A high-throughput screening RT-qPCR assay for quantifying surrogate markers of immunity from PBMCs.

Immunoassays that quantitate cytokines and other surrogate markers of immunity from peripheral blood mononuclear cells (PBMCs), such as flow cytometry or Enzyme-Linked Immunosorbent Spot (ELIspot), allow highly sensitive measurements of immune effector function. However, those assays consume relatively high numbers of cells and expensive reagents, precluding comprehensive analyses and high-throughput screening (HTS). To address this issue, we developed a sensitive and specific reverse transcription-quantitative PCR (RT-qPCR)-based HTS assay, specifically designed to quantify surrogate markers of immunity from very low numbers of PBMCs. We systematically evaluated the volumes and concentrations of critical reagents within the RT-qPCR protocol, miniaturizing the assay and ultimately reducing the cost by almost 90% compared to current standard practice. We assessed the suitability of this cost-optimized RT-qPCR protocol as an HTS tool and determined the assay exceeds HTS uniformity and signal variance testing standards. Furthermore, we demonstrate this technique can effectively delineate a hierarchy of responses from as little as 50,000 PBMCs stimulated with CD4+ or CD8+ T cell peptide epitopes. Finally, we establish that this HTS-optimized protocol has single-cell analytical sensitivity and a diagnostic sensitivity equivalent to detecting 1:10,000 responding cells (i.e., 100 Spot Forming Cells/106 PBMCs by ELIspot) with over 90% accuracy. We anticipate this assay will have widespread applicability in preclinical and clinical studies, especially when samples are limited, and cost is an important consideration.

Memory CD8+ T cell compartment associated with delayed onset of Plasmodium falciparum infection and better parasite control in sickle-cell trait children.

OBJECTIVES: Study of individuals with protection from Plasmodium falciparum (Pf) infection and clinical malaria, including individuals affected by the sickle-cell trait (HbAS), offers the potential to identify cellular targets that could be translated for therapeutic development. We previously reported the first involvement of cellular immunity in HbAS-associated relative protection and identified a novel subset of memory-activated NK cells that was enriched in HbAS children and associated with parasite control. We hypothesised that other memory cell subsets might distinguish the baseline profile of HbAS children and children with normal haemoglobin (HbAA). METHODS: Subsets of memory T cells and NK cells were analysed by flow cytometry in paired samples collected from HbAS and HbAA children, at baseline and during the first malaria episode of the ensuing transmission season. Correlations between cell frequencies and features of HbAS-mediated protection from malaria were determined. RESULTS: HbAS children displayed significantly higher frequency of memory CD8+ T cells at baseline than HbAA children. Baseline frequency of memory CD8+ T cells correlated with features of HbAS-mediated protection from malaria. Exploration of memory CD8+ T cell subsets revealed that central memory CD8+ T cell frequency was higher in HbAS children than in HbAA children. CONCLUSION: This study shows that HbAS children develop a larger memory CD8+ T cell compartment than HbAA children, and associates this compartment with better control of subsequent onset of infection and parasite density. Our data suggest that central memory CD8+ T cells may play an important role in the relative protection against malaria experienced by HbAS individuals, and further work to investigate this is warranted.

An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells.

Reliable extraction and sensitive detection of RNA from human peripheral blood mononuclear cells (PBMCs) is critical for a broad spectrum of immunology research and clinical diagnostics. RNA analysis platforms are dependent upon high-quality and high-quantity RNA; however, sensitive detection of specific responses associated with high-quality RNA extractions from human samples with limited PBMCs can be challenging. Furthermore, the comparative sensitivity between RNA quantification and best-practice protein quantification is poorly defined. Therefore, we provide herein a critical evaluation of the wide variety of current generation of RNA-based kits for PBMCs, representative of several strategies designed to maximize sensitivity. We assess these kits with a reverse transcription quantitative PCR (RT-qPCR) assay optimized for both analytically and diagnostically sensitive cell-based RNA-based applications. Specifically, three RNA extraction kits, one post-extraction RNA purification/concentration kit, four SYBR master-mix kits, and four reverse transcription kits were tested. RNA extraction and RT-qPCR reaction efficiency were evaluated with commonly used reference and cytokine genes. Significant variation in RNA expression of reference genes was apparent, and absolute quantification based on cell number was established as an effective RT-qPCR normalization strategy. We defined an optimized RNA extraction and RT-qPCR protocol with an analytical sensitivity capable of single cell RNA detection. The diagnostic sensitivity of this assay was sufficient to show a CD8+ T cell peptide epitope hierarchy with as few as 1 × 104 cells. Finally, we compared our optimized RNA extraction and RT-qPCR protocol with current best-practice immune assays and demonstrated that our assay is a sensitive alternative to protein-based assays for peptide-specific responses, especially with limited PBMCs number. This protocol with high analytical and diagnostic sensitivity has broad applicability for both primary research and clinical practice.

A novel population of memory-activated natural killer cells associated with low parasitaemia in Plasmodium falciparum-exposed sickle-cell trait children.

OBJECTIVES: The sickle-cell trait phenotype is associated with protection from malaria. Multiple molecular mechanisms have been proposed to explain this protection, but the role of the host immune system has been poorly investigated. We hypothesised that cellular immunity to malaria may develop differently in sickle-cell trait children (HbAS) and children with normal haemoglobin (HbAA) repeatedly exposed to Plasmodium falciparum (Pf). METHODS: Paired samples collected prior to the Pf transmission season and during the first malaria episode of the ensuing transmission season from HbAS and HbAA children were analysed by multiplex bead-based assay and comprehensive multi-dimensional flow cytometry profiling. RESULTS: Cellular immune profiles were enriched in HbAS relative to HbAA children before the start of the Pf transmission season, with a distinct NK subset. These cells were identified as a novel subset of memory-activated NK cells characterised by reduced expression of the ecto-enzyme CD38 as well as co-expression of high levels of HLA-DR and CD45RO. The frequency of this NK subset before the transmission season was negatively correlated with parasite density quantified during the first malaria episode of the ensuing transmission season. Functional assessment revealed that these CD38dim CD45RO+ HLA-DR+ NK cells represent a important source of IFN-γ. CONCLUSION: Our data suggest that this novel memory-activated NK cell subset may contribute to an accelerated and enhanced IFN-γ-mediated immune response and to control of parasite density in individuals with the sickle-cell trait. This distinct cellular immune profile may contribute to predispose HbAS children to a relative protection from malaria.

Targeted insertion of an anti-CD2 monoclonal antibody transgene into the GGTA1 locus in pigs using FokI-dCas9.

Xenotransplantation from pigs has been advocated as a solution to the perennial shortage of donated human organs and tissues. CRISPR/Cas9 has facilitated the silencing of genes in donor pigs that contribute to xenograft rejection. However, the generation of modified pigs using second-generation nucleases with much lower off-target mutation rates than Cas9, such as FokI-dCas9, has not been reported. Furthermore, there have been no reports on the use of CRISPR to knock protective transgenes into detrimental porcine genes. In this study, we used FokI-dCas9 with two guide RNAs to integrate a 7.1 kilobase pair transgene into exon 9 of the GGTA1 gene in porcine fetal fibroblasts. The modified cells lacked expression of the αGal xenoantigen, and secreted an anti-CD2 monoclonal antibody encoded by the transgene. PCR and sequencing revealed precise integration of the transgene into one allele of GGTA1, and a small deletion in the second allele. The cells were used for somatic cell nuclear transfer to generate healthy male knock-in piglets, which did not express αGal and which contained anti-CD2 in their serum. We have therefore developed a versatile high-fidelity system for knocking transgenes into the pig genome for xenotransplantation purposes.

Anti-apoptotic proteins BCL-2, MCL-1 and A1 summate collectively to maintain survival of immune cell populations both in vitro and in vivo.

Survival of various immune cell populations has been proposed to preferentially rely on a particular anti-apoptotic BCL-2 family member, for example, naive T cells require BCL-2, while regulatory T cells require MCL-1. Here we examined the survival requirements of multiple immune cell subsets in vitro and in vivo, using both genetic and pharmacological approaches. Our findings support a model in which survival is determined by quantitative participation of multiple anti-apoptotic proteins rather than by a single anti-apoptotic protein. This model provides both an insight into how the sum of relative levels of anti-apoptotic proteins BCL-2, MCL-1 and A1 influence survival of T cells, B cells and dendritic cells, and a framework for ascertaining how these different immune cells can be optimally targeted in treatment of immunopathology, transplantation rejection or hematological cancers.

Cognate antigen engagement on parenchymal cells stimulates CD8+ T cell proliferation in situ.

T-cell responses are initiated upon cognate presentation by professional antigen presenting cells in lymphoid tissue. T cells then migrate to inflamed tissues, but further T-cell stimulation in these parenchymal target sites is not well understood. Here we show that T-cell expansion within inflamed tissues is a distinct phase that is neither a classical primary nor classical secondary response. This response, which we term 'the mezzanine response', commences within days after initial antigen encounter, unlike the secondary response that usually occurs weeks after priming. A further distinction of this response is that T-cell proliferation is driven by parenchymal cell antigen presentation, without requiring professional antigen presenting cells, but with increased dependence on IL-2. The mezzanine response might, therefore, be a new target for inhibiting T-cell responses in allograft rejection and autoimmunity or for enhancing T-cell responses in the context of microbial or tumour immunity.

Plasmacytoid dendritic cells are short-lived: reappraising the influence of migration, genetic factors and activation on estimation of lifespan.

Plasmacytoid dendritic cells (pDCs) play an important role in immunity to certain pathogens and immunopathology in some autoimmune diseases. They are thought to have a longer lifespan than conventional DCs (cDCs), largely based on a slower rate of BrdU labeling by splenic pDCs. Here we demonstrated that pDC expansion and therefore BrdU labeling by pDCs occurs in bone marrow (BM). The rate of labeling was similar between BM pDCs and spleen cDCs. Therefore, slower BrdU labeling of spleen pDCs likely reflects the "migration time" (∼2 days) for BrdU labeled pDCs to traffic to the spleen, not necessarily reflecting longer life span. Tracking the decay of differentiated DCs showed that splenic pDCs and cDCs decayed at a similar rate. We suggest that spleen pDCs have a shorter in vivo lifespan than estimated utilizing some of the previous approaches. Nevertheless, pDC lifespan varies between mouse strains. pDCs from lupus-prone NZB mice survived longer than C57BL/6 pDCs. We also demonstrated that activation either positively or negatively impacted on the survival of pDCs via different cell-death mechanisms. Thus, pDCs are also short-lived. However, the pDC lifespan is regulated by genetic and environmental factors that may have pathological consequence.

Prosurvival Bcl-2 family members reveal a distinct apoptotic identity between conventional and plasmacytoid dendritic cells.

Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.

Bcl-2 antagonists kill plasmacytoid dendritic cells from lupus-prone mice and dampen interferon-α production.

OBJECTIVE: Interferon-α (IFNα)-producing plasmacytoid dendritic cells (PDCs) are implicated in the pathogenesis of systemic lupus erythematosus (SLE). IFNα-related genes are highlighted among SLE susceptibility alleles and are characteristically expressed in the blood of patients with SLE, while in mouse models of lupus, PDC numbers and IFNα production are increased. This study was undertaken to investigate the effects of inhibitors that selectively target different antiapoptotic molecules on the survival of PDCs. METHODS: PDC numbers, in vitro survival, and expression of antiapoptotic molecules were evaluated in lupus-prone (NZB × NZW)F1 (NZB/NZW) mice. The impact of Bcl-2 antagonists and glucocorticoids on PDCs was evaluated in vitro and in vivo. IFNα production by NZB/NZW mice was evaluated before and after treatment with Bcl-2 antagonists. RESULTS: PDCs, but not lymphoid tissue-resident conventional DCs, largely relied on the antiapoptotic protein Bcl-2 for survival. The enlarged PDC compartment in NZB/NZW mice was associated with selectively prolonged survival and increased Bcl-2 transcription. Functionally, this resulted in enhanced production of IFNα. Bcl-2 inhibitors selectively killed mouse and human PDCs, including PDCs from SLE patients, but not conventional DCs, dampened IFNα production by PDCs, and synergized with glucocorticoids to kill activated PDCs. CONCLUSION: Enhanced PDC survival is a likely contributing factor to enhanced IFNα production by lupus PDCs. Bcl-2 antagonists potently and selectively kill PDCs and reduce IFNα production. Thus, we believe that they are attractive candidates for treating PDC-associated diseases.

The closely related CD103+ dendritic cells (DCs) and lymphoid-resident CD8+ DCs differ in their inflammatory functions.

Migratory CD103+ and lymphoid-resident CD8+ dendritic cells (DCs) share many attributes, such as dependence on the same transcription factors, cross-presenting ability and expression of certain surface molecules, such that it has been proposed they belong to a common sub-lineage. The functional diversity of the two DC types is nevertheless incompletely understood. Here we reveal that upon skin infection with herpes simplex virus, migratory CD103+ DCs from draining lymph nodes were more potent at inducing Th17 cytokine production by CD4+ T cells than CD8+ DCs. This superior capacity to drive Th17 responses was also evident in CD103+ DCs from uninfected mice. Their differential potency to induce Th17 differentiation was reflected by higher production of IL-1ß and IL-6 by CD103+ DCs compared with CD8+ DCs upon stimulation. The two types of DCs from isolated lymph nodes also differ in expression of certain pattern recognition receptors. Furthermore, elevated levels of GM-CSF, typical of those found in inflammation, substantially increased the pool size of CD103+ DCs in lymph nodes and skin. We argue that varied levels of GM-CSF may explain the contrasting reports regarding the positive role of GM-CSF in regulating development of CD103+ DCs. Together, we find that these two developmentally closely-related DC subsets display functional differences and that GM-CSF has differential effect on the two types of DCs.

GM-CSF-responsive monocyte-derived dendritic cells are pivotal in Th17 pathogenesis.

Although multiple dendritic cell (DC) subsets have the potential to induce Th17 differentiation in vitro, the key DC that is critical in Th17 induction and Th17-mediated disease remains moot. In this study, we revealed that CCR2(+) monocyte-derived DCs (moDCs), but not conventional DCs, were critical for in vivo Th17 induction and autoimmune inflammation. Functional comparison in vitro indicated that moDCs are the most potent type of Th17-inducing DCs compared with conventional DCs and plasmacytoid DCs. Furthermore, we demonstrated that the importance of GM-CSF in Th17 induction and Th17-mediated disease is its endowment of moDCs to induce Th17 differentiation in vivo, although it has little effect on moDC numbers. Our findings identify the in vivo cellular targets that can be selectively manipulated to ameliorate Th17-mediated inflammatory diseases, as well as the mechanism of GM-CSF antagonism in such diseases.

Preclinical screening for acute toxicity of therapeutic monoclonal antibodies in a hu-SCID model.

Monoclonal antibodies (mAbs) have been a spectacular clinical and commercial success in the treatment of cancer and autoimmune diseases. Many of these mAbs (for example, OKT3, Campath-1H, rituximab and infliximab) are against surface or secreted products of lymphocytes. However, mAbs can have a variety of adverse effects including fever, chills and nausea. This is probably a result of cytokine release, which is most seriously manifested as a 'cytokine storm' as highlighted by the TGN1412 (anti-CD28) trial. Prediction of adverse effects of mAbs would be clinically advantageous and numerous in vitro assays attempting to predict adverse effects have been reported. Here, we report an in vivo humanized mouse model to detect adverse effects in response to OKT3, Campath-1H or the polyclonal Ab preparation anti-thymocyte globulin. We found that the administration of each of these Abs to humanized mice led to acute clinical symptoms such as piloerection, hypomotility and hypothermia, particularly when delivered via the intravenous route. A cytokine storm occurred in the humanized mice receiving OKT3. This model system is a potentially useful tool to predict adverse effects and select initial doses for first-in-human trials. We would advocate this in vivo model, in addition to current in vitro preclinical testing, as a more representative and robust means of assessing potential adverse effects of mAb before their human use.

Anti-CD2 producing pig xenografts effect localized depletion of human T cells in a huSCID model.

BACKGROUND: We investigated whether graft produced anti-human CD2, mediated by adenovirus (Adv) transduction of pig neonatal islet cell clusters (pNICC), would protect xenografts in a humanized mouse model from immune attack and whether such immunosuppression would remain local. METHODS: A mouse anti-human CD2 Ab (CD2hb11) previously generated by us was genetically engineered to produce chimeric and humanized versions. The three forms of CD2hb11 were named dilimomab (mouse), diliximab (chimeric) and dilizumab (humanized). All 3 forms of CD2hb11 Ab were tested for their ability to bind CD3(+) human T cells and to inhibit a human anti-pig xenogeneic mixed lymphocyte reaction (MLR). They were administered systemically in a humanized mouse model in order to test their ability to deplete human CD3(+) T cells and whether they induced a cytokine storm. An adenoviral vector expressing diliximab was generated for transduction of pNICC. Humanized mice were transplanted with either control-transduced pNICC or diliximab-transduced pNICC and human T cells within grafts and spleens were enumerated by flow cytometry. RESULTS: Dilimomab and diliximab inhibited a human anti-pig xenogeneic response but dilizumab did not. All 3 forms of CD2hb11 Ab bound human T cells in vitro though dilimomab and diliximab exhibited 300-fold higher avidity than dilizumab. All 3 anti-CD2 Abs could deplete human CD3(+) T cells in vivo in a humanized mouse model without inducing upregulation of activation markers or significant release of cytokines. Humanized mice transplanted with diliximab-transduced pNICC afforded depletion of CD3(+) T cells at the graft site leaving the peripheral immune system intact. CONCLUSIONS: Local production of a single Ab against T cells can reduce graft infiltration at the xenograft site and may reduce the need for conventional, systemic immunosuppression.

Influenza-induced, helper-independent CD8+ T cell responses use CD40 costimulation at the late phase of the primary response.

The helper-dependent pathway of priming CD8(+) T cells involves "licensing" of DCs by CD40L on CD4(+) T cells. The helper-independent ("helpless") pathways elicited by many viruses, including influenza, are less widely understood. We have postulated that CD40L can be up-regulated on DCs by such viruses, and this promotes priming of CD8(+) T cells via CD40. Most studies on costimulation have been performed in the presence of CD4(+) T cells, and so the role of CD40L costimulation under helpless circumstances has not been fully elucidated. Here, we investigated such a role for CD40L using CD40L KO mice. Although the number of influenza-specific CD8(+) T cells was unaffected by the absence of CD4(+) T cells, it was markedly decreased in the absence of CD40L. Proliferation (the number of CD44(+)BrdU(+) influenza-specific CD8(+) T cells) in the primary response was diminished in CD40L KO mice at Day 8 but not at Day 5 after infection. MLR studies indicated that CD40L expression on DCs was critical for CD8(+) T cell activation. Adoptive transfer of CD40 KO CD8(+) T cells compared with WT cells confirmed that CD40 on such cells was critical for the generation of primary anti-influenza CD8(+) T cell responses. The late effect also corresponded with the late expression of CD40 by influenza-specific CD8(+) T cells. We suggest that costimulation via CD40L on DCs and CD40 on CD8(+) T cells is important in optimizing primary CD8(+) T cell responses during influenza infection.

Unlike CD4+ T-cell help, CD28 costimulation is necessary for effective primary CD8+ T-cell influenza-specific immunity.

The importance of costimulation on CD4(+) T cells has been well documented. However, primary CTLs against many infections including influenza can be generated in the absence of CD4(+) T-cell help. The role of costimulation under such "helpless" circumstances is not fully elucidated. Here, we investigated such a role for CD28 using CTLA4Ig transgenic (Tg) mice. To ensure valid comparison across the genotypes, we showed that all mice had similar naïve precursor frequencies and similar peak viral loads. In the absence of help, viral clearance was significantly reduced in CTLA4Ig Tg mice compared with WT mice. CD44(+) BrdU(+) influenza-specific CD8(+) T cells were diminished in CTLA4Ig Tg mice at days 5 and 8 postinfection. Adoptive transfer of ovalbumin-specific transgenic CD8(+) T cells (OT-I)-I cells into WT or CTLA4Ig Tg mice revealed that loss of CD28 costimulation resulted in impairment in OT-I cell division. As shown previously, neither viral clearance nor the generation of influenza-specific CD8(+) T cells was affected by the absence of CD4(+) T cells alone. In contrast, both were markedly impaired by CD28 blockade of "helpless" CD8(+) T cells. We suggest that direct CD28 costimulation of CD8(+) T cells is more critical in their priming during primary influenza infection than previously appreciated.

Versatile co-expression of graft-protective proteins using 2A-linked cassettes.

BACKGROUND: Expression of multiple graft-protective proteins targeted to different locations (i.e., intracellular, cell surface, and secreted) has become an increasingly important goal in xenotransplantation. The 2A "ribosome skip" signal is used as a linker to enable transgene co-expression, but some studies have shown that post-translational modification and trafficking of 2A-linked proteins may be adversely affected depending on their position relative to 2A. We tested whether several relevant proteins, subject to a range of processing and localization mechanisms, could be efficiently co-expressed using the 2A system. METHODS: Six expression cassettes were constructed, each containing up to four 2A-linked open reading frames, encoding combinations of human CD55, thrombomodulin (TBM), CD39, CTLA4-Ig and hygromycin resistance. Each linker incorporated a furin cleavage site to remove the carboxy-terminal extension that remains on upstream proteins after 2A processing. The cassettes were used to produce vectors for transfection, adenoviral transduction and transgenesis. Expression was detected by flow cytometry and/or Western blotting. RESULTS: All proteins were expressed in the appropriate location following transient transfection of COS-7 cells, irrespective of the number of linked genes. The percentage of stable transfectants expressing a linked gene was increased 10-fold (from 4-5% to 58-67%) by incorporating the hygromycin resistance gene into the cassette. Stable transfection of transgenic GalT KO pig fibroblasts with a hygromycin- TBM-CD39 construct resulted in surface expression of both TBM and CD39 by the majority of hygromycin-resistant cells. Expression was maintained after flow cytometric sorting and expansion. Adenoviral transduction of NIT-1 mouse insulinoma cells with a TBM-CD39 construct resulted in strong expression of both genes on the cell surface. Mice transgenic for 3-gene (CD55- TBM-CD39) or 4-gene (CD55- TBM-CTLA4Ig-CD39) constructs expressed all genes except CD55. CONCLUSIONS: These results confirm the versatility of the 2A system, and demonstrate that careful construct design can minimize potential problems with post-translational modification and trafficking. In addition, incorporation of a selection marker into the 2A-linked chain can dramatically increase the proportion of stable transfectants expressing proteins of interest. This provides a powerful method for the rapid modification of existing genetically modified pigs.

In situ protection against islet allograft rejection by CTLA4Ig transduction.

BACKGROUND: Immunosuppression focused at or near the graft site would reduce the need for systemic immunosuppression thus educing fewer side effects. We investigated whether locally produced CTLA4Ig, mediated by adenovirus (Adv) transduction of mouse islets, would protect allografts and whether such immunosuppression would remain localized. METHODS: Adv-CTLA4Ig- or Adv-control-transduced islets were grafted under the kidney capsule of fully allogeneic diabetic mice. CTLA4Ig secreted from the grafted islets was detected by enzyme immunoassay of blood or immunohistochemistry of graft sections. Graft survival was monitored by blood glucose measurement. Histologic scores of graft sections stained with Gomori aldehyde fuchsin to detect insulin granules or hematoxylin-eosin to detect inflammation were used to compare grafts placed at different sites within the same mouse. RESULTS: Adv-CTLA4Ig-transduced islet grafts secreted CTLA4Ig that was detected transiently in the circulation but persistently at the graft site. Survival of these grafts was significantly enhanced compared with control Adv-transduced and untransduced grafts. The kidney graft site availed elucidation of the site of action of CTLA4Ig. Histologic scores indicated that CTLA4Ig-producing grafts were protected by comparison with control grafts on the contralateral kidney. Hence, graft protection was not attributable to general systemic immunosuppression. Indeed, survival of CTLA4Ig-producing grafts was enhanced over control grafts placed at the opposite pole of the same kidney, indicating that graft protection was not solely due to inhibition of priming in the common draining lymph nodes. CONCLUSIONS: Islet allografts are protected by locally produced CTLA4Ig-disrupting immune interactions at the effector site.

Resident and monocyte-derived dendritic cells become dominant IL-12 producers under different conditions and signaling pathways.

IL-12 is such a pivotal cytokine that it has been called the third signal for T cell activation, TCR engagement being the first and costimulation being the second. It has been generally viewed that the resident CD8(+) dendritic cell (DC) subset is the predominant IL-12-producing cell type. In this study, we found, although this is so under steady state conditions, under inflammatory conditions monocyte-derived DC (mDC) became a major cell type producing IL-12. Depletion of either type of DC resulted in reduced production of IL-12 in vivo. For CD8(+) DC, IL-12 production could be stimulated by various pathways viz. signaling through MyD88, Trif, or nucleotide-binding oligomerization domain (Nod)-like receptors. In contrast, for mDC, IL-12 production was mainly dependent on MyD88 signaling. Thus, conventional DCs and mDCs use different pathways to regulate IL-12 production.