Despite extensive study, the role of phosphatidylinositol 3-kinase (PI3-kinase) activation in CD28 function has been highly contentious. To definitively address this question, we generated knock-in mice expressing mutations in two critical domains of the cytoplasmic tail of CD28. Mutation of the proximal tyrosine motif interrupted PI3-kinase binding and prevented CD28-dependent phosphorylation of protein kinase B (PKB)/ Akt; however, there was no detectable effect on interleukin-2 (IL-2) secretion, expression of Bcl-X L , or on T-cell function in vivo. Furthermore, we demonstrate that signaling initiated by the C-terminal proline motif is directly responsible for tyrosine phosphorylation of phosphoinosotide-dependent kinase 1, protein kinase C, and glycogen synthase kinase 3␤, as well as contributing to threonine phosphorylation of PKB. T cells mutated in this domain were profoundly impaired in IL-2 secretion, and the mice had marked impairment of humoral responses as well as less severe disease manifestations in experimental allergic encephalomyelitis. These data demonstrate that the distal proline motif initiates a critical nonredundant signaling pathway, whereas direct activation of PI3-kinase by the proximal tyrosine motif of CD28 is not required for normal T-cell function.CD28 and T-cell receptor (TCR)-derived signals act synergistically, leading to optimal T-cell proliferation, cytokine secretion, and cell survival (for a review, see reference 32). The importance of CD28 in vivo is evidenced by impaired responses of CD28-deficient mice in a number of model systems, including allergic airway inflammation and experimental allergic encephalomyelitis (EAE) (13,34). In addition, the recent development of inhibitors of CD28 as effective therapeutics for autoimmune disease and transplant immunosuppression further emphasizes the critical role of this receptor in human disease (21,57).Despite extensive study, the biochemical mechanism(s) that mediates CD28 function remains incompletely understood. Specific motifs within the cytoplasmic tail of CD28 have been identified that trigger distinct signaling pathways. Binding and activation of Src family kinases to the distal proline motif (sequence PYAP) initiates signaling, whereas the proximal tyrosine motif (sequence YMNM) binds and activates the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) as well as other adaptor proteins, including Grb2 and GADS (12,27,28,33,42,48,51). Studies have suggested that both motifs contribute to CD28-dependent interleukin-2 (IL-2) secretion and proliferation but that the upregulation of Bcl-X L is uniquely dependent on PI3-kinase activation by the proximal tyrosine at position 170 (11,25,43). The potential for extensive overlap between pathways initiated by each motif exists, as well as overlap between CD28 and TCR-derived signals, making it unclear as to whether CD28 initiates any critical, nonredundant signaling pathway.We generated gene-targeted knock-in mice expressing either wild-type CD28 or mutations in the proximal tyr...

182 Protective immunity against infection requires sustained antibody production by long-lived plasma cells (LLPC) that survive for years/decades within specialized niches. What regulates/supports this survival remains largely unknown. However, it has been shown that normal and transformed (human multiple myeloma) LLPC are critically dependent on the bone marrow microenvironment, including cell-to-cell interactions. This lead us to hypothesize that modulating these interactions could either enhance antibody production for vaccine development or, conversely, compromise the survival of transformed/normal LLPC in the bone marrow microenvironment. We have shown that the T cell costimulatory receptor CD28 expressed on both normal and transformed LLPC, plays an essential role in survival. While LLPC and short-lived plasma cells (SLPC) both express CD28, its activation in vitro only significantly increases survival and IgG production in LLPC. Consistent with these findings, we show in vivo, vaccinated bone marrow CD28−/−:μMT chimeras had significantly reduced long-term antibody titers and decreased LLPC (but not SLPC) t1/2 from 426 to 63 days. These findings demonstrate the existence of a distinct bone marrow (BM) LLPC subset necessary to sustain antibody titers, and establish a central role for CD28 function in the maintenance of plasma cells and humoral immunity. While CD28 signaling has been shown to play an important role in maintaining long-term humoral immune responses, the mechanism by which CD28 signaling affects PC function has not yet been determined. To further elucidate CD28 signaling in BM PC, we utilized CD28 conditional knock-in mice. In these mice, the CD28 cytoplasmic tail is mutated at either the YMNM or proline-rich motifs, resulting in an inhibition of PI3K or vav signaling, respectively. We found that CD28-vav signaling deficient BM PC were selectively depleted in vivo and could not be rescued by CD28 activation in in vitro serum starvation conditions. Furthermore, anti-CD28 mAb drove a 1.5 fold increase in Blimp-1 expression in BM PC, compared to control. This increase was regulated through the CD28-vav signaling pathway, as CD28 activation in CD28-vav signaling deficient BM PC did not increase Blimp-1 expression. To further determine if CD28 is acting directly on the Blimp-1 promoter, we examined in silico for a CD28RE composite element, previously reported to transcriptionally regulate IL-2 production in T cells and IL-8 production in myeloma cells. To our surprise, we found a CD28RE “like” site 4712bp upstream of the Blimp-1 start site. To confirm CD28 transcriptionally regulates Blimp-1 promoter activity, we transfected the CD28+ plasmacytoma cell line J558 with full-length or truncated Blimp-1 promoter constructs (i.e. 7000bp, 4500bp, 1500bp). We found CD28 activation enhances Blimp-1 activity in J558 cells transfected with full-length-Blimp-1, and this activity was lost when the promoter was truncated. Using site-directed mutagenesis, we confirmed the CD28RE is required for induction of Blimp-1 in PC. Furthermore, we show CD28 activation of Blimp-1 increases the BCMA receptor in BM PC. Taken together, our data suggests the CD28-vav signaling pathway in PC induces a CD28RE composite element, which is necessary for the induction of the key PC transcriptional regulator Blimp-1, required to maintain LLPC and humoral immunity. Disclosures: No relevant conflicts of interest to declare.

The T cell costimulatory molecule CD28 plays an important role in the thymic generation of Foxp3+ regulatory T cells (Tregs) essential for the maintenance of self-tolerance. In this study, we show that a cell-intrinsic signal from CD28 is involved in the generation of cytokine-responsive Foxp3− precursors using studies of mixed bone marrow chimeras as well as TCR-specific generation of Foxp3+ cells using intrathymic transfer of TCR-transgenic thymocytes expressing a natural Treg TCR. Contrary to a previous report, the analysis of CD28 mutant knockin mice revealed that this cell-intrinsic signal is only partially dependent on the Lck-binding PYAP motif. Surprisingly, even though the absence of CD28 resulted in a 6-fold decrease in thymic Tregs, the TCR repertoires of CD28-deficient and sufficient cells were largely overlapping. Thus, these data suggest that CD28 does not operate by markedly enlarging the repertoire of TCRs available for Treg development, but rather by improving the efficiency of Treg development of thymocytes expressing natural Treg TCRs.

Pancreatic adenocarcinoma is the fourth leading cause of cancer death with an overall 5-year survival of less than 5%. As there is ample evidence that pancreatic adenocarcinomas elicit antitumor immune responses, identification of pancreatic cancer-associated antigens has spurred the development of vaccination-based strategies for treatment. While promising results have been observed in animal tumor models, most clinical studies have found only limited success. As most trials were performed in patients with advanced pancreatic cancer, the contribution of immune suppressor mechanisms should be taken into account. In this article, we detail recent work in tumor antigen vaccination and the recently identified mechanisms of immune suppression in pancreatic cancer. We offer our perspective on how to increase the clinical efficacy of vaccines for pancreatic cancer.

One strategy in cancer immunotherapy is to capitalize on the key immunoregulatory and antigen presenting capabilities of dendritic cells (DCs). This approach is dependent on efficient delivery of tumor specific antigens to DCs, which subsequently induce an anti-tumor T-cell mediated immune response. Human adenovirus serotype 5 (HAdV5) has been used in human studies for gene delivery, but has limited infection in DCs, which lack the proper receptors. Addition of the porcine fiber knob (PK) from porcine adenovirus type 4 to HAdV5 allows the virus to deliver genetic material via binding to glycosylated surface proteins and bypasses the coxsackie-and-adenovirus receptor required by wild-type HAdV5. In this study we explored the potential therapeutic applications of an adenovirus with PK-based tropism against cancers expressing mesothelin. Infectivity and gene transfer assays were used to compare Ad5-PK to wild-type HAdV5. Mouse models were used to demonstrate peptide specificity and T-cell responses. We show that the PK modification highly augmented infection of DCs, including the CD141+ DC subset, a key subset for activation of naïve CD8+ T-cells. We also show that Ad5-PK increases DC infectivity and tumor specific antigen expression. Finally, vaccination of mice with the Ad5-PK vector resulted in enhanced T-cell-mediated interferon gamma (IFN-γ) release in response to both mesothelin peptide and a tumor line expressing mesothelin. Ad5-PK is a promising tool for cancer immunotherapy as it improves infectivity, gene transfer, protein expression, and subsequent T-cell activation in DCs compared to wild-type HAdV5 viruses.

T cell activation requires signaling through the TcR and engagement of a costimulatory receptor. CD28 is the most important costimulatory receptor; however, the signaling pathways initiated by CD28 remain highly controversial. To examine this, we generated gene targeted knock-in mice that express mutations in either the 1) proximal YMNM (Y170F), 2) distal PYAP (AYAA), or 2) both the YMNM and PYAP (Y170F/AYAA) motifs of the cytoplasmic tail of CD28. Mutation of the PYAP motif resulted in impaired proliferation, cytokine secretion and B cell help. In contrast, the Y170F mutant had normal to slightly increased proliferation and IL-2 production in vitro, as well as a prolonged duration of inflammation in vivo. T cells from both mutant knock-in mice upregulated the activation markers CD25, CD69 and CD95. Interestingly, the CD28 knock-out and CD28-AYAA knock-in mice both fail to induce the expression of ICOS on T cells, a critical component for germinal center production. To determine if additional motifs mediate CD28-dependent functions, we tested the responses of the double mutant. The phenotype of the Y170F/AYAA double mutant was not substantially different than the single AYAA mutant. These data strongly suggest that the residual T cell responses present in the AYAA mouse are not due to signaling initiated by the YMNM motif, and that additional signaling pathways mediate the remaining CD28-dependent effects.