Induction of adaptive immunity by flagellin does not require robust activation of innate immunity

The ability of TLR agonists to promote adaptive immune responses is attributed to their ability to robustly activate innate immunity. However, it has been observed that, for adjuvants in actual use in research and vaccination, TLR signaling is dispensable for generating humoral immunity. Here, we examined the role of TLR5 and MyD88 in promoting innate and humoral immunity to flagellin using a prime/boost immunization regimen. We observed that eliminating TLR5 greatly reduced flagellin‐induced cytokine production, except for IL‐18, and ablated DC maturation but did not significantly impact flagellin's ability to promote humoral immunity. Elimination of MyD88, which will ablate signaling through TLR and IL‐1β/IL‐18 generated by Nod‐like receptors, reduced, but did not eliminate flagellin's promotion of humoral immunity. In contrast, loss of the innate immune receptor for profilin‐like protein (PLP), TLR11, greatly reduced the ability of PLP to elicit humoral immunity. Together, these results indicate that, firstly, the degree of innate immune activation induced by TLR agonists may be in great excess of that needed to promote humoral immunity and, secondly, there is considerable redundancy in mechanisms that promote the humoral immune response upon innate immune recognition of flagellin. Thus, it should be possible to design innate immune activators that are highly effective vaccine adjuvants yet avoid the adverse events associated with systemic TLR activation.     

TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity

Summary: The TIM (T cell/transmembrane, immunoglobulin, and mucin) gene family plays a critical role in regulating immune responses, including allergy, asthma, transplant tolerance, autoimmunity, and the response to viral infections. The unique structure of TIM immunoglobulin variable region domains allows highly specific recognition of phosphatidylserine (PtdSer), exposed on the surface of apoptotic cells. TIM-1, TIM-3, and TIM-4 all recognize PtdSer but differ in expression, suggesting that they have distinct functions in regulating immune responses. TIM-1, an important susceptibility gene for asthma and allergy, is preferentially expressed on T-helper 2 (Th2) cells and functions as a potent costimulatory molecule for T-cell activation. TIM-3 is preferentially expressed on Th1 and Tc1 cells, and generates an inhibitory signal resulting in apoptosis of Th1 and Tc1 cells. TIM-3 is also expressed on some dendritic cells and can mediate phagocytosis of apoptotic cells and cross-presentation of antigen. In contrast, TIM-4 is exclusively expressed on antigen-presenting cells, where it mediates phagocytosis of apoptotic cells and plays an important role in maintaining tolerance. TIM molecules thus provide a functional repertoire for recognition of apoptotic cells, which determines whether apoptotic cell recognition leads to immune activation or tolerance, depending on the TIM molecule engaged and the cell type on which it is expressed.     

The protein tyrosine kinase inhibitor herbimycin A, but not genistein, specifically inhibits signal transduction by the T cell antigen receptor.

Several lines of evidence implicate a regulatory tyrosine phosphorylation in the activation of phospholipase C (PLC) by the T cell antigen receptor (TCR). These include studies using inhibitors of protein tyrosine kinases (PTKs). In Jurkat T cells expressing the heterologous human muscarinic receptor (HM1), PLC activity can be induced by either the TCR or HM1. HM1 activates PLC via a guanine nucleotide binding protein. We have studied the selectivity of the effects of the PTK inhibitors, herbimycin A and genistein, in this system. The results indicate that these inhibitors have different mechanisms of action, and suggest that herbimycin A, but not genistein, is a specific inhibitor of PTKs in T cells. Herbimycin A markedly inhibited both the resting and induced levels of phosphotyrosine-containing proteins, including the gamma 1 isozyme of PLC and the zeta chain of the TCR, and prevented activation of PLC by anti-TCR mAb. Herbimycin A did not inhibit activation of PLC by HM1. Genistein had a much less pronounced effect than herbimycin A on the appearance of tyrosine phosphoproteins. Moreover, genistein inhibited activation of PLC by both the TCR and HM1, and inhibition was only partial. Genistein was cytotoxic and markedly inhibited protein synthesis in both Jurkat cells and human peripheral lymphocytes. Herbimycin A was not cytotoxic. These findings confirm the role of a regulatory tyrosine phosphorylation in activation of PLC by the TCR. Herbimycin A was a selective inhibitor of a subclass of PTKs in Jurkat cells. In contrast, inhibition of signal transduction and later events in T cells by genistein may be due to effects other than direct inhibition of PTK activity.     

CD1 proteins: targets of T cell recognition in innate and adaptive immunity

The CD1 family consists of antigen presenting molecules encoded by genes located outside of the major histocompatibility complex. CD1 proteins are conserved among mammalian species and are expressed on the surface of cells involved in antigen presentation. The CD1 system has been shown to be involved in activation of cell-mediated responses, and T cells specific for either CD1 molecules or antigens presented by CD1 have been isolated. Structural and biochemical analyses demonstrate that antigens presented by CD1 are nonpeptide lipid or glycolipid structures, including examples found in the cell walls of pathogenic mycobacteria. The hydrophobic part of these antigens most likely binds in the CD1 ligand-binding groove, whereas the polar headgroup of these antigens appears to make direct contact with the T cell receptor and determines specific recognition. Presentation of antigens by CD1 molecules requires uptake and intracellular processing by antigen presenting cells and can be achieved for both exogenous and endogenous antigens. T cells recognizing CD1 restricted antigens have a broad range of functional activities that suggest that the CD1 system is involved in both innate and adaptive immune responses against microbial infections.     

Inhibition of T lymphocyte heparanase by heparin prevents T cell migration and T cell-mediated immunity

Previously we reported that activated T lymphocytes express a heparanase enzyme that degrades the heparan sulfate moiety of the proteoglycan of the extracellular matrix (ECM). Expression of the heparanase enzyme was found to be associated with the ability of activated T lymphocytes to penetrate blood vessel walls and accumulate in target organs. We recently found that relatively low doses of heparin administered to mice or rats inhibited T cell-mediated immune reactions. In the present study we investigated the effects in vitro and in vivo of the heparanase inhibitor, heparin, on the expression of T lymphocyte heparanase and on the ability of T lymphocytes to mediate a delayed-type hypersensitivity (DTH) reaction. We found that heparanase was induced by immunizing mice with antigen in vivo or by activating T lymphocytes with concanavalin A in vitro. Relatively low doses of heparin administered once daily in vivo (5 micrograms) or present in vitro (0.1 microgram/ml) inhibited the expression of heparanase induced by immunization or by concanavalin A incubation. Higher or lower doses of heparin did not have these effects. The same doses of heparin that inhibited expression of heparanase also inhibited the ability of the lymph node cells to migrate to a site of antigen and adoptively produce a DTH reaction. These findings suggest that modulation of cell-mediated immune reactions may be achieved by relatively low doses of heparin which inhibit expression of T lymphocyte heparanase.     

IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity

IFNalpha/beta, IL-12, and IL-15 regulate NK cell activation and expansion, but signals triggering resolution of the NK response upon induction of adaptive immunity remain to be defined. We now report that IL-21, a product of activated T cells, may serve this function. Mice lacking IL-21R (IL-21R(-/-)) had normal NK cell development but no detectable responses to IL-21. IL-21 enhanced cytotoxic activity and IFNgamma production by activated murine NK cells but did not support their viability, thus limiting their duration of activation. Furthermore, IL-21 blocked IL-15-induced expansion of resting NK cells, thus preventing the initiation of further innate responses. In contrast, IL-21 enhanced the proliferation, IFNgamma production, and cytotoxic function of CD8(+) effector T cells in an allogeneic MLR. These observations suggest that IL-21 promotes the transition between innate and adaptive immunity.     

Attenuation of edema and infarct volume following focal cerebral ischemia by early but not delayed administration of a novel small molecule KDR kinase inhibitor

Vascular endothelial growth factor (VEGF) may mediate increases in vascular permeability and hence plasma extravasation and edema following cerebral ischemia. To better define the role of VEGF in edema, we examined the effectiveness of a novel small molecule KDR kinase inhibitor Compound-1 in reducing edema and infarct volume following focal cerebral ischemia in studies utilizing treatment regimens initiated both pre- and post-ischemia, and with study durations of 24-72 h. Rats were subjected to 90 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. Pretreatment with Compound-1 (40 mg/kg p.o.) starting 0.5h before occlusion significantly reduced infarct volume at 72 h post-MCAO (vehicle, 194.1+/-22.9 mm(3) vs. Compound-1, 127.6+/-22.8mm(3) and positive control MK-801, 104.4+/-22.6mm(3), both p<0.05 compared to vehicle control), whereas Compound-1 treatment initiated at 2h after occlusion did not affect infarct volume. Compound-1 pretreatment also significantly reduced brain water content at 24h (vehicle, 80.3+/-0.2% vs. Compound-1, 79.7+/-0.2%, p<0.05) but not at 72 h after MCAO. These results demonstrate that early pretreatment administration of a KDR kinase inhibitor elicited an early, transient decrease in edema and subsequent reduction in infarct volume, implicating VEGF as a mediator of stroke-related vascular permeability and ischemic injury.     

The Structure of the 18S rRNA, a molecule that might be evolutionarily related to some receptors of innate immunity

Comparisons between the sequences of insect and vertebrate 18S rRNAs and the sequences of mammalian formyl peptide and some vertebrate chemokine receptor mRNAs demonstrated non-random structural similarities between these two groups of RNAs. It has been proposed that sections of the more ancient and conserved rRNA genes could have participated in the building of these more recent genes involved in immune responses. Here we analyze the sequence architecture of the 18S rRNA in insects (Drosophila simulans) and vertebrates (man), in terms of similarities between selected segments within the individual molecules. The insect and vertebrate 18S rRNAs are basically similar, but show specific insertions/deletions and base changes. In spite of these differences, in both sequences a significantly higher-than-expected (by random occurrence) number of 7-or-more-base oligonucleotide repeats was observed between segments roughly corresponding to nt 350-1050 and nt 1150-1850, with mutual between-repeats distances comprised in the range 700-900 nt. Based on this result we performed a multialignment of segments 317-1035 of Drosophila, 360-1005 of man, 1096-1864 of Drosophila, and 1066-1736 of man, the first two segments covering the region of first occurrence of the repeats and the last two the region of recurrences. At both ends of these segments the four sequences could be aligned with relatively minor gaps and the number of base identities in all four sequences was significantly higher than expected by random coincidences. These results support the hypothesis that an ancestral gene structure, composed of a chain of about 700 nt, duplicated to form a two-unit tandem repeat which still represents the most substantial part of the 18S rRNA molecule in extant insects and vertebrates.     

T cell recognition of cell-surface antigens. II. Antigen recognition is necessary yet not sufficient for triggering T cell-mediated immunity

We investigated whether recognition per se of cell surface antigens by lymphocytes is both necessary and sufficient to trigger sensitization of T cells. We assayed sensitization of normal rat lymphocytes against monolayers of mouse fibroblasts by measuring the acquisition of cytotoxicity against target monolayers syngeneic to the sensitizing monolayer, following 5 days of culturing on living or glutaraldehyde-fixed fibroblasts. Monolayers of either living or glutaraldehyde-treated fibroblasts were used as cellular immunoadsorbents to assay recognition. Glutaraldehyde treatment of mouse fibroblasts did not seem to alter the cell surface antigens detectable by alloantibodies. Yet, rat lymphocytes cultured on such monolayers did not undergo sensitization. This was not due to the lack of feeder effect by the glutaraldehyde-fixed monolayers, since the addition of living rat fibroblasts, which sustained the survival of the lymphocytes, did not result in sensitization. Not even when living fibroblasts were added to syngeneic fixed monolayers did sensitization of the rat lymphocytes against the monolayer cells occur. Yet, addition of living fibroblasts to fixed allogeneic monolayers resulted in sensitization against the living fibroblasts, but not against the fixed cells. To test whether lymphocytes recognize surface antigens of glutaraldehyde-treated monolayers, the capacity of the latter to specifically adsorb lymphocytes possessing receptors for the fibroblast antigen was measured. The nonadhering rat lymphocytes, seeded on glutaraldehyde-treated C3H fibroblasts, lost their capacity to become sensitized against fresh C3H cells, but retained their capacity to react against BALB/c fibroblasts. Recognition of C3H antigen did take place, yet this was not sufficient to trigger sensitization. Thus, rat lymphocytes seem to recognize the antigens of fixed monolayers, but cannot respond to them.     

Inhibition of spontaneous but not antibody-dependent cell-mediated cytotoxicity by simple sugars: evidence that endogenous lectins may mediate spontaneous cell-mediated cytotoxicity.

Using Chang and K-562 cell line cells as targets, we have observed that a number of sugars are capable of inhibiting spontaneous cell-mediated cytotoxicity (SCMC) but not antibody-dependent cell-mediated cytotoxicity (ADCC). The sugars D(-)ribose, beta-gentiobiose, N-acetyl-D-galatosamine, and alpha-lactose all significantly inhibited SCMC of Chang and K-562 cell line cells. Because these same sugars caused no inhibition of ADCC against either Chang or K-562 cell line cells in assays run simultaneously, the results do not appear to be due to a non-specific toxic effect of the sugars against the effector cells. These studies add to the evidence that ADCC and SCMC are mediated by separate receptors. Furthermore, they provide evidence that endogenous lectin receptors or lectin-like molecules may be involved in the recognition and/or effector stages leading to SCMC. Thus, NK cells may recognize targets by virtue of receptors capable of interacting with monosaccharide, disaccharide, or oligosaccharide sequences present alone, as glycolipids, and/or as glycoproteins on the target cell surface.     

The subcellular location of antigen expressed by adenoviral vectors modifies adaptive immunity but not dependency on cross-presenting dendritic cells

Adenoviral (Ad) vaccine vectors can generate protective immunity to various pathogens in animal studies. However, recent failures in clinical vaccine trials have underscored the need for a better understanding of how mucosal immune responses to Ad-encoded vaccine Ags are generated in vivo. In this study, we addressed whether directing Ad-encoded ovalbumin (OVA) to different subcellular compartments influences the generation of OVA-specific acquired immunity and the APCs required following i.n. immunization of mice. We show that both secreted and membrane-anchored OVA activate CD4(+) T cells, induce cytotoxic CD8(+) T lymphocytes (CTLs) and generate serum IgG. Additionally, vaginal IgG is induced when OVA is expressed at these subcellular locations, but only the secreted form generates a significant IgA response in the lungs. On the contrary, intracellular expression of OVA efficiently expands CD8(+) T cells but fails to activate CD4(+) T cells, results in poor CTL activity, and does not generate Abs. Finally, we show that regardless of the subcellular localization of OVA, conventional DCs (cDCs) are required for the activation of T cells. However, the direct transduction of conventional DCs is not essential. These findings have important implications for the improvement of Ad vector design and vaccine-induced mucosal immunity.     

The messenger between worlds: the regulation of innate and adaptive type‐2 immunity by innate lymphoid cells

Although type‐2 immune responses evolved primarily to defend against extracellular helminths, in part through the co‐opting of tissue repair and remodeling mechanisms, they are often inappropriately directed towards relatively innocuous allergens resulting in conditions including asthma, allergic rhinitis, food allergy, and atopic dermatitis. The recent discovery of group 2 innate lymphoid cells (ILC2) has increased our understanding of the initiation of these responses and the roles played by CD4⁺ T helper (Th) 2 cells in their modulation. This review focuses on the important messenger role of ILC2 in translating epithelial‐derived alarmins into downstream adaptive type‐2 responses via dendritic cells and T cells, with special emphasis on their roles in allergic disease.     

Inhibition of cathepsin S reduces allogeneic T cell priming but not graft-versus-host disease against minor histocompatibility antigens

Cathepsin (Cathepsin) S, L, and B proteases mediate antigen presentation on major histocompatibility complex (MHC) class II by degrading the invariant chain Ii, which blocks peptide loading. The ability of the Cathepsin S inhibitor LHVS (morpholinurea-leucine-homophenylalanine-vinylsulfone phenyl) to impede antigen presentation has led its development as a therapy for autoimmune diseases. There is substantial evidence that donor T cell recognition of host minor histocompatibility antigens (miHA) and subsequent destruction of host tissue mediates graft-versus-host disease (GVHD). We hypothesized that enzymes involved in antigen presentation may play a role in the development of GVHD. Using the C57BL/6 → BALB.B minor mismatch acute GVHD (aGVHD) model, we found that the cathepsin S activity of spleens from allogenetically transplanted mice were significantly increased 1 week after transplantation compared with syngeneic mice. Although LHVS decreased T cell priming responses against both single OVA antigen and miHA in?vitro, LHVS did not reduce the severity of aGVHD. In fact, LHVS exacerbated a CD4(+)-T cell-dependent model of GVHD similar to chronic GVHD. This suggests that cytokines rather than T cells may mediate much of the damage in the aGVHD model and that therapeutics based on inhibition of antigen presentation for GVHD must be approached with caution.     

The induction of resting B cell differentiation does not require T cell contact.

A T cell clone as well as immediately ex vivo CD4+ lymph node T cells are shown to support the differentiation of co-cultured resting B cells in the absence of T cell-B cell contact. Antibodies specific for class II products of the major histocompatibility complex inhibit the transactivation of resting but not activated B cells. This differential inhibition pattern indicates that the responses obtained from resting B cell populations are not due to their contamination with B cell blasts. Further, supernatants prepared from an activated T cell clone induce resting B cell differentiation. Two lines of evidence suggest that the activity contained in these supernatants can be attributed to interleukin (IL)-5. Activity is neutralized by monoclonal anti-IL-5; and both recombinant and affinity-purified IL-5 induce the differentiation of immediately ex vivo resting and activated B cells with comparable efficiency. Taken together, these results demonstrate that contact with T cells does not provide prerequisite signals for the induction of resting B cell differentiation.     

T cell-mediated immunity to oncornavirus-induced tumors IV. Preliminary evidence for a specific suppression of anti-Moloney cell-mediated immune response by autoimmune T cells

Previous reports have demonstrated that adult C 57 BL/6 mice infected with murine sarcoma virus (MSV) develop a strong cell-mediated immune response against Friend, Moloney, Rauscher virus-induced type-specific (FMR) antigens and reject their tumors. To demonstrate a possible role for auto-anti-MSV T blasts, syngeneic C 57 BL/6 mice were immunized with highly enriched anti-FMR cytolytic T cells. One of 3 pools of these autoimmune T cells prepared from 12 surviving immunized mice (a) inhibited specifically the in vitro anti-MSV cytolysis generation and (b) enhanced drastically the MSV tumor growth in vivo. The possibility for such an immunization procedure to induce anti-idiotype T cells, the repeatability of this effect and the relationship of the suppressor cells with antigen-specific suppressor cells and other components of the anti-MSV immune response are discussed.     

Blocking T cell co‐stimulation using a CD80 blocking small molecule reduces delayed type hypersensitivity responses in rhesus monkeys

Blockade of co-stimulation signals between T cells and antigen-presenting cells could be an important approach for treatment of autoimmune diseases and transplant rejection. Recently a series of small compound inhibitors which bind human CD80 (B7-1) and inhibit T cell co-stimulation has been described. To investigate their potency for clinical use, one of these compounds, RhuDex™, was evaluated for reactivity with rhesus monkey CD80. The in vitro biological effect on rhesus monkey lymphocytes, the potency for suppression of an inflammatory recall response and the protein-induced delayed type hypersensitivity (DTH) response in the skin were studied. In a rhesus monkey T cell co-stimulation assay RhuDex™ inhibited proinflammatory cytokine release and cellular proliferation with micromolar potency. Systemic administration of RhuDex™ to rhesus monkeys inhibited the DTH response significantly, indicating that this compound may inhibit autoimmune mediated inflammatory processes where the target, CD80, is up-regulated.     

Suppressor T cell induction by soluble OKT3 antibody does not require cross-linking of the T3 molecule

The monoclonal antibody OKT3 can induce T suppressor cells of the in vitro antibody response when used at concentrations as low as 10 ng/ml and in cultures containing as few as 4% OKT3-pretreated T cells. These suppressor cells are radioresistant, cyclosporin sensitive and belong to the T4 as well as the T8 subpopulations. In contrast with the mitogenic effect of OKT3 the suppressor activity can be induced and expressed in highly monocyte-depleted cultures. Moreover F(ab')2 fragments of OKT3, although not mitogenic, are able to induce suppressor cells. Thus cross-linking of the T3 molecule (such as that realized when intact OKT3 IgG and monocytes are present) is not required to induce suppressor cells among resting normal T cells.     

Genetic control of cell-mediated immunity in the rat. III. T cells restricted by the RT1.A locus recognize viable Listeria but not isolated bacterial antigens

This study investigates the discordance between the restriction criteria required for the transfer of cellular resistance to Listeria monocytogenes (LM) and those for the transfer of delayed type hypersensitivity to Listeria antigens. Infective bacteria elicit both RT1.A-restricted T cells and RT1.B-restricted T cells. Both populations of T cells mediate lymphoblast localization and macrophage accumulation, which are reactions characteristic of delayed type hypersensitivity (DTH), and cause macrophage activation with rapid and efficient bacterial elimination, which is an expression of cellular resistance. If alcohol-killed Listeria organisms (pLMA) are injected, only the RT1.B-restricted T cell subset is triggered. Direct comparison of lymphoblast localization in LM infection sites and the expression of resistance revealed that efficient resistance may be mediated by small numbers of lymphoblasts and that below a certain threshold there is no correlation between lymphoblast localization and the level of resistance.     

The small molecule inhibitor g6 significantly reduces bone marrow fibrosis and the mutant burden in a mouse model of jak2-mediated myelofibrosis

Philadelphia chromosome-negative myeloproliferative neoplasms, including polycythemia vera, essential thrombocytosis, and myelofibrosis, are disorders characterized by abnormal hematopoiesis. Among these myeloproliferative neoplasms, myelofibrosis has the most unfavorable prognosis. Furthermore, currently available therapies for myelofibrosis have little to no efficacy in the bone marrow and hence, are palliative. We recently developed a Janus kinase 2 (Jak2) small molecule inhibitor called G6 and found that it exhibits marked efficacy in a xenograft model of Jak2-V617F-mediated hyperplasia and a transgenic mouse model of Jak2-V617F-mediated polycythemia vera/essential thrombocytosis. However, its efficacy in Jak2-mediated myelofibrosis has not previously been examined. Here, we hypothesized that G6 would be efficacious in Jak2-V617F-mediated myelofibrosis. To test this, mice expressing the human Jak2-V617F cDNA under the control of the vav promoter were administered G6 or vehicle control solution, and efficacy was determined by measuring parameters within the peripheral blood, liver, spleen, and bone marrow. We found that G6 significantly reduced extramedullary hematopoiesis in the liver and splenomegaly. In the bone marrow, G6 significantly reduced pathogenic Jak/STAT signaling by 53%, megakaryocytic hyperplasia by 70%, and the Jak2 mutant burden by 68%. Furthermore, G6 significantly improved the myeloid to erythroid ratio and significantly reversed the myelofibrosis. Collectively, these results indicate that G6 is efficacious in Jak2-V617F-mediated myelofibrosis, and given its bone marrow efficacy, it may alter the natural history of this disease.