Eduardo Davila

Repeated Administration of Cytosine-Phosphorothiolated Guanine-Containing Oligonucleotides Together with Peptide/Protein Immunization Results in Enhanced CTL Responses with Anti-Tumor Activity

The development of therapeutic anti-cancer vaccines designed to elicit CTL responses with anti-tumor activity has become a reality thanks to the identification of several tumor-associated Ags and their corresponding peptide T cell epitopes. However, peptide-based vaccines, in general, fail to elicit sufficiently strong CTL responses capable of producing therapeutic anti-tumor effects (i.e., prolongation of survival, tumor reduction). Here we report that repeated administration of synthetic oligonucleotides containing foreign cytosine-phosphorothiolated guanine (CpG) motifs increased 10- to 100-fold the CTL response to immunization with various synthetic peptides corresponding to well-known T cell epitopes. Moreover, repeated CpG administration allowed the induction of CTL to soluble protein even in the absence of additional adjuvant. Our results indicate that the potentiating effect of CpG in CTL responses required the participation of Th lymphocytes. Repeated CpG administration resulted in overt splenomegaly and lymphadenopathy with a significant increase in the numbers of CTL precursors and dendritic cells. Protein vaccination in combination with repeated CpG therapy was effective in delaying tumor cell growth and extending survival in mice bearing melanoma tumors. These findings support the contention that repeated administration of CpG-oligonucleotides enhances the effect of peptide and protein vaccines leading to potent anti-tumor responses, presumably through the induction of Th1 and dendritic cells, which are essential for optimal CTL responses. The immunostimulatory properties of CpG motifs may be key in inducing a consistent long term immunity to tumor-associated Ags when using peptides or proteins as T cell-inducing vaccines.

Proteomic identification of non-Gal antibody targets after pig-to-primate cardiac xenotransplantation.

Abstract:  Background:  Experience with non‐antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non‐Gal endothelial cell (EC) antigens.

Cell-Based Immunotherapy with Suppressor CD8+ T Cells in Rheumatoid Arthritis

The chronic persistence of rheumatoid synovitis, an inflammation driven by activated T cells, macrophages, and fibroblasts causing irreversible joint damage, suggests a failure in physiologic mechanisms that down-regulate and terminate chronic immune responses. In vitro CD8+CD28−CD56+ T cells tolerize APCs, prevent the priming of naive CD4+ T cells, and suppress memory CD4+ T cell responses. Therefore, we generated CD8+CD28−CD56+ T cell clones from synovial tissues, expanded them in vitro, and adoptively transferred them into NOD-SCID mice engrafted with synovial tissues from patients with rheumatoid arthritis. Adoptively transferred CD8+CD28−CD56+ T cells displayed strong anti-inflammatory activity. They inhibited production of IFN-γ, TNF-α, and chemokines in autologous and HLA class I-matched heterologous synovitis. Down-regulation of costimulatory ligands CD80 and CD86 on synovial fibroblasts was identified as one mechanism of immunosuppression. We propose that rheumatoid synovitis can be suppressed by cell-based immunotherapy with immunoregulatory CD8+ T cells.

Avoiding Tolerance Against Prostatic Antigens With Subdominant Peptide Epitopes.

A potential novel therapy for prostate cancer is the induction of immune responses to normal prostate-associated antigens (PAA). One approach is to use synthetic peptides from PAA to educate T cells as a means of developing a defined and specific immunotherapy for prostate cancer. A likely major hurdle when using normal PAA for this type of therapy is the tolerance that the immune system may already have for PAA. To evaluate mechanisms for overcoming tolerance, the authors assessed the level of tolerance to SV40T antigen in a transgenic mouse. The SV40T antigen is selectively expressed in the prostates of mice from the transgenic adenocarcinoma mouse prostate (TRAMP) model. The authors have shown that TRAMP mice are tolerant to a dominant cytotoxic T-lymphocyte (CTL) epitope from the SV40T antigen compared with nontransgenic littermates. The tolerance was exhibited as early as 4 weeks and as late as 24 weeks. The use of multiple injections of an oligonucleotide that contains an unmethylated CpG induced high levels of hematopoiesis but did not overcome the tolerance. Injection of an antibody to activate CD40 increased the CTL response in normal mice but also did not overcome tolerance. However, tolerance in the TRAMP mice was avoided when an epitope that had previously been characterized as a subdominant epitope was administered. The authors are investigating the potential of subdominant epitopes to induce prostatitis and antitumor responses. The results of this work should facilitate the development of immune-based therapies for prostate cancer.

T‐cell responses during pig‐to‐primate xenotransplantation

Abstract: Xenotransplantation using porcine organs may resolve a chronic shortage of donor organs for clinical transplantation if significant immunological barriers can be overcome. To determine the potential role of T lymphocytes in Xenograft (Xg) rejection, we transplanted transgenic hCD46 porcine hearts heterotopically into baboon recipients. Methods: Recipients were treated to deplete anti‐Gal antibody with a non‐antigenic α‐Gal polyethylene glycol polymer (TPC) (n=2), TPC plus rituximab (anti‐CD20) (n=1) or were untreated (n=1). None of the recipients received T‐cell immunosuppression. Results: All Xgs failed within 7 days and showed evidence of a mixed humoral and cellular rejection process. Cellular infiltration consisting primarily of CD4+ T cells and few CD8+ T cells. Proliferation and cytotoxicity assays showed sensitization of CD4+ and CD8+ T cells that reacted with porcine IFN‐γ (pIFN‐γ)‐stimulated porcine aortic endothelial cells (PAEC). The CD4+ lymphocytes displayed greater cytotoxicity than CD8+ cells. An increased frequency of PAEC‐specific interleukin (IL) 2 and IFN‐γ‐secreting T cells was observed, suggesting a Th1 cytokine bias. An increase in the percentage of circulating CD4+CD28− cells was observed at the time of rejection and over 50% of the CD4+ cells recovered from residual pig tissue at necropsy lacked CD28 expression. Conclusions: These findings show that lymphocytes are efficiently stimulated by PAEC antigens and can mediate direct tissue destruction. These studies (1) provide an insight into the potential of cellular‐mediated cardiac Xg rejection, (2) show for the first time the induction of cytotoxic pig‐specific CD4+CD28− lymphocytes and (3) provide a rational basis for determining different modes of immunosuppression to treat Xg rejection.

Amplifying TLR-MyD88 Signals within Tumor-Specific T Cells Enhances Antitumor Activity to Suboptimal Levels of Weakly Immunogenic Tumor Antigens

The efficacy of T cell-based immunotherapy to treat cancer patients remains a challenge partly because of the weak activity toward subdominant tumor antigens (TAg) and to tumors expressing suboptimal TAg levels. Recent reports indicate that Toll-like receptor (TLR) stimulation on T cells can lower the activation threshold. In this study, we examined the antitumor activity and survival of TLR2-MyD88-stimulated CD8 T cells derived from melanoma patients and T-cell receptor transgenic pmel mice. TLR2-stimulated pmel CD8 T cells, but not TLR2(-/-)pmel or MyD88(-/-)pmel T cells, responded to significantly lower TAg levels and resulted in increased production of effector molecules and cytotoxicity. Wild-type or MyD88(-/-) mice treated with TLR2 ligand and pmel T cells, but not TLR2(-/-)pmel or MyD88(-/-)pmel T cells, showed tumor regression of an established melanoma tumor. Overexpressing TLR2 in TAg-specific T cells eradicated tumors; four times fewer cells were needed to generate antitumor responses. The enhanced antitumor activity of TLR2-MyD88-stimulated T cells was associated with increased effector function but perhaps more importantly with improved survival of T cells. Activating TLR-MyD88 signals in patient-derived T cells also reduced the activation threshold to several weakly immunogenic TAgs, resulting in increased cytokine production, expansion, and cytotoxicity. These data highlight a previously unappreciated role for activating TLR-MyD88 signals in tumor-reactive T lymphocytes.

Biology of T lymphocytes

T cells constitute one arm of the adaptive immune system. The accumulating information on various aspects of T-cell biology shows the intricacies in the regulation of immune responses. How we translate the cellular and molecular details of this regulation into innovation and development of therapies for disease management remains a fundamental, but exciting, challenge.

MHC-Binding Peptides as Immunotherapeutics for Cancer

Cytotoxic and helper T lymphocytes react with peptides associated to MHC class I and class I1 molecules respectively. Although most T-cell responses appear to be directed towards antigens derived from infectious agents, there are many examples of T lymphocytes recognizing and destroying tumor cells. Tumor-infiltrating lymphocyte (TIL) therapy in metastatic advanced cancer patients has shown in a limited number of cases, to be an efficient way of destroying large tumor masses and achieve remissions. However, probably because the large heterogeneity in cell population and potency between various TIL preparation, positive results with this type of therapy have been very inconsistent. Thus, it would be of benefit to develop ways to produce Tcell preparations for adoptive therapy that uniformly display high anti-tumor activity and specificity.