Danya Liu

Intracellular protein therapy with SOCS3 inhibits inflammation and apoptosis

Suppressor of cytokine signaling (SOCS) 3 attenuates proinflammatory signaling mediated by the signal transducer and activator of transcription (STAT) family of proteins. But acute inflammation can occur after exposure to pathogen-derived inducers staphylococcal enterotoxin B (SEB) and lipopolysaccharide (LPS), or the lectin concanavalin A (ConA), suggesting that physiologic levels of SOCS3 are insufficient to stem proinflammatory signaling under pathogenic circumstances. To test this hypothesis, we developed recombinant cell-penetrating forms of SOCS3 (CP-SOCS3) for intracellular delivery to counteract SEB-, LPS- and ConA-induced inflammation. We found that CP-SOCS3 was distributed in multiple organs within 2 h and persisted for at least 8 h in leukocytes and lymphocytes. CP-SOCS3 protected animals from lethal effects of SEB and LPS by reducing production of inflammatory cytokines and attenuating liver apoptosis and hemorrhagic necrosis. It also reduced ConA-induced liver apoptosis. Thus, replenishing the intracellular stores of SOCS3 with CP-SOCS3 effectively suppresses the devastating effects of acute inflammation.

2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

Blockade of CD28 signals results in the up-regulation of 2B4 on primary CD8+ effectors and plays a critical role in controlling antigen-specific CD8+ T cell responses.

Suppression of Acute Lung Inflammation by Intracellular Peptide Delivery of a Nuclear Import Inhibitor

Acute lung inflammation is a potentially life-threatening complication of infections due to community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), a worldwide emerging pathogen, which causes necrotizing pneumonia and acute respiratory distress syndrome (ARDS). MRSA virulence factors encompass immunotoxins termed superantigens that contribute to lung inflammation. In this study, we demonstrate that staphylococcal enterotoxin B (SEB)-induced lung inflammation is attenuated by a cell-penetrating peptide nuclear import inhibitor of nuclear factor (NF)-kappaB and other stress-responsive transcription factors (SRTFs). This inhibitor suppressed production of a wide spectrum of cytokines and chemokines induced by direct SEB airway exposure. Consequently, trafficking of neutrophils, monocytes/macrophages, and lymphocytes to the bronchoalveolar space was significantly reduced while vascular injury, manifested by increased permeability and protein leakage, was attenuated. Moreover, induction of systemic proinflammatory cytokines and chemokines in response to direct SEB airway exposure was reduced. Thus, intracellular delivery of a nuclear import inhibitory peptide suppresses respiratory and systemic expression of key mediators of lung inflammation evoked by SEB.

An anti-CD154 domain antibody prolongs graft survival and induces Foxp3(+) iTreg in the absence and presence of CTLA-4 Ig.

The use of monoclonal antibodies targeting the CD154 molecule remains one of the most effective means of promoting graft tolerance in animal models, but thromboembolic complications during early clinical trials have precluded their use in humans. Furthermore, the role of Fc‐mediated deletion of CD154‐expressing cells in the observed efficacy of these reagents remains controversial. Therefore, determining the requirements for anti‐CD154‐induced tolerance will instruct the development of safer but equally efficacious treatments. To investigate the mechanisms of action of anti‐CD154 therapy, two alternative means of targeting the CD40–CD154 pathway were used: a nonagonistic anti‐CD40 antibody and an Fc‐silent anti‐CD154 domain antibody. We compared these therapies to an Fc‐intact anti‐CD154 antibody in both a fully allogeneic model and a surrogate minor antigen model in which the fate of alloreactive cells could be tracked. Results indicated that anti‐CD40 mAbs as well as Fc‐silent anti‐CD154 domain antibodies were equivalent to Fc‐intact anti‐CD154 mAbs in their ability to inhibit alloreactive T cell expansion, attenuate cytokine production of antigen‐specific T cells and promote the conversion of Foxp3+ iTreg. Importantly, iTreg conversion observed with Fc‐silent anti‐CD154 domain antibodies was preserved in the presence of CTLA4‐Ig, suggesting that this therapy is a promising candidate for translation to clinical use.

Age-Associated B Cells Express a Diverse Repertoire of VH and Vκ Genes with Somatic Hypermutation

The origin and nature of age-associated B cells (ABCs) in mice are poorly understood. In this article, we show that their emergence required MHC class II and CD40/CD40L interactions. Young donor B cells were adoptively transferred into congenic recipients and allowed to remain for 1 mo in the absence of external Ag. B cells expressing the T-bet transcription factor, a marker for ABCs, were generated after multiple cell divisions from C57BL/6 donors but not from MHC class II– or CD40-deficient donors. Furthermore, old CD154 (CD40L)-deficient mice did not accrue ABCs, confirming that they arise primarily through T-dependent interactions. To determine what Igs ABCs express, we sequenced VH and Vκ rearranged genes from unimmunized 22-mo-old C57BL/6 mice and showed that they had a heterogeneous repertoire, which was comparable to that seen in old follicular and marginal zone B cell subsets. However, in contrast to the follicular and marginal zone cells, ABCs displayed significant somatic hypermutation. The mutation frequency was lower than found in germinal center cells after deliberate immunization, suggesting that ABCs have undergone mild stimulation from endogenous Ags over time. These observations show that quiescent ABCs are Ag-experienced cells that accumulate during T cell–dependent responses to diverse Ags during the life of an individual.

CD40/CD154 Blockade Inhibits Dendritic Cell Expression of Inflammatory Cytokines but Not Costimulatory Molecules

Blockade of the CD40/CD154 pathway remains one of the most effective means of promoting graft survival following transplantation. However, the effects of CD40/CD154 antagonism on dendritic cell (DC) phenotype and functionality following transplantation remain incompletely understood. To dissect the effects of CD154/CD40 blockade on DC activation in vivo, we generated hematopoietic chimeras in mice that expressed a surrogate minor Ag (OVA). Adoptive transfer of OVA-specific CD4+ and CD8+ T cells led to chimerism rejection, which was inhibited by treatment with CD154 blockade. Surprisingly, CD154 antagonism did not alter the expression of MHC and costimulatory molecules on CD11c+ DCs compared with untreated controls. However, DCs isolated from anti-CD154–treated animals exhibited a significant reduction in inflammatory cytokine secretion. Combined blockade of inflammatory cytokines IL-6 and IL-12p40 attenuated the expansion of Ag-specific CD4+ and CD8+ T cells and transiently inhibited the rejection of OVA-expressing cells. These results suggest that a major effect of CD154 antagonism in vivo is an impairment in the provision of signal three during donor-reactive T cell programming, as opposed to an impact on the provision of signal two. We conclude that therapies designed to target inflammatory cytokines during donor-reactive T cell activation may be beneficial in attenuating these responses and prolonging graft survival.

In vivo islet protection by a nuclear import inhibitor in a mouse model of type 1 diabetes.

Background Insulin-dependent Type 1 diabetes (T1D) is a devastating autoimmune disease that destroys beta cells within the pancreatic islets and afflicts over 10 million people worldwide. These patients face life-long risks for blindness, cardiovascular and renal diseases, and complications of insulin treatment. New therapies that protect islets from autoimmune destruction and allow continuing insulin production are needed. Increasing evidence regarding the pathomechanism of T1D indicates that islets are destroyed by the relentless attack by autoreactive immune cells evolving from an aberrant action of the innate, in addition to adaptive, immune system that produces islet-toxic cytokines, chemokines, and other effectors of islet inflammation. We tested the hypothesis that targeting nuclear import of stress-responsive transcription factors evoked by agonist-stimulated innate and adaptive immunity receptors would protect islets from autoimmune destruction. Principal Findings Here we show that a first-in-class inhibitor of nuclear import, cSN50 peptide, affords in vivo islet protection following a 2-day course of intense treatment in NOD mice, which resulted in a diabetes-free state for one year without apparent toxicity. This nuclear import inhibitor precipitously reduces the accumulation of islet-destructive autoreactive lymphocytes while enhancing activation-induced cell death of T and B lymphocytes derived from autoimmune diabetes-prone, non-obese diabetic (NOD) mice that develop T1D. Moreover, in this widely used model of human T1D we noted attenuation of pro-inflammatory cytokine and chemokine production in immune cells. Conclusions These results indicate that a novel form of immunotherapy that targets nuclear import can arrest inflammation-driven destruction of insulin-producing beta cells at the site of autoimmune attack within pancreatic islets during the progression of T1D.

Candida-Elicited Murine Th17 Cells Express High CTLA-4 Compared with Th1 Cells and Are Resistant to Costimulation Blockade

Effector and memory T cells may cross-react with allogeneic Ags to mediate graft rejection. Whereas the costimulation properties of Th1 cells are well studied, relatively little is known about the costimulation requirements of microbe-elicited Th17 cells. The costimulation blocker CTLA-4 Ig has been ineffective in the treatment of several Th17-driven autoimmune diseases and is associated with severe acute rejection following renal transplantation, leading us to investigate whether Th17 cells play a role in CD28/CTLA-4 blockade-resistant alloreactivity. We established an Ag-specific model in which Th1 and Th17 cells were elicited via Mycobacterium tuberculosis and Candida albicans immunization, respectively. C. albicans immunization elicited a higher frequency of Th17 cells and conferred resistance to costimulation blockade following transplantation. Compared with the M. tuberculosis group, C. albicans–elicited Th17 cells contained a higher frequency of IL-17+IFN-γ+ producers and a lower frequency of IL-10+ and IL-10+IL-17+ cells. Importantly, Th17 cells differentially regulated the CD28/CTLA-4 pathway, expressing similarly high CD28 but significantly greater amounts of CTLA-4 compared with Th1 cells. Ex vivo blockade experiments demonstrated that Th17 cells are more sensitive to CTLA-4 coinhibition and therefore less susceptible to CTLA-4 Ig. These novel insights into the differential regulation of CTLA-4 coinhibition on CD4+ T cells have implications for the immunomodulation of pathologic T cell responses during transplantation and autoimmunity.

Inhibition of CD8+ T Cell–Derived CD40 Signals Is Necessary but Not Sufficient for Foxp3+ Induced Regulatory T Cell Generation In Vivo

Current models of CD4+ T cell help suggest a major role for CD154 binding to CD40 expressed on dendritic cells, with a lesser role for direct T:T interactions via CD40 expressed on CD8+ T cells. However, the contribution of CD8+ T cell–derived CD40 signals during the donor-reactive T cell response to a transplant has never been studied. In this study, we examined the graft-rejection kinetics and CD4+ and CD8+ donor-reactive T cell responses under conditions in which CD40 was genetically ablated only on APC, as well as under conditions in which CD40 was genetically ablated only on donor-reactive CD8+ T cells. Our results revealed a significant role for CD8+ T cell–expressed CD40 in the augmentation of donor-reactive CD8+ T cell responses following transplantation and showed that CD40 expressed on CD8+ T cells must be inhibited to allow conversion of CD4+ T cells into induced regulatory T cells. Thus, this study identifies a major role for CD8+ T cell–derived CD40 signals as a critical switch factor that both promotes optimal differentiation of cytokine-producing CD8+ effector T cell responses and inhibits the differentiation of Ag-specific Foxp3+ induced regulatory T cells in vivo.

Low-Affinity Memory CD8+ T Cells Mediate Robust Heterologous Immunity

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide–MHC, the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming, low-affinity priming elicited fully differentiated memory T cells with a CD45RBhi status. High CD45RB status enabled robust secondary responses in vivo, as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity–primed mice, but not in high affinity–primed mice. Mechanistically, low affinity–primed memory CD8+ T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RBhi status was also a stable marker of priming affinity within polyclonal CD8+ T cell populations. Following high-affinity rechallenge, low affinity–primed CD45RBhi cells became CD45RBlo, demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8+ T cells. Thus, these data establish a novel mechanism by which CD45 isoforms tune low affinity–primed memory CD8+ T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency, low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge.