Two polymorphisms in the TIM‐4 gene are associated with asthma in a Chinese Han population

The gene family of the T cell immunoglobulin and mucin domain (TIM) proteins encodes cell surface receptors that are involved in the regulation of Th1‐ and Th2‐cell‐mediated immunity. TIM‐1 gene has been found to be associated with asthma in several populations. TIM‐4, the natural ligand for TIM‐1, may influence the susceptility to asthma.To investigate the association of the TIM‐4 gene polymorphisms with asthma in a Chinese Han population. Three single nucleotide polymorphisms (SNPs) in TIM‐4 gene, rs6882076, rs12658558 and rs4702747, were genotyped in 551 unrelated asthma patients and 549 healthy controls. We found that two SNPs of the TIM‐4 gene, rs6882076 and rs4702747, were associated with asthma susceptibility in our study population (with P‐values = 0.009 and 0.005 respectively). No association was observed between asthma and rs12658558. Our results suggest that TIM‐4 gene polymorphisms are associated with asthma in a Chinese Han population.     

Enhanced suppressor function of TIM‐3+FoxP3+ regulatory T cells

T‐cell immunoglobulin and mucin domain 3 (TIM‐3) is an Ig‐superfamily member expressed on IFN‐γ‐secreting Th1 and Tc1 cells and was identified as a negative regulator of immune tolerance. TIM‐3 is expressed by a subset of activated CD4⁺ T cells, and anti‐CD3/anti‐CD28 stimulation increases both the level of expression and the number of TIM‐3⁺ T cells. In mice, TIM‐3 is constitutively expressed on natural regulatory T (Treg) cells and has been identified as a regulatory molecule of alloimmunity through its ability to modulate CD4⁺ T‐cell differentiation. Here, we examined TIM‐3 expression on human Treg cells to determine its role in T‐cell suppression. In contrast to mice, TIM‐3 is not expressed on Treg cells ex vivo but is upregulated after activation. While TIM‐3⁺ Treg cells with increased gene expression of LAG3, CTLA4, and FOXP3 are highly efficient suppressors of effector T (Teff) cells, TIM‐3^? Treg cells poorly suppressed Th17 cells as compared with their suppression of Th1 cells; this decreased suppression ability was associated with decreased STAT‐3 expression and phosphorylation and reduced gene expression of IL10, EBI3, GZMB, PRF1, IL1Rα, and CCR6. Thus, our results suggest that TIM‐3 expression on Treg cells identifies a population highly effective in inhibiting pathogenic Th1‐ and Th17‐cell responses.     

Role of a histamine 4 receptor as an anti‐inflammatory target in carrageenan‐induced pleurisy in mice

The histamine 4 receptor (H4R) is expressed primarily on cells involved in inflammation and immune responses. Despite much research into inflammatory diseases, no drugs with favourable safety profiles are yet available for their treatment. The aim of the present study was to determine the potential anti‐inflammatory effect of 4‐methylhistamine (4‐MeH) or JNJ77777120 (JNJ) and to explore the role of H4R in a mouse model of carrageenan (Cg) ‐induced pleurisy. A single dose of 4‐MeH or JNJ (30 mg/kg) was administered intraperitoneally 1 hr before Cg administration. The results illustrate that both the numbers of CD4⁺, CD25⁺, CD4⁺ CD25⁺, GITR⁺, GITR⁺ IL‐17A⁺‐expressing T cells and the levels of T helper type 1 (Th1)/Th17 cytokines were markedly increased in both the Cg‐treated and 4‐MeH‐treated groups, whereas the cytokines produced by Th2 cells were significantly decreased in the same groups. However, JNJ treatment significantly decreased both the number of T‐cell subsets and GITR⁺, GITR⁺ IL‐17A⁺‐expressing T cells, and the production of Th1/Th17 cytokines. Further, JNJ up‐regulated the expression of the Th2 cytokines. RT‐PCR analysis revealed an increased expression of interleukin‐1β, tumour necrosis factor‐α, monocyte chemoattractant protein‐1 and intercellular adhesion molecule‐1 in the Cg‐treated and 4‐MeH‐treated groups, which was reduced by treatment with JNJ in lung tissues. Moreover, histological examinations revealed anti‐inflammatory effects of JNJ, whereas 4‐MeH worsened Cg‐induced inflammation. In conclusion, the results of the present work clearly indicate that JNJ possesses important anti‐inflammatory properties that are increased in 4‐MeH‐treated mice, suggesting that H4R are involved in pleurisy and that JNJ has an anti‐inflammatory effect in associated disease conditions.     

Dendritic cells, pro-inflammatory responses, and antigen presentation in a rodent malaria infection

Summary: An infection of mice with Plasmodium chabaudi is characterized by a rapid and marked inflammatory response with a rapid but regulated production of interleukin‐12 (IL‐12), tumor necrosis factor‐α (TNF‐α), and interferon‐γ (IFN‐γ). Recent studies have shown that dendritic cells (DCs) are activated in vivo in the spleen, are able to process and present malaria antigens during infection, and may provide a source of cytokines that contribute to polarization of the CD4 T‐cell response. P. chabaudi‐infected erythrocytes are phagocytosed by DCs, and peptides of malaria proteins are presented on major histocompatibility complex (MHC) class II. The complex disulfide‐bonded structure of some malaria proteins can impede their processing in DCs, which may affect the magnitude of the CD4 T‐cell response and influence T‐helper 1 (Th1) or Th2 polarization. DCs exhibit a wide range of responses to parasite‐infected erythrocytes depending on their source, their maturational state, and the Plasmodium species or strain. P. chabaudi‐infected erythrocytes stimulate an increase in the expression of costimulatory molecules and MHC class II on mouse bone marrow‐derived DCs, and they are able to induce the production of pro‐inflammatory cytokines such as IL‐12, TNF‐α, and IL‐6, thus enhancing the Th1 response of naïve T cells. IFN‐γ and TNF‐α play a role in both protective immunity and the pathology of the infection, and the inflammatory disease may be regulated by IL‐10 and transforming growth factor‐β. It will therefore be important to elucidate the host and parasite molecules that are involved in activation or suppression of the DCs and to understand the interplay between these opposing forces on the host response in vivo during a malaria infection.     

Dendritic cells activated by an anti‐inflammatory agent induce CD4+ T helper type 2 responses without impairing CD8+ memory and effector cytotoxic T‐lymphocyte responses

Prevalence of pro‐inflammatory diseases is rising in developed country populations. The increase in these diseases has fuelled the search for new, immune suppressive, anti‐inflammatory therapies, which do not impact, or minimally impact, CD4⁺ and/or CD8⁺ T‐cell‐mediated immunity. The goal of this study was to determine if antigen‐presenting cells (APCs) activated by the anti‐inflammatory oligosaccharide, lacto‐N‐fucopentaose III (LNFPIII), would have an impaired ability to drive CD4⁺ T helper (Th) or CD8⁺ memory and effector T‐cell responses. To investigate this we activated splenic dendritic cells (SDCs) with LNFPIII and examined their ability to drive antigen‐specific CD4⁺ Th, and CD8⁺ memory and cytotoxic T‐cell (CTL) responses compared with lipopolysaccharide (LPS) ‐stimulated SDCs. The LNFPIII‐activated SDCs had altered co‐stimulatory molecule expression compared with LPS‐stimulated SDCs, while the levels of SDC chemokines following activation by either compound were similar. LNFPIII‐activated SDCs produced significantly lower levels of interleukin‐12 but surprisingly higher levels of interleukin‐6 than LPS‐activated SDCs. Similar to previous studies using bone‐marrow‐derived DCs, LNFPIII‐activated SDCs induced strong Th2 responses in vivo and ex vivo. LNFPIII activation of APCs was independent of the Toll‐interleukin‐1 receptor adaptor myeloid differentiating factor 88. Importantly, LNFPIII‐matured DCs induced CD8⁺ memory and effector CTL responses similar to those driven by LPS‐matured DCs, including the frequency of interferon‐γ‐producing CD8⁺ T cells and induction of CTL effectors. Treatment of APCs by the anti‐inflammatory glycan LNFPIII did not impair their ability to drive CD8⁺ effector and memory cell‐mediated immunity.     

Promoter polymorphisms of the TIM‐4 gene are correlated with disease activity in patients with systemic lupus erythematosus

Although the TIM gene family plays important roles in immune responses, little is known about TIM regulation in the development of systemic lupus erythematosus (SLE). This study aimed to investigate the association of two TIM‐4 single nucleotide polymorphisms (SNPs) rs6874202 (?1419G>A) and rs62382402 (?1609G>A) with SLE susceptibility in a Chinese Han population. The results showed no significant differences between patients with SLE and control group for rs6874202 and rs62382402 (p = .72, .53 respectively). However, the anti‐dsDNA levels in serum from SLE patients with GG genotype of TIM‐4 gene at ‐1419 site were significantly higher than those with GA and AA genotype (p = .0335), and C3 levels of SLE patients with GG and GA genotype were much lower than those with AA genotypes (p = .0187). Moreover, the apoptotic cell levels of SLE patients with AA and GG genotypes were significantly higher than those with GA genotypes in patients with SLE (p = .0393). In addition, the C3 concentration of SLE patients with the GG genotype of TIM‐4 gene at ‐1609 site was found to be significantly higher than those with the GA genotype (p = .0129). The results imply that GG genotype of the TIM‐4 gene at ‐1419 site might be associated with the disease activity of SLE.     

CD4+ T-cell inhibitory ligands: a tool for characterizing dysfunctional CD4+ T cells during chronic infection

T helper type 17 (Th17) lymphocytes are found in high frequency in tumour‐burdened animals and cancer patients. These lymphocytes, characterized by the production of interleukin‐17 and other pro‐inflammatory cytokines, have a well‐defined role in the development of inflammatory and autoimmune pathologies; however, their function in tumour immunity is less clear. We explored possible opposing anti‐tumour and tumour‐promoting functions of Th17 cells by evaluating tumour growth and the ability to promote tumour infiltration of myeloid‐derived suppressor cells (MDSC), regulatory T cells and CD4⁺ interferon‐γ⁺ cells in a retinoic acid‐like orphan receptor γt (RORγt) ‐deficient mouse model. A reduced percentage of Th17 cells in the tumour microenvironment in RORγt‐deficient mice led to enhanced tumour growth, that could be reverted by adoptive transfer of Th17 cells. Differences in tumour growth were not associated with changes in the accumulation or suppressive function of MDSC and regulatory T cells but were related to a decrease in the proportion of CD4⁺ T cells in the tumour. Our results suggest that Th17 cells do not affect the recruitment of immunosuppressive populations but favour the recruitment of effector Th1 cells to the tumour, thereby promoting anti‐tumour responses.     

Opposing effects of pro‐ and anti‐inflammatory cytokine gene polymorphisms on the risk for breast cancer in western Indian women: a pilot study

In an earlier study, the genotypes associated with higher level of transforming growth factor‐β1 (TGF‐β1) were found to reduce the risk for breast cancer in western Indian women. This observation implied that gene polymorphisms affecting the levels of pro‐ and anti‐inflammatory cytokines may influence the risk for breast cancer in this population. Hence, we performed genotyping for three more functional single‐nucleotide polymorphisms (SNPs) responsible for variations in the levels of cytokines associated with inflammation. To that effect, polymorphisms in genes coding for IL‐4 (IL‐4 C‐590T; rs2243250), IFN‐γ (IFN‐G A + 874T; rs2430561) and MCP‐1 (MCP‐1 A‐2578G; rs1024611) were examined in premenopausal, healthy women (N = 239) and patients with breast cancer (N = 182) from western India. In carriers of the IL‐4*590T allele, a reduced risk for the disease (dominant model; OR = 0.61, 95% CI 0.37–0.98) was seen similar to that seen in TGF‐B1*10C carriers. An opposite trend was observed with respect to the alleles associated with higher expression of MCP‐1 or IFN‐γ. In individuals positive for three or more alleles associated with higher levels of either pro‐ or anti‐inflammatory cytokines, an additive effect on the modulation of risk for the disease was evident (for TGF‐B1 & IL‐4, OR = 0.33, 95% CI 0.12–0.87; for IFN‐G & MCP‐1, OR = 2.29, 95% CI 0.95–5.51). In the context of contrasting observations in other populations, these results indicate a significant contribution of anti‐inflammatory genotypes in the modulation of risk for breast cancer in western Indian women.     

Inhibition of Interferon Regulatory Factor 4 Suppresses Th1 and Th17 Cell Differentiation and Ameliorates Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T‐cell‐mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. Interferon regulatory factor 4 (IRF4) is expressed predominantly in the immune system and plays an important role in its development and function. Recent study demonstrated that IRF4 was critical for the generation of IL‐17‐producing Th17 cells. However, the effect of IRF4 on experimental autoimmune encephalomyelitis (EAE), an animal model of MS, needs to be further investigated. In our current study, inhibition of IRF4 with IRF4 siRNA (SiIRF4) decreases EAE scores and infiltration of Th1 and Th17 cells, but increases Treg infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. In our DC‐T‐cell coculture system, SiIRF4‐treated DCs resulted in significantly less IFN‐γ and IL‐17 production from T cells. Next, we adoptively transfer CD11c⁺ DCs from SiIRF4‐treated mice into recipient mice and found that these CD11c⁺ DCs ameliorated EAE. Furthermore, CD11c⁺ DCs from SiIRF4‐treated naive mice exhibited significantly reduced expression of pro‐inflammatory cytokines TNF‐α, IL‐1β, IL‐6 and IL‐12/IL‐23 (p40), and a corresponding increase in anti‐inflammatory IL‐10 expression. In conclusion, inhibition of IRF4 suppresses Th1 and Th17 cell differentiation and ameliorates EAE, via a direct regulation of DCs.     

Type 1 innate lymphoid cell aggravation of atherosclerosis is mediated through TLR4

ILC populations elaborate a similar cytokine expression pattern with helper T cell subsets Th1, Th2 and Th17. Recent studies indicate that CD 25+ILC 2 could alleviate atherosclerosis by altering lipid metabolism, whereas the depletion of CD 90‐expressing ILC s had no influence on atherosclerosis. Thus, these findings raise the question of whether ILC 1 cells react on atherosclerosis. Hence, our group attempted to explore the role of ILC 1 cells in atherosclerosis. We found that ILC 1 cells have a high Th1‐like gene expression of T‐bet and IFN ‐γ, which is distinct from ILC 2, ILC 3 or conventional NK (cNK ) cells. Moreover, atherosclerotic lesions were greatly reduced in ApoE−/−Rag1−/− mice treated with anti‐NK 1.1 mA bs for depleting ILC 1 cells (ILC 1+cNK cells), compared to ApoE−/−Rag1−/− mice treated with anti‐IL ‐15R mA bs for depleting cNK cells, and these effects could be fully rescued through the adoptive transfer of ILC 1 cells sorted from the spleen of ApoE−/−TLR 4+/+ mice into ApoE−/−Rag1−/− mice treated with anti‐NK 1.1 mA bs. However, the adoptive transfer of ILC 1 cells sorted from the spleen of ApoE−/−TLR 4−/− mice into ApoE−/−Rag1−/− mice treated with anti‐NK 1.1 mA bs blocked the progression of atherosclerosis, indicating that the pro‐atherosclerotic role of ILC 1 cells is dependent on TLR 4. Furthermore, oxLDL ‐induced increase in IFN ‐γ expression from ApoE−/− ILC 1 cells was correlated with the decrease in BACH 2 expression. Taken together, ILC 1 cells exist in atherosclerosis and aggravate atherosclerosis via increasing pro‐inflammatory cytokine expression in a TLR 4/BACH 2‐dependent manner.     

Using two phases of the CD4 T cell response to blood-stage murine malaria to understand regulation of systemic immunity and placental pathology in Plasmodium falciparum infection

Plasmodium falciparum infection and malaria remain a risk for millions of children and pregnant women. Here, we seek to integrate knowledge of mouse and human T helper cell (Th) responses to blood-stage Plasmodium infection to understand their contribution to protection and pathology. Although there is no complete Th subset differentiation, the adaptive response occurs in two phases in non-lethal rodent Plasmodium infection, coordinated by Th cells. In short, cellular immune responses limit the peak of parasitemia during the first phase; in the second phase, humoral immunity from T cell–dependent germinal centers is critical for complete clearance of rapidly changing parasite. A strong IFN-γ response kills parasite, but an excess of TNF compared with regulatory cytokines (IL-10, TGF-β) can cause immunopathology. This common pathway for pathology is associated with anemia, cerebral malaria, and placental malaria. These two phases can be used to both understand how the host responds to rapidly growing parasite and how it attempts to control immunopathology and variation. This dual nature of T cell immunity to Plasmodium is discussed, with particular reference to the protective nature of the continuous generation of effector T cells, and the unique contribution of effector memory T cells.     

Plasmodium falciparum infection of the placenta impacts on the T helper type 1 (Th1)/Th2 balance of neonatal T cells through CD4+CD25+ forkhead box P3+ regulatory T cells and interleukin‐10

Placental malaria infection affects the T helper type 1 (Th1)/Th2 balance in neonatal children. We investigated a potential role of regulatory T cells in this balance by comparing T cell responses of cord blood mononuclear cells (CBMC) from parasitized and non‐parasitized placenta of Gambian women. CBMC were depleted of CD4⁺CD25⁺ forkhead box P3 (FoxP3)⁺ regulatory T cells and analysed in vitro for their ability to produce interferon (IFN)‐γ, sCD30 and interleukin (IL)‐10 in response to phytohaemagglutinin (PHA), live Plasmodium falciparum, schizont extracts and the recombinant P. falciparum blood stage antigen merozoite surface protein 1 (MSP1₁₉). As expected, lower IFN‐γ and higher sCD30 responses were observed for the cells from the parasitized group. In addition, higher IL‐10 levels were produced by CBMC from the parasitized group. Depletion of regulatory T cells decreased IL‐10 production, which resulted in a restoration of IFN‐γ expression in response to all stimuli. The Th2 marker sCD30 remained significantly higher in the parasitized group in response to malaria protein antigens while similar levels were recovered between both groups in response to live P. falciparum. Similar effects were observed by adding an antibody that blocks IL‐10 function. These results suggest that the impact of P. falciparum infection on Th1 differentiation of neonatal T cells can be ascribed to regulatory T cells through production of IL‐10.     

TIM‐1 rs41297579 G>A (−1454) and TIM‐4 rs7700944 gene polymorphisms as possible risk factor for rheumatoid arthritis: relation to activity and severity

T his study was aimed to evaluate the impact of both TIM‐1 rs41297579 G>A (?1454) and TIM‐4 rs7700944 polymorphisms on susceptibility to rheumatoid arthritis (RA) in a cohort of Egyptian population and to evaluate for the first time their relation to activity, severity, disease‐related disability and erosion. TIM‐1 rs41297579 G>A (?1454) and TIM‐4 rs7700944 gene polymorphisms were typed by RFLP for 128 patients with RA and 125 healthy controls. The A allele, A‐containing genotypes (GA and AA) of the TIM‐4 and GA haplotype were present with significant higher frequency in patients with RA than healthy controls (P_c < 0.001). These findings suggest that the A allele, A‐containing genotypes (GA and AA) and GA haplotype may be a susceptibility risk factor for RA [OR = 5.83 (3.6–9.4), OR = 9.41 (5.0–17.6) and OR = 4.21 (1.07–19.2), respectively]. No associations were found between TIM genotypes and disease activity, severity or presence of erosion. However, the RA patients with GA genotype of TIM‐4 have higher grade of rheumatoid factor (RF) positivity (P = 0.018), and have worse disease‐related disability (P = 0.007) and worse pain (0.025). TIM‐4 rs7700944 and not TIM‐1 rs41297579 G>A (?1454) is associated with RA in the present cohort of Egyptian and may be a risk factor for development of RA in Egyptian. Both SNPs have no effect on disease activity, severity or erosion. However, TIM‐4 GA genotype is associated with higher grade of RF positivity and worse disease‐related disability and pain.     

Multiple functions of human T cells generated by experimental malaria challenge

Protective immunity generated following malaria infection may be comprised of Ab or T cells against malaria Ag of different stages; however, the short‐lived immunity that is observed suggests deficiency in immune memory or regulatory activity. In this study, cellular immune responses were investigated in individuals receiving Plasmodium falciparum sporozoite challenge by the natural (mosquito bite) route as part of a malaria vaccine efficacy trial. Parasitemia, monitored by blood film microscopy and PCR, was subsequently cleared with drugs. All individuals demonstrated stable IFN‐γ, IL‐2 and IL‐4 ex vivo ELISPOT effector responses against P. falciparum‐infected RBC (iRBC) Ag, 28 and 90 days after challenge. However, infected RBC‐specific central memory responses, as measured by IFN‐γ cultured ELISPOT, were low and unstable over time, despite CD4⁺ T cells being highly proliferative by CFSE dilution, and showed an inverse relationship to parasite density. In support of the observation of poor memory, co‐culture experiments showed reduced responses to common recall Ag, indicating malaria‐specific regulatory activity. This activity could not be accounted for by the expression of IL‐10, TGF‐β, FOXP3 or CTLA‐4, but proliferating T cells expressed high levels of CD95, indicating a pro‐apoptotic phenotype. Lastly, there was an inverse relationship between FOXP3 expression, when measured 10 days after challenge, and ex vivo IFN‐γ measured more than 100 days later. This study shows that malaria infection elicits specific Th1 and Th2 effector cells, but concomitant weak central memory and regulatory activity, which may help to explain the short‐lived immunity observed.     

Adoptive transfer of murine T cells expressing a chimeric‐PD1‐Dap10 receptor as an immunotherapy for lymphoma

Adoptive transfer of T cells is a promising cancer therapy and expression of chimeric antigen receptors can enhance tumour recognition and T‐cell effector functions. The programmed death protein 1 (PD1) receptor is a prospective target for a chimeric antigen receptor because PD1 ligands are expressed on many cancer types, including lymphoma. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 extracellular domain fused to the cytoplasmic domain of CD3ζ. Additionally, chimeric antigen receptor therapies use various co‐stimulatory domains to enhance efficacy. Hence, the inclusion of a Dap10 or CD28 co‐stimulatory domain in the chPD1 receptor was compared to determine which domain induced optimal anti‐tumour immunity in a mouse model of lymphoma. The chPD1 T cells secreted pro‐inflammatory cytokines and lysed RMA lymphoma cells. Adoptive transfer of chPD1 T cells significantly reduced established tumours and led to tumour‐free survival in lymphoma‐bearing mice. When comparing chPD1 receptors containing a Dap10 or CD28 domain, both receptors induced secretion of pro‐inflammatory cytokines; however, chPD1‐CD28 T cells also secreted anti‐inflammatory cytokines whereas chPD1‐Dap10 T cells did not. Additionally, chPD1‐Dap10 induced a central memory T‐cell phenotype compared with chPD1‐CD28, which induced an effector memory phenotype. The chPD1‐Dap10 T cells also had enhanced in vivo persistence and anti‐tumour efficacy compared with chPD1‐CD28 T cells. Therefore, adoptive transfer of chPD1 T cells could be a novel therapy for lymphoma and inclusion of the Dap10 co‐stimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile and T‐cell differentiation phenotype for anti‐tumour therapies.