Zhenhua Dai

Natural CD8+CD122+ T Cells Are More Potent in Suppression of Allograft Rejection Than CD4+CD25+ Regulatory T Cells

Despite extensive studies on CD4+CD25+ regulatory T cells (Tregs), their application in adoptive transfer therapies is still not optimal in immune‐competent wild‐type (WT) animal models. Therefore, it is compelling to search for more potent Tregs for potential clinical application. Mounting evidence has shown that naturally occurring CD8+CD122+ T cells are also Tregs. However, their suppression in allograft rejection, efficiency in suppression and underlying mechanisms remain unclear. Using a murine allotransplantation model, we reported here that CD8+CD122+ Tregs were actually more potent in suppression of allograft rejection and underwent more rapid homeostatic proliferation than their CD4+CD25+ counterparts. Moreover, they produced more IL‐10 and were more potent in suppressing T cell proliferation in vitro. Deficiency in IL‐10 in CD4+CD25+ and CD8+CD122+ Tregs resulted in their reduced but equal suppression in vivo and in vitro, suggesting that IL‐10 is responsible for more effective suppression by CD8+CD122+ than CD4+CD25+ Tregs. Importantly, transfer of CD8+CD122+ Tregs together with the administration of recombinant IL‐15 significantly prolonged allograft survival in WT mice. Thus, for the first time, we demonstrate that naturally arising CD8+CD122+ Tregs not only inhibit allograft rejection but also exert this suppression more potently than their CD4+CD25+ counterparts. This novel finding may have important implications for tolerance induction.

CD8+CD122+ T-Cells: A Newly Emerging Regulator with Central Memory Cell Phenotypes

CD8+CD122+ T-cells have been traditionally described as antigen-specific memory T-cells that respond to previously encountered antigens more quickly and vigorously than their naïve counterparts. However, mounting evidence has demonstrated that murine CD8+CD122+ T-cells exhibit a central memory phenotype (CD44highCD62Lhigh), regulate T cell homeostasis, and act as regulatory T-cells (Treg) by suppressing both autoimmune and alloimmune responses. Importantly, naturally occurring murine CD8+CD122+ Tregs are more potent in immunosuppression than their CD4+CD25+ counterparts. They appear to be acting in an antigen-non-specific manner. Human CD8+CXCR3+ T-cells are the equivalent of murine CD8+CD122+ Tregs and also exhibit central memory phenotypes. In this mini-review article, we will summarize recent progresses in their phenotypes, homeostatic expansion, antigen-specificity, roles in the suppression of alloimmune and autoimmune responses, and the mechanisms underlying their inhibitory function.

Editorial: Memory T Cells: Effectors, Regulators, and Implications for Transplant Tolerance

The Editorial on the Research Topic Memory T Cells: Effectors, Regulators, and Implications for Transplant Tolerance Memory T-cells respond to previously encountered antigens more rapidly and vigorously than their naive counterparts. They are divided into three subsets: central memory, effector memory, and tissue-resident memory T-cells. They are somewhat resistant to immunosuppressive treatments and are generally believed to be a threat to transplant survival. However, mounting evidence has demonstrated that memory CD8+CD122+ T-cells with central memory cell phenotypes (CD45RA−CD44highCD62LhighCCR7+) can regulate T-cell homeostasis and suppress both autoimmune and alloimmune responses. Therefore, memory T-cells, especially CD8+CD122+ T-cells, may respond as either aggressive memory or regulatory T-cells (Treg). This research topic may shed light on when they act as memory versus Treg cells, and how to target memory T-cells or otherwise utilize memory-like Tregs to promote long-term allograft survival. Memory T-cells are considered to be a major barrier to long-term transplant survival or tolerance (1). Targeting allospecific T-cell memory appears to be required for transplant tolerance induction. Then, the question is whether blocking conventional T-cell costimulation would inhibit memory T-cell responses. Previous studies have shown that memory T-cells are resistant to CD40/CD154 costimulatory blockade (2, 3). It is also generally accepted that B7-CD28 costimulation is not required for memory T-cell activation (4). They are either less dependent on or totally independent of CD28 costimulation (5, 6). Therefore, it is likely that blocking B7–CD28 is insufficient for preventing allograft rejection in the face of memory T-cells. Perhaps that is why a high incidence of acute rejection of renal allografts, despite CTLA4-Ig treatments, has occurred in clinic due to the cross-reactivity of memory T-cells, derived from pathogen-specific immune responses, with an alloantigen (7). However, recent studies using animal models have shown that optimal elaboration of secondary T-cell responses is dependent on B7–CD28 interactions in the context of anti-infectious immunity (Ville et al.). Interestingly, selectively targeting CD28 with FR104 is more potent in suppression of allograft rejection than targeting CD80/86 with CTLA4-Ig (Ville et al.), suggesting that selective blockade of CD28 signaling alone presents an advantage of allowing immunoregulatory signals mediated by CTLA4. Furthermore, blocking OX-40 costimulatory signal prolongs secondary heart allograft survival in the presence of CD40/CD40L and LFA-1/ICAM-1 blockade (8), indicating that additional blockade of OX-40 signaling is required for abrogating memory T cell responses. Memory T-cells can rapidly trigger alloimmune responses (9). It has been known that early infiltration of CD8+ memory T-cells into allografts facilitates allograft rejection and presents a hurdle to achieving long-term allograft survival (10–12). Signaling pathways for memory T-cell migration to an inflamed graft include G protein-coupled chemokine receptor signaling and cognate antigen-engaged TCR signaling as both signals trigger downstream integrin activation (Zhang and Lakkis). Interestingly, cognate antigen presence is necessary for driving antigen-specific memory T-cell migration into the peripheral tissue even without acute inflammation (13). Blocking integrin with anti-LFA-1 or anti-VLA-4 mAb prevents memory T-cell migration to a graft, attenuates alloreactive memory T-cell recall responses, and suppresses allograft rejection (14, 15). However, indiscriminately blocking LFA-1, though preventing memory and effector T-cell migration, increases the chance of developing post-transplant EBV-associated lymphoproliferative diseases while targeting VLA-4 may result in reactivation of fatal infections (16). Therefore, it is important to seek new strategies, instead of the universal blockade of major chemokines, to prevent donor-specific memory T-cell migration without increasing the risk of infections. One potential strategy to do so is to target the inside-out signaling pathway downstream of the TCR but not chemokine receptors (Zhang and Lakkis), such as SKAP1, leading to the suppression of antigen-driven but not chemokine-driven memory T-cell migration to a graft. Recently, there has been a renewed interest in immune metabolism in CD8+ T-cells. Their proliferation and function require a metabolic adaptation to meet their needs for energy and biosynthesis (Yap et al.). Activated CD8+ T-cells reprogram their metabolism from OXPHOS to aerobic glycolysis and glutaminolysis (17), supporting their rapid growth with sufficient energy as well as metabolic intermediates. Since glycolysis and glutaminolysis are two major metabolic pathways that are essential for CD8+ effector cell function, blocking metabolic pathways could lead to the discovery of new immunosuppressive drugs for preventing allograft rejection, although these approaches likely cause significant side effects. For instance, 2-Deoxy-d-glucose (2-DG) inhibits glycolysis by blocking hexokinase function and hence suppresses cytotoxic function of effector CD8 T-cells while blocking glutaminolysis with a glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) inhibits T-cell proliferation (Yap et al.). More studies are urgently needed to seek metabolic antagonists that are effective in immunosuppression but result in less severe side effects. Previous studies have shown that CD8+CD122+ T-cells with central memory phenotypes regulate T-cell homeostasis (18) while more recent data have suggested that they suppress conventional T-cell responses (18–24) and control autoimmune diseases (25, 26). We have found that memory CD8+CD122+ T-cells and bystander central memory CD8+ T-cells inhibit murine allograft rejection (27, 28). Moreover, others have shown that central memory CD8+ T-cells mediate lung allograft acceptance (29). Importantly, we have demonstrated that memory-like CD8+CD122+ Tregs are more potent in their suppression of allograft rejection than their CD4+CD25+ counterparts (30). Hence, not only are memory CD8+CD122+ T-cells regulatory cells, they can also boost the strength of Treg-mediated suppression. The exact mechanisms underlying their suppression are still not well understood, although CD8+CD122+ Tregs may restrict immune responsiveness by production of IL-10, TGFβ1, and IFNγ. In order to utilize them in clinic transplantation, more extensive studies are required to fully understand their mechanisms of action and their safety. Furthermore, we have revealed that PD-1 expression on CD8+CD122+ T-cells is critical for their regulatory function. Therefore, memory-like CD8+CD122+PD-1+ T-cells could be one of the best Treg subsets for the induction of long-term allograft survival or tolerance. It remains to be determined whether this subset of Tregs can be expanded in vitro.

A New Immunosuppressive Molecule Emodin Induces both CD4+FoxP3+ and CD8+CD122+ Regulatory T Cells and Suppresses Murine Allograft Rejection

Due to vigorous alloimmunity, an allograft is usually rejected without any conventional immunosuppressive treatment. However, continuous global immunosuppression may cause severe side effects, including tumors and infections. Mounting evidence has shown that cyclosporine (CsA), a common immunosuppressant used in clinic, impedes allograft tolerance by dampening regulatory T cells (Tregs), although it inhibits allograft rejection at the same time. Therefore, it is necessary to seek an alternative immunosuppressive drug that spares Tregs with high efficiency in suppression but low toxicity. In this study, we investigated the capacity of emodin, an anthraquinone molecule originally extracted from certain natural plants, to prolong transplant survival in a mouse model and explored the cellular and molecular mechanisms underlying its action. We found that emodin significantly extended skin allograft survival and hindered CD3+ T cell infiltration in the allograft, accompanied by an increase in CD4+Foxp3+ and CD8+CD122+ Treg frequencies and numbers but a reduction in effector CD8+CD44highCD62Llow T cells in recipient mice. Emodin also inhibited effector CD8+ T cells proliferation in vivo. However, CD4+CD25+, but not CD8+CD122+, Tregs derived from emodin-treated recipients were more potent in suppression of allograft rejection than those isolated from control recipients, suggesting that emodin also enhances the suppressive function of CD4+CD25+ Tregs. Interestingly, depleting CD25+ Tregs largely reversed skin allograft survival prolonged by emodin while depleting CD122+ Tregs only partially abrogated the same allograft survival. Furthermore, we found that emodin hindered dendritic cell (DC) maturation and reduced alloantibody production posttransplantation. Finally, we demonstrated that emodin inhibited in vitro proliferation of T cells and blocked their mTOR signaling as well. Therefore, emodin may be a novel mTOR inhibitor that suppresses alloimmunity by inducing both CD4+FoxP3+ and CD8+CD122+ Tregs, suppressing alloantibody production, and hindering DC maturation. Thus, emodin is a newly emerging immunosuppressant and could be utilized in clinical transplantation in the future.

PSORI-CM02 Formula Increases CD4+ Foxp3+ Regulatory T Cell Frequency and Ameliorates Imiquimod-Induced Psoriasis in Mice

Psoriasis is an autoimmune and inflammatory disease, which is estimated to affect 2–3% of the population in the world. PSORI-CM02 is an empirical formula of Chinese medicine optimized from Yin Xie Ling, which is widely used to treat psoriasis in China for decades. However, its antipsoriatic mechanisms are still not well understood. Here, we explored the therapeutic effects of PSORI-CM02 on psoriasis and its mechanisms of action in imiquimod-induced psoriasis-like mouse models and human HaCaT cells. In experiments in vitro, PSORI-CM02 significantly inhibited HaCaT cell proliferation in dose-dependent and time-dependent manners. Furthermore, it hindered the progression of HaCaT cell cycle and arrested HaCaT cells at G1 phase. On the other hand, our in vivo studies demonstrated that PSORI-CM02 dramatically reduced psoriasis area and severity index scores and lesion temperature in imiquimod-induced psoriatic mice. The antioxidative activities of glutathione, catalase, and superoxide dismutase were increased while oxidative activity of malonaldehyde was markedly decreased after treatments with PSORI-CM02. PSORI-CM02 also suppressed the mRNA expression of proinflammatory cytokines, including TNF-α, IL-6, and IL-17, and lowered their protein levels in the serum as well. In addition, PSORI-CM02 could reduce the expression of IKKα and NF-κB in psoriatic skin tissue. It also upregulated the proportion of CD4+ Foxp3+ regulatory T cells (Tregs) in both lymph nodes and spleens and promoted CD4+ CD25+ Treg proliferation in vitro. Taken together, our research demonstrated that PSORI-CM02 inhibited HaCaT cell proliferation by arresting them at G1 phase and alleviated systemic inflammation and psoriasis in mice via altering the oxidative/anti-oxidative status, tipping the balance between Th17 responsiveness and CD4+ Foxp3+ Treg generation, and suppressing the expression of proinflammatory cytokines as well as NF-κB signaling.

Effects of Cigarette Smoking on Transplant Survival: Extending or Shortening It?

Cigarette smoking (CS) regulates both innate and adaptive immunity and causes numerous diseases, including cardiovascular, respiratory, and autoimmune diseases, allergies, cancers, and transplant rejection. Therefore, smoking poses a serious challenge to the healthcare system worldwide. Epidemiological studies have always shown that CS is one of the major risk factors for transplant rejection, even though smoking plays redundant roles in regulating immune responses. The complex roles for smoking in immunoregulation are likely due to molecular and functional diversities of cigarette smoke components, including carbon monoxide (CO) and nicotine. Especially, CO has been shown to induce immune tolerance. Although CS has been shown to impact transplantation by causing complications and subsequent rejection, it is overlooked whether CS interferes with transplant tolerance. We have previously demonstrated that cigarette smoke exposure reverses long-term allograft survival induced by costimulatory blockade. Given that CS impacts both adaptive and innate immunity and that it hinders long-term transplant survival, our perspective is that CS impacts transplant tolerance. Here, we review impacts of CS on major immune cells that are critical for transplant outcomes and propose the cellular and molecular mechanisms underlying its effects on alloimmunity and transplant survival. Further investigations are warranted to fully understand why CS exerts deleterious rather than beneficial effects on transplant survival even if some of its components are immunosuppressive.

Transcription Factor Retinoid-Related Orphan Receptor γt: A Promising Target for the Treatment of Psoriasis

Psoriasis, which is a common chronic inflammatory skin disease, endangers human health and brings about a major economic burden worldwide. To date, treatments for psoriasis remain unsatisfied because of their clinical limitations and various side effects. Thus, developing a safer and more effective therapy for psoriasis is compelling. Previous studies have explicitly shown that psoriasis is an autoimmune disease that is predominantly mediated by T helper 17 (Th17) cells, which express high levels of interleukin-17 (IL-17) in response to interleukin-23 (IL-23). The discovery of the IL-23–Th17–IL-17 axis in the development of psoriasis has led to the paradigm shift of understanding pathogenesis of psoriasis. Although anti-IL-17 antibodies show marked clinical efficacy in treating psoriasis, compared with antibodies targeting IL-17A or IL-17R alone, targeting Th17 cells themselves may have a maximal benefit by affecting multiple proinflammatory cytokines, including IL-17A, IL-17F, IL-22, and granulocyte-macrophage colony-stimulating factor, which likely act synergistically to drive skin inflammation in psoriasis. In this review, we mainly focus on the critical role of Th17 cells in the pathogenesis of psoriasis. Especially, we explore the small molecules that target retinoid-related orphan receptor γt (RORγt), a vital transcription factor for Th17 cells. Given that RORγt is the lineage-defining transcription factor for Th17 cell differentiation, targeting RORγt via small molecular inverse agonists may be a promising strategy for the treatment of Th17-mediated psoriasis.

Esculetin ameliorates psoriasis-like skin disease in mice by inducing CD4+Foxp3+ regulatory T cells

Psoriasis is an autoimmune and inflammatory skin disease affecting around 2–3% of the worlds population. Patients with psoriasis need extensive treatments with global immunosuppressive agents that may cause severe side effects. Esculetin, a type of coumarins, is an active ingredient extracted mainly from the bark of Fraxinus rhynchophylla, which has been used to treat inflammatory and autoimmune diseases in China. However, the antipsoriatic effects of esculetin have not been reported. In this study, we aimed to investigate the effects of esculetin on psoriatic skin inflammation in a mouse model and explored the potential molecular mechanisms underlying its action. We found that esculetin ameliorated the skin lesion and reduced PASI scores as well as weight loss in imiquimod-induced psoriasis-like mice, accompanied with weakened proliferation and differentiation of keratinocytes and T cell infiltration in esculetin-treated psoriatic mice. In addition, esculetin reduced the frequency of CD8+CD44highCD62Llow effector T cells in psoriatic mice. In contrast, it increased the frequency of CD4+Foxp3+ Tregs in both lymph nodes and spleens of the psoriatic mice while promoting the differentiation of CD4+CD25− T cells into CD4+Foxp3+ Tregs in vitro. Interestingly, depleting CD4+Foxp3+ Tregs largely reversed esculetin-mediated reduction in PASI scores, indicating that esculetin attenuates murine psoriasis mainly by inducing CD4+Foxp3+ Tregs. Furthermore, the mRNA levels of proinflammatory cytokines in the psoriatic mouse skin, including IL-6, IL-17A, IL-22, IL-23, TNF-α, and IFN-γ, were dramatically decreased by the treatment with esculetin. Finally, we found that esculetin inhibited the phosphorylation of IKKα and P65 in the psoriatic skin, suggesting that it inhibits the activation of NF-κB signaling. Thus, we have demonstrated that esculetin attenuates psoriasis-like skin lesion in mice and may be a potential therapeutic candidate for the treatment of psoriasis in clinic.

Chimeric Antigen Receptor (CAR) Treg: A Promising Approach to Inducing Immunological Tolerance

Cellular therapies with polyclonal regulatory T-cells (Tregs) in transplantation and autoimmune diseases have been carried out in both animal models and clinical trials. However, The use of large numbers of polyclonal Tregs with unknown antigen specificities has led to unwanted effects, such as systemic immunosuppression, which can be avoided via utilization of antigen-specific Tregs. Antigen-specific Tregs are also more potent in suppression than polyclonal ones. Although antigen-specific Tregs can be induced in vitro, these iTregs are usually contaminated with effector T cells during in vitro expansion. Fortunately, Tregs can be efficiently engineered with a predetermined antigen-specificity via transfection of viral vectors encoding specific T cell receptors (TCRs) or chimeric antigen receptors (CARs). Compared to Tregs engineered with TCRs (TCR-Tregs), CAR-modified Tregs (CAR-Tregs) engineered in a non-MHC restricted manner have the advantage of widespread applications, especially in transplantation and autoimmunity. CAR-Tregs also are less dependent on IL-2 than are TCR-Tregs. CAR-Tregs are promising given that they maintain stable phenotypes and functions, preferentially migrate to target sites, and exert more potent and specific immunosuppression than do polyclonal Tregs. However, there are some major hurdles that must be overcome before CAR-Tregs can be used in clinic. It is known that treatments with anti-tumor CAR-T cells cause side effects due to cytokine “storm” and neuronal cytotoxicity. It is unclear whether CAR-Tregs would also induce these adverse reactions. Moreover, antibodies specific for self- or allo-antigens must be characterized to construct antigen-specific CAR-Tregs. Selection of antigens targeted by CARs and development of specific antibodies are difficult in some disease models. Finally, CAR-Treg exhaustion may limit their efficacy in immunosuppression. Recently, innovative CAR-Treg therapies in animal models of transplantation and autoimmune diseases have been reported. In this mini-review, we have summarized recent progress of CAR-Tregs and discussed their potential applications for induction of immunological tolerance.

Mangiferin Attenuates Murine Lupus Nephritis by Inducing CD4 + Foxp3 + Regulatory T Cells via Suppression of mTOR Signaling

Background/Aims: Lupus nephritis (LN) is an autoimmune glomerulonephritis that frequently develops secondary to systemic lupus erythematosus. Patients with LN require extensive treatments with global immunosuppressive agents. However, long-term treatment with conventional immunosuppressants may cause various side effects. Therefore, it’s important to seek alternative drugs for treating LN. Here we aimed to investigate the immunoregulatory effects of mangiferin (MG), an ingredient that was originally extracted from natural herbs, including Mangifera Indica Linn. and Rhizoma Anemarrhenae. Methods: FasL-deficient B6/ gld mice were used as a spontaneous LN model. The serum anti-dsDNA Ab and creatinine levels were analyzed via ELISA. Renal histology and immunopathology were determined using H&E and PAS staining, immunofluorescence (IgG and C3), and IHC staining (CD3 and a-SMA). Cytokine gene expression was measured by RT-PCR assays while effector T cells and Tregs were enumerated by flow analysis. Finally, the proliferation and apoptosis of T cells were measured by CFSE staining and flow analysis while their mTOR signaling was detected through Western blotting. Results: We found that administration of MG ameliorated LN in lupus-prone B6/gld mice by reducing the urinary protein and serum creatinine levels, diminishing T cell infiltration in kidneys and improving renal immunopathology. MG also significantly lowered the percentages of CD44highCD62Llow effector T cells in B6/gld mice. Importantly, treatments with MG augmented CD4+FoxP3+ Treg frequencies in spleens, lymph nodes and kidneys of B6/gld mice. It also induced CD4+FoxP3+ Tregs from CD3+ T cells in vitro and promoted Treg proliferation. Furthermore, it inhibited CD3+ T cell proliferation in vitro and suppressed their phosphorylation of mTOR and its downstream P70S6K. However, MG did not promote T cell apoptosis, implying that it is not cytotoxic. Depletion of CD4+CD25+FoxP3+ Tregs in B6/gld mice abrogated its therapeutic effects on LN. Conclusion: MG exerts a novel therapeutic effect on murine LN via upregulating CD4+FoxP3+ Tregs, downregulating mTOR/p70S6K pathway and improving renal immunopathology. It may be useful for treating LN in clinic.