Tat mammaglobin fusion protein transduced dendritic cells stimulate mammaglobin-specific CD4 and CD8 T cells

Abstract

Proteins can be efficiently introduced into cells when fused to a protein transduction domain, such as Tat from the human immunodeficiency virus. We recently reported that dendritic cells transduced with a Tat fusion protein containing the extracellular domain of Her2/neu (Tat-Her2/neu) induced CD8 cytotoxic T lymphocytes (CTL) that specifically lysed Her2/neu-expressing breast and ovarian cancer cells. In the current study we further investigated the mechanism of protein transduction, utilizing the breast cancer-associated protein, mammaglobin-A, which is expressed in about 80% of breast cancers. Using a Tat-mammaglobin fusion protein, we tested the ability of Tat-mammaglobin transduced dendritic cells to stimulate antigen-specific CD4 and CD8 T cells. Low levels of serum considerably improved protein transduction as determined by Western blot, and also improved presentation of antigenic peptide as evidenced by functional studies using antigen-specific T cells. Confocal microscope analyses of antigen-presenting cells (APC) incubated with Tat-mammaglobin showed localized distribution in addition to diffuse distribution in the cytosol. In contrast, mammaglobin lacking Tat showed only a localized distribution. Simultaneous incubation with both proteins resulted in overlapping localized distributions, suggesting Tat fusion proteins are processed through both the MHC class I and class II pathways. Indeed, stimulation of T cells with Tat-mammaglobin transduced dendritic cells led to an expansion of mammaglobin-specific CD4 T helper-1 lymphocytes along with CD8 CTL. We conclude that Tat-mammaglobin transduced dendritic cells can induce both CD4 and CD8 mammaglobin-specific T cells. These findings could be further exploited for the development of a mammaglobin-based vaccine for breast cancer.

Enhanced antigen presentation and CTL activity by transduction of mature rather than immature dendritic cells with octaarginine-modified liposomes

“…4), consistent with the high in vitro pMHC presentation. Our results were in agreement with many studies, which reported that transduction of mDCs with recombinant fusion proteins containing HIV-Tat was preferential over transduction of iDCs [8,12,13]. The longevity of pMHC on the surface of mDCs might be an advantage of pulsing Ag to DCs after their maturation [27,28].…”

“…This pathway is currently called "cross-presentation" pathway [4,21]. In an attempt to enhance the cellular uptake and promote the cross-presentation pathway in DCs, many investigators perused the recombinant fusion protein or synthetic peptides which contain CPPs tandemly linked to the Ag [7][8][9][10][11][12][13][14]. However the construction of such a recombinant proteins/ peptides are specific for a single Ag, limiting the usefulness of this technology.…”

“…Since then various research groups, including our own, have applied CPP transduction to DCs as a method of stimulating Ag-specific T cells. Many studies reported that mice immunized with DCs transduced with recombinant CPP-Ag protein suppressed the tumor growth significantly [8][9][10][11][12][13][14][15]. Apart from the recombinant fusion protein, synthetic polypeptide containing CPP tandemly linked to peptide epitopes is another striking approach [14].…”

The Tat-conjugated N-terminal region of mucin antigen 1 (MUC1) induces protective immunity against MUC1-expressing tumours

“…Recombinant proteins, peptides, viral vectors, RNA, immune complexes and killed tumour cell lysates have been used in this capacity [5], among which the use of the protein transduction domain (PTD) has been given much attention, because it is safer than viral vectors yet equally effective in loading MUC1 [8,9]. It has been hypothesized that the intracellular delivery of tumour-associated antigens (TAAs) into mature DCs by a PTD, such as the human immunodeficiency virus (HIV) Tat peptide, allows DCs to process and present the internalized antigens to T cells by major histocompatibility complex (MHC) class I and II molecules efficiently [10,11]. DCs that are pulsed with Tat-TAA have been demonstrated to induce antigen-specific CD4 T cells effectively as well as cytotoxic T lymphocytes (CTLs) [11,12].…”

“…It has been hypothesized that the intracellular delivery of tumour-associated antigens (TAAs) into mature DCs by a PTD, such as the human immunodeficiency virus (HIV) Tat peptide, allows DCs to process and present the internalized antigens to T cells by major histocompatibility complex (MHC) class I and II molecules efficiently [10,11]. DCs that are pulsed with Tat-TAA have been demonstrated to induce antigen-specific CD4 T cells effectively as well as cytotoxic T lymphocytes (CTLs) [11,12].…”