Francesca Fallarino

Blockade of T Cell Activation Using a Surface-Linked Single-Chain Antibody to CTLA-4 (CD152)

CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by engaging CTLA-4 has been limited by sharing of its natural ligands with the costimulatory protein CD28. In the present study, a CTLA-4-specific single-chain Ab (scFv) was developed and expressed on the cell surface to promote selective engagement of this regulatory molecule. Transfectants expressing anti-CTLA-4 scFv at their surface bound soluble CTLA-4 but not soluble CD28. Coexpression of anti-CTLA-4 scFv with anti-CD3ε and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated bulk T cells as well as CD4+ and CD8+ T cell subsets. Importantly, expression of anti-CTLA-4 scFv on the same cell surface as the TCR ligand was essential for the inhibitory effects of CTLA-4-specific ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of components of the proximal TCR signaling apparatus was similarly dependent on coexpression of TCR and CTLA-4 ligands on the same surface. These findings support a predominant role for CTLA-4 function in the modification of the proximal TCR signal. Using T cells from DO11.10 and 2C TCR transgenic mice, negative regulatory effects of selective CTLA-4 ligation were also demonstrated during the stimulation of Ag-specific CD4+ and CD8+ T cells by MHC/peptide complexes. Together these studies demonstrate that selective ligation of CTLA-4 using a membrane-bound scFv results in attenuated T cell responses only when coengaged with the TCR during T cell/APC interaction and define an approach to harnessing the immunomodulatory potential of CTLA-4-specific ligation.

Improved efficacy of dendritic cell vaccines and successful immunization with tumor antigen peptide-pulsed peripheral blood mononuclear cells by coadministration of recombinant murine interleukin-12

The well‐characterized P815 tumor model was used to optimize anti‐tumor immunization approaches in mice. Tumor peptides derived from antigens P198 or P1A were targeted to antigen‐presenting cells (APC) by ex vivo pulsing. Initial experiments with irradiated pulsed splenic dendritic cells (sDC) injected weekly in the hind footpads for 3 weeks demonstrated cytolytic T lymphocyte (CTL) generation in 10–20% of mice. Because of the importance of interleukin‐12 (IL‐12) in tumor rejection responses, pulsed sDCs also were given together with recombinant murine IL‐12 (rmIL‐12). This strategy induced peptide‐specific CTL in 100% of the mice. The IL‐12 had to be injected in the footpads on days 0, 1 and 2 of each immunization week to achieve an optimal effect. The improvement seen with the addition of IL‐12 prompted examination of other sources of APC. Purified resting B cells, lipopolysaccharide (LPS) blasts and non‐fractionated splenocytes or peripheral blood mononuclear cells (PBMC) were pulsed with peptide and administered with the same schedule of rmIL‐12. Because these cell types appeared to bind peptides less avidly than did DC, increasing peptide doses were used during pulsing. Interestingly, immunization with each of these APC also induced specific CTL in 100% of mice, provided rmIL‐12 was coadministered. CTLs were detected both in the spleen and in the peripheral blood. Immunization with irradiated, P1A‐pulsed PBMC plus rmIL‐12 resulted in protection against challenge with tumors expressing the specific antigen in all mice. The ease by which human patient PBMCs can be prepared provides a straightforward vaccination approach to be used in clinical trials of peptide‐based immunization in melanoma. Int. J. Cancer 80:324–333, 1999.

Mechanisms of CTLA-4-Ig in tolerance induction

The size of the peripheral T lymphocyte pool remains relatively constant throughout adult life, but individual populations undergo expansion and contraction upon antigen encounter due to signals delivered by members of the B7-CD28 family of costimulatory molecules. This family includes receptors on T cells that can provide either activating or inhibitory signals. In general, activation occurs in response to pathogens, when lymphocyte expansion and acquisition of effector functions is appropriate. Conversely inhibitory receptors provide down-modulating signals that help terminate immune responses and maintain self-tolerance. The activating receptor CD28 engages the same B7-1 and B7-2 molecules as the inhibitory receptor cytotoxic T lymphocyte antigen 4 (CTLA-4), although with reduced affinity than CTLA-4. In addition to this direct competitive mechanism, CTLA-4 can directly inhibit T cell receptor (TCR) signals independently of CD28 expression and recent findings indicate that CTLA-4 may also operate through reverse signaling on ligand-expressing cells. Fusion proteins between the extracellular domain of CTLA-4 and an immunoglobulin Fc portion have been created that have potent immunosuppressive properties in animal models of transplantation and autoimmunity and that show great promise in clinical trials. Like CTLA-4, CTLA-Ig, is thought to selectively prevent activation of CD28 by interacting with B7-1 and B7-2. In addition, CTLA-4-Ig can bind to B7 molecules expressed on dendritic cells and activate a pathway of tryptophan catabolism that can lead to indirect inhibition of lymphocyte activation and T cell death. In this review, we will focus on the current knowledge of the mechanisms of action of CTLA-4 and CTLA-4-Ig.

Melanoma presenting as circulating tumor cells associated with failed angiogenesis

The ability of cancer cells to promote angiogenesis has been associated with a transition from a micrometastatic phenotype to a state in which large solid metastases can form. We describe a case of metastatic melanoma that presented with large numbers of circulating tumor cells and tissue infiltration without large solid metastases. Immunohistochemistry and reverse transcriptase-PCR was used to study the expression of melanoma tumor markers in circulating tumor cells. A tumor cell line was established from the patient and analyzed for adhesion molecule expression, expression of vascular endothelial growth factor, and the ability to inhibit solid tumor formation by other melanoma cells implanted as a xenograft. Examination of the circulating tumor cells confirmed they were of melanoma origin. Analysis of the tumor cell line obtained from this patient demonstrated intact expression of &agr; and &bgr; integrins but poor production of vascular endothelial growth factor, and also the ability to inhibit solid tumor formation of third-party melanoma tumors, suggesting a strong antiangiogenic activity. Our results indicate that lethal metastatic melanoma can occur even if tumor angiogenesis is defective, an observation that has implications for potential limits of antiangiogenic therapies.