Mutagenesis-Based Characterization and Improvement of a Novel Inclusion Body Tag.

Whereas, bacterial inclusion bodies (IBs) for long were regarded as undesirable aggregates emerging during recombinant protein production, they currently receive attention as promising nanoparticulate biomaterials with diverse applications in biotechnology and biomedicine. We previously identified ssTorA, a signal sequence that normally directs protein export via the Tat pathway in E. coli, as a tag that induces the accumulation of fused proteins into IBs under overexpression conditions. Here, we used targeted mutagenesis to identify features and motifs being either critical or dispensable for IB formation. We found that IB formation is neither related to the function of ssTorA as a Tat-signal sequence nor is it a general feature of this family of signal sequences. IB formation was inhibited by co-overexpression of ssTorA binding chaperones TorD and DnaK and by amino acid substitutions that affect the propensity of ssTorA to form an α-helix. Systematic deletion experiments identified a minimal region of ssTorA required for IB formation in the center of the signal sequence. Unbiased genetic screening of a library of randomly mutagenized ssTorA sequences for reduced aggregation properties allowed us to pinpoint residues that are critical to sustain insoluble expression. Together, the data point to possible mechanisms for the aggregation of ssTorA fusions. Additionally, they led to the design of a tag with superior IB-formation properties compared to the original ssTorA sequence.

Sustained Rap1 activation in autoantigen-specific T lymphocytes attenuates experimental autoimmune encephalomyelitis.

Altered Ras superfamily guanine nucleotide triphosphatase signaling may contribute to the activation of autoreactive T cells in diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here, we show that transgenic expression of activated Rap1, a Ras-related protein which is protective in murine arthritis, in both wildtype (WT) and 2D2 mice, enhances autoreactive T cell activation by myelin oligodendrocyte glycoprotein peptide in vitro and in vivo. However, RapV12 reduces the number of autoreactive T cells in both WT and 2D2 mice, and increases murine survival in experimental autoimmune encephalitis, suggesting Rap1 activation restricts autoimmune T cell-mediated pathology through enhancing tolerance.

Sustained T cell Rap1 signaling is protective in the collagen-induced arthritis model of rheumatoid arthritis.

OBJECTIVE: Defective activation of T cell receptor-proximal signaling proteins, such as the small GTPase Rap1, is thought to contribute to the pathologic behavior of rheumatoid arthritis (RA) synovial T cells. This study was undertaken to determine whether maintaining Rap1 signaling in murine T cells modifies disease onset or severity in collagen-induced arthritis (CIA). METHODS: CIA experiments were conducted using wild-type and RapV12-transgenic mice, which express an active mutant of Rap1 in the T cell compartment. Mice were assessed using macroscopic, microscopic, and radiologic measures, and serum levels of anticollagen antibodies were measured by enzyme-linked immunosorbent assay. Phenotypic and functional characterization of wild-type and RapV12-transgenic T cells under homeostatic conditions and during disease onset was performed by flow cytometry. RESULTS: Disease incidence and severity, synovial infiltration, joint destruction, and anticollagen antibody production were significantly reduced in RapV12-transgenic mice. Although the numbers and percentages of CD3+, CD4+, and CD8+ (naive, effector, and memory) T cells, Treg cells, and Th17 cells were equivalent in wild-type and RapV12-transgenic mice, a significant decrease in the percentage of tumor necrosis factor α-secreting CD8+ T cells was observed in RapV12-transgenic mice during CIA. RapV12-transgenic T cells also inefficiently expressed inducible costimulator and CD40L costimulatory proteins involved in B cell immunoglobulin class switching. CONCLUSION: Our findings indicate that maintenance of T cell Rap1 signaling in murine T cells reduces disease incidence and severity in CIA, which are associated with specific defects in T cell effector function. Therefore, the restoration of Rap1 function in RA synovial T cells may have therapeutic benefit in RA.

Synergy between IL-15 and Id2 promotes the expansion of human NK progenitor cells, which can be counteracted by the E protein HEB required to drive T cell development.

The cytokine IL-15 and the inhibitor of DNA binding (Id)2, which negatively regulates the activity of basic helix-loop-helix transcription factors, have been shown to play key roles in NK cell development. Consistent with this, exogenous IL-15 added to human thymic progenitor cells stimulated their development into NK cells at the expense of T cells both in fetal thymic organ culture and in coculture with stromal cells expressing the Notch ligand Delta-like 1. Overexpression of Id2 in thymic progenitor cells stimulated NK cell development and blocked T cell development. This, in part, is attributed to inhibition of the transcriptional activity of the E protein HEB, which we show in this study is the only E protein that enhanced T cell development. Notably, Id2 increased a pool of lineage CD1a-CD5+ progenitor cells that in synergy with IL-15 furthered expansion and differentiation into NK cells. Taken together, our findings point to a dualistic function of Id2 in controlling T/NK cell lineage decisions; T cell development is impaired by Id2, most likely by sequestering HEB, whereas NK cell development is promoted by increasing a pool of CD1a-CD5+ NK cell progenitors, which together with IL-15 differentiate into mature NK cells.

A Rac1 inhibitory peptide suppresses antibody production and paw swelling in the murine collagen-induced arthritis model of rheumatoid arthritis.

INTRODUCTION: The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests. RESULTS: Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor alpha, interferon gamma and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help. CONCLUSIONS: The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be shown.

Isolation and in vitro generation of gene-manipulated human plasmacytoid and conventional dendritic cells.

Our understanding of human lymphocyte development has increased significantly over the past 20 years. In particular, our insight into human T- and B-cell development has improved (1, 2). Nonetheless, there are many gaps in our understanding, particularly regarding the early stages of development of hematopoietic progenitor cells (HPCs) into downstream lineage-biased and lineage-restricted precursors and the molecular mechanisms underlying these activities. The same holds true for our knowledge of human dendritic cell (DC) development. While the amount of data on the different subsets of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) rapidly increases in mice (3, 4), the developmental stages of different DC subsets in humans remain poorly defined (2). The relatively easy access to patient material and therefore human precursor cells that can be isolated from these tissues combined with the availability of in vitro and in vivo differentiation assays allows studies in the field of human hematopoietic development, including that of DCs. In addition, the opportunities to manipulate gene expression, by stable overexpression of a gene of interest or RNA interference-mediated knockdown, generate valuable information about the mechanisms underlying lineage commitment and differentiation.

IL-7 enhances thymic human T cell development in "human immune system" Rag2-/-IL-2Rgammac-/- mice without affecting peripheral T cell homeostasis.

IL-7 is a central cytokine in the development of hematopoietic cells, although interspecies discrepancies have been reported. By coculturing human postnatal thymus hematopoietic progenitors and OP9-huDL1 stromal cells, we found that murine IL-7 is approximately 100-fold less potent than human IL-7 for supporting human T cell development in vitro. We investigated the role of human IL-7 in newborn BALB/c Rag2(-/-)gamma(c)(-/-) mice transplanted with human hematopoietic stem cells (HSC) as an in vivo model of human hematopoiesis using three approaches to improve IL-7 signaling: administration of human IL-7, ectopic expression of human IL-7 by the transplanted human HSC, or enforced expression of a murine/human chimeric IL-7 receptor binding murine IL-7. We show that premature IL-7 signaling at the HSC stage, before entrance in the thymus, impeded T cell development, whereas increased intrathymic IL-7 signaling significantly enhanced the maintenance of immature thymocytes. Increased thymopoiesis was also observed when we transplanted BCL-2- or BCL-x(L)-transduced human HSC. Homeostasis of peripheral mature T cells in this humanized mouse model was not improved by any of these strategies. Overall, our results provide evidence for an important role of IL-7 in human T cell development in vivo and highlight the notion that IL-7 availability is but one of many signals that condition peripheral T cell homeostasis.

Human thymus regeneration and T cell reconstitution.

The thymus supports the development of T cells throughout life from hematopoietic progenitor cells migrating from the bone marrow. During the early years after birth thymic activity is highest, but progressively declines resulting in diminished naïve T cell output. Underlying causes of thymic involution may be degeneration of the stromal thymic network, providing survival and differentiation factors for developing T cells, or insufficiency of the progenitor cells to home and/or develop in the aged thymus. In young people the reduced thymic output is insignificant, since the peripheral T cell compartment is under compensatory homeostatic control. However, in more or less immunocompromised individuals, including aged people and patients depleted of T cells due to conditioning regimens before bone marrow transplantation or HIV infection, the thymus is necessary to replenish the peripheral T cell compartment. This may require rejuvenation of the thymus. Alternatively, approaches to generate mature T cells independent of the thymus have gained considerable interest.

Stimulated plasmacytoid dendritic cells impair human T-cell development.

Thymic plasmacytoid dendritic cells (pDCs) are located predominantly in the medulla and at the corticomedullary junction, the entry site of bone marrow-derived multipotential precursor cells into the thymus, allowing for interactions between thymic pDCs and precursor cells. We demonstrate that in vitro-generated pDCs stimulated with CpG or virus impaired the development of human autologous CD34(+)CD1a(-) thymic progenitor cells into the T-cell lineage. Rescue by addition of neutralizing type I interferon (IFN) antibodies strongly implies that endogenously produced IFN-alpha/beta is responsible for this inhibitory effect. Consistent with this notion, we show that exogenously added IFN-alpha had a similar impact on IL-7- and Notch ligand-induced development of thymic CD34(+)CD1a(-) progenitor cells into T cells, because induction of CD1a, CD4, CD8, and TCR/CD3 surface expression and rearrangements of TCRbeta V-DJ gene segments were severely impaired. In addition, IL-7-induced proliferation but not survival of the developing thymic progenitor cells was strongly inhibited by IFN-alpha. It is evident from our data that IFN-alpha inhibits the IL-7R signal transduction pathway, although this could not be attributed to interference with either IL-7R proximal (STAT5, Akt/PKB, Erk1/2) or distal (p27(kip1), pRb) events.

Delta-like1-induced Notch1 signaling regulates the human plasmacytoid dendritic cell versus T-cell lineage decision through control of GATA-3 and Spi-B.

Human early thymic precursors have the potential to differentiate into multiple cell lineages, including T cells and plasmacytoid dendritic cells (pDCs). This decision is guided by the induction or silencing of lineage-specific transcription factors. The ETS family member Spi-B is a key regulator of pDC development, whereas T-cell development is critically dependent on GATA-3. Here we show that triggering of the Notch1 signaling pathway by Delta-like1 controls the T/pDC lineage decision by regulating the balance between these factors. CD34+ CD1a- thymic progenitor cells express Notch1, but down-regulate this receptor when differentiating into pDCs. On coculture with stromal cell lines expressing either human Delta-like1 (DL1) or Jagged1 (Jag1) Notch ligands, thymic precursors express GATA-3 and develop into CD4+ CD8+ TCRalphabeta+ T cells. On the other hand, DL1, but not Jag1, down-regulates Spi-B expression, resulting in impaired development of pDCs. The Notch1-induced block in pDC development can be relieved through the ectopic expression of Spi-B. These data indicate that DL1-induced activation of the Notch1 pathway controls the lineage commitment of early thymic precursors by altering the levels between Spi-B and GATA-3.