral-protein and a tolerizing self-tumor protein. Encounter with peripheral self-antigen rendered dual-TCR T cells tolerant to self, but these cells responded normally through the virus-specific TCR. Moreover, proliferation induced by virus rescued function of tolerized self-tumor-reactive TCR, restoring anti-tumor activity. These studies demonstrate that peripheral CD8(+) T cell tolerance to self-proteins can be regulated at the level of the self-reactive TCR complex rather than by central cellular inactivation and suggest an alternate strategy to enhance adoptive T cell immunotherapy.

The antiapoptotic herpes simplex virus glycoprotein J localizes to multiple cellular organelles and induces reactive oxygen species formation.

The Us5 gene of herpes simplex virus (HSV) encodes glycoprotein J (gJ). The only previously reported function of gJ was its ability to inhibit apoptosis. However, the mechanism by which gJ prevents apoptosis is not understood, and it is not known whether gJ mediates additional cellular effects. In this study, we evaluated the expression, localization, and cellular effects of Us5/gJ. Us5 was first expressed 4 h after infection. gJ was detectable at 6 h and was expressed in glycosylated and unglycosylated forms. Us5 was regulated as a late gene, with partial dependency on DNA replication for expression. Us5 expression was delayed in the absence of ICP22; furthermore, expression of Us5 in trans protected cells from apoptosis induced by an HSV mutant with deletion of ICP27, suggesting that the antiapoptotic effects of ICP22 and ICP27 are mediated in part through effects on gJ expression. Within HSV-infected or Us5-transfected cells, gJ was distributed widely, especially to the endoplasmic reticulum, trans-Golgi network, and early endosomes. gJ interacted with F(o)F(1) ATP synthase subunit 6 by a yeast two-hybrid screen and had strong antiapoptotic effects, which were mediated by protein rather than mRNA. Antiapoptotic activity required the extracellular and transmembrane domains of gJ, but not the intracellular domain. Consistent with inhibition of F(o)F(1) ATP synthase function, Us5 was required for HSV-induced reactive oxygen species (ROS) formation, and gJ was sufficient to induce ROS in Us5-transfected cells. Thus, HSV gJ is a multifunctional protein, modulating other cellular processes in addition to inhibition of apoptosis.

Facilitating matched pairing and expression of TCR chains introduced into human T cells.

Adoptive transfer of T lymphocytes is a promising treatment for a variety of malignancies but often not feasible due to difficulties generating T cells that are reactive with the targeted antigen from patients. To facilitate rapid generation of cells for therapy, T cells can be programmed with genes encoding the alpha and beta chains of an antigen-specific T-cell receptor (TCR). However, such exogenous alpha and beta chains can potentially assemble as pairs not only with each other but also with endogenous TCR alpha and beta chains, thereby generating alphabetaTCR pairs of unknown specificity as well as reducing the number of exogenous matched alphabetaTCR pairs at the cell surface. We demonstrate that introducing cysteines into the constant region of the alpha and beta chains can promote preferential pairing with each other, increase total surface expression of the introduced TCR chains, and reduce mismatching with endogenous TCR chains. This approach should improve both the efficacy and safety of ongoing efforts to use TCR transfer as a strategy to generate tumor-reactive T cells.