Angela Giorgini

Blockade of chronic graft-versus-host disease by alloantigen-induced CD4+CD25+Foxp3+ regulatory T cells in nonlymphopenic hosts.

CD4+CD25+ regulatory T cells (Tregs) are well known to suppress immunopathology induced in lymphopenic animals following T cell reconstitution, including acute graft‐versus‐host disease (GVHD) post‐bone marrow transplantation. The regulatory potential of this subset in nonlymphopenic hosts and in chronic, Th2‐mediated GVHD is less clear. We have generated alloantigen‐specific cells from CD4+CD25+ populations stimulated with MHC‐disparate dendritic cells and found them to express a stable Treg forkhead box p3+ phenotype with enhanced suppressive activity mediated by cell contact. When transferred into nonlymphopenic F1 hosts, nonspecific Tregs proliferated as rapidly as CD4+CD25− cells but displayed distinct growth kinetics in vitro. Tregs, expanded in response to alloantigen in vitro, displayed greatly enhanced suppressive activity, which was partially antigen‐specific. They were effective inhibitors of chronic GVHD, blocking donor cell engraftment, splenomegaly, autoantibody production, and glomerulonephritis. CD25+ and CD25− cells were equally susceptible to inhibition by immunosuppressive drugs targeting TCR signaling and rapamycin, but Tregs were resistant to inhibition by dexamethasone. The data indicate that alloantigen‐driven expansion, rather than homeostatic proliferation, is key to the effectiveness of CD4+CD25+ Tregs in GVHD and suggest that cellular therapy with alloantigen‐induced Tregs in combination with glucocorticoid treatment would be effective in prevention of chronic GVHD after immune reconstitution.

FcγRIII and FcγRIV are indispensable for acute glomerular inflammation induced by switch variant monoclonal antibodies

The relative ability of IgG subclasses to cause acute inflammation and the roles of specific effector mechanisms in this process are not clear. We explored this in an in vivo model of glomerular inflammation in the mouse. Trinitrophenol was planted on the glomerular basement membrane after conjugation to nephrotoxic Ab. The relative nephritogenicity of anti-trinitrophenol switch variant mAbs was then explored and shown to be IgG2a > IgG2b, with no disease caused by IgG1. Using knockout mice, we showed that FcγRIII was necessary for both neutrophil influx and glomerular damage induced by IgG2a and IgG2b. Surprisingly, IgG1 did not cause disease although it binds to FcγRIII. Using blocking Abs, we showed that this was explained by an additional requirement for FcγRIV, which does not bind to IgG1. IgG2a- or IgG2b-induced neutrophil influx was not affected by deficiency of either FcγRI or C3. Bone marrow chimeras were constructed to test the effect of combined deficiency of FcγRI and C3, and there was no effect on IgG2a- or IgG2b-mediated neutrophil influx. However, IgG2b-induced albuminuria and thrombosis were reduced in C3-deficient mice, showing an additional role for complement in IgG2b-mediated glomerular damage. The results show that IgG2a and IgG2b are the pathogenic subclasses in acute neutrophil-mediated glomerular inflammation, with an indispensable role for both FcγRIII and FcγRIV. Additionally, complement contributes to IgG2b-induced glomerular injury.

The Use of Filamentous Bacteriophage fd to Deliver MAGE-A10 or MAGE-A3 HLA-A2-Restricted Peptides and to Induce Strong Antitumor CTL Responses

Delivery of tumor-associated Ag-derived peptides in a high immunogenic form represents one of the key issues for effective peptide-based cancer vaccine development. We report herein the ability of nonpathogenic filamentous bacteriophage fd virions to deliver HLA-A2-restricted MAGE-A10254–262- or MAGE-A3271–279-derived peptides and to elicit potent specific CTL responses in vitro and in vivo. Interestingly, human anti-MAGE-A3271–279-specific CTLs were able to kill human MAGE-A3+ tumor cells, even if these cells naturally express a low amount of MAGE-A3271–279 peptide-HLA epitope surface complexes and are usually not recognized by CTLs generated by conventional stimulation procedures. MAGE-A3271–279-specific/CD8+ CTL clones were isolated from in vitro cultures, and their high avidity for Ag recognition was assessed. Moreover, in vivo tumor protection assay showed that vaccination of humanized HHD (HLA-A2.1+/H2-Db+) transgenic mice with phage particles expressing MAGE-A3271–279-derived peptides hampered tumor growth. Overall, these data indicate that engineered filamentous bacteriophage virions increase substantially the immunogenicity of delivered tumor-associated Ag-derived peptides, thus representing a novel powerful system for the development of effective peptide-based cancer vaccines.

Transferred Antigen-Specific TH17 but not TH1 Cells Induce Crescentic Glomerulonephritis in Mice

To explore the role of antigen-specific CD4(+) T cells in glomerulonephritis, we administered ovalbumin 323-339 peptide conjugated to glomerular-binding polyclonal antibody and induced disease in RAG1(-/-) mice with CD4(+) T cells from OT2 × RAG1(-/-) mice. These OT2 × RAG1(-/-) mice have a transgenic T-cell receptor specific for this peptide. When CD4(+) T cells were primed in vivo, crescentic glomerulonephritis developed after 21 days in mice given peptide-conjugated glomerular-binding antibody but not unconjugated antibody control. We then investigated the relative roles of T(H)1 and T(H)17 cells, using Fab(2) fragments of glomerular-binding antibody to exclude a role for antibody in this model. T cells from OT2 × RAG1(-/-) mice were polarized in vitro, and T(H)1 or T(H)17 cell lines were injected into mice that were also given peptide-conjugated Fab(2) or unconjugated Fab(2) control, giving four experimental groups. After 21 days crescentic glomerulonephritis was seen in mice receiving T(H)17 cells and peptide-conjugated Fab(2) but in none of the other three groups. These results suggest that T(H)17 but not T(H)1 cells can induce crescentic glomerulonephritis.

Toll-Like Receptor 4 Stimulation Triggers Crescentic Glomerulonephritis by Multiple Mechanisms Including a Direct Effect on Renal Cells

A role for toll-like receptor 4 (TLR4) has been suggested in previous studies of glomerulonephritis, but the complex integration of these effects has not been explored. To separate effects on the innate and adaptive immune responses, we use the autologous nephrotoxic nephritis model with two disease induction protocols. First, we give a TLR4 ligand at the time of immunization and show the effects are mediated via TLR4 by comparing wild-type and TLR4-deficient mice. In wild-type mice histological measures of disease and serum creatinine are all at least twice as high as TLR4-deficient mice, due to an enhanced immune response to the nephritogenic sheep IgG. Second, we stimulate TLR4 later in the course of disease development and construct four groups of bone marrow chimeric or sham chimeric mice to study the role of TLR4 on bone marrow or renal cells. The most striking finding is that renal cell TLR4 stimulation increases glomerular crescent formation, with a mean of 21% and 25% in the two groups of mice with renal cell TLR4 compared with 0.1% and 0.6% in the two groups without, with differences mirrored by changes in serum creatinine. These findings, in a single disease model, illustrate that TLR4 stimulation triggers crescentic glomerulonephritis by effects on both the adaptive and innate immune response, with a crucial direct effect on renal cells.

Toll-Like Receptor 2 or Toll-Like Receptor 4 Deficiency Does Not Modify Lupus in MRLlpr Mice

Systemic lupus erythematosus is an autoimmune disease with a high morbidity and nephritis is a common manifestation. Previous studies in murine lupus models have suggest a role for Toll-like receptor 2 and 4. We examined the role of these molecules in MRL lpr mice which is one of the most established and robust murine models. We compared disease parameters in Toll-like receptor 2 or Toll-like receptor 4 deficient mice with their littermate controls. We found no difference in the severity of glomerulonephritis as assessed by histology, serum creatinine and albuminuria when Toll-like receptor 2 or Toll-like receptor 4 deficient MRLlpr mice were compared with Toll-like receptor sufficient controls. We also found similar levels of anti-dsDNA and anti-ssDNA antibodies. These results show that Toll-like receptor 2 and Toll-like receptor 4 do not play a significant role in MRLlpr mice, and therefore they may not be important in human lupus.