Immunological and histological evaluation of clinical samples from psoriasis patients treated with anti-CD6 itolizumab

Psoriasis is a chronic inflammatory disease with a prevalence of approximately 2–3% in the general population. The majority of diagnosed patients have plaque psoriasis, and about 20% have moderate-to-severe disease. Itolizumab, a new monoclonal antibody specific for the CD6 molecule mainly expressed on T lymphocytes, has demonstrated to inhibit in vitro ligand-induced proliferation and pro-inflammatory cytokine production. We assessed the immunological and histopathological effect of the antibody using clinical samples taken from 26 patients with moderate-to-severe psoriasis included in a clinical trial. The precursor frequency of lymphocytes activated with anti-CD2/CD3/CD28 beads, as well as the number of interferon (IFN)-γ-secreting T cells after stimulation, were measured at different time points of the study. Serum cytokine levels and anti-idiotypic antibody response to itolizumab were also evaluated. Additionally, lymphocyte infiltration and epidermis hyperplasia were studied in five patients. A significant reduction in T cell proliferation capacity and number of IFN-γ-producing T cells was found in treated patients. Serum levels of interleukin-6, tumor necrosis factor and IFN-γ showed an overall trend toward reduction. No anti-idiotypic antibody response was detected. A significant reduction in the epidermis hyperplasia was observed in analyzed patients. These results support the relevance of the CD6 molecule as a therapeutic target for the treatment of this disease.     

Immunological evaluation of rheumatoid arthritis patients treated with itolizumab

Rheumatoid arthritis is an autoimmune disease characterized by joint inflammation that affects approximately 1% of the general population. Itolizumab, a monoclonal antibody specific for the human CD6 molecule mainly expressed on T lymphocytes, has been shown to inhibit proliferation of T cells and proinflammatory cytokine production in psoriasis patients. We have now assessed the immunological effect of itolizumab in combination with methotrexate in rheumatoid arthritis by analyzing clinical samples taken from 30 patients enrolled in a clinical trial. T and B cell subpopulations were measured at different time points of the study. Plasma cytokine levels and anti-idiotypic antibody response to itolizumab were also evaluated. The combined treatment of itolizumab and methotrexate led to a reduction in the frequency of T cell subpopulations, and plasma levels of proinflammatory cytokines showed a significant decrease up to at least 12 weeks after treatment ended. No anti-idiotypic antibody response was detected. These results support the relevance of the CD6 molecule as a therapeutic target for the treatment of this disease.     

IL-21 promotes skin recruitment of CD4(+) cells and drives IFN-γ-dependent epidermal hyperplasia

Psoriasis is a chronic inflammatory disorder of the skin characterized by epidermal hyperplasia and infiltration of leukocytes into the dermis and epidermis. T cell-derived cytokines, such as IFN-γ and IL-17A, play a major role in the psoriasis-associated epidermal hyperplasia, even though factors/mechanisms that regulate the production of these cytokines are not fully understood. We have recently shown that IL-21 is synthesized in excess in psoriatic skin lesions and causes epidermal hyperplasia when injected intradermally in mice. Moreover, in the human psoriasis SCID mouse model, neutralization of IL-21 reduces both skin thickening and expression of inflammatory molecules, thus supporting the pathogenic role of IL-21 in psoriasis. However, the basic mechanism by which IL-21 promotes skin pathology remains unknown. In this study, we show that CD4(+) cells accumulate early in the dermis of IL-21-treated mice and mediate the development of epidermal hyperplasia. Indeed, IL-21 fails to induce skin damage in RAG1-deficient mice and CD4(+) cell-depleted wild-type mice. The majority of CD4(+) cells infiltrating the dermis of IL-21-treated mice express IFN-γ and, to a lesser extent, IL-17A. Studies in cytokine knockout mice show that IFN-γ, but not IL-17A, is necessary for IL-21-induced epidermal hyperplasia. Finally, we demonstrate that IFN-γ-producing CD4(+) cells infiltrating the human psoriatic plaque express IL-21R, and abrogation of IL-21 signals reduces IFN-γ expression in cultures of psoriatic CD4(+) cells. Data indicate that IL-21 induces an IFN-γ-dependent pathogenic response in vivo, thus contributing to elucidate a mechanism by which IL-21 sustains skin-damaging inflammation.     

CD8+ T cells induce thyroid epithelial cell hyperplasia and fibrosis

CD8(+) T cells can be important effector cells in autoimmune inflammation, generally because they can damage target cells by cytotoxicity. This study shows that activated CD8(+) T cells induce thyroid epithelial cell hyperplasia and proliferation and fibrosis in IFN-γ(-/-) NOD.H-2h4 SCID mice in the absence of CD4(+) T cells. Because CD8(+) T cells induce proliferation rather than cytotoxicity of target cells, these results describe a novel function for CD8(+) T cells in autoimmune disease. In contrast to the ability of purified CD8(+) T cells to induce thyrocyte proliferation, CD4(+) T cells or CD8 T cell-depleted splenocytes induced only mild thyroid lesions in SCID recipients. T cells in both spleens and thyroids highly produce TNF-α. TNF-α promotes proliferation of thyrocytes in vitro, and anti-TNF-α inhibits development of thyroid epithelial cell hyperplasia and proliferation in SCID recipients of IFN-γ(-/-) splenocytes. This suggests that targeting CD8(+) T cells and/or TNF-α may be effective for treating epithelial cell hyperplasia and fibrosis.     

siRNA沉默t-bet基因对CD4、CD8T淋巴细胞亚群IFN-γ产生的影响

利用化学合成针对人t-bet基因的小干扰RNA（siRNA）,转染体外培养的人外周血单个核细胞,利用流式细胞仪分选纯化CD4^＋、CD8^＋T淋巴细胞,半定量RT-PCR检测CD4^＋、CD8^＋T淋巴细胞中t-betmRNA的表达变化,流式细胞术检测转染前后IFN-γ产生的变化情况。探讨转录因子t-bet对人CD4^＋、CD8^＋T淋巴细胞亚群IFN-γ产生的调控作用。与对照组相比,转染后的人CD4^＋、CD8^＋T淋巴细胞t-betmRNA表达水平明显下降;转染siRNA后,CD4＋T淋巴细胞中IFN-γ＋细胞比例为（18.46±6.86）%,与对照组（50.20±5.91）%比较有显著性差异（P〈0.01）;CD8＋T淋巴细胞IFN-γ＋细胞比例为（74.18±9.33）%,和对照组（76.51±6.49）%比较差异不明显（P〉0.05）。体外转录合成的siRNA可有效降低人CD4^＋、CD8^＋T淋巴细胞t-bet的基因表达;转录因子t-bet对人不同淋巴细胞亚群IFN-γ的产生所起作用不同。     

Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states

T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p?=?0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.     

免疫反应及相关细胞因子在EV71 相关手足口病 合并肺水肿中的作用

目的:探讨细胞、体液免疫应答以及相关的细胞因子在EV71相关手足口病合并肺水肿中的作用。方法:将90例经鉴定为EV71感染患儿分为手足口病合并肺水肿组38例,手足口病无并发症组52例,并设查体健康对照组28例。ELISA法检测90例EV71感染引起的手足口病患儿血清IL-6、IL-10、TNF-α及IFN-γ含量;采用流式细胞仪对血液中CD3+T、CD4+及CD8+T细胞百分比进行检测;采用免疫比浊法对90例EV71感染引起的手足口病患儿血清免疫球蛋白(IgG、IgA及IgM)及补体C3、C4含量进行检测。结果:手足口病合并肺水肿组患儿血清IL-6、IL-10及IFN-γ含量明显升高,同时伴随CD4+及CD8+T细胞百分比下降;手足口病合并肺水肿组,无并发症组及健康对照组患儿血清IgM分别为1.85±0.73,1.46±0.790和0.88±0.39,三组之间差别具有统计学意义(F=14.967,P<0.05)。而IgG与IgA在三组之间无明显变化(X2=5.535,P>0.05;F=1.988,P>0.05);手足口病患儿血清C3及C4含量均明显低于健康对照组(F=46.079;62.794,P<0.05)。结论:由IL-10及IFN-γ的异常释放而引起的广泛的中枢及外周神经系统炎症反应和T淋巴细胞衰竭是引起EV71合并肺水肿病程进展的重要原因;在EV71感染后引发的手足口病进程中存在IgM的大量释放,且伴随补体C3、C4的消耗。     

The role of gamma interferon in DNA vaccine-induced tumor immunity targeting simian virus 40 large tumor antigen

The central role of CD4+ T lymphocytes in mediating DNA vaccine-induced tumor immunity against the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory. In the present study, we extend our previous findings by examining the roles of IFN-γ and Th1-associated effector cells within the context of DNA immunization in a murine model of pulmonary metastasis. Immunization of BALB/c mice with plasmid DNA encoding SV40 Tag (pCMV-Tag) generated IFN-γ-secreting T lymphocytes that produced this cytokine upon in vitro stimulation with mKSA tumor cells. The role of IFN-γ as a mediator of protection against mKSA tumor development was assessed via in vivo IFN-γ neutralization, and these experiments demonstrated a requirement for this cytokine in the induction immune phase. Neutralization of IFN-γ was associated with a reduction in Th1 cytokine-producing CD4+ and CD8+ splenocytes, as assessed by flow cytometry analysis, and provided further evidence for the role of CD4+ T lymphocytes as drivers of the cellular immune response. Depletion of NK cells and CD8+ T lymphocytes demonstrated the expendability of these cell types individually, but showed a requirement for a resident cytotoxic cell population within the immune effector phase. Our findings demonstrate the importance of IFN-γ in the induction of protective immunity stimulated by pCMV-Tag DNA-based vaccine and help to clarify the general mechanisms by which DNA vaccines trigger immunity to tumor cells.     

Robust antigen specific th17 T cell response to group A Streptococcus is dependent on IL-6 and intranasal route of infection

Group A streptococcus (GAS, Streptococcus pyogenes) is the cause of a variety of clinical conditions, ranging from pharyngitis to autoimmune disease. Peptide-major histocompatibility complex class II (pMHCII) tetramers have recently emerged as a highly sensitive means to quantify pMHCII-specific CD4+ helper T cells and evaluate their contribution to both protective immunity and autoimmune complications induced by specific bacterial pathogens. In lieu of identifying an immunodominant peptide expressed by GAS, a surrogate peptide (2W) was fused to the highly expressed M1 protein on the surface of GAS to allow in-depth analysis of the CD4+ helper T cell response in C57BL/6 mice that express the I-A(b) MHCII molecule. Following intranasal inoculation with GAS-2W, antigen-experienced 2W:I-A(b)-specific CD4+ T cells were identified in the nasal-associated lymphoid tissue (NALT) that produced IL-17A or IL-17A and IFN-γ if infection was recurrent. The dominant Th17 response was also dependent on the intranasal route of inoculation; intravenous or subcutaneous inoculations produced primarily IFN-γ+ 2W:I-A(b+) CD4+ T cells. The acquisition of IL-17A production by 2W:I-A(b)-specific T cells and the capacity of mice to survive infection depended on the innate cytokine IL-6. IL-6-deficient mice that survived infection became long-term carriers despite the presence of abundant IFN-γ-producing 2W:I-A(b)-specific CD4+ T cells. Our results suggest that an imbalance between IL-17- and IFN-γ-producing CD4+ T cells could contribute to GAS carriage in humans.     

Interferon-inducible immunity-related GTPase Irgm1 regulates IFNγ-dependent host defense,lymphocyte survival and autophagy

IFN-γ is a pleiotropic cytokine that plays a key role in host resistance, yet when not properly regulated can become detrimental to the host. The interferon-inducible Immunity Related GTPase family M member 1 (Irgm1), previously characterized as an effector molecule required for macrophage microbicidal activity, has been shown recently to control IFN-γ-dependent cell survival and host resistance. Irgm1 regulates the expansion/survival of mature effector CD4⁺ T lymphocytes by protecting them from IFN-γ-induced autophagic cell death. Importantly, mice deficient in both IFN-γ and Irgm1 were rescued from the lymphocyte depletion and increased mortality that typically occurs in Irgm1^–/– animals following pathogen exposure. We propose that Irgm1 plays a major role in maintaining T lymphocyte homeostasis during host IFN-γ responses by protecting these cells from autophagy-dependent cell death.     

Identification of a novel proinflammatory human skin-homing Vγ9Vδ2 T cell subset with a potential role in psoriasis

γδ T cells mediate rapid tissue responses in murine skin and participate in cutaneous immune regulation including protection against cancer. The role of human γδ cells in cutaneous homeostasis and pathology is characterized poorly. In this study, we show in vivo evidence that human blood contains a distinct subset of proinflammatory cutaneous lymphocyte Ag and CCR6-positive Vγ9Vδ2 T cells, which is rapidly recruited into perturbed human skin. Vγ9Vδ2 T cells produced an array of proinflammatory mediators including IL-17A and activated keratinocytes in a TNF-α- and IFN-γ-dependent manner. Examination of the common inflammatory skin disease psoriasis revealed a striking reduction of circulating Vγ9Vδ2 T cells in psoriasis patients compared with healthy controls and atopic dermatitis patients. Decreased numbers of circulating Vγ9Vδ2 T cells normalized after successful treatment with psoriasis-targeted therapy. Taken together with the increased presence of Vγ9Vδ2 T cells in psoriatic skin, these data indicate redistribution of Vγ9Vδ2 T cells from the blood to the skin compartment in psoriasis. In summary, we report a novel human proinflammatory γδ T cell involved in skin immune surveillance with immediate response characteristics and with potential clinical relevance in inflammatory skin disease.     

γδ T lymphocytes from cystic fibrosis patients and healthy donors are high TNF-α and IFN-γ-producers in response to Pseudomonas aeruginosa

Background

γδ T cells have an important immunoregulatory and effector function through cytokine release. They are involved in the responses to Gram-negative bacterium and in protection of lung epithelium integrity. On the other hand, they have been implicated in airway inflammation.

Methods

The aim of the present work was to study intracytoplasmic IL-2, IL-4, IFN-γ and TNF-α production by γδ and αβ T lymphocytes from cystic fibrosis patients and healthy donors in response to Pseudomonas aeruginosa (PA). Flow cytometric detection was performed after peripheral blood mononuclear cells (PBMC) culture with a cytosolic extract from PA and restimulation with phorbol ester plus ionomycine. Proliferative responses, activation markers and receptor usage of γδ T cells were also evaluated.

Results

The highest production of cytokine was of TNF-α and IFN-γ, γδ being better producers than αβ. No differences were found between patients and controls. The Vγ9δ2 subset of γδ T cells was preferentially expanded. CD25 and CD45RO expression by the αβ T subset and PBMC proliferative response to PA were defective in cystic fibrosis lymphocytes.

Conclusion

Our results support the hypothesis that γδ T lymphocytes play an important role in the immune response to PA and in the chronic inflammatory lung reaction in cystic fibrosis patients. They do not confirm the involvement of a supressed Th1 cytokine response in the pathogenesis of this disease.     

异型流感病毒感染相关T淋巴细胞增殖活性及IFN-γ阳性Ｔ淋巴细胞水平研究

为探讨机体异型流感病毒间交叉保护作用机制,将实验动物随机分成实验组和对照组,测定异型流感病毒感染后病毒载量,T淋巴细胞增殖活性和IFN-γ阳性CD3＋CD8＋及CD3＋CD4＋淋巴细胞水平的变化。结果显示,异型流感病毒感染后产生的交叉免疫应答反应可能与T淋巴细胞增殖有关;与CTL及Th1类淋巴细胞水平相关,并有时间限制性;IL-2可以加强异型流感病毒感染后IFN-γ阳性CD3＋CD8＋淋巴细胞水平。本研究为制备能够抵御变异流感病毒感染的疫苗提供了理论基础。     

Apilimod inhibits the production of IL-12 and IL-23 and reduces dendritic cell infiltration in psoriasis

Psoriasis is characterized by hyperplasia of the epidermis and infiltration of leukocytes into both the dermis and epidermis. IL-23, a key cytokine that induces T(H)17 cells, has been found to play a critical role in the pathogenesis of psoriasis. Apilimod is a small-molecule compound that selectively suppresses synthesis of IL-12 and IL-23. An open-label clinical study of oral administration of apilimod was conducted in patients with psoriasis. Substantial improvements in histology and clinical measurements were observed in patients receiving 70 mg QD. The expression of IL-23p19 and IL-12/IL-23p40 in skin lesions was significantly reduced in this dose group, with a simultaneous increase in IL-10 observed. A decrease in the levels of T(H)1 and T(H)17 cytokines/chemokines in skin lesions followed these p19 and p40 changes. In parallel, a reduction in skin-infiltrating CD11c(+) dendritic cells and CD3(+) T cells was seen, with a greater decrease in the CD11c(+) population. This was accompanied by increases in T and B cells, and decreases in neutrophils and eosinophils in the periphery. This study demonstrates the immunomodulatory activity of apilimod and provides clinical evidence supporting the inhibition of IL-12/IL-23 synthesis for the treatment of T(H)1- and T(H)17-mediated inflammatory diseases.     

Murine Peyer's patch dendritic cells prime naïve CD4+ T cells to produce interferon-γ

We investigated the role of Peyer's patch (PP) dendritic cells (DCs) in the production of interferon (IFN)-γ from naïve CD4⁺ T cells of T cell receptor transgenic mice. PP DCs were found to prime naïve CD4⁺ T cells for the production of higher levels of IFN-γ, when compared to spleen (SP) DCs. However, a similar level of interleukin-12 (IL-12) production was observed for PP and SP DCs stimulated via the CD40 molecule. In addition, PP DCs expressed slightly higher levels of B7.2 (CD86) compared to SP DCs. This data demonstrates that PP DCs have a distinct function in the induction of IFN-γs and suggests that PP DCs may enhance IFN-γ production via another cytokine or costimulatory molecule, in addition to IL-12.     

Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load

High Ag load in chronic viral infections has been associated with impairment of Ag-specific T cell responses; however, the relationship between Ag load in chronic Mycobacterium tuberculosis infection and functional capacity of M. tuberculosis-specific T cells in humans is not clear. We compared M. tuberculosis-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads-that is, persons with latent M. tuberculosis infection (LTBI), with smear-negative pulmonary tuberculosis (TB), and smear-positive TB. Patients with smear-positive TB had decreased polyfunctional IFN-γ(+)IL-2(+)TNF-α(+) and IL-2-producing specific CD4 T cells and increased TNF-α single-positive cells, when compared with smear-negative TB and LTBI. TB patients also had increased frequencies of M. tuberculosis-specific CD8 T cells, compared with LTBI. M. tuberculosis-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear-positive TB, and correlated positively with ex vivo IFN-γ(+)IL-2(+)TNF-α(+) CD4 T cells, and inversely with TNF-α single-positive CD4 T cells. During 6 mo of anti-TB treatment, specific IFN-γ(+)IL-2(+)TNF-α(+) CD4 and CD8 T cells increased, whereas TNF-α and IFN-γ single-positive T cells decreased. These results suggest progressive impairment of M. tuberculosis-specific T cell responses with increasing mycobacterial load and recovery of responses during therapy. Furthermore, these data provide a link between specific cytokine-producing subsets and functional capacity of M. tuberculosis-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. These data have potentially significant applications for the diagnosis of TB and for the identification of T cell correlates of TB disease progression.     

TNF inhibition rapidly down-regulates multiple proinflammatory pathways in psoriasis plaques

The mechanisms of action of marketed TNF-blocking drugs in lesional tissues are still incompletely understood. Because psoriasis plaques are accessible to repeat biopsy, the effect of TNF/lymphotoxin blockade with etanercept (soluble TNFR) was studied in ten psoriasis patients treated for 6 months. Histological response, inflammatory gene expression, and cellular infiltration in psoriasis plaques were evaluated. There was a rapid and complete reduction of IL-1 and IL-8 (immediate/early genes), followed by progressive reductions in many other inflammation-related genes, and finally somewhat slower reductions in infiltrating myeloid cells (CD11c+ cells) and T lymphocytes. The observed decreases in IL-8, IFN-gamma-inducible protein-10 (CXCL10), and MIP-3alpha (CCL20) mRNA expression may account for decreased infiltration of neutrophils, T cells, and dendritic cells (DCs), respectively. DCs may be less activated with therapy, as suggested by decreased IL-23 mRNA and inducible NO synthase mRNA and protein. Decreases in T cell-inflammatory gene expression (IFN-gamma, STAT-1, granzyme B) and T cell numbers may be due to a reduction in DC-mediated T cell activation. Thus, etanercept-induced TNF/lymphotoxin blockade may break the potentially self-sustaining cycle of DC activation and maturation, subsequent T cell activation, and cytokine, growth factor, and chemokine production by multiple cell types including lymphocytes, neutrophils, DCs, and keratinocytes. This results in reversal of the epidermal hyperplasia and cutaneous inflammation characteristic of psoriatic plaques.