Downregulation of IL-12 and a novel negative feedback system mediated by CD25+CD4+ T cells

CD25(+)CD4(+) regulatory T cells suppress immune responses and are believed to play roles in preventing autoimmune diseases. However, the mechanism(s) underlying the suppression and the regulation of their homeostasis remain to be elucidated. Here we show that these regulatory T cells downregulated CD25(-)CD4(+) T-cell-mediated production of IL-12 from antigen-presenting cells, which can act as a growth factor for CD25(-)CD4(+) T cells. We further found that CD25(+)CD4(+) T cells, despite their well-documented "anergic" nature, proliferate significantly in vitro only when CD25(-)CD4(+) T cells are present. Notably, this proliferation was strongly dependent on IL-2 and relatively independent of IL-12. Thus, CD25(+)CD4(+) T cells suppress CD25(-)CD4(+) T-cell responses, at least in part, by inhibiting IL-12 production while they themselves can undergo proliferation with the mediation of CD25(-)CD4(+) T cells in vitro. These results offer a novel negative feedback system involving a tripartite interaction among CD25(+)CD4(+) and CD25(-)CD4(+) T cells, and APCs that may contribute to the termination of immune responses.     

Concomitant blockade of A2AR and CTLA-4 by siRNA-loaded polyethylene glycol-chitosan-alginate nanoparticles synergistically enhances antitumor T-cell responses

Inhibitory immune checkpoint (ICP) molecules are important immunosuppressive factors in a tumor microenvironment (TME). They can robustly suppress T-cell-mediated antitumor immune responses leading to cancer progression. Among the checkpoint molecules, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) is one of the critical inhibitors of anticancer T-cell responses. Besides, the expression of adenosine receptor (A2AR) on tumor-infiltrating T cells potently reduces their function. We hypothesized that concomitant silencing of these molecules in T cells might lead to enhanced antitumor responses. To examine this assumption, we purified T cells from the tumor, spleen, and local lymph nodes of CT26 colon cancer-bearing mice and suppressed the expression of A2AR and CTLA-4 using the small interfering RNA (siRNA)-loaded polyethylene glycol-chitosan-alginate (PCA) nanoparticles. The appropriate physicochemical properties of the produced nanoparticles (NPs; size of 72 nm, polydispersive index [PDI] < 0.2, and zeta potential of 11 mV) resulted in their high efficiency in transfection and suppression of target gene expression. Following the silencing of checkpoint molecules, various T-cell functions, including proliferation, apoptosis, cytokine secretion, differentiation, and cytotoxicity were analyzed, ex vivo. The results showed that the generated nanoparticles had optimal physicochemical characteristics and significantly suppressed the expression of target molecules in T cells. Moreover, a concomitant blockade of A2AR and CTLA-4 in T cells could synergistically enhance antitumor responses through the downregulation of PKA, SHP2, and PP2Aα signaling pathways. Therefore, this combination therapy can be considered as a novel promising anticancer therapeutic strategy, which should be further investigated in subsequent studies.     

IL-4 and IL-13 receptors: Roles in immunity and powerful vaccine adjuvants

The roles of interleukin (IL)-4 and IL-13 during both innate and adaptive Th2 mediated immunity have received considerable scrutiny, however, mechanisms by which these cytokines influence the cellular interactions involved in negatively modulating the development of effective Th1 immunity are poorly characterized. In this article we discuss the recent advances in IL-4/IL-13 biology, mainly (i) role of these cytokines in allergic inflammation, atopic dermatitis, cancer, transplant rejection, bacterial/viral infections, and specifically the therapeutic potential of IL-13Rα2, (ii) insights into how “alarmin” stimulation activate IL-4/IL-13 at the lung mucosae, (iii) how these two cytokines modulate antigen-specific CD8⁺ T cell quality/avidity in a vaccine route dependent manner and (iv) finally discuss the potential of using transient inhibition of IL-4 and/or IL-13 at the vaccination site as a platform vaccine technology to induce strong sustained high quality CD8⁺ T cell immunity for protection against many chronic mucosal pathogens such as HIV-1.     

Selective expansion and enhanced anti-tumor effect of antigen-specific CD4+ T cells by retrovirus-mediated IL-15 expression

Mounting evidence has demonstrated that CD4⁺ T cells play an important role in anti-tumor immune responses. Thus, adoptive transfer of these cells may have great potential for anti-cancer therapy. However, due to the difficulty to generate sufficient tumor-specific CD4⁺ T cells, the use of CD4⁺ T cells in tumor therapy is limited. It has been found that IL-15 transfection enhances the proliferation and anti-tumor activity of tumor-specific CD8⁺ T cells, but the effect of IL-15 transfection on CD4⁺ T cells remains unknown. Here, the effects of retrovirus-mediated IL-15 expression in Ova-specific CD4⁺ T cells from Do11.10 mice were evaluated and it was discovered that IL-15 transfected CD4⁺ T cells expressed both soluble and membrane-bound IL-15. Retrovirus-mediated IL-15 expression led to a selective expansion of antigen-specific CD4⁺ T cells by inhibiting their apoptosis. Invivo IL-15 transfected CD4⁺ T cells were more effective in suppressing tumor growth than control retroviral vector transfected ones. To ensure the safety of the method, the employment of thymidine kinase gene made it possible to eliminate these transgenic CD4⁺ T cells following ganciclovir treatment. Together, we show that IL-15 transfection induced a selective expansion of antigen-specific CD4⁺ T cells ex vivo and enhanced their tumor-suppression effects in vivo. This has an important significance for improving the efficacy of adoptive T cell therapy.     

The expression of T-cell surface antigens CTLA-4, CD26, and CD28 is modulated by inhibition of dipeptidylpeptidase IV (DPP IV,CD26) activity in murine stress-induced abortions

Inhibition of DPP IV has been shown to abrogate the stress-related increase in murine abortions and a concomitant increase in gamma-interferon. The aim of the present study was to investigate a potential impact of the DPP IV inhibitor Isoleucine Cyanopyrrolidide on the expression of surface antigens involved in T-cell responses. DBA/2-mated CBA mice were stressed on day 5.5 of pregnancy and received injections of a DPP IV inhibitor. On day 13 of gestation, the animals were sacrificed and the percentage of abortions was determined. As shown before, stress failed to boost the abortion rate in mice receiving the DPP IV inhibitor. In stressed animals, a lower surface density of CTLA-4 on decidual CD26-positive lymphocytes was observed than in non-stressed animals. Inhibition of DPP IV restored CTLA-4 surface density to normal and decreased surface expression of CD26 and CD28 on decidual lymphocytes irrespective of stress exposure. These observations suggest that a modulation of T-cell surface antigens expression due to inhibition of DPP IV activity may contribute to the potent anti-abortogenic effect observed here.     

CD8+ cytotoxic T lymphocytes in cancer immunotherapy: A review

CD8⁺ cytotoxic T lymphocytes (CTLs) are preferred immune cells for targeting cancer. During cancer progression, CTLs encounter dysfunction and exhaustion due to immunerelated tolerance and immunosuppression within the tumor microenvironment (TME), with all favor adaptive immune-resistance. Cancer-associated fibroblasts (CAFs), macrophage type 2 (M2) cells, and regulatory T cells (Tregs) could make immunologic barriers against CD8 ⁺ T cell-mediated antitumor immune responses. Thus, CD8 ⁺ T cells are needed to be primed and activated toward effector CTLs in a process called tumor immunity cycle for making durable and efficient antitumor immune responses. The CD8 ⁺ T cell priming is directed essentially as a corroboration work between cells of innate immunity including dendritic cells (DCs) and natural killer (NK) cells with CD4 ⁺ T cells in adoptive immunity. Upon activation, effector CTLs infiltrate to the core or invading site of the tumor (so-called infiltrated–inflamed [I–I] TME) and take essential roles for killing cancer cells. Exogenous reactivation and/or priming of CD8 ⁺ T cells can be possible using rational immunotherapy strategies. The increase of the ratio for costimulatory to coinhibitory mediators using immune checkpoint blockade (ICB) approach. Programmed death-1 receptor (PD-1)–ligand (PD-L1) and CTL-associated antigen 4 (CTLA-4) are checkpoint receptors that can be targeted for relieving exhaustion of CD8 ⁺ T cells and renewing their priming, respectively, and thereby eliminating antigen-expressing cancer cells. Due to a diverse relation between CTLs with Tregs, the Treg activity could be dampened for increasing the number and rescuing the functional potential of CTLs to induce immunosensitivity of cancer cells.     

Interleukin-2 dose,blood monocyte and CD25+ lymphocyte counts as predictors of clinical response to interleukin-2 therapy in patients with renal cell carcinoma

Summary The purpose of this study was to determine immunological parameters in the peripheral blood that correlate with the clinical effect of interleukin-2 (IL-2) in patients with metastatic renal cell cancer. A group of 26 patients with metastatic renal cell cancer underwent IL-2 treatment using a 36-day schedule with continuous intravenous IL-2 infusion (3 × 10⁶ units m^–2 day^–1) administered from days 1 to 5 and days 12 to 16. The white blood cell count and the absolute and relative number of neutrophils, lymphocytes, eosinophils and monocytes were recorded six times in peripheral blood during the treatment. Also the blood counts of T cell and NK cell subsets and cells expressing the T cell activation markers IL-2R and VLA-1 were measured. The lymphokine-activated killer (LAK) cell cytotoxicity was measured either with or without additional in vitro stimulation by IL-2. Multivariate statistical analysis showed that the clinical responses were related to the administered dose of IL-2, to a low number of blood cells expressing IL-2 receptors and to a reduction in the blood monocyte count (P <0.05).     

Apoptosis of CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript> T cells in response to Sirolimus requires activation of T cell receptor and is modulated by IL-2

Targeted molecular therapies inhibit proliferation and survival of cancer cells but may also affect immune cells. We have evaluated the effects of Sirolimus and Sorafenib on proliferation and survival of lymphoid cell subsets. Both drugs were cytotoxic to CD4⁺CD25^high T cells, and were growth inhibitory for CD4⁺ and CD8⁺ T cells. Cytotoxicity depended on CD3/CD28 stimulation and was detectable within 12 h, with 80–90% of CD4⁺CD25^high cells killed by 72 h. Cell death was due to apoptosis, based on Annexin V and 7AAD staining. Addition of IL-2 prevented the apoptotic response to Sirolimus, potentially accounting for reports that Sirolimus can enhance proliferation of CD4⁺CD25^high cells. These results predict that Sirolimus or Sorafenib would reduce CD4⁺CD25^high cells if administered prior to antigenic stimulation in an immunotherapy protocol. However, administration of IL-2 protects CD4⁺CD25^high T cells from cytotoxic effects of Sirolimus, a response that may be considered in design of therapeutic protocols.     

Characterization of chicken interleukin 2 receptor alpha chain, a homolog to mammalian CD25

To identify chicken IL-2R alpha chain (chCD25), the cDNA of chCD25 was cloned and mapped onto chicken chromosome 1. The polyclonal and monoclonal antibodies raised from the recombinant chCD25 specifically bound to the cell surface of splenic mononuclear cells (SMC) and inhibited chicken IL-2-dependent proliferation of T cells. Flow cytometry analysis revealed that chCD25 molecules could be expressed on the surface of monocytes/macrophages, thrombocytes, CD4+ and CD8+ cells as well as tissue cells. Importantly, the CD4+CD25+ and CD8+CD25+ cells were upregulated dramatically in chickens infected with H9N2 avian influenza virus. These results confirm that the cloned cDNA is the nucleotide sequence of chicken IL-2R, and suggest that chicken CD4+CD25+ and CD8+CD25+ cells may play an important role in immune responses induced by H9N2 virus, and the monoclonal antibodies to chCD25 may be useful for investigating biological functions of chicken regulatory T cells.     

Suppression of IL‐2 Production and Proliferation of CD4+ T Cells by Tuberostemonine O

Tuberostemonine stereoisomers are natural alkaloids found in Stemona tuberosa, that are known to have anti‐inflammatory and anti‐infective properties. Tuberostemonine alkaloids inhibit inflammation by suppressing the expression of inflammatory mediators such as cyclooxygenase and nitric oxide synthase. However, the direct immunomodulatory properties of tuberostemonine alkaloids in T cells have not been elucidated so far. In this study, the activities in T cells of tuberostemonine N (TbN) and a novel alkaloid, tuberostemonine O (TbO), isolated from S. tuberosa, were investigated. Although TbN did not have a significant effect on cytokine production in splenic T cells, TbO selectively suppressed interleukin (IL)‐2 production. Moreover, TbO, but not TbN, significantly inhibited IL‐2 production by primary CD4⁺ T cells and delayed the T‐cell proliferation in a dose‐dependent manner. Addition of excess recombinant IL‐2 restored the decreased cell‐division rates in TbO‐treated CD4⁺ T cells to control levels. Collectively, these findings suggest that the immunomodulatory effects of TbO occurred by the suppression of IL‐2 expression and IL‐2‐induced T‐cell proliferation, suggesting a potential beneficial role of tuberostemonine alkaloids for the control of chronic inflammatory and autoimmune diseases caused by hyperactivated T cells.     

Expansion of NK cells by engineered K562 cells co-expressing 4-1BBL and mMICA,combined with soluble IL-21

NK cells hold promise for protecting hosts from cancer and pathogen infection through direct killing and expressing immune-regulatory cytokines. In our study, a genetically modified K562 cell line with surface expression of 4-1BBL and MICA was constructed to expand functional NK cells in vitro for further adoptive immunotherapy against cancer. After a long-term up to 21 day co-culture with newly isolated peripheral blood mononuclear cells (PBMCs) in the presence of soluble IL-21 (sIL-21), notable increase in proportion of expanded NK cells was observed, especially the CD56^brightCD16⁺ subset. Apparent up-regulation of activating receptors CD38, CD69 and NKG2D was detected on expanded NK cells, so did inhibitory receptor CD94; the cytotoxicity of expanded NK cells against target tumor cells exceeded that of NK cells within fresh PBMCs. The intracellular staining showed expanded NK cells produced immune-regulatory IFN-γ. Taken together, we expanded NK cells with significant up-regulation of activating NKG2D and moderate enhancement of cytotoxicity, with IFN-γ producing ability and a more heterogeneous population of NK cells. These findings provide a novel perspective on expanding NK cells in vitro for further biology study and adoptive immunotherapy of NK cells against cancer.     

Cord blood CD4CD25 regulatory T cells fail to inhibit cord blood NK cell functions due to insufficient production and expression of TGF-beta1

Although CD4⁺CD25⁺ Treg (Treg) cells are known to modulate NK cell functions, the modulation mechanism of these cells in cord blood has not been fully clarified. The purpose of this study was to clarify the mechanism whereby cord blood Treg cells modulate cord NK cells. By performing various cultures of purified NK cells with or without autologous Treg cells, diminished inhibitory effects of cord Treg cells towards cord NK cell functions, including activation, cytokine production, and cytotoxicity, were observed. We also observed lower secretion of sTGF-beta1 and lower expression of mTGF-beta1 by cord Treg cells than by adult Treg cells. These data revealed the capability of adult Treg cells to suppress rhIL-2-stimulated NK cell function by TGF-beta1, both membrane-bound and soluble types. The reduced inhibitory capabilities of cord Treg cells compared with adult Treg cells is thought to be due to insufficient expression of TGF-beta1.     

Heligmosomoides polygyrus antigens inhibit the intrinsic pathway of apoptosis by overexpression of survivin and Bcl-2 protein in CD4 T cells

Many laboratory studies and epidemiological observations confirm that nematodes prevent some immune-mediated diseases. The development of immunologically well-defined laboratory models of intestinal nematode infection has allowed significant advances to be made in understanding the immunological basis of effector mechanisms operating during infection under controlled laboratory conditions. The Heligmosomoides polygyrus- mouse system is used for studies of parasite immunomodulation. H. polygyrus causes a chronic, asymptomatic intestinal infection and effectively maintains both local and systemic tolerance to reduce allergic and autoimmune inflammation. However, exposure of mice to H. polygyrus antigen reduced spontaneous and glucocorticoid-induced apoptosis of CD4- positive T cells in mesenteric lymph node (MLN). In this study we evaluate the proliferation, cytokine secretion, cell cycle progression and expression of apoptosis related genes in MLN CD4 T cells of uninfected and H. polygyrus infected mice ex vivo and in vitro after restimulation with parasite excretory secretory antigen (ESAg), somatic antigen (SAg) and fraction 9 (F9Ag) of somatic antigen. For the first time we explain the influence of H. polygyrus antigens on the intrinsic pathway of apoptosis. We found that the proliferation provoked by fraction 9 and inhibition of apoptosis was dependent on a low Bax/Bcl-2 ratio, dramatical upregulation of survivin, D1 cyclin, P-glycoprotein, and loss of p27Kip1 protein with inhibition of active caspase-3 but not caspase- 8.     

Plasmodium yoelii: distinct CD4(+)CD25(+) regulatory T cell responses during the early stages of infection in susceptible and resistant mice

The outcome of experimental murine infection with different strains of malaria parasites, ranging from spontaneous cure to death, depends largely on the establishment of effective Th1 responses during the early stages of infection. Here we describe the disparity in CD4(+)CD25(+) regulatory T cell (Treg) responses during the early stages of infection with the highly virulent Plasmodium yoelii 17XL strain in susceptible (BALB/c) and resistant (DBA/2) mice. An increased proportion of Tregs 3-4 days post inoculation, co-occurring with elevated IL-10 levels, is observed in BALB/c but not in DBA/2 mice. These findings suggest that Treg proliferation might be causally associated with the suppression of Th1 responses during early malaria infection, leading to increase parasitemia and mortality in BALB/c mice, possibly in an IL-10-dependent manner.     

Differential role of reactive oxygen species in the activation of mitogen-activated protein kinases and Akt by key receptors on B-lymphocytes: CD40, the B cell antigen receptor, and CXCR4

Background Antibodies produced by B-lymphocytes play a key role in the host defense against infection. The development, survival, and activation of B cell is regulated by multiple receptors including the B cell antigen receptor (BCR), which detects the presence of pathogens, CD40, which binds co-stimulatory molecules on activated T cells, and chemokines such as SDF-1 (CXCL12) that play key roles in B cell development and trafficking. Signaling by many receptors results in the generation of reactive oxygen species (ROS) that function as second messengers by regulating the activity of redox-sensitive kinases and phosphatases. We investigated the role of ROS in signaling by the BCR, CD40, and CXCR4, the receptor for SDF-1. We focused on activation of ERK, JNK, p38, and Akt, kinases that regulate multiple processes including cell survival, proliferation, and migration. Results Using the anti-oxidants N-acetyl L-cysteine (NAC) and ebselen to deplete intracellular ROS, we identified a differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by these receptors. We found that CD40 activated JNK, p38, and Akt via redox-dependent pathways that were sensitive to ROS depletion by NAC and ebselen. In contrast, BCR-induced activation of ERK, JNK, p38, and Akt was not affected by ROS depletion. We also found that CXCR4-induced Akt activation was ROS-dependent even though activation of the ERK, JNK, and p38 MAP kinases by CXCR4 occurred via ROS-independent pathways. Conclusion The differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by the BCR, CD40, and CXCR4 likely reflects the multiplicity of upstream activators for each of these kinases, only some of which may be regulated in a redox-dependent manner. These findings support the idea that ROS are important second messengers in B cells and suggest that oxidants or anti-oxidants could be used to modulate B cell activation.     

CD4+ lymphocytes require platelets for adhesion to immobilized fibronectin in flow: role of beta(1) (CD29)-, beta(2) (CD18)-related integrins and non-integrin receptors

The role of platelets in T-lymphocytes adhesion is not clear yet. Herpesvirus saimiri (HVS)-infected CD4(+) T-lymphocytes were placed into polystyrene plates pre-coated with fibronectin. The adherent T-cells were enumerated by image analysis. Under static condition, 38+/-10cells/mm(2) adhered and addition of gel-filtered platelets (GFP) and PMA enhanced cell adhesion 4.3- and 4.1-fold. Using PMA plus GFP 11.9-fold enhancement in cell adhesion was achieved. In contrast, under flow (200s(-1)), neither basal adhesion nor following separate addition of PMA or GFP was observed, whereas combined addition of PMA and GFP induced noticeable adhesion (34cells/mm(2)). The adhesion was inhibited by blockade of alpha(5)-integrin (CD49e, 87%), beta(2)-integrin (CD18, 78%), CD40L (60%), PSGL-1 (CD162, 60%), and CD40L plus PSGL-1 (83%). Thus, activated platelets promote activated T-cell adhesion to fibronectin under flow via integrins (alpha(5)beta(1), and alpha(L)beta(2)), CD40-CD40L and P-selectin-PSGL-1 mediated interactions.