Giuseppe Penna

1α,25-Dihydroxyvitamin D3 Inhibits Differentiation, Maturation, Activation, and Survival of Dendritic Cells Leading to Impaired Alloreactive T Cell Activation

1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D3, is a potent immunomodulatory agent. Here we show that dendritic cells (DCs) are major targets of 1,25(OH)2D3-induced immunosuppressive activity. 1,25(OH)2D3 prevents the differentiation in immature DCs of human monocytes cultured with GM-CSF and IL-4. Addition of 1,25(OH)2D3 during LPS-induced maturation maintains the immature DC phenotype characterized by high mannose receptor and low CD83 expression and markedly inhibits up-regulation of the costimulatory molecules CD40, CD80, and CD86 and of class II MHC molecules. This is associated with a reduced capacity of DCs to activate alloreactive T cells, as determined by decreased proliferation and IFN-γ secretion in mixed leukocyte cultures. 1,25(OH)2D3 also affects maturing DCs, leading to inhibition of IL-12p75 and enhanced IL-10 secretion upon activation by CD40 ligation. In addition, 1,25(OH)2D3 promotes the spontaneous apoptosis of mature DCs. The modulation of phenotype and function of DCs matured in the presence of 1,25(OH)2D3 induces cocultured alloreactive CD4+ cells to secrete less IFN-γ upon restimulation, up-regulate CD152, and down-regulate CD154 molecules. The inhibition of DC differentiation and maturation as well as modulation of their activation and survival leading to T cell hyporesponsiveness may explain the immunosuppressive activity of 1,25(OH)2D3.

Cutting Edge: Selective Usage of Chemokine Receptors by Plasmacytoid Dendritic Cells

The existence of dendritic cell (DC) subsets is firmly established, but their trafficking properties are virtually unknown. In this study, we show that myeloid (M-DCs) and plasmacytoid (P-DCs) DCs isolated from human blood differ widely in the capacity to migrate to chemotactic stimuli. The pattern of chemokine receptors expressed by blood M-DCs and P-DCs, with the exception of CCR7, is similar. However, most chemokine receptors of P-DCs, in particular those specific for inflammatory chemokines and classical chemotactic agonists, are not functional in circulating cells. Following maturation induced by CD40 ligation, the receptors for inflammatory chemokines are down-regulated, and CCR7 on P-DCs becomes coupled to migration. The drastically impaired capacity of blood P-DCs to migrate in response to inflammatory chemotactic signals contrasts with the response to lymph node-homing chemokines, indicating a propensity to migrate to secondary lymphoid organs rather than to sites of inflammation.

The control of T cell responses by dendritic cell subsets

Dendritic cells are known as the most efficient antigen-presenting cell type to activate naïve T cells; however, they are able to do more than just efficiently present antigen to T cells. They are key modulators of the immune response that can influence Th cell differentiation by preferentially inducing Th type 1 or 2 cell responses, and the differential polarisation of CD4(+) T cells appears to be mediated by discrete dendritic cell subsets.

Intrathecal Delivery of IFN-γ Protects C57BL/6 Mice from Chronic-Progressive Experimental Autoimmune Encephalomyelitis by Increasing Apoptosis of Central Nervous System-Infiltrating Lymphocytes

The exclusive detrimental role of proinflammatory cytokines in demyelinating diseases of the CNS, such as multiple sclerosis, is controversial. Here we show that the intrathecal delivery of an HSV-1-derived vector engineered with the mouse IFN-γ gene leads to persistent (up to 4 wk) CNS production of IFN-γ and inhibits the course of a chronic-progressive form of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice by myelin oligodendrocyte glycoprotein (MOG)35–55. Mice treated with the IFN-γ-containing vector before EAE onset showed an earlier onset but a milder course of the disease compared with control mice treated with the empty vector. In addition, 83% of IFN-γ-treated mice completely recovered within 25 days post immunization, whereas control mice did not recover up to 60 days post immunization. Mice treated with the IFN-γ-containing vector within 1 wk after EAE onset partially recovered from the disease within 25 days after vector injection, whereas control mice worsened. Recovery from EAE in mice treated with IFN-γ was associated with a significant increase of CNS-infiltrating lymphocytes undergoing apoptosis. During the recovery phase, the mRNA level of TNFR1 was also significantly increased in CNS-infiltrating cells from IFN-γ-treated mice compared with controls. Our results further challenge the exclusive detrimental role of IFN-γ in the CNS during EAE/multiple sclerosis, and indicate that CNS-confined inflammation may induce protective immunological countermechanisms leading to a faster clearance of encephalitogenic T cells by apoptosis, thus restoring the immune privilege of the CNS.

Functional Maturation of Adult Mouse Resting Microglia into an APC Is Promoted by Granulocyte-Macrophage Colony-Stimulating Factor and Interaction with Th1 Cells

A precise knowledge of the early events inducing maturation of resting microglia into a competent APC may help to understand the involvement of this cell type in the development of CNS immunopathology. To elucidate whether signals from preactivated T cells are sufficient to induce APC features in resting microglia, microglia from the adult BALB/c mouse CNS were cocultured with Th1 and Th2 lines from DO11.10 TCR transgenic mice to examine modulation of APC-related molecules and Ag-presenting capacity. Upon Ag-specific interaction with Th1, but not Th2, cells, microglia strongly up-regulated the surface expression of MHC class II, CD40, and CD54 molecules. Induction of CD86 on mouse microglia did not require T cell-derived signals. Acutely isolated adult microglia stimulated Th1 cells to secrete IFN-γ and, to a lesser extent, IL-2, but were inefficient stimulators of IL-4 secretion by Th2 cells. Microglia exposed in vitro to IFN-γ showed enhanced expression of MHC class II, CD40, and CD54 molecules and became able to restimulate Th2 cells. In addition to IFN-γ, GM-CSF increased the ability of microglia to activate Th1, but not Th2, cells without up-regulating MHC class II, CD40, or CD54 molecules. These results suggest that interaction with Th1 cells and/or Th1-secreted soluble factors induces the functional maturation of adult mouse microglia into an APC able to sustain CD4+ T cell activation. Moreover, GM-CSF, a cytokine secreted by T cells as well as reactive astrocytes, could prime microglia for Th1-stimulating capacity, possibly by enhancing their responsiveness to Th1-derived signals.

Relative efficiency of microglia, astrocytes, dendritic cells and B cells in naive CD4+ T cell priming and Th1/Th2 cell restimulation

We have compared the efficiency of central nervous system and peripheral antigen‐presenting cells (APC) in T cell priming and restimulation. OVA peptide 323u2009–u2009339‐dependent activation of DO11.10 TCR‐transgenic naive CD4+ and polarized Th1 or Th2 cells was assessed in the presence of microglia and astrocytes from the neonatal mouse brain as well as dendritic cells (DC) and B cells purified from adult mouse lymph nodes. DC were the most efficient in inducing naive T cell proliferation, IL‐2 secretion and differentiation into Th1 cells, followed by IFN‐γ‐preactivated microglia, large and small B cells. Astrocytes failed to activate naive T cells. IFN‐γ‐pretreated microglia were as efficient as DC in the restimulation of Th1 cells, whereas IFN‐γ‐pretreated astrocytes, large and small B cells were much less efficient. Conversely, Th2 cells were efficiently restimulated by all the APC types examined. During T cell priming, DC secreted more IL‐12 than microglia but similar amounts of IL‐12 were secreted by the two cell types upon interaction with Th1 cells. The hierarchy of APC established in this study indicates that DC and microglia are the most efficient in the stimulation of naive CD4+ T cells and in the restimulation of Th1 cells, suggesting that activated microglia may effectively contribute to Th1 responses leading to central nervous system inflammation and tissue damage. These potentially pathogenic responses could be counteracted by the high efficiency of astrocytes as well as microglia in restimulating Th2 cells.

Th1 cells induce and Th2 inhibit antigen-dependent IL-12 secretion by dendritic cells

Dendritic cells are the most relevant antigen‐presenting cells (APC) for presentation of antigens administered in adjuvant to CD4+ T cells. Upon interaction with antigen‐specific T cells, dendritic cells (DC) expressing appropriate peptide‐MHC class II complexes secrete IL‐12, a cytokine that drives Th1 cell development. To analyze the T cell‐mediated regulation of IL‐12 secretion by DC, we have examined their capacity to secrete IL‐12 in response to stimulation by antigen‐specific Th1 and Th2 DO11.10 TCR‐transgenic cells. These cells do not differ either in TCR clonotype or CD40 ligand (CD40L) expression. Interaction with antigen‐specific Th1, but not Th2 cells, induces IL‐12 p40 and p75 secretion by DC. The induction of IL‐12 production by Th1 cells does not depend on their IFN‐γ secretion, but requires direct cell‐cell contact mediated by peptide/MHC class II‐TCR and CD40‐CD40L interactions. Th2 cells not only fail to induce IL‐12 secretion, but they inhibit its induction by Th1 cells. Unlike stimulation by Th1, inhibition of IL‐12 production by Th2 cells is mediated by soluble molecules, as demonstrated by transwell cultures. Among Th2‐derived cytokines, IL‐10, but not IL‐4 inhibit Th1‐driven IL‐12 secretion. IL‐10 produced by Th2 cells appears to be solely responsible for the inhibition of Th1‐induced IL‐12 secretion, but it does not account for the failure of Th2 cells to induce IL‐12 production by DC. Collectively, these results demonstrate that Th1 cells up‐regulate IL‐12 production by DC via IFN‐γ‐independent cognate interaction, whereas this is inhibited by Th2‐derived IL‐10. The inhibition of Th1‐induced IL‐12 production by Th2 cells with the same antigen specificity represents a novel mechanism driving the polarization of CD4+ T cell responses.

Early Th1 Response in Unprimed Nonobese Diabetic Mice to the Tyrosine Phosphatase-Like Insulinoma-Associated Protein 2, an Autoantigen in Type 1 Diabetes

The insulinoma-associated protein 2 (IA-2) is a phosphatase-like autoantigen inducing T and B cell responses associated with human insulin-dependent diabetes mellitus (IDDM). We now report that T cell responses to IA-2 can also be detected in the nonobese diabetic (NOD) mouse, a model of human IDDM. Cytokine secretion in response to purified mouse rIA-2, characterized by high IFN-γ and relatively low IL-10 and IL-6 secretion, was elicited in spleen cells from unprimed NOD mice. Conversely, no response to IA-2 was induced in spleen cells from BALB/c, C57BL/6, or Biozzi AB/H mice that express, like NOD, the I-Ag7 class II molecule, but are not susceptible to spontaneous IDDM. The IA-2-induced IFN-γ response in NOD spleen cells could already be detected at 3 wk and peaked at 8 wk of age, whereas the IL-10 secretion was maximal at 4 wk of age and then waned. IA-2-dependent IFN-γ secretion was induced in CD4+ cells from spleen as well as pancreatic and mesenteric lymph nodes. It required Ag presentation by I-Ag7 molecules and engagement of the CD4 coreceptor. Interestingly, cytokines were produced in the absence of cell proliferation and IL-2 secretion. The biological relevance of the response to IA-2 is indicated by the enhanced IDDM following a single injection of the recombinant protein emulsified in IFA into 18-day-old NOD mice. In addition, IFN-γ production in response to IA-2 and IDDM acceleration could be induced by IL-12 administration to 12-day-old NOD mice. These results identify IA-2 as an early T cell-inducing autoantigen in the NOD mouse and indicate a role for the IA-2-induced Th1 cell response in IDDM pathogenesis.

The involvement of IL-12 in murine experimentally induced autoimmune thyroid disease

Experimental autoimmune thyroid disease (EAT) can be induced experimentally in mice following immunization with mouse thyroglobulin (mTg) and the adjuvants lipopolysaccharide (LPS) or complete Freunds adjuvant (CFA). EAT can also be transferred to naive recipients by CD4+ T cells from mTg‐primed mice. Here we demonstrate a role for IL‐12 in the development of EAT by the ability of neutralizing antibody to IL‐12 to reduce disease severity and by the lack of significant levels of thyroid infiltration in IL‐12p40‐deficient mice following immunization with mTg and CFA. A single injection of 300u2004ng IL‐12 at the time of initial immunization with mTg and LPS was able to increase the degree of thyroid infiltration. These data are all consistent with EAT being a Th1‐mediated disease. Conversely, however, administration of IL‐12 over a prolonged period markedly inhibited the induction of EAT by mTg and CFA and, if given to recipients, inhibited the transfer of EAT by mTg‐primed lymphnode cells. The development of an autoantibody response to mTg was also inhibited when IL‐12 was administered throughout the experimental period, suggesting that sustained exposure to IL‐12 can be immunosuppressive.

IL-12 Administration Reveals Diabetogenic T Cells in Genetically Resistant I-Eα-Transgenic Nonobese Diabetic Mice: Resistance to Autoimmune Diabetes Is Associated with Binding of Eα-Derived Peptides to the I-Ag7 Molecule

Nonobese diabetic (NOD) and NOD-DRα transgenic (tg) mice, expressing Aαd:Aβg7 and Aαd:Aβg7 plus DRα:Eβg7 class II molecules, respectively, both develop insulin-dependent diabetes mellitus (IDDM), whereas NOD-Eα tg mice expressing Aαd:Aβg7 plus Eα:Eβg7 are protected. We show that IL-12 administration induces rapid IDDM onset in NOD-DRα but fails to provoke insulitis and diabetes in NOD-Eα tg mice. Nevertheless, T cells from IL-12-treated NOD-Eα tg mice secrete IFN-γ and transfer IDDM to NOD-SCID and NOD-Eα-SCID recipients, demonstrating the presence of peripheral diabetogenic Th1 cells in the protected mice. Surprisingly, regulatory cells were undetectable. Moreover, Eα:Eβg7 could substitute for DRα:Eβg7 in Ag presentation, arguing against mechanisms of protection involving capture of diabetogenic I-Ag7-restricted epitopes by Eα:Eβg7molecules. Interestingly, the expression of naturally processed epitopes derived from DRα- and Eα-chains bound to I-Ag7 is different in the two strains of tg mice, and the difference is enhanced by IL-12 administration. I-Ag7 molecules from both NOD-DRα and NOD-Eα tg mice present the conserved DRα/Eα 52-68 sequence, at high and low levels, respectively. In addition, only IDDM-resistant NOD-Eα tg mice possess APCs bearing Eα65-77/I-Ag7 complexes, which tolerize the specific T cells. This is associated with the selective inhibition of the response to insulinoma-associated protein 2 (IA-2), an autoantigen in IDDM. Our results support protective mechanisms based on I-Ag7 blockade by peptides unique to the Eα-chain, such as Eα65-77 and/or tolerance of diabetogenic T cells cross-reactive with Eα-peptide/I-Ag7 complexes.