ADAM17-dependent proteolysis of L-selectin promotes early clonal expansion of cytotoxic T cells.

L-selectin on T-cells is best known as an adhesion molecule that supports recruitment of blood-borne naïve and central memory cells into lymph nodes. Proteolytic shedding of the ectodomain is thought to redirect activated T-cells from lymph nodes to sites of infection. However, we have shown that activated T-cells re-express L-selectin before lymph node egress and use L-selectin to locate to virus-infected tissues. Therefore, we considered other roles for L-selectin proteolysis during T cell activation. In this study, we used T cells expressing cleavable or non-cleavable L-selectin and determined the impact of L-selectin proteolysis on T cell activation in virus-infected mice. We confirm an essential and non-redundant role for ADAM17 in TCR-induced proteolysis of L-selectin in mouse and human T cells and show that L-selectin cleavage does not regulate T cell activation measured by CD69 or TCR internalisation. Following virus infection of mice, L-selectin proteolysis promoted early clonal expansion of cytotoxic T cells resulting in an 8-fold increase over T cells unable to cleave L-selectin. T cells unable to cleave L-selectin showed delayed proliferation in vitro which correlated with lower CD25 expression. Based on these results, we propose that ADAM17-dependent proteolysis of L-selectin should be considered a regulator of T-cell activation at sites of immune activity.

Purity of transferred CD8(+) T cells is crucial for safety and efficacy of combinatorial tumor immunotherapy in the absence of SHP-1.

Adoptive transfer of tumor-specific cytotoxic T cells is a promising advance in cancer therapy. Similarly, checkpoint inhibition has shown striking clinical results in some patients. Here we combine adoptive cell transfer with ablation of the checkpoint protein Src homology 2-domain-containing phosphatase 1 (SHP-1, Ptpn6). Naturally occurring motheaten mice lack SHP-1 and do not survive weaning due to extensive immunopathology. To circumvent this limitation, we created a novel SHP-1(null) mouse that is viable up to 12 weeks of age by knocking out IL1r1. Using this model, we demonstrate that the absence of SHP-1 augments the ability of adoptively transferred CD8(+) T cells to control tumor growth. This therapeutic effect was only observed in situations where T-cell numbers were limited, analogous to clinical settings. However, adoptive transfer of non-CD8(+) SHP-1(null) hematopoietic cells resulted in lethal motheaten-like pathology, indicating that systemic inhibition of SHP-1 could have serious adverse effects. Despite this caveat, our findings support the development of SHP-1 inhibition strategies in human T cells to complement adoptive transfer therapies in the clinic.

Genetic modification of primary chronic lymphocytic leukemia cells with a lentivirus expressing CD38.

Studies of the role of individual genes in chronic lymphocytic leukemia (CLL) have been hampered by the inability to consistently transfect primary tumor cells. Here, we describe a highly efficient method of genetically modifying primary CLL cells using a VSVG pseudotyped lentiviral vector. We transduced CD38 negative CLL cells with a lentiviral vector encoding CD38 which caused increased surface CD38 expression in all the samples tested (n=17) with no evidence of plasmacytoid differentiation. The mean percentage of positive cells expressing CD38 was 87%+/-8.5% and the mean cell viability 74%+/-17%. This high level of transduction of all the CLL cell samples tested demonstrates the utility of this technique which should prove applicable for the introduction and analysis of other genes in these non-dividing cells.

Antigen specificity determines the pro- or antitumoral nature of CD8+ T cells.

Although CD8+ T cells are usually considered antitumoral, several recent studies report that the cells can also promote tumor progression. Using the melanoma cell line B16 as a murine model of pulmonary metastasis, we examined whether the pro- versus antitumoral effects of CD8+ T cells relate to their Ag specificity. Results of the study indicate that although CD8+ T cells specific for tumor Ags promote tumor rejection, CD8+ T cells specific for unrelated Ags promote tumor progression. We found the effect to be partly attributable to CD8+ T cells dampening effective antitumor NK cell responses. Notably, activation of CD8+ T cell responses by an unrelated stimulus, in this case infection with influenza virus, increased the number of pulmonary tumor nodules. These data provide a rationale for previously unexplained data identifying contrasting roles for CD8+ T cells in tumor progression.

Loss of Src homology region 2 domain-containing protein tyrosine phosphatase-1 increases CD8+ T cell-APC conjugate formation and is associated with enhanced in vivo CTL function.

Extensive evidence has been accumulated to implicate the intracellular protein tyrosine phosphatase, Src homology region 2 domain-containing protein tyrosine phosphatase-1 (SHP-1), as a negative regulator of TCR-signaling thresholds. Specifically, T cells from the SHP-1-deficient mouse, motheaten, exhibit a hyperproliferative phenotype when activated by cognate peptide-pulsed APCs. However, the cellular basis for this phenotype has not been fully explained. Using the intracellular fluorescent dye, CFSE, we show that a greater proportion of motheaten vs control naive CD8(+) T cells undergo cell division when activated by peptide-pulsed APCs. Furthermore, there is a greater likelihood of TCRs on SHP-1-deficient vs control T cells binding to peptide/MHC ligands on APCs when using TCR down-regulation as an indirect measure of TCR engagement. In addition, T cell-APC conjugate assays provide direct evidence that a greater proportion of SHP-1-deficient T cells are capable of forming stable conjugates with APCs and this may explain, at least in part, their hyperproliferative response to TCR-triggered stimulation. The physiological relevance of the combined in vitro observations is demonstrated by the significantly enhanced in vivo expansion and CTL capacity generated in mice receiving adoptively transferred SHP-1-deficient naive CD8(+) T cells when compared with control T cells.

CD22 is a functional ligand for SH2 domain-containing protein-tyrosine phosphatase-1 in primary T cells.

The intracellular Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase (SHP-1) has been characterized as a negative regulator of T cell function, contributing to the definition of T cell receptor signaling thresholds in developing and peripheral mouse T lymphocytes. The activation of SHP-1 is achieved through the engagement of its tandem SH2 domains by tyrosine-phosphorylated proteins; however, the identity of the activating ligand(s) for SHP-1, within mouse primary T cells, is presently unresolved. The identification of SHP-1 ligand(s) in primary T cells would provide crucial insight into the molecular mechanisms by which SHP-1 contributes to in vivo thresholds for T cell activation. Here we present a combination of biochemical and yeast genetic analyses indicating CD22 to be a T cell ligand for the SHP-1 SH2 domains. Based on these observations we have confirmed that CD22 is indeed expressed on mouse primary T cells and capable of associating with SHP-1. Significantly, CD22-deficient T cells demonstrate enhanced proliferation in response to anti-CD3 or allogeneic stimulation. Furthermore, the co-engagement of CD3 and CD22 results in a raising of TCR signaling thresholds hence demonstrating a previously unsuspected functional role for CD22 in primary T cells.

Normal B-1 cell development but defective BCR signaling in Lck-/- mice.

Mature B cells are grouped into two major subsets, B-1 and B-2, believed to derive from separate lineages. We have recently shown that B-1 cells, which are characterized by CD5 surface expression, specifically exhibit significant levels of the tyrosine kinase Lck in man. Here we show that also in mice Lck expression is restricted to B-1 cells and address the potential role of Lck in B-1 cell development and activation. Using as a model an Lck-/- mouse, we show that, while dispensable for B-1 cell development, Lck is required for full and sustained activation of the tyrosine phosphorylation and MAP kinase cascades triggered by the BCR in CD5+, B-1 cells. The data suggest a potential role for Lck in the achievement of the higher activation threshold required for productive BCR signaling in B-1 as compared to B-2 cells.

Gene delivery into primary T cells: overview and characterization of a transgenic model for efficient adenoviral transduction.

Technologies for transfer of exogenous genes into primary T cells have been limited until recently. The introduction of new approaches for gene transfer via different viral vectors has expanded the options for genetic manipulation of primary T cells and has provided powerful tools for studies of T cell activation and differentiation. We provide a brief overview of the systems currently available and contrast the advantages and disadvantages of each. We also describe a new transgenic model that enables highly efficient gene delivery into primary T cells by nonreplicating adenoviral vectors.

Efficient adenovirus-mediated gene transfer into primary T cells and thymocytes in a new coxsackie/adenovirus receptor transgenic model.

BACKGROUND: Gene transfer studies in primary T cells have suffered from the limitations of conventional viral transduction or transfection techniques. Replication-defective adenoviral vectors are an attractive alternative for gene delivery. However, naive lymphocytes are not readily susceptible to infection with adenoviruses due to insufficient expression of the coxsackie/adenovirus receptor. RESULTS: To render T cells susceptible to adenoviral gene transfer, we have developed three new murine transgenic lines in which expression of the human coxsackie/adenovirus receptor (hCAR) with a truncated cytoplasmic domain (hCAR(Delta)cyt) is limited to thymocytes and lymphocytes under direction of a human CD2 mini-gene. hCAR(Delta)cyt.CD2 transgenic mice were crossed with DO11.10 T cell receptor transgenic mice (DO11.hCAR(Delta)cyt) to allow developmental studies in a defined, clonal T cell population. Expression of hCAR(Delta)cyt enabled adenoviral transduction of resting primary CD4+ T cells, differentiated effector T cells and thymocytes from DO11.hCAR(Delta)cyt with high efficiency. Expression of hCAR(Delta)cyt transgene did not perturb T cell development in these mice and adenoviral transduction of DO11.hCAR(Delta)cyt T cells did not alter their activation status, functional responses or differentiative potential. Adoptive transfer of the transduced T cells into normal recipients did not modify their physiologic localization. CONCLUSION: The DO11.hCAR(Delta)cyt transgenic model thus allows efficient gene transfer in primary T cell populations and will be valuable for novel studies of T cell activation and differentiation.