Autoantibodies against DNA topoisomerase I promote renal allograft rejection by increasing alloreactive T cell responses.

Antibodies reactive to self-antigens are an important component of posttransplant immune responses. The generation requirements and functions of autoantibodies, as well as the mechanisms of their influence on alloimmune responses, still remain to be determined. Our study investigated the contribution of autoimmunity during rejection of renal allografts. We have previously characterized a mouse model in which the acute rejection of a life-supporting kidney allograft is mediated by antibodies. At rejection, recipient sera screening against >4000 potential autoantigens revealed DNA topoisomerase I peptide 205-219 (TI-I205-219) as the most prominent epitope. Subsequent analysis showed TI-I205-219-reactive autoantibodies are induced in nonsensitized recipients of major histocompatibility complex-mismatched kidney allografts in a T cell-dependent manner. Immunization with TI-I205-219 broke self-tolerance, elicited TI-I205-219 immunoglobin G autoantibodies, and resulted in acute rejection of allogeneic but not syngeneic renal transplants. The graft loss was associated with increased priming of donor-reactive T cells but not with donor-specific alloantibodies elevation. Similarly, passive transfer of anti-TI-I205-219 sera following transplantation increased donor-reactive T cell activation with minimal effects on donor-specific alloantibody levels. The results identify DNA topoisomerase I as a novel self-antigen in transplant settings and demonstrate that autoantibodies enhance activation of donor-reactive T cells following renal transplantation.

Macrophage-inducible C-type lectin activates B cells to promote T cell reconstitution in heart allograft recipients.

Diminishing homeostatic proliferation of memory T cells is essential for improving the efficacy of lymphoablation in transplant recipients. Our previous studies in a mouse heart transplantation model established that B lymphocytes secreting proinflammatory cytokines are critical for T cell recovery after lymphoablation. The goal of the current study was to identify mediators of B cell activation following lymphoablation in allograft recipients. Transcriptome analysis revealed that macrophage-inducible C-type lectin (Mincle, Clec4e) expression is up-regulated in B cells from heart allograft recipients treated with murine anti-thymocyte globulin (mATG). Recipient Mincle deficiency diminishes B cell production of pro-inflammatory cytokines and impairs T lymphocyte reconstitution. Mixed bone marrow chimeras lacking Mincle only in B lymphocytes have similar defects in T cell recovery. Conversely, treatment with a synthetic Mincle ligand enhances T cell reconstitution after lymphoablation in non-transplanted mice. Treatment with agonistic CD40 mAb facilitates T cell reconstitution in CD4 T cell-depleted, but not in Mincle-deficient, recipients indicating that CD40 signaling induces T cell proliferation via a Mincle-dependent pathway. These findings are the first to identify an important function of B cell Mincle as a sensor of damage-associated molecular patterns released by the graft and demonstrate its role in clinically relevant settings of organ transplantation.

Anti-donor MHC Class II Alloantibody Induces Glomerular Injury in Mouse Renal Allografts Subjected to Prolonged Cold Ischemia.

BACKGROUND: The mechanisms underlying the effects of prolonged cold-ischemia storage on kidney allografts are poorly understood. METHODS: To investigate effects of cold ischemia on donor-reactive immune responses and graft pathology, we used a mouse kidney transplantation model that subjected MHC-mismatched BALB/c kidney allografts to cold-ischemia storage for 0.5 or 6 hours before transplant into C57BL/6 mice. RESULTS: At day 14 post-transplant, recipients of allografts subjected to 6 versus 0.5 hours of cold-ischemia storage had increased levels of anti-MHC class II (but not class I) donor-specific antibodies, increased donor-reactive T cells, and a significantly higher proportion of transplant glomeruli infiltrated with macrophages. By day 60 post-transplant, allografts with a 6 hour cold-ischemia time developed extensive glomerular injury compared with moderate pathology in allografts with 0.5 hour of cold-ischemia time. Pathology was associated with increased serum levels of anti-class 2 but not anti-class 1 donor-specific antibodies. Recipient B cell depletion abrogated early macrophage recruitment, suggesting augmented donor-specific antibodies, rather than T cells, increase glomerular pathology after prolonged cold ischemia. Lymphocyte sequestration with sphingosine-1-phosphate receptor 1 antagonist FTY720 specifically inhibited anti-MHC class II antibody production and abrogated macrophage infiltration into glomeruli. Adoptive transfer of sera containing anti-donor MHC class II antibodies or mAbs against donor MHC class II restored early glomerular macrophage infiltration in FTY720-treated recipients. CONCLUSIONS: Post-transplant inflammation augments generation of donor-specific antibodies against MHC class II antigens. Resulting MHC class II-reactive donor-specific antibodies are essential mediators of kidney allograft glomerular injury caused by prolonged cold ischemia.

Interleukin-27 promotes CD8+ T cell reconstitution following antibody-mediated lymphoablation.

Antibody-mediated lymphoablation is used in solid organ and stem cell transplantation and autoimmunity. Using murine anti-thymocyte globulin (mATG) in a mouse model of heart transplantation, we previously reported that the homeostatic recovery of CD8+ T cells requires help from depletion-resistant memory CD4+ T cells delivered through CD40-expressing B cells. This study investigated the mechanisms by which B cells mediate CD8+ T cell proliferation in lymphopenic hosts. While CD8+ T cell recovery required MHC class I expression in the host, the reconstitution occurred independently of MHC class I, MHC class II, or CD80/CD86 expression on B cells. mATG lymphoablation upregulated the B cell expression of several cytokine genes, including IL-15 and IL-27, in a CD4-dependent manner. Neither treatment with anti-CD122 mAb nor the use of IL-15Rα-/- recipients altered CD8+ T cell recovery after mATG treatment, indicating that IL-15 may be dispensable for T cell proliferation in our model. Instead, IL-27 neutralization or the use of IL-27Rα-/- CD8+ T cells inhibited CD8+ T cell proliferation and altered the phenotype and cytokine profile of reconstituted CD8+ T cells. Our findings uncover what we believe is a novel role of IL-27 in lymphopenia-induced CD8+ T cell proliferation and suggest that targeting B cell-derived cytokines may increase the efficacy of lymphoablation and improve transplant outcomes.

Memory CD4 T Cells Induce Antibody-Mediated Rejection of Renal Allografts.

Despite advances in immunosuppression, antibody-mediated rejection is a serious threat to allograft survival. Alloreactive memory helper T cells can induce potent alloantibody responses and often associate with poor graft outcome. Nevertheless, the ability of memory T cells to elicit well characterized manifestations of antibody-mediated rejection has not been tested. We investigated helper functions of memory CD4 T cells in a mouse model of renal transplantation. Whereas the majority of unsensitized C57Bl/6 recipients spontaneously accepted fully MHC-mismatched A/J renal allografts, recipients containing donor-reactive memory CD4 T cells rapidly lost allograft function. Increased serum creatinine levels, high serum titers of donor-specific alloantibody, minimal T cell infiltration, and intense C4d deposition in the grafts of sensitized recipients fulfilled all diagnostic criteria for acute renal antibody-mediated rejection in humans. IFNγ neutralization did not prevent the renal allograft rejection induced by memory helper T cells, and CD8 T cell depletion at the time of transplantation or depletion of both CD4 and CD8 T cells also did not prevent the renal allograft rejection induced by memory helper T cells starting at day 4 after transplantation. However, B cell depletion inhibited alloantibody generation and significantly extended allograft survival, indicating that donor-specific alloantibodies (not T cells) were the critical effector mechanism of renal allograft rejection induced by memory CD4 T cells. Our studies provide direct evidence that recipient T cell sensitization may result in antibody-mediated rejection of renal allografts and introduce a physiologically relevant animal model with which to investigate mechanisms of antibody-mediated rejection and novel therapeutic approaches for its prevention and treatment.

IFN-γ production by memory helper T cells is required for CD40-independent alloantibody responses.

Cognate T-B cell interactions and CD40-CD154 costimulation are essential for productive humoral immunity against T-dependent Ags. We reported that memory CD4 T cells can deliver help to B cells and induce pathogenic IgG alloantibodies in the absence of CD40-CD154 interactions. To determine cytokine requirements for CD40-independent help, we used CD40(-/-) mice containing differentiated subsets of donor-reactive memory Th cells as heart allograft recipients. Th1 and Th17, but not Th2, memory CD4 T cells elicited high titers of anti-donor Ab. Abs induced by Th17 memory CD4 T cells had decreased reactivity against donor MHC class I molecules and inferior ability to cause complement deposition in heart allografts compared with Abs induced by Th1 cells, suggesting a requirement for IFN-γ during CD40-independent help. IFN-γ neutralization inhibited helper functions of memory CD4 T cells in both CD40(-/-) recipients and wild type recipients treated with anti-CD154 mAb. Our results suggest that IFN-γ secreted by pre-existing memory helper cells determines both isotype and specificity of donor-reactive alloantibodies and can thus affect allograft pathology. This information may be valuable for identifying transplant patients at risk for de novo development of pathogenic alloantibodies and for preventing alloantibody production in T cell-sensitized recipients.

Novel CD8 T cell alloreactivities in CCR5-deficient recipients of class II MHC disparate kidney grafts.

Recipient CD4 T regulatory cells inhibit the acute T cell-mediated rejection of renal allografts in wild-type mice. The survival of single class II MHC-disparate H-2(bm12) renal allografts was tested in B6.CCR5(-/-) recipients, which have defects in T regulatory cell activities that constrain alloimmune responses. In contrast to wild-type C57BL/6 recipients, B6.CCR5(-/-) recipients rejected the bm12 renal allografts. However, donor-reactive CD8 T cells rather than CD4 T cells were the primary effector T cells mediating rejection. The CD8 T cells induced to bm12 allografts in CCR5-deficient recipients were reactive to peptides spanning the 3 aa difference in the I-A(bm12) versus I-A(b) ß-chains presented by K(b) and D(b) class I MHC molecules. Allograft-primed CD8 T cells from CCR5-deficient allograft recipients were activated during culture either with proinflammatory cytokine-stimulated wild-type endothelial cells pulsed with the I-A(bm12) peptides or with proinflammatory cytokine-simulated bm12 endothelial cells, indicating their presentation of the I-A(bm12) ß-chain peptide/class I MHC complexes. In addition to induction by bm12 renal allografts, the I-A(bm12) ß-chain-reactive CD8 T cells were induced in CCR5-deficient, but not wild-type C57BL/6, mice by immunization with the peptides. These results reveal novel alloreactive CD8 T cell specificities in CCR5-deficient recipients of single class II MHC renal allografts that mediate rejection of the allografts.

CD40-independent help by memory CD4 T cells induces pathogenic alloantibody but does not lead to long-lasting humoral immunity.

CD40/CD154 interactions are essential for productive antibody responses to T-dependent antigens. Memory CD4 T cells express accelerated helper functions and are less dependent on costimulation when compared with naïve T cells. Here, we report that donor-reactive memory CD4 T cells can deliver help to CD40-deficient B cells and induce high titers of IgG alloantibodies that contribute to heart allograft rejection in CD40-/- heart recipients. While cognate interactions between memory helper T and B cells are crucial for CD40-independent help, this process is not accompanied by germinal center formation and occurs despite inducible costimulatory blockade. Consistent with the extrafollicular nature of T/B cell interactions, CD40-independent help fails to maintain stable levels of serum alloantibody and induce differentiation of long-lived plasma cells and memory B cells. In summary, our data suggest that while CD40-independent help by memory CD4 T cells is sufficient to induce high levels of pathogenic alloantibody, it does not sustain long-lasting anti-donor humoral immunity and B cell memory responses. This information may guide the future use of CD40/CD154 targeting therapies in transplant recipients containing donor-reactive memory T cells.

Interleukin-17 promotes early allograft inflammation.

Acute cellular rejection of organ transplants is executed by donor-reactive T cells, which are dominated by interferon-gamma-producing cells. As interferon-gamma is dispensable for graft destruction, we evaluated the contribution of interleukin-17A (IL-17) to intragraft inflammation in major histocompatibility complex-mismatched heart transplants. A/J (H-2(a)) cardiac allografts placed into wild-type BALB/c (H-2(d)) mice induced intragraft IL-17 production on day 2 after transplant. Allografts placed into BALB/c IL-17(-/-) recipients demonstrated diminished production of the chemokines CXCL1 and CXCL2 and delayed neutrophil and T cell recruitment. However, by day 7 after transplant, allografts from IL-17(-/-) and wild-type recipients had comparable levels of cellular infiltration. The priming of donor-specific T cells was not affected by the absence of IL-17, and the kinetics of cardiac allograft rejection were similar in wild-type and IL-17(-/-) recipients. In contrast, IL-17(-/-) mice depleted of CD8 T cells rejected A/J allografts in a delayed fashion compared with CD8-depleted wild-type recipients. Although donor-reactive CD4 T cells were efficiently activated in both groups, the infiltration of effector T cells into allografts was impaired in IL-17(-/-) recipients. Our data indicate that locally produced IL-17 amplifies intragraft inflammation early after transplantation and promotes tissue injury by facilitating T cell recruitment into the graft. Targeting the IL-17 signaling network in conjunction with other graft-prolonging therapies may decrease this injury and improve the survival of transplanted organs.

The interferon-gamma-induced murine guanylate-binding protein-2 inhibits rac activation during cell spreading on fibronectin and after platelet-derived growth factor treatment: role for phosphatidylinositol 3-kinase.

Exposure of cells to certain cytokines can alter how these same cells respond to later cues from other agents, such as extracellular matrix or growth factors. Interferon (IFN)-gamma pre-exposure inhibits the spreading of fibroblasts on fibronectin. Expression of the IFN-gamma-induced GTPase murine guanylate-binding protein-2 (mGBP-2) can phenocopy this inhibition and small interfering RNA knockdown of mGBP-2 prevents IFN-gamma-mediated inhibition of cell spreading. Either IFN-gamma treatment or mGBP-2 expression inhibits Rac activation during cell spreading. Rac is required for cell spreading. mGBP-2 also inhibits the activation of Akt during cell spreading on fibronectin. mGBP-2 is incorporated into a protein complex containing the catalytic subunit of phosphatidylinositol 3-kinase (PI3-K), p110. The association of mGBP-2 with p110 seems important for the inhibition of cell spreading because S52N mGBP-2, which does not incorporate into the protein complex with p110, is unable to inhibit cell spreading. PI3-K activation during cell spreading on fibronectin was inhibited in the presence of mGBP-2. Both IFN-gamma and mGBP-2 also inhibit cell spreading initiated by platelet-derived growth factor treatment, which is also accompanied by inhibition of Rac activation by mGBP-2. This is the first report of a novel mechanism by which IFN-gamma can alter how cells respond to subsequent extracellular signals, by the induction of mGBP-2.

Disruption of the circadian clock due to the Clock mutation has discrete effects on aging and carcinogenesis.

The mammalian circadian system has been implicated in the regulation of various biological processes including those involved in genotoxic stress responses and tumor suppression. Here we report that mice with the functional deficiency in circadian transcription factor CLOCK (Clock/Clock mutant mice) do not display predisposition to tumor formation both during their normal lifespan or when challenged by gamma- radiation. This phenotype is consistent with high apoptotic and low proliferation rate in lymphoid tissues of Clock mutant mice and is supported by the gene expression profiling of a number of apoptosis and cell cycle-related genes, as well as by growth inhibition of cells with CLOCK downregulation. At the same time, Clock mutant mice respond to low-dose irradiation by accelerating their aging program, and develop phenotypes that are reminiscent of those in Bmal1-deficient mice. Taken together, our results demonstrate the dichotomy in biological consequences of the disruption of the circadian clock with respect to ageing and cancer. They also highlight the existence of a complex interconnection between ageing, carcinogenesis and individual components of the circadian clock machinery.

The role of mammalian circadian proteins in normal physiology and genotoxic stress responses.

The last two decades have significantly advanced our understanding of the organization of the circadian system at all levels of regulation-molecular, cellular, tissue, and systemic. It has been recognized that the circadian system represents a complex temporal regulatory network, which plays an important role in synchronizing various biological processes within an organism and coordinating them with the environment. It is believed that deregulation of this synchronization may result in the development of various pathologies. However, recent studies using various circadian mutant mouse models have demonstrated that at least some of the components of the molecular oscillator are actively involved in physiological processes not directly related to their role in the circadian clock. The growing amount of evidence suggests that, in addition to their circadian function, circadian proteins are important in maintaining tissue homeostasis under normal and stress conditions. In this chapter, we will summarize recent data about the regulation of the mammalian molecular circadian oscillator and will focus on a new role of the circadian system and individual circadian proteins in the organism's physiology and response to genotoxic stress in connection with diseases treatment and prevention.

Early aging and age-related pathologies in mice deficient in BMAL1, the core componentof the circadian clock.

Mice deficient in the circadian transcription factor BMAL1 (brain and muscle ARNT-like protein) have impaired circadian behavior and demonstrate loss of rhythmicity in the expression of target genes. Here we report that Bmal1(-/-) mice have reduced lifespans and display various symptoms of premature aging including sarcopenia, cataracts, less subcutaneous fat, organ shrinkage, and others. The early aging phenotype correlates with increased levels of reactive oxygen species in some tissues of the Bmal1(-/- )animals. These findings, together with data on CLOCK/BMAL1-dependent control of stress responses, may provide a mechanistic explanation for the early onset of age-related pathologies in the absence of BMAL1.

Post-translational regulation of circadian transcriptional CLOCK(NPAS2)/BMAL1 complex by CRYPTOCHROMES.

Mammalian CLOCK(NPAS2), BMAL1 and CRYPTOCHROMEs are core components of the circadian oscillatory mechanism. The active CLOCK/BMAL1 or NPAS2/BMAL1 complexes regulate expression of numerous genes including two Cryptochromes. The products of these genes, CRY1 and CRY2, in turn repress CLOCK/BMAL1 transcriptional activity by an unknown mechanism. We have examined the effect of CRYPTOCHROMEs on posttranslational modifications and intracellular distribution of endogenous and ectopically expressed CLOCK(NPAS2) and BMAL1 proteins. We found that ectopic coexpression with CRY led to stabilization and nuclear accumulation of unphosphorylated forms of the proteins, which directly correlated with the inhibition of their transcriptional activity. This effect was CRY-specific, as other known repressors of CLOCK/BMAL1 and NPAS2/ BMAL1 transcriptional activity were not able to induce similar effects. CRYs had no effect on CLOCK(NPAS2)/BMAL1 complex formation or its ability to bind DNA. Altogether, these results demonstrate that CRYs regulate the functional activity of circadian transcriptional complex at the posttranslational level. Importantly, the posttranslational modifications and intracellular distribution of CLOCK and BMAL1 proteins were critically impaired in the tissues of mice with targeted disruption of both Cry genes, thus confirming the suggested role of CRY in clock function in vivo. Based on these findings we propose a modified model of the circadian transcriptional control, which implies CRY-mediated periodic rotation of transcriptionally active and inactive forms of CLOCK/BMAL1 on the promoter. This model provides mechanistic explanation for previously reported dual functional activity of CLOCK/BMAL1 and highlights the involvement of the circadian system in modulating the organism's response to various types of genotoxic stress, including chemotherapy and radiation.

Dual role of the CLOCK/BMAL1 circadian complex in transcriptional regulation.

The basic helix-loop-helix (bHLH) -PAS domain containing transcription factors CLOCK and BMAL1 are two major components of the circadian molecular oscillator. It is known that the CLOCK/BMAL1 complex positively regulates the activity of E-box containing promoters. Here we demonstrate that the CLOCK/BMAL1 complex can also suppress the activity of some promoters upon its interaction with CRYPTOCHROME (CRY). Such a dual function of the circadian transcriptional complex provides a mechanistic explanation for the unpredicted pattern of circadian gene expression in the tissues of Bmal1 null mice. We speculate that the switch from transcriptional activation to transcriptional repression may provide a highly efficient mechanism for circadian control of gene expression. We also show that CLOCK/BMAL1 can interfere with promoter regulation by other, non-circadian, transcription factors including N-MYC and ETS, leading to attenuation or abrogation of transcription of CLOCK/BMAL1-controlled stress-induced genes. We propose that, based upon these results, both circadian repression and activation of the transcription of different target genes are required for circadian responses to various external stimuli, including genotoxic stress induced by anticancer treatment.

Circadian sensitivity to the chemotherapeutic agent cyclophosphamide depends on the functional status of the CLOCK/BMAL1 transactivation complex.

The circadian clock controls many aspects of mammalian physiology, including responses to cancer therapy. We find that wild-type and circadian mutant mice demonstrate striking differences in their response to the anticancer drug cyclophosphamide (CY). While the sensitivity of wild-type mice varies greatly, depending on the time of drug administration, Clock mutant and Bmal1 knockout mice are highly sensitive to treatment at all times tested. On the contrary, mice with loss-of-function mutations in Cryptochrome (Cry1-/-Cry2-/- double knockouts) were more resistant to CY compared with their wild-type littermates. Thus, both time-of-day and allelic-dependent variations in response to chemotherapy correlate with the functional status of the circadian CLOCK/BMAL1 transactivation complex. Pharmacokinetic analysis of plasma concentration of different CY metabolites shows that, in contrast to the traditional view, circadian variations in drug sensitivity cannot be attributed to the changes in the rates of CY metabolic activation and/or detoxification. At the same time, mice of different circadian genotypes demonstrate significant differences in B cell responses to toxic CY metabolites: B cell survival/recovery rate was directly correlated with the in vivo drug sensitivity. Based on these results, we propose that the CLOCK/BMAL1 transcriptional complex affects the lethality of chemotherapeutic agents by modulating the survival of the target cells necessary for the viability of the organism.

BMAL1-dependent circadian oscillation of nuclear CLOCK: posttranslational events induced by dimerization of transcriptional activators of the mammalian clock system.

Mammalian CLOCK and BMAL1 are two members of bHLH-PAS-containing family of transcription factors that represent the positive elements of circadian autoregulatory feedback loop. In the form of a heterodimer, they drive transcription from E-box enhancer elements in the promoters of responsive genes. We have examined abundance, posttranslational modifications, cellular localization of endogenous and ectopically expressed CLOCK and BMAL1 proteins. Nuclear/cytoplasm distribution of CLOCK was found to be under circadian regulation. Analysis of subcellular localization of CLOCK in embryo fibroblasts of mice carrying different germ-line circadian mutations showed that circadian regulation of nuclear accumulation of CLOCK is BMAL1-dependent. Formation of CLOCK/BMAL1 complex following ectopic coexpression of both proteins is followed by their codependent phosphorylation, which is tightly coupled to CLOCK nuclear translocation and degradation. This binding-dependent coregulation is specific for CLOCK/BMAL1 interaction, as no other PAS domain protein that can form a complex with either CLOCK or BMAL1 was able to induce similar effects. Importantly, all posttranslational events described in our study are coupled with active transactivation complex formation, which argues for their significant functional role. Altogether, these results provide evidence for an additional level of circadian system control, which is based on regulation of transcriptional activity or/and availability of CLOCK/BMAL1 complex.

The interferon (IFN)-induced GTPase, mGBP-2. Role in IFN-gamma-induced murine fibroblast proliferation.

To investigate the function of mGBP-2, a member of the interferon (IFN)-induced guanylate-binding protein family of GTPases, NIH 3T3 fibroblasts were generated that constitutively expressed mGBP-2. mGBP-2 induced a faster growth rate, with the highest expressing clones showing approximately a 50% reduction in doubling time. mGBP-2-expressing cells also grew to higher density and exhibited partial loss of contact growth inhibition, as evidenced by the formation of foci in post-confluent cultures. In addition, mGBP-2-expressing cells showed decreased dependence on serum-derived growth factors. However, they did not lose the requirement for anchorage-dependent growth. Finally, NIH 3T3 cells expressing mGBP-2 formed tumors in athymic mice. An mGBP-2 protein carrying a point mutation (S52N) that reduced GTP binding failed to produce these phenotypes when expressed at the same levels as wild type. The additional finding that IFN-gamma treatment of NIH 3T3 cells resulted in an increase in proliferation similar to that observed for mGBP-2 in the absence of other IFN-induced proteins suggests that mGBP-2 may indeed be important for these growth changes.