B lymphocyte depletion by CD20 monoclonal antibody prevents diabetes in nonobese diabetic mice despite isotype-specific differences in Fc gamma R effector functions.

NOD mice deficient for B lymphocytes from birth fail to develop autoimmune or type 1 diabetes. To assess whether B cell depletion influences type 1 diabetes in mice with an intact immune system, NOD female mice representing early and late preclinical stages of disease were treated with mouse anti-mouse CD20 mAbs. Short-term CD20 mAb treatment in 5-wk-old NOD female mice reduced B cell numbers by approximately 95%, decreased subsequent insulitis, and prevented diabetes in >60% of littermates. In addition, CD20 mAb treatment of 15-wk-old NOD female mice significantly delayed, but did not prevent, diabetes onset. Protection from diabetes did not result from altered T cell numbers or subset distributions, or regulatory/suppressor T cell generation. Rather, impaired CD4+ and CD8+ T cell activation in the lymph nodes of B cell-depleted NOD mice may delay diabetes onset. B cell depletion was achieved despite reduced sensitivity of NOD mice to CD20 mAbs compared with C57BL/6 mice. Decreased B cell depletion resulted from deficient FcgammaRI binding of IgG2a/c CD20 mAbs and 60% reduced spleen monocyte numbers, which in combination reduced Ab-dependent cellular cytotoxicity. With high-dose CD20 mAb treatment (250 microg) in NOD mice, FcgammaRIII and FcgammaRIV compensated for inadequate FcgammaRI function and mediated B cell depletion. Thereby, NOD mice provide a model for human FcgammaR polymorphisms that reduce therapeutic mAb efficacy in vivo. Moreover, this study defines a new, clinically relevant approach whereby B cell depletion early in the course of disease development may prevent diabetes or delay progression of disease.

The type and frequency of immunoregulatory CD4+ T-cells govern the efficacy of antigen-specific immunotherapy in nonobese diabetic mice.

Antigen-specific immunotherapy, an approach to selectively block autoimmune diabetes, generally declines in nonobese diabetic (NOD) mice as disease progresses. To define the parameters influencing the efficacy of antigen-specific immunotherapy once diabetes is established, plasmid DNA (pDNA) vaccination was used to suppress autoimmune-mediated destruction of syngeneic islet grafts in diabetic NOD recipients. pDNAs encoding a glutamic acid decarboxylase 65 (GAD65)-Ig molecule (pGAD65), interleukin (IL)-4 (pIL4), and IL-10 (pIL10) significantly delayed the onset of recurrent diabetes compared with pGAD65+pIL10-vaccinated recipients. Despite differences in efficacy, a similar frequency of GAD65-specific CD4(+) T-cells secreting IL-4, IL-10, or interferon-gamma were detected in mice treated with pGAD65+pIL4+pIL10 and pGAD65+pIL10. However, the frequency of FoxP3-expressing CD4(+)CD25(+)CD62L(hi) T-cells was increased in the renal and pancreatic lymph nodes of diabetic recipients vaccinated with pGAD65+pIL4+pIL10. These immunoregulatory CD4(+)CD25(+) T-cells (CD4(+)CD25(+) Treg) exhibited enhanced in vivo and in vitro suppressor activity that partially was transforming growth factor-beta dependent. Furthermore, duration of islet graft protection in pGAD65+pIL4+pIL10-vaccinated diabetic recipients correlated with the persistence of CD4(+)CD25(+) Treg. These data demonstrate that the frequency and maintenance of FoxP3-expressing CD4(+)CD25(+) Treg influence antigen-induced suppression of ongoing beta-cell autoimmunity in diabetic recipients.

Low-avidity CD8lo T cells induced by incomplete antigen stimulation in vivo regulate naive higher avidity CD8hi T cell responses to the same antigen.

We have previously reported that multiple injections of soluble MHC class I tetramers assembled with wild-type HY peptide induces unresponsiveness to male skin grafts in naive female C57BL/6 (B6) mice. Induction of unresponsiveness is dependent on a population of unresponsive allospecific CD8(lo )T cells. Reduced expression of CD8 acts to limit a T cell response to HY peptide by limiting the avidity window of effective signal transduction. We and others have demonstrated that CD8(lo) T cells are an alternative stable phenotype of CD8alphabeta(+) T cells in vitro and in vivo after antigen stimulation. We show here that CD8(lo) T cells can suppress naive CD8(+) T cell responses to HY antigen in vitro and male skin graft rejection in vivo after adoptive transfer into female recipients. These novel regulatory T cells express surface TGF-beta1 and secrete T cytotoxic 2 cytokines after antigen-specific stimulation. Anti-TGF-beta antibody and latency-associated peptide inhibit the suppressive effects in vitro. We also show that HY-specific memory CD8(+) T cells overcome regulation by CD8(lo) T cells. These data define a novel peripheral regulatory CD8(+ )T cell population that arises after repeated antigen encounter in vivo. These cells have implications in the maintenance of tolerance and memory.

Pneumocystis: immune recognition and evasion.

Pulmonary infection caused by the opportunistic fungal organism Pneumocystis continues to be a leading AIDS defining illness. The initiation of highly active antiretroviral therapy (HAART) in the HIV-infected population has led to a significant reduction in the incidence of Pneumocystis pneumonia (PCP), although recent trends suggest the incidence has plateaued rather than decreased. Host defense against Pneumocystis involves a delicate, concerted balance between the inflammatory response and immune-mediated clearance. Innate cellular immunity is a cornerstone in this response as it provides the initial recognition event that precipitates an immune response, ultimately leading to clearance of the organism from the host. This review will focus on carbohydrate moieties found in the Pneumocystis cell wall and the immune events that occur following their recognition.

The beta-glucan receptor dectin-1 recognizes specific morphologies of Aspergillus fumigatus.

Alveolar macrophages represent a first-line innate host defense mechanism for clearing inhaled Aspergillus fumigatus from the lungs, yet contradictory data exist as to which alveolar macrophage recognition receptor is critical for innate immunity to A. fumigatus. Acknowledging that the A. fumigatus cell wall contains a high beta-1,3-glucan content, we questioned whether the beta-glucan receptor dectin-1 played a role in this recognition process. Monoclonal antibody, soluble receptor, and competitive carbohydrate blockage indicated that the alveolar macrophage inflammatory response, specifically the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, CXCL2/macrophage inflammatory protein-2 (MIP-2), CCL3/macrophage inflammatory protein-1alpha (MIP-1alpha), granulocyte-colony stimulating factor (G-CSF), and granulocyte monocyte-CSF (GM-CSF), to live A. fumigatus was dependent on recognition via the beta-glucan receptor dectin-1. The inflammatory response was triggered at the highest level by A. fumigatus swollen conidia and early germlings and correlated to the levels of surface-exposed beta glucans, indicating that dectin-1 preferentially recognizes specific morphological forms of A. fumigatus. Intratracheal administration of A. fumigatus conidia to mice in the presence of a soluble dectin-Fc fusion protein reduced both lung proinflammatory cytokine/chemokine levels and cellular recruitment while modestly increasing the A. fumigatus fungal burden, illustrating the importance of beta-glucan-initiated dectin-1 signaling in defense against this pathogen. Collectively, these data show that dectin-1 is centrally required for the generation of alveolar macrophage proinflammatory responses to A. fumigatus and to our knowledge provides the first in vivo evidence for the role of dectin-1 in fungal innate defense.

Single cell analysis shows decreasing FoxP3 and TGFbeta1 coexpressing CD4+CD25+ regulatory T cells during autoimmune diabetes.

Natural CD4(+)CD25(+) regulatory T (CD4(+)CD25(+) T reg) cells play a key role in the immunoregulation of autoimmunity. However, little is known about the interactions between CD4(+)CD25(+) T reg cells and autoreactive T cells. This is due, in part, to the difficulty of using cell surface markers to identify CD4(+)CD25(+) T reg cells accurately. Using a novel real-time PCR assay, mRNA copy number of FoxP3, TGFbeta1, and interleukin (IL)-10 was measured in single cells to characterize and quantify CD4(+)CD25(+) T reg cells in the nonobese diabetic (NOD) mouse, a murine model for type 1 diabetes (T1D). The suppressor function of CD4(+)CD25(+)CD62L(hi) T cells, mediated by TGFbeta, declined in an age-dependent manner. This loss of function coincided with a temporal decrease in the percentage of FoxP3 and TGFbeta1 coexpressing T cells within pancreatic lymph node and islet infiltrating CD4(+)CD25(+)CD62L(hi) T cells, and was detected in female NOD mice but not in NOD male mice, or NOR or C57BL/6 female mice. These results demonstrate that the majority of FoxP3-positive CD4(+)CD25(+) T reg cells in NOD mice express TGFbeta1 but not IL-10, and that a defect in the maintenance and/or expansion of this pool of immunoregulatory effectors is associated with the progression of T1D.