Dendritic cell–based (DC-based) immunotherapy represents a promising approach to the prevention and treatment of many diseases, including cancer, but current strategies have met with only limited success in clinical and preclinical studies. Previous studies have demonstrated that a TAT peptide derived from the HIV TAT protein has the ability to transduce peptides or proteins into various cells. Here, we describe the use of TAT-mediated delivery of T cell peptides into DCs to prolong antigen presentation and enhance T cell responses. While immunization of mice with DCs pulsed with an antigenic peptide derived from the human TRP2 protein generated partial protective immunity against B16 tumor, immunization with DCs loaded with a TAT-TRP2 peptide resulted in complete protective immunity, as well as significant inhibition of lung metastases in a 3-day tumor model. Although both DC/TRP2 and DC/TAT-TRP2 immunization increased the number of TRP2-specific CD8+ T cells detected by Kb/TRP2 tetramers, T cell activity elicited by DC/TAT-TRP2 was three- to tenfold higher than that induced by DC/TRP2. Furthermore, both CD4+ and CD8+ T cells were required for antitumor immunity demonstrated by experiments with antibody depletion of subsets of T cells, as well as with various knockout mice. These results suggest that a TAT-mediated antigen delivery system may have important clinical applications for cancer therapy.
Helen Y. Wang, Tihui Fu, Gang Wang, Gang Zeng, Donna M. Perry-Lalley, James C. Yang, Nicholas P. Restifo, Patrick Hwu, Rong-Fu Wang
The Th1 cytokines IL-2 and IFN-γ, which inhibit T cell proliferation and promote activation induced cell death, may be required to diminish alloreactive T cell numbers and to foster tolerance across full allogeneic barriers. However, we hypothesized that these cytokines might be dispensable when the alloreactive T cell clone size is relatively small, as is seen in recipients of minor-mismatched grafts. We show that alloreactive T cell clone size of C57BL/6 mice against multiple minor-mismatched 129X1/sv mice was ∼4–9-fold smaller than that against MHC-mismatched BALB/c mice. In the MHC-mismatched combination, CD28-B7 blockade by CTLA4Ig induced long-term graft survival in wild-type recipients, but this treatment was ineffective in IFNγ–/– or IL-2–/– recipients. In contrast, in the minor-mismatched combination, CTLA4Ig induced long-term allograft survival in wild-type, IFNγ–/–, and IL-2–/– recipients. Bcl-xL transgenic animals, which are defective in "passive" T cell death, are likewise sensitive to the effects of CTLA4Ig only in the setting of the minor-mismatch grafts. Therefore, the alloreactive T cell clone size is an important determinant affecting the need for Th1 cytokines and T cell death in tolerance induction. These data have implications for the design of tolerance strategies in transplant recipients with varying degrees of MHC mismatching.
Koji Kishimoto, Sigrid Sandner, Jaime Imitola, Masayuki Sho, Yongsheng Li, Peter B. Langmuir, David M. Rothstein, Terry B. Strom, Laurence A. Turka, Mohamed H. Sayegh
To mount an effective immune response, T cells must divide in response to antigen contact. To maintain tolerance, mucosal lamina propria T cells (LPTs) may adapt their cycling to an antigen-rich gut stimulatory environment. Here, we compared the cell cycle kinetics of LPTs and peripheral blood T cells (PBTs) before and after CD3- and CD2-mediated activation. While CD3-activated naive (CD45RA+) and memory (CD45RO+) PBTs peaked in the S and G2/M phase at 2–3 days, CD3-activated LPTs peaked at 4–6 days. In contrast, CD2 activation induced modest PBT but vigorous LPT cycling. The doubling time of CD3-activated PBTs was 1 day, while that of CD3- or CD2-activated LPTs was 2 days. LPTs failed to upregulate cyclin-dependent kinase 4 and cyclin D3, but Rb phosphorylation and cyclin A and B1 upregulation were induced by CD2 engagement. The extents of clonal expansion in LPT and PBT were comparable, indicating that LPTs’ slow replication delays but does not hinder cell division. CD2-activated LPTs displayed a striking upregulation of p53, whose blockade by antisense oligonucleotides accelerated their S phase transit time to that of CD3-activated PBTs. By slowing LPT cycling, p53 may act as a negative regulator of mucosal immunity, promoting immunological tolerance by preventing excessive T cell replication.
Andreas Sturm, Jugoh Itoh, James W. Jacobberger, Claudio Fiocchi
Vissia Viglietta, Sally C. Kent, Tihamer Orban, David A. Hafler
To understand the relationship between host antigen-presenting cells (APCs) and donor T cells in initiating graft-versus-host disease (GVHD), we followed the fate of host dendritic cells (DCs) in irradiated C57BL/6 (B6) recipient mice and the interaction of these cells with minor histocompatibility antigen- (miHA-) mismatched CD8+ T cells from C3H.SW donors. Host CD11c+ DCs were rapidly activated and aggregated in the T cell areas of the spleen within 6 hours of lethal irradiation. By 5 days after irradiation, <1% of host DCs were detectable, but the activated donor CD8+ T cells had already undergone as many as seven divisions. Thus, proliferation of donor CD8+ T cells preceded the disappearance of host DCs. When C3H.SW donor CD8+ T cells were primed in vivo in irradiated B6 mice or ex vivo by host CD11c+ DCs for 24–36 hours, they were able to proliferate and differentiate into IFN-γ–producing cells in β2-microglobulin–deficient (β2m–/–) B6 recipients and to mediate acute GVHD in β2m–/– → B6 chimeric mice. These results indicate that, although host DCs disappear rapidly after allogeneic bone marrow transplantation, they prime donor T cells before their disappearance and play a critical role in triggering donor CD8+ T cell–mediated GVHD.
Yi Zhang, Jean-Pierre Louboutin, Jiang Zhu, Adam J. Rivera, Stephen G. Emerson
Toll-like receptor 2 (TLR2) and TLR4 play important roles in the early innate immune response to microbial challenge. To clarify the functional roles of TLRs 2 and 4 in mast cells, we examined bone marrow–derived mast cells (BMMCs) from TLR2 or TLR4 gene-targeted mice. Peptidoglycan (PGN) from Staphylococcus aureus stimulated mast cells in a TLR2-dependent manner to produce TNF-α, IL-4, IL-5, IL-6, and IL-13, but not IL-1β. In contrast, LPS from Escherichia coli stimulated mast cells in a TLR4-dependent manner to produce TNF-α, IL-1β, IL-6, and IL-13, but not IL-4 nor IL-5. Furthermore, TLR2- but not TLR4-dependent mast cell stimulation resulted in mast cell degranulation and Ca2+ mobilization. In a mast cell–dependent model of acute sepsis, TLR4 deficiency of BMMCs in mice resulted in significantly higher mortality because of defective neutrophil recruitment and production of proinflammatory cytokines in the peritoneal cavity. Intradermal injection of PGN led to increased vasodilatation and inflammation through TLR2-dependent activation of mast cells in the skin. Taken together, these results suggest that direct activation of mast cells via TLR2 or TLR4 by respective microligands contributes to innate and allergic immune responses.
Volaluck Supajatura, Hiroko Ushio, Atsuhito Nakao, Shizuo Akira, Ko Okumura, Chisei Ra, Hideoki Ogawa
Leukotrienes are lipid mediators that evoke primarily proinflammatory responses by activating receptors present on virtually all cells. The production of leukotrienes is tightly regulated, and expression of 5-lipoxygenase, the enzyme required for the first step in leukotriene synthesis, is generally restricted to leukocytes. Arachidonic acid released from the cell membrane of activated leukocytes is rapidly converted to LTA4 by 5-lipoxygenase. LTA4 is further metabolized to either LTC4 or LTB4 by the enzyme LTC4 synthase or LTA4 hydrolase, respectively. Unlike 5-lipoxygenase, these enzymes are expressed in most tissues. This observation previously has led to the suggestion that LTA4 produced by leukocytes may, in some cases, be delivered to other cell types before being converted into LTC4 or LTB4. While in vitro studies indicate that this process, termed transcellular biosynthesis, can lead to the production of leukotrienes, it has not been possible to determine the significance of this pathway in vivo. Using a series of bone marrow chimeras generated from 5-lipoxygenase– and LTA4 hydrolase–deficient mice, we show here that transcellular biosynthesis contributes to the production of leukotrienes in vivo and that leukotrienes produced by this pathway are sufficient to contribute significantly to the physiological changes that characterize an ongoing inflammatory response.
Jean-Etienne Fabre, Jennifer L. Goulet, Estelle Riche, MyTrang Nguyen, Kenneth Coggins, Steven Offenbacher, Beverly H. Koller
Rotaviruses are the leading cause of severe diarrheal disease in young children. Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA mAb’s directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice. We investigated whether the anti-VP6 IgA (7D9) mAb could inhibit rotavirus replication inside epithelial cells and found that 7D9 acted at an early stage of infection to neutralize rotavirus following antibody lipofection. Using electron cryomicroscopy, we determined the three-dimensional structure of the virus-antibody complex. The attachment of 7D9 IgA to VP6 introduces a conformational change in the VP6 trimer, rendering the particle transcriptionally incompetent and preventing the elongation of initiated transcripts. Based on these observations, we suggest that anti-VP6 IgA antibodies confers protection in vivo by inhibiting viral transcription at the start of the intracellular phase of the viral replication cycle.
Ningguo Feng, Jeffrey A. Lawton, Joana Gilbert, Nelly Kuklin, Phuoc Vo, B.V. Venkataram Prasad, Harry B. Greenberg
While the pathologic mechanisms responsible for organ-specific tissue damage in primary biliary cirrhosis (PBC) remain an enigma, it has been suggested that the pathology is mediated by autoreactive T cells infiltrating the intrahepatic bile ducts. Previously, we have documented that there is 100-fold enrichment in the frequency of CD4+ autoreactive T cells in the liver that are specific for peptides encoded by the E2 components of the pyruvate dehydrogenase complexes (PDC-E2). We have also recently characterized the first MHC class I–restricted epitope for PDC-E2, namely amino acid 159–167, a region very similar to the epitope recognized by MHC class II–restricted CD4+ cells and by autoantibodies. The effector functions of these PDC-E2159-167–specific CD8+ cytotoxic T lymphocytes (CTLs) are not well understood. We have taken advantage of tetramer technology and report herein that there is tenfold increase in the frequency of PDC-E2159-167–specific CTLs in the liver as compared with the blood in PBC. In addition, the precursor frequency of the CTLs in blood was significantly higher in early-stage PBC. Of interest was the fact that, upon stimulation with the peptide, the response of PDC-E2159-167 tetramer-positive cells is heterogeneous with respect to IFN-γ synthesis. These data, we believe for the first time, document the enrichment of autoantigen-specific CD8+ T cells in the PBC liver, suggesting that CD8+ T cells play a significant role in the immunopathogenesis of PBC.
Hiroto Kita, Shuji Matsumura, Xiao-Song He, Aftab A. Ansari, Zhe-Xiong Lian, Judy Van de Water, Ross L. Coppel, Marshall M. Kaplan, M. Eric Gershwin
Older bone marrow transplantation (BMT) recipients are at heightened risk for acute graft-versus-host disease (GVHD) after allogeneic BMT, but the causes of this association are poorly understood. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older mice. GVHD mortality, morbidity, and pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice, which also secreted more TNF-α and IL-12 after LPS stimulation. In a B6 → B6D2F1 model, CD4+ donor T cells but not CD8+ T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using B6D2F1 BM chimeras created with either old or young BM. Four months after chimera creation, allogeneic BMT from B6 donors caused significantly worse GVHD in old BM chimeras. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. These data demonstrate a hitherto unsuspected mechanism of amplified donor T cell responses by aged allogeneic host APCs that increases acute GVHD in aged recipients in this BMT model.
Rainer Ordemann, Raymond Hutchinson, Jeffrey Friedman, Steven J. Burakoff, Pavan Reddy, Ulrich Duffner, Thomas M. Braun, Chen Liu, Takanori Teshima, James L.M. Ferrara