Barbara Valzasina

Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria.

Penetration of the gut mucosa by pathogens expressing invasion genes is believed to occur mainly through specialized epithelial cells, called M cells, that are located in Peyers patches. However, Salmonella typhimurium that are deficient in invasion genes encoded by Salmonella pathogenicity island 1 (SPI1) are still able to reach the spleen after oral administration. This suggests the existence of an alternative route for bacterial invasion, one that is independent of M cells. We report here a new mechanism for bacterial uptake in the mucosa tissues that is mediated by dendritic cells (DCs). DCs open the tight junctions between epithelial cells, send dendrites outside the epithelium and directly sample bacteria. In addition, because DCs express tight-junction proteins such as occludin, claudin 1 and zonula occludens 1, the integrity of the epithelial barrier is preserved.

OX40 triggering blocks suppression by regulatory T cells and facilitates tumor rejection.

Regulatory T (T reg) cells are the major obstacle to cancer immunotherapy, and their depletion promptly induces conversion of peripheral precursors into T reg cells. We show that T reg cells can be functionally inactivated by OX40 triggering. In tumors, the vast majority of CD4+ T cells are Foxp3+ and OX40bright. However, intratumor injection of the agonist anti-OX40 monoclonal antibody (mAb) OX86, but not anti-CD25 mAb, induces tumor rejection in 80% of mice, an effect that is abrogated by CD8 depletion. Upon intratumor OX40 triggering, increased numbers of infiltrating dendritic cells (DCs) migrate to draining lymph nodes and generate a new wave of tumor-specific cytotoxic T lymphocytes, as detected by tetramer and CD44 staining of node CD8+ T lymphocytes. Tumor-bearing Rag1-knockout (KO) mice reconstituted with OX40-deficient T reg cells and wild-type (WT) effector T cells, or the reciprocal combination, showed that both T reg and effector T cells must be triggered via OX40 for the tumor to be rejected. Accordingly, WT but not OX40-KO mice receiving intratumor coinjection of OX86 and ovalbumin protein were able to revert tumor-induced tolerization of adoptively transferred OX40-competent OTII T lymphocytes. In conclusion, OX40-mediated inactivation of T reg cell function unleashes nearby DCs, allowing them to induce an adaptive immune response. In addition, the known OX40-dependent delivery of fitness signals to activated T cells is boosted by concurrent T reg cell inhibition. OX40 triggering thus has multiple effects that converge to mediate tumor rejection.

Dendritic Cells Shuttle Microbes Across Gut Epithelial Monolayers

Understanding the mechanisms governing the type of induced immune response after microbial invasion, could be of crucial importance for the rational design of a bacteria-based vaccine. Targeting a vaccine directly to dendritic cells (DCs), which are considered the most powerful antigen presenting cells, could be extremely effective. Here we describe that CD11b+CD8alpha- dendritic cells are involved in the direct bacterial uptake across mucosal surfaces. DCs are widely spread in the lamina propria of the gut and are recruited at the site of infection. DCs open the tight junctions between epithelial cells, send dendrites outside of the epithelium and sample bacteria. Moreover, the integrity of the epithelial barrier is preserved because DCs express tight junction proteins, such as occludin, claudin 1 and Junctional Adhesion Molecule (JAM) and can establish tight junctions-like structures with neighbouring epithelial cells.

Tumor-Induced Expansion of Regulatory T Cells by Conversion of CD4+CD25− Lymphocytes Is Thymus and Proliferation Independent

The CD25- and CD25+ CD4 T-lymphocyte compartments are tightly regulated. We show here that tumors break such balance, increasing the number of CD4+CD25+ T cells in draining lymph node and spleen but not contralateral node of tumor-bearing mice. Tumor injection in thymectomized and CD25-depleted mice shows that CD4+CD25+ T-cell expansion occurs even in the absence of the thymus and independently from proliferation of preexisting CD25+ T cells. These newly generated cells are bona fide regulatory T cells (T reg) in terms of Foxp3 expression and suppression of CD3-stimulated or allogeneic effector cell proliferation. Transfer of congenic Thy1.1 CD4+CD25- T cells, from mice treated or not with vinblastine, into tumor-bearing or tumor-free mice and analysis of recovered donor lymphocytes indicate that conversion is the main mechanism for acquiring the expression of CD25 and Foxp3 through a process that does not require proliferation. Although conversion of CD4+CD25- T cells for generation of T regs has been described as a natural process that maintains peripheral T-reg population, this process is used by the tumor for immune escape. The prompt recovery of T regs from monoclonal antibody-mediated CD25 depletion in tumor-bearing mice suggests attempts able to inactivate rather than deplete them when treating existing tumors.

CD40/CD40L interaction regulates CD4+CD25+ T reg homeostasis through dendritic cell‐produced IL‐2

CD4+CD25+ regulatory T cells (T reg) development and homeostasis require IL‐2 and costimulation through same TNF‐receptor family members. CD40KO mice have reduced number of T reg in peripheral blood, thymus and spleen. Herein we show that naive T reg express low basal level of CD40L that is upregulated upon TCR‐triggered mediated activation. Treatment of wt mice with Ab blocking CD40/CD40L interaction results in a fast decrease in T reg number that rapidly recovers upon Ab withdrawal. CFSE‐labeled T reg from wt mice injected into CD40KO, but not wild‐type (wt) mice, showed reduced survival and proliferation in homeostatic setting. In vitro, dendritic cells from CD40KO mice but not wt mice produce diminished amount of IL‐2 upon T reg encounter and are impaired in expanding T reg, a defect corrected by the addition of rIL‐2. Accordingly, four daily IL‐2 administrations to CD40KO mice normalize T reg number by promoting both their survival and homeostatic proliferation. Such IL‐2 effect is transient since T reg number returns to the low constitutive level described in CD40KO mice within 5 days upon IL‐2 withdrawal thus suggesting that IL‐2 is persistently needed to assure T reg homeostasis.

Toll-like receptor 4 is not required for the full maturation of dendritic cells or for the degradation of Gram-negative bacteria

Toll‐like receptor 4 (TLR4) has been recently associated with cellular responses to lipopolysaccharide (LPS), and mice mutated in tlr4, such as C57BL/10ScCr or C3H/HeJ mice, become hyporesponsive to LPS. In this study, we have analyzed the capacity of bone marrow‐derived dendritic cells (BMDC) from C57BL/10ScCr (ScCr‐BMDC) or C3H/HeJ (HeJ‐BMDC) mice to respond to LPS or to Gram‐negative bacteria. We show that ScCr‐ or HeJ‐BMDC are insensitive to LPS, but can mature in response to live and killed Gram‐negative bacteria. Interestingly, only ScCr‐BMDC but not HeJ‐BMDC, stimulated with bacteria, have reduced capacity to produce pro‐ and anti‐inflammatory cytokines as compared to BMDC from control mice, probably due to genetic defects unrelated to the tlr4 mutation. Nevertheless, ScCr‐BMDC and ScCr BM‐macrophages (BM‐Mϕ) phagocytose Salmonella typhimurium similarly to control cells, indicating that TLR4 is not compulsory for bacterial uptake. Moreover, BM‐Mϕ, but not BM‐DC from B10ScCr or C3H/HeJ mice, are impaired in their capacity to kill intracellular bacteria and to produce NO as compared to wild type controls. However, the bacteria killing property of BM‐Mϕ is completely restored by pretreating the cells with IFN‐γ. Hence, TLR4 plays different roles in DC versus Mϕ.