Sarah L. Buchan

Electroporation as a “Prime/Boost” Strategy for Naked DNA Vaccination against a Tumor Antigen

We have developed novel DNA fusion vaccines encoding tumor Ags fused to pathogen-derived sequences. This strategy activates linked T cell help and, using fragment C of tetnus toxin, amplification of anti-tumor Ab, CD4+, and CD8+ T cell responses is achievable in mice. However, there is concern that simple DNA vaccine injection may produce inadequate responses in larger humans. To overcome this, we tested electroporation as a method to increase the transfection efficiency and immune responses by these tumor vaccines in vivo in mice. Using a DNA vaccine expressing the CTL epitope AH1 from colon carcinoma CT26, we confirmed that effective priming and tumor protection in mice are highly dependent on vaccine dose and volume. However, suboptimal vaccination was rendered effective by electroporation, priming higher levels of AH1-specific CD8+ T cells able to protect mice from tumor growth. Electroporation during priming with our optimal vaccination protocol did not improve CD8+ T cell responses. In contrast, electroporation during boosting strikingly improved vaccine performance. The prime/boost strategy was also effective if electroporation was used at both priming and boosting. For Ab induction, DNA vaccination is generally less effective than protein. However, prime/boost with naked DNA followed by electroporation dramatically increased Ab levels. Thus, the priming qualities of DNA fusion vaccines, integrated with the improved Ag expression offered by electroporation, can be combined in a novel homologous prime/boost approach, to generate superior antitumor immune responses. Therefore, boosting may not require viral vectors, but simply a physical change in delivery, facilitating application to the cancer clinic.

DNA Fusion Vaccine Designed to Induce Cytotoxic T Cell Responses Against Defined Peptide Motifs: Implications for Cancer Vaccines

DNA vaccination offers a strategy to induce immune attack on cancer cells, but tumor Ags are often weak. Inclusion of a “foreign” protein increases immunogenicity, and we found previously that fusion of the fragment C (FrC) of tetanus toxin to the tumor Ag sequence promotes Ab and CD4+ responses against B cell tumors. For CTL responses, use of the full two-domain FrC may be less helpful, because known immunogenic MHC class I-binding peptides in the second domain could compete with attached tumor-derived epitopes. Therefore, we removed the second domain, retaining the N-terminal domain, which contains a “universal” helper epitope. We investigated the ability to induce CTL responses of candidate peptides placed at the C terminus of this domain. As test peptides, we repositioned the two known CTL motifs from the second domain to this site. Strong CTL responses to each peptide were induced by the engineered construct, as compared with the native FrC construct. Induced CTLs were able to specifically kill tumor cells transfected with FrC as a surrogate tumor Ag both in vitro and in vivo. Further reduction of the domain to a short helper epitope generated only weak CTL responses against fused peptides, and synthetic peptides mixed with the plasmid containing the first domain were ineffective. The single FrC domain-peptide vaccine design also was able to induce high levels of CTLs against a known epitope from carcinoembryonic Ag. Response to peptide was suppressed if two FrC domains were present, consistent with immunodominance. These principles and designs may have relevance for cancer vaccines delivered via DNA.

Critical Components of a DNA Fusion Vaccine Able to Induce Protective Cytotoxic T Cells Against a Single Epitope of a Tumor Antigen

DNA vaccines can activate immunity against tumor Ags expressed as MHC class I-associated peptides. However, priming of CD8+ CTL against weak tumor Ags may require adjuvant molecules. We have used a pathogen-derived sequence from tetanus toxin (fragment C (FrC)) fused to tumor Ag sequences to promote Ab and CD4+ T cell responses. For induction of CD8+ T cell responses, the FrC sequence has been engineered to remove potentially competitive MHC class I-binding epitopes and to improve presentation of tumor epitopes. The colon carcinoma CT26 expresses an endogenous retroviral gene product, gp70, containing a known H2-Ld-restricted epitope (AH1). A DNA vaccine encoding gp70 alone was a poor inducer of CTL, and performance was not significantly improved by fusion of full-length FrC. However, use of a minimized domain of FrC, with the AH1 sequence fused to the 3′ position, led to rapid induction of high levels of CTL. IFN-γ-producing epitope-specific CTL were detectable ex vivo and these killed CT26 targets in vitro. The single epitope vaccine was more effective than GM-CSF-transfected CT26 tumor cells in inducing an AH1-specific CTL response and equally effective in providing protection against tumor challenge. Levels of AH1-specific CTL in vivo were increased following injection of tumor cells, and CTL expanded in vitro were able to kill CT26 cells in tumor bearers. Pre-existing immunity to tetanus toxoid had no effect on the induction of AH1-specific CTL. These data demonstrate the power of epitope-specific CTL against tumor cells and illustrate a strategy for priming immunity via a dual component DNA vaccine.

Sustained TL1A expression modulates effector and regulatory T-cell responses and drives intestinal goblet cell hyperplasia

The tumor necrosis factor (TNF) superfamily protein TNF-like 1A (TL1A) is the ligand for death receptor 3 (DR3). TL1A is induced on activated dendritic cells (DCs) and its expression has been linked to human inflammatory bowel disease. To address how TL1A might influence intestinal inflammation, we generated transgenic mice that constitutively express TL1A on DCs. TL1A transgenic mice developed striking goblet cell hyperplasia in the ileum that was associated with elevated interleukin (IL)-13 levels in the small intestine. IL-13- and IL-17-producing small intestinal lamina propria T cells were increased in TL1A transgenic mice. TL1A also enhanced regulatory T (Treg) cell turnover in vivo and directly stimulated Treg cell proliferation in vitro. The presence of TL1A attenuated the ability of Treg cells to suppress conventional T cells, an effect that required DR3 signaling in either conventional T cells or Treg cells. Our findings identify mechanisms by which chronic DR3 signaling could promote pathogenesis in inflammatory bowel disease.

DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance

The Wilms tumor antigen, WT1, is associated with several human cancers, including leukemia. We evaluated WT1 as an immunotherapeutic target using our proven DNA fusion vaccine design, p.DOM-peptide, encoding a minimal tumor-derived major histocompatibility complex (MHC) class I-binding epitope downstream of a foreign sequence of tetanus toxin. Three p.DOM-peptide vaccines, each encoding a different WT1-derived, HLA-A2-restricted epitope, induced cytotoxic T lymphocytes (CTLs) in humanized transgenic mice expressing chimeric HLA-A2, without affecting hematopoietic stem cells. Mouse CTLs killed human leukemia cells in vitro, indicating peptide processing/presentation. Low numbers of T cells specific for these epitopes have been described in cancer patients. Expanded human T cells specific for each epitope were lytic in vitro. Focusing on human WT1(37-45)-specific cells, the most avid of the murine responses, we demonstrated lysis of primary leukemias, underscoring their clinical relevance. Finally, we showed that these human CTL kill target cells transfected with the relevant p.DOM-peptide DNA vaccine, confirming that WT1-derived epitopes are presented to T cells similarly by tumors and following DNA vaccination. Together, these data link mouse and human studies to suggest that rationally designed DNA vaccines encoding WT1-derived epitopes, particularly WT1(37-45), have the potential to induce/expand functional tumor-specific cytotoxic responses in cancer patients.

Common Patterns of B Cell Perturbation and Expanded V4-34 Immunoglobulin Gene Usage in Autoimmunity and Infection

Features of the lymphocyte population in systemic lupus erythematosus (SLE) include a disordered B cell profile and production of autoantibodies. An additional distinctive perturbation is the over-expression of V4-34-encoded serum immunoglobulins (Ig). A similar rise in V4-34-encoded Ig occurs in normal subjects following infection with c ertain herpesviruses, and is found in Epstein–Barr virus (EBV)-associated infectious mononucleosis (IM). To assess common and distinctive features of B cells in patients with SLE and IM, we compared the B cell profile and V4-34 gene involvement in patients with SLE and IM. B cell profiles from patients with IM paralleled those of patients with SLE, showing a differential loss of naïve and memory B cells and the maintenance of plasmablast/early plasma cells. Class-switched V4-34-encoded IgG from plasmablast/early plasma cells was evident both in patients with SLE and IM and revealed common features of oligoclonal expansions with most having undergone somatic hypermutation. It has been proposed that, in healthy individuals, expression of the V4-34 gene is specifically censored prior to isotype switch as a control on autoreactivity. If so, censoring is bypassed following EBV infection, after which equilibrium is restored. Continuing high serum levels in SLE may arise either by disordered regulation, or by subclinical reactivation of endogenous virus.

OX40- and CD27-Mediated Costimulation Synergizes with Anti–PD-L1 Blockade by Forcing Exhausted CD8+ T Cells To Exit Quiescence

Exhaustion of chronically stimulated CD8+ T cells is a significant obstacle to immune control of chronic infections or tumors. Although coinhibitory checkpoint blockade with anti–programmed death ligand 1 (PD-L1) Ab can restore functions to exhausted T cell populations, recovery is often incomplete and dependent upon the pool size of a quiescent T-bethigh subset that expresses lower levels of PD-1. In a model in which unhelped, HY-specific CD8+ T cells gradually lose function following transfer to male bone marrow transplantation recipients, we have explored the effect of shifting the balance away from coinhibition and toward costimulation by combining anti–PD-L1 with agonistic Abs to the TNFR superfamily members, OX40 and CD27. Several weeks following T cell transfer, both agonistic Abs, but especially anti-CD27, demonstrated synergy with anti–PD-L1 by enhancing CD8+ T cell proliferation and effector cytokine generation. Anti-CD27 and anti–PD-L1 synergized by downregulating the expression of multiple quiescence-related genes concomitant with a reduced frequency of T-bethigh cells within the exhausted population. However, in the presence of persistent Ag, the CD8+ T cell response was not sustained and the overall size of the effector cytokine-producing pool eventually contracted to levels below that of controls. Thus, CD27-mediated costimulation can synergize with coinhibitory checkpoint blockade to switch off molecular programs for quiescence in exhausted T cell populations, but at the expense of losing precursor cells required to maintain a response.

DNA fusion vaccines induce targeted epitope-specific CTLs against minor histocompatibility antigens from a normal or tolerized repertoire

We have designed DNA fusion vaccines able to induce high levels of epitope-specific CD8+ T cells, using linked CD4+ T cell help. Such vaccines can activate effective immunity against tumor Ags. To model performance against minor histocompatibility (H) Ags important in allogeneic hemopoietic stem cell transplantation, responses against the H2Db-restricted Uty and Smcy male HY epitopes have been investigated. Vaccination of females induced high levels of tetramer-specific, IFN-γ-producing CD8+ T cells against each epitope. Vaccines incorporating a single epitope primed effector CTL able to kill male splenocytes in vitro and in vivo, and HYDbUty-specific vaccination accelerated rejection of syngeneic male skin grafts. Priming against either epitope established long-term memory, expandable by injection of male cells. Expanded CD8+ T cells remained specific for the priming HY epitope, with responses to the second suppressed. To investigate vaccine performance in a tolerized repertoire, male mice were vaccinated with the fusion constructs. Strikingly, this also generated epitope-specific IFN-γ-producing CD8+ T cells with cytotoxic function. However, numbers and avidity were lower than in vaccinated females, and vaccinated males failed to reject CFSE-labeled male splenocytes in vivo. Nevertheless, these findings indicate that DNA fusion vaccines can mobilize CD8+ T cells against endogenous minor H Ags, even from a profoundly tolerized repertoire. In the transplantation setting, vaccination of donors could prime and expand specific T cells for in vivo transfer. For patients, vaccination could activate a potentially less tolerized repertoire against similar Ags that may be overexpressed by tumor cells, for focused immune attack.

Disturbances in peripheral blood B cell subpopulations in autoimmune patients.

A variety of cell surface markers are being used to identify B cell subpopulations in peripheral blood. Currently at least eight subpopulationshave been identified. Analyses of healthy individuals indicate that in general the various B cell subpopulations exist in relatively similar ratios in unrelated individuals. It has been demonstrated that B lymphocyte homeostasis is disturbed during infection and autoimmune disease. In this review we compare the distribution of B cell subpopulations in the peripheral blood of patients with systemic lupus erythematosus, rheumatoid arthritis and primary Sjogrens syndrome with each other, and with healthy individuals. The different autoimmune diseases have distinct changes in the B cell subpopulations. Understanding the nature of these B subpopulation signatures will potentially impact understanding the mechanisms of disease, diagnosis and therapy.

Death receptor 3 is essential for generating optimal protective CD41 T-cell immunity against Salmonella

The TNF receptor superfamily member death receptor 3 (DR3) exacerbates Th2‐ and Th17‐cell‐mediated inflammatory and autoimmune conditions, yet no role in host defence has been reported. Here, we examined the role of DR3 during infection with Salmonella enterica serovar Typhimurium. Infection resulted in protracted expression of the DR3 ligand TL1A but not the related TNF superfamily proteins OX40L or CD30L. TL1A expression was localized to splenic F4/80+ macrophages where S. enterica Typhimurium replicates, and temporally coincided with the onset of CD4+‐cell expansion. To address the relevance of the TL1A‐DR3 interaction, we examined immune responses to S. enterica Typhimurium in mice lacking DR3. Infected DR3‐/‐ mice harboured reduced numbers of antigen‐experienced and proliferating CD4+ T cells compared with WT mice. Furthermore, the frequency of IFN‐γ+ CD4+ T cells in DR3‐/‐ mice was lower throughout the time of bacterial clearance. Importantly, bacterial clearance, which is dependent on Th1 cells, was also impaired in DR3‐/‐ mice. This defect was intrinsic to CD4+ T cells as evidenced by an increase in bacterial burden in RAG2‐deficient mice receiving DR3‐/‐ CD4+ T cells compared with WT CD4+‐cell recipients. These data establish for the first time a role for DR3 in a host defence responce.