Dendritic cell antigen presentation drives simultaneous cytokine production by effector and regulatory T cells in inflamed skin.

Effector (Teff) and regulatory (Treg) T cells produce cytokines that balance immunity and immunopathology at sites of infection. It is not known how this balance is achieved. Here, we show that Treg and Teff cells specific for the same foreign peptide:major histocompatibility complex II (pMHCII) ligand accumulated preferentially in a subcutaneous site injected with the relevant antigen plus an adjuvant. Some of the Treg cells in this site were producing IL-10 12 days after injection, whereas a similar fraction of the Teff cells were producing IFN-gamma. Acute ablation of Treg cells increased the fraction of IFN-gamma-producing Teff cells, indicating that Teff function was limited by the Treg cells. Production of cytokines by both populations was driven by pMHCII presentation by local CD11b(hi) dermal dendritic cells. Therefore, balanced production of microbicidal and suppressive cytokines in inflamed skin is achieved by simultaneous dendritic cell antigen presentation to Teff and Treg cells.

CD4+ T cells that enter the draining lymph nodes after antigen injection participate in the primary response and become central-memory cells.

We explored the relationship between the time of naive CD4+ T cell exposure to antigen in the primary immune response and the quality of the memory cells produced. Naive CD4+ T cells that migrated into the skin-draining lymph nodes after subcutaneous antigen injection accounted for about half of the antigen-specific population present at the peak of clonal expansion. These late-arriving T cells divided less and more retained the central-memory marker CD62L than the T cells that resided in the draining lymph nodes at the time of antigen injection. The fewer cell divisions were related to competition with resident T cells that expanded earlier in the response and a reduction in the number of dendritic cells displaying peptide-major histocompatibility complex (MHC) II complexes at later times after antigen injection. The progeny of late-arriving T cells possessed the phenotype of central-memory cells, and proliferated more extensively during the secondary response than the progeny of the resident T cells. The results suggest that late arrival into lymph nodes and exposure to antigen-presenting cells displaying lower numbers of peptide-MHC II complexes in the presence of competing T cells ensures that some antigen-specific CD4+ T cells divide less in the primary response and become central-memory cells.

A protease-dependent mechanism for initiating T-dependent B cell responses to large particulate antigens.

Ab production is critical for antimicrobial immunity, and the initial step in this process is the binding of Ag to the BCR. It has been shown that small soluble proteins can directly access the lymph node follicles to reach naive B cells, but virus particles must be translocated into follicles via subcapsular sinus macrophages. In this article, we explore how large particulate Ags generate humoral immune responses. Ag-specific follicular B cells rapidly acquired Ag, presented peptide:MHC class II ligands, and produced T-dependent Ab responses following s.c. injection of 1-mum, Ag-linked microspheres, despite the microspheres being confined to the subcapsular sinus. The mechanism of Ag acquisition did not require dendritic cells, subcapsular sinus macrophages, or B cell movement to the subcapsular sinus. Rather, B cell Ag acquisition was protease-dependent, suggesting that some protein Ags are cleaved from the surface of particles to directly initiate humoral immune responses.

Esterification of cholesterol by a type III secretion effector during intracellular Salmonella infection.

Survival of Salmonella typhimurium within a vacuole in host cells depends on secreted virulence factors encoded by the Salmonella pathogenicity island 2 (SPI-2). High levels of cholesterol are detected at the Salmonella-containing vacuole (SCV). Here we show that the SPI-2 effector SseJ esterifies cholesterol in vitro, in cells and during infection. Intracellular infections with wild-type as compared with DeltasseJ bacteria led to higher levels of cholesterol ester production in HeLa cells and RAW macrophages and were shown to increase levels of lipid droplets (structures enriched in cholesterol esters). Ectopic expression of SseJ reduced cholesterol levels in cellular membranes and antagonized a major membrane activity of a second bacterial effector known to be important to the stability of the SCV. Previous studies in mouse models of infection have established a virulence defect in DeltasseJ bacteria and have suggested a role for SseJ in regulating SCV dynamics. Our data indicating the molecular activity of SseJ suggest that cholesterol and its esterification at the SCV are functionally important for intracellular bacterial survival.

Proliferating CD4+ T cells undergo immediate growth arrest upon cessation of TCR signaling in vivo.

To investigate the role of TCR signaling in the exit of CD4+ T cells from cell cycle, we took advantage of a low frequency TEa T cell adoptive transfer technique as well as the Y-Ae mAb to interrupt Ag/MHC recognition before the completion of clonal expansion. Termination of TCR signaling after 36 h of Ag exposure caused an immediate reduction in cell size and deceleration of G1->SG2M phase cell cycle progression. As a consequence, clonal expansion in the absence of durable TCR signaling decreased by two-thirds. Thus, CD4+ T cells scan for the presence Ag throughout their clonal expansion response, and continuously adjust their rate of cell growth and G1->S phase transition to match their intensity of TCR signaling.

The humoral immune response is initiated in lymph nodes by B cells that acquire soluble antigen directly in the follicles.

The initial step in a humoral immune response involves the acquisition of antigens by B cells via surface immunoglobulin. Surprisingly, anatomic studies indicate that lymph-borne proteins do not have access to the follicles where naive B cells reside. Thus, it is unclear how B cells acquire antigens that drain to lymph nodes. By tracking a fluorescent antigen and a peptide:MHC II complex derived from it, we show that antigen-specific B cells residing in the follicles acquire antigen within minutes of injection, first in the region closest to the subcapsular sinus where lymph enters the lymph node. Antigen acquisition, presentation, and subsequent T cell-dependent activation did not require B cell migration through the T cell area or exposure to dendritic cells. These results indicate that the humoral response is initiated as soluble antigens diffuse directly from lymph in the subcapsular sinus to be acquired by antigen-specific B cells in the underlying follicles.

Visualizing the first 50 hr of the primary immune response to a soluble antigen.

Recently, static and dynamic imaging methods have produced the first glimpses of the interactions between antigen-specific T cells and peptide-MHC-bearing antigen-presenting cells in the lymph nodes. Using data from these experiments, we produced a numerically, spatially, and temporally scaled simulation of the first 50 hr of the primary T cell-dependent immune response. The simulation highlights how lymph node structure facilitates antigen presentation to rare, naive, antigen-specific CD4+ T cells.

Salmonella enterica serovar Typhimurium requires nonsterol precursors of the cholesterol biosynthetic pathway for intracellular proliferation.

We have previously shown that Salmonella enterica serovar Typhimurium infection perturbs the host cholesterol biosynthetic pathway. Here we show that inhibiting the first step of this pathway (3-hydroxy-3-methylglutaryl coenzyme A reductase) reduces the growth of intracellular S. enterica serovar Typhimurium and has no effect on extracellular bacterial growth. Selectively inhibiting synthesis of downstream sterol components has no effect on infection, suggesting that the effect of statins on host nonsterol intermediates is detrimental to bacterial growth. Furthermore, statins also reduce bacterial proliferation in the S. enterica serovar Typhimurium mouse model. This suggests that blocking the production of nonsterol precursors in the host cell can be used to reduce infection.

The Salmonella-containing vacuole is a major site of intracellular cholesterol accumulation and recruits the GPI-anchored protein CD55.

Intracellular, pathogenic Salmonella typhimurium avoids phago-lysosome fusion, and exists within a unique vacuolar niche that resembles a late endosome. This model has emerged from studying the trafficking of host proteins to the Salmonella-containing vacuole (SCV). Very little is known about the role of major host lipids during infection. Here, we show using biochemical analyses as well as fluorescence microscopy, that intracellular infection perturbs the host sterol biosynthetic pathway and induces cholesterol accumulation in the SCV. Cholesterol accumulation is seen in both macrophages and epithelial cells: at the terminal stages of infection, as much as 30% of the total cellular cholesterol resides in the SCV. We find that accumulation of cholesterol in the SCV is linked to intracellular bacterial replication and may be dependent on Salmonella pathogenicity island 2 (SPI-2). Furthermore, the construction of a three-dimensional space-filling model yields novel insights into the structure of the SCV: bacteria embedded in cholesterol-rich membranes. Finally, we show that the glycosylphosphatidylinositol (GPI)-anchored protein CD55 is recruited to the SCV. These data suggest that, in contrast to prevailing models, the SCV accumulates components of cholesterol-rich early endocytic pathways during intracellular bacterial replication.