Detection of melanoma-reactive CD4+ HLA-class I-restricted cytotoxic T cell clones with long-term assay and pretreatment of targets with interferon-γ

Twenty-five CD4⁺ cytotoxic T lymphocyte (CTL) clones were obtained from the peripheral blood or tumor tissues of melanoma patients undergoing active specific immunotherapy. Melanoma-reactive T cells were cloned by limiting dilution using either autologous or allogeneic melanoma cells to stimulate their proliferation. Sixteen of the clones reacted against autologous melanoma cells but not against the autologous lymphoblastoid cell line, which we defined as melanoma-specific. Optimal demonstration of the lytic activity of CD4⁺ CTL required a 16-h incubation period and an effectortarget cell ratio of 401. In addition, a 24-h pre-incubation of the target melanoma cells with 100 U interferon (IFN) consistently augmented lysis by these CD4⁺ CTL, increasing it from a mean level of 20% to one of 52%. Lysis by 8 of the 11 melanoma-reactive CD4⁺ T cell clones was exclusively HLA-class-I-restricted, as judged by blocking with monoclonal antibodies (mAb). Five of these HLA class-I-restricted clones were reactive only with the autologous melanoma cells, while the other 3 clones were also reactive with allogeneic melanoma cells. In all cases, the T cells and melanoma targets shared at least one HLA class I allele, usually HLA-A2, HLA-C3 or HLA-B62. Interestingly, lysis by 2 of the 11 clones was inhibited by both anti-HLA-class-I or -HLA-class-II mAb, while lysis by 1 other clone was inhibited by neither. HLA class I molecules and several accessory molecules were maximally expressed by the melanoma target cells, both in terms of distribution and copy number before IFN treatment. Thus, IFN may have acted by increasing the expression of melanoma-associated epitopes as presented by HLA class I (or HLA class II) molecules. A proportion of human CD4⁺CTL appeared to recognize melanoma-associated epitopes presented by the HLA class I molecule, although their lytic potency may be less than that of their CD8⁺ counterparts.This work was supported by USPHS grant R01-CA 36233, and a grant from the Concern Foundation for Cancer Research.     

Interleukin-4 plus tumor necrosis factor α augments the antigenicity of melanoma cells

Immune cytokines are important regulators of the immune response to neoplastic cells. We previously reported that interleukin 4 (IL-4) and either tumor necrosis factor α (TNF) or interferon γ (IFN) synergistically inhibit melanoma cell growth and induce cell differentiation. In the present study we used various combinations of IL-4, IFN and TNF to enhance the antigenicity of melanoma cells. IL-4 plus TNF significantly increased the ability of melanoma cells to stimulate cytotoxic T cells (CTL) and act as targets of these CTL; IL-4 plus IFN was somewhat less effective, while TNF plus IFN was not as effective. IL-4 plus TNF also increased the expression of HLA class I and HLA-DR antigens on melanoma cells. The CTL lines examined in this study were CD3⁺CD4⁺ and oligoclonal. These preclinical results suggest that the immune response to melanoma whole-cell vaccines might be enhanced by pretreating vaccine cells with IL-4 plus TNF.     

Th17 and Th17-stimulated CD8<Superscript>+</Superscript> T cells play a distinct role in Th17-induced preventive and therapeutic antitumor immunity

CD4⁺ Th17 cells induce antitumor immunity leading to the eradication of established tumors. However, the mechanism of antitumour immunity and CTL activation by Th17 cells and the distinct role of Th17 and Th17-activated CTLs in antitumor immunity are still elusive. In this study, we generated ovalbumin (OVA)-specific Th17 cells by cultivating OVA-pulsed dendritic cells with CD4⁺ T cells derived from transgenic OTII mice in the presence of IL-6, IL-23, TGF-β, and anti-IFN-γ antibody. We demonstrated that Th17 cells acquired major histocompatibility complex/peptide (pMHC)-I and expressed RORγt, IL-17, and IL-2. Th17 cells did not have any direct in vitro tumor cell–killing activity. However, Th17 cells were able to stimulate CD8⁺ CTL responses via IL-2 and pMHC I, but not IL-17 signaling, which play a major role in Th17-induced preventive immunity against OVA-expressing B16 melanoma. Th17 cells stimulated the expression of CCL2 and CCL20 in lung tumor microenvironments promoting the recruitment of various inflammatory leukocytes (DCs, CD4⁺, and CD8⁺ T cells) stimulating more pronounced therapeutic immunity for early-stage (5-day lung metastases or 3 mm, s.c.) tumor than for well-established (6 mm, s.c.) tumor. The therapeutic effect of Th17 cells is associated with IL-17 and is mediated by Th17-stimulated CD8⁺ CTLs and other inflammatory leukocytes recruited into B16 melanoma via Th17-stimulated CCL20 chemoattraction. Taken together, our data elucidate a distinct role of Th17 and Th17-stimulated CD8⁺ CTLs in the induction of preventive and therapeutic antitumor immunity, which may greatly impact the development of Th17-based cancer immunotherapy.     

T cell precursor frequency differentially affects CTL responses under different immune conditions

Generation of effective CTL responses is the goal of many vaccination protocols. However, to what extant T cell precursor frequencies will generate a CD8⁺ CTL response has not been elucidated properly. In this study, we employed a model system, in which naive CD4⁺ and CD8⁺ T cells derived from ovalbumin (OVA)-specific TCR transgenic OT II and OT I mice were used for adoptive transfer into wild-type, Ia^b−/− gene knockout and transgenic RIP-mOVA mice, and assessed OVA-pulsed DC (DC_OVA)-stimulated CD8⁺ CTL responses in these mice. We demonstrated that (i) a critical threshold exists above which T cells precursor frequency cannot enhance the CTL responses in wild-type C57BL/6 mice, (ii) increasing CD8⁺ T cell precursors is required to generate CTL responses but with functional memory defect in absence of CD4⁺ T cell help, and (iii) increasing CD4⁺ and CD8⁺ T cell precursors overcomes immune suppression to DC_OVA-stimulated CD8⁺ CTL responses in transgenic RIP-mOVA mice with OVA-specific self immune tolerance. Taken together, these findings may have important implications for optimizing immunotherapy against cancer.     

Elimination of CD4+ T cells in mice bearing an advanced sarcoma augments the antitumor action of interleukin-2

Experiments were undertaken to determine whether the depletion of CD4⁺ T cells from mice bearing an advanced immunogenic SA-1 sarcoma would result in an enhanced ability of interleukin-2 (IL-2) to cause tumor regression. The results show that whereas IL-2 therapy given as a 5-day course starting on day 10 of tumor growth caused complete regression of the tumor, it failed to cause regression if started on day 15 of tumor growth. However, in mice depleted of CD4⁺ T cells by treatment with anti-CD4 monoclonal antibody (mAb), IL-2 therapy started on day 15 resulted in appreciable tumor regression in most animals, and the therapeutic effect was greatly increased if two consccutive courses of anti-CD4 mAb and IL-2 therapy were given. On the other hand, treatment with anti-CD4 mAb alone had no effect on tumor growth. It was shown that the therapeutic action of combination therapy with anti-CD4 mAb and IL-2 was mediated by CD8⁺ T cells, because the therapeutic effect was completely ablated in mice depleted of CD8⁺ T cells with anti-CD8 mAb. Taken together these results suggest that, at a late stage of growth of an immunogenic tumor, depletion of CD4⁺ T cells can enhance the antitumor effect of IL-2 therapy by releasing CD8⁺-T-cell-mediated immunity from T-cell-mediated suppression.     

Cytotoxic T cell lines recognize autologous and allogeneic melanomas with shared or cross-reactive HLA-A

Summary Cytotoxic T lymphocytes (CTL), CD3⁺, / T-cell-receptor-positive, are important effector cells with specific immunity in melanoma patients. The establishment and expansion in vitro of CTL of a specific phenotype to tumor cells strongly depends on the method of activation and sensitization with tumor cells. We generated CD3⁺ CTL lines to melanoma by co-culturing peripheral blood lymphocytes with autologous irradiated melanoma cells and repetitive stimulation with high-dose interleukin-4 in a cocktail culture medium. CTL lines were investigated for their specificity to kill autologous and allogeneic melanoma. Histocompatibility locus antigen (HLA) class I (A, B) molecules are important restrictive recognition antigens for CTL. Although these antigens are highly polymorphic, they can share a similar immunogenic molecular epitope(s) and can be immunologically cross-reactive. The CTL lines generated were found to kill not only autologous melanoma, but also allogeneic melanomas having class I HLA-A antigens shared or cross-reactive with autologous HLA-A. These CTL lines were poor killers of melanomas bearing non-shared or non-cross-reactive HLA-A. Cold-target inhibition assays demonstrated this CTL cross-reactivity to allogeneic melanoma specificity. Epstein-Barr-virus-transformed autologous and allogeneic B lymphoblastoid cell lines failed to block autologous melanoma killing, indicating that CTL were not recognizing major histocompatibility complex antigens, serum proteins or culture medium products as the primary target antigen. HLA-A2 was the major shared HLA-A antigen recognized by CTL lines on the melanoma lines studied. CTL lines also recognized shared HLA-A11 and A24 on allogeneic melanoma. There were no CTL lines showing restriction to HLA-B. These results suggest that common tumor-associated antigens are present on melanomas and are recognized in association with distinct HLA-A epitopes by CTL.This study was supported by grant CA12 582 awarded by the National Cancer Institute, USA     

Interleukin-4 is effective in restoring cytotoxic T cell activity that declines during in vivo progression of a murine B lymphoma

 We previously reported [Chakrabarti et al. (1992) Cell Immunol 142:54; 144:455] that, in a murine B lymphoma model 2C3, idiotype (Id)-specific CD8⁺ cytotoxic T lymphocytes (CTL) are generated in mice following hyperimmunization with irradiated tumor cells, and that they are effective in tumor rejection. The present study reveals that 2C3-specific CTL are also induced in spleens during tumor progression, but are not sustained. At the early stage of tumor growth, the splenic T cells following a 5-day incubation in vitro with killed 2C3 tumor targets, produce high levels of cytokines, namely interleukin-4 (IL-4), IL-10 and interferon γ (IFNγ). Their cytotoxic T lymphocyte (CTL) activity and cytokine levels, except IL-2, sharply decline at the late stage when the mice are increasingly moribund. Although the decline in cytokine level is also evident with CD4⁺ T cells, a precipitous and concurrent decrease occurs primarily in the IL-4 level with both CD4⁺ and CD8⁺ T cells of late-tumor-bearing animals (TBA). Study with the unseparated splenocytes also reveals that sevenfold less IL-4 is produced at the late stage. Furthermore, the cytotoxicity of CTL from late TBA can be effectively restored by addition of supernatants from the splenocyte culture of early TBA, or by IL-4, but not by IFNγ and IL-10. In addition, only IL-4-activated CD8⁺ T cells from the late TBA are found, by Winn assay, to be protective in vivo. Thus it appears that IL-4, required to sustain antitumor CTL activity, is consumed by T and possibly other cells at the late stage of tumor growth, thereby compromising host immunity against the tumor. We contend that induction or maintenance of protective immunity depends not only on the tumor antigen but also on the specific cytokine milieu in a tumor-bearing host. Received: 8 February 1997 / Accepted: 24 April 1997     

血清IL-6、IL-8、IgM抗体及T细胞亚群水平对新生儿先天性梅毒的诊断价值

目的:探讨血清白介素-6(IL-6)、白介素-8(IL-8)、IgM抗体及T细胞亚群对先天性梅毒新生儿的诊断价值。方法:选择2015年5月至2017年5月在我院进行临床治疗的先天性梅毒新生儿81例为观察组,另选同期来我院进行健康体检81例新生儿为对照组。比较两组患者血清IL-6、IL-8、T细胞亚群中CD~(3+)、CD~(4+)、CD~(8+)、CD~(4+)/CD~(8+)细胞及IgM抗体的阳性率。结果:治疗后,观察组血清IL-6、IL-8水平均明显高于对照组(P0.05),T细胞亚群中CD~(3+)、CD~(4+)、CD~(4+)/CD~(8+)明显低于对照组,而CD~(8+)T细胞比例高于对照组(P0.05)。19S-IgM-TP ELISA法检测出IgM的阳性率92.59%,明显高于TRUST法(74.07%)及TP-ELSA法(70.37%)(P0.05)。ROC曲线中,血清IL-8特异度为88.34%明显高于血清IL-6特异度81.48%、IgM抗体特异度60.13%、T细胞亚群特异度65.34%;IgM抗体的曲线面积88.91 cm~2明显大于IL-6的曲线面积45.09 cm~2、IL-8的曲线面积76.19 cm~2、T细胞亚群的曲线面积77.35 cm~2;T细胞亚群准备性67.89%明显高于IL-6准确性60.39%、IL-8准确性51.09%、IgM抗体准确性50.12;IgM抗体的灵敏度60.13%高于IL-6灵敏度59.19%、IL-8灵敏度42.35%、T细胞亚群灵敏度59.37%。具有比较意义(P0.05)。结论:血清IL-6、IL-8水平、T细胞亚群中CD~(3+)、CD~(4+)、CD~(8+)、CD~(4+)/CD~(8+)及IgM抗体阳性率是诊断先天性梅毒新生儿的重要指标。     

Substantial increase in the frequency of circulating CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade 1

Background

The NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8⁺ cytotoxic T cells and is classically considered absent in CD4⁺ T cells. However, recent studies have identified a distinctive population of CD4⁺ T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4⁺ T cell-immunocompromised individuals suggests that CD4⁺ T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4⁺ T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4⁺NKG2D⁺ T cell expansion were also measured.

Results

Close to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4⁺ T cells and CD8⁺ T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4⁺ T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4⁺NKG2D⁺ T cells was seen in patients with CIN 1, despite the overall levels of CD4⁺ T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4⁺NKG2D⁺ T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group.

Conclusions

Taken together, our results suggest that the significant increase within the CD4⁺NKG2D⁺ T cell population in CIN 1 patients might be the result of a chronic exposure to viral and/or pro-inflammatory factors, and concomitantly might also influence the clearance of CIN 1-type lesion.     

Natural CD825 regulatory T cell-secreted exosomes capable of suppressing cytotoxic T lymphocyte-mediated immunity against B16 melanoma

Natural CD4⁺25⁺ and CD8⁺25⁺ regulatory T (Tr) cells have been shown to inhibit autoimmune diseases. Immune cells secrete exosomes (EXOs), which are crucial for immune regulation. However, immunomodulatory effect of natural Tr cell-secreted EXOs is unknown. In this study, we purified natural CD8⁺25⁺ Tr cells from C57BL/6 mouse naive CD8⁺ T cells, and in vitro amplified them with CD3/CD28 beads. EXOs (EXO_Tr) were purified from Tr cell’s culture supernatants by differential ultracentrifugation and analyzed by electron microscopy, Western blot and flow cytometry. Our data showed that EXO_Tr had a “saucer” or round shape with 50–100 nm in diameter, contained EXO-associated markers LAMP-1 and CD9, and expressed natural Tr cell markers CD25 and GITR. To assess immunomodulatory effect, we i.v. immunized C57BL/6 mice with ovalbumin (OVA)-pulsed DCs (DC_OVA) plus Tr cells or EXO_Tr, and then assessed OVA-specific CD8⁺ T cell responses using PE-H-2K^b/OVA tetramer and FITC-anti-CD8 antibody staining by flow cytometry and antitumor immunity in immunized mice with challenge of OVA-expressing BL6–10_OVA melanoma cells. We demonstrated that DC_OVA-stimulated CD8⁺ T cell responses and protective antitumor immunity significantly dropped from 2.52% to 1.08% and 1.81% (p < 0.05), and from 8/8 to 2/8 and 5/8 mice DC_OVA (p < 0.05) in immunized mice with co-injection of Tr cells and EXO_Tr, respectively. Our results indicate that natural CD8⁺25⁺ Tr cell-released EXOs, alike CD8⁺25⁺ Tr cells, can inhibit CD8⁺ T cell responses and antitumor immunity. Therefore, EXOs derived from natural CD4⁺25⁺ and CD8⁺25⁺ Tr cells may become an alternative for immunotherapy of autoimmune diseases.     

Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy

To study in vivo activated cytolytic T cells, CD8⁺ T cells clones were isolated from a melanoma patient (HLA A2, A11) treated with active specific immunotherapy for 5 years. CD8⁺ T lymphocytes, purified by fluorescence-activated cell sorting, were cloned directly from the peripheral blood without antigen-presenting cells in the presence of irradiated autologous melanoma cells and recombinant interleukin-2 (IL-2) and IL-4. These conditions were inhibitory to de novo in vitro immunization. Of the 28 cytolytic CD8⁺ T cell clones, 21 lysed the autologous melanoma cell line (M7) but not the autologous lymphoblastoid cell line (LCL-7) nor the two melanoma cell lines, M1 (HLA A28) and M2 (HLA A28, A31), used to immunize the patient. The remaining 7 clones were also melanoma-specific, although their reactivities were broader, lysing several melanoma cell lines but not HLA-matched lymphoblastoid cells. Eight clones from the first group, ostensibly self-MHC-restricted, were expanded for further analysis. All expressed cluster determinants characteristic of mature, activated T cells, but not those of thymocytes, naive T cells, B cells or natural killer (NK) cells. They also expressed CD13, a myeloid marker. Of the 8 clones, 3 expressed both CD4 and CD8, but dual expression was not correlated with specificity of lysis. Two CD8⁺ and 2 CD4⁺ CD8⁺ clones were specific for the autologous melanoma cells, the other 4 were also reactive against other HLA-A2-positive melanomas. Cytotoxicity for both singly and doubly positive clones was restricted by HLA class I but not class II antigens. Analysis of the RNA expression of the T cell receptor (TCR) V and V gene segments revealed heterogeneous usage by the A2-restricted clones and, perhaps, also by the broadly melanoma-specific clones. Apparent TCR-restricted usage was noted for the self-MHC-restricted clones; 2 of the 4 expressed the V17/V7 dimer. Since the T cell clones were derived from separate precursors of circulating cytotoxic T lymphocytes (CTL), the V17/V7 TCR was well represented in the peripheral blood lymphocytes of this patient. In summary, we show that melanoma cells presented their own antigens to stimulate the proliferation of melanoma-reactive CD8⁺ CTL. CTL with a range of melanoma specificities and different TCR dimers were encountered in this patient, perhaps as a result of hyperimmunization. Restricted TCR gene usage was noted only for classical self-MHC-restricted CD8⁺ T cell clones, although lysis of the autologous melanoma cells was effected by a variety of TCR structures. Molecular definition of the TCR repertoire of well-characterized T cell clones in this and other patients should provide new insight into the human antitumor immune response.Supported by National Institutes of Health research grants CA 36233 and EY 9031, the Lucy Adams Memorial Fund and a grant from the Concern Foundation     

Induction of interleukin-2 receptor by tumor necrosis factor α on cultured ovarian tumor-associated lymphocytes

Summary We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor (TNF) in the induction, expansion, long-term proliferation and lytic function of CD8⁺ TAL. TNF up-regulated the IL-2 receptor (IL-2R) chain (Tac antigen) on the surface of CD3⁺ CD8⁺ CD4^– TAL, enhanced the proliferation of autologous tumor-specific CTL, and potentiated their lytic function in long-term cultures. Furthermore, in the induction and expansion phase of CD8⁺ TAL, the presence of TNF was associated with a selective increase in CD8⁺ IL-2R⁺ (Tac⁺) cells, and subsequent decrease in CD4⁺ IL-2R⁺ (Tac⁺) cells. These results suggest that the observed facilitation of the outgrowth of CD8⁺ cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNF in the analysis of signalling in autologous tumor-reactive CTL.     

Accumulation of cytolytic CD8 T cells in B16-melanoma and proliferation of mature T cells in TIS21-knockout mice after T cell receptor stimulation

In vivo and in vitro effects of TIS21 gene on the mature T cell activation and antitumor activities were explored by employing MO5 melanoma orthograft and splenocytes isolated from the TIS21-knockout (KO) ² mice. Proliferation and survival of mature T cells were significantly increased in the KO than the wild type (WT) ³ cells, indicating that TIS21 inhibits the rate of mature T cell proliferation and its survival. In MO5 melanoma orthograft model, the KO mice recruited much more CD8⁺ T cells into the tumors at around day 14 after tumor cell injection along with reduced tumor volumes compared with the WT. The increased frequency of granzyme B⁺ CD8⁺ T cells in splenocytes of the KO mice compared with the WT may account for antitumor-immunity of TIS21 gene in the melanoma orthograft. In contrast, reduced frequencies of CD107a⁺ CD8⁺ T cells in the splenocytes of KO mice may affect the loss of CD8⁺ T cell infiltration in the orthograft at around day 19. These results indicate that TIS21 exhibits antiproliferative and proapoptotic effects in mature T cells, and differentially affects the frequencies of granzyme B⁺ CD8⁺ T-cells and CD107a⁺ CD8⁺ T-cells, thus transiently regulating in vivo anti-tumor immunity.     

Interleukin-4 impairs granzyme-mediated cytotoxicity of Simian virus 40 large tumor antigen-specific CTL in BALB/c mice

In this report we analyzed the impact of interleukin-4 (IL-4) on tumor-associated simian virus 40 (SV40) large T-antigen (TAg)-specific CD8⁺ cytotoxic T cells during rejection of syngeneic SV40 transformed mKSA tumor cells in BALB/c mice. Strikingly, challenge of naïve mice with low doses of mKSA tumor cells revealed a CD8⁺ T cell-dependent prolonged survival time of naïve IL-4^?/? mice. In mice immunized with SV40 TAg we observed in IL-4^?/? mice, or in wild type mice treated with neutralizing anti-IL-4 monoclonal antibody, a strongly enhanced TAg-specific cytotoxicity of tumor associated CD8⁺ T cells. The enhanced cytotoxicity in IL-4^?/? mice was accompanied by a significant increase in the fraction of CD8⁺ tumor associated T-cells expressing the cytotoxic effector molecules granzyme A and B and in granzyme B-specific enzymatic activity. The data suggest that endogenous IL-4 can suppress the generation of CD8⁺ CTL expressing cytotoxic effector molecules especially when the antigen induces only a very weak CTL response.     

Cigarette smoke increases TLR4 and TLR9 expression and induces cytokine production from CD8+ T cells in chronic obstructive pulmonary disease

Background

Cigarette smoke is a major risk factor for chronic obstructive pulmonary disease (COPD), an inflammatory lung disorder. COPD is characterized by an increase in CD8⁺ T cells within the central and peripheral airways. We hypothesized that the CD8⁺ T cells in COPD patients have increased Toll-like receptor (TLR) expression compared to control subjects due to the exposure of cigarette smoke in the airways.

Methods

Endobronchial biopsies and peripheral blood were obtained from COPD patients and control subjects. TLR4 and TLR9 expression was assessed by immunostaining of lung tissue and flow cytometry of the peripheral blood. CD8⁺ T cells isolated from peripheral blood were treated with or without cigarette smoke condensate (CSC) as well as TLR4 and TLR9 inhibitors. PCR and western blotting were used to determine TLR4 and TLR9 expression, while cytokine secretion from these cells was detected using electrochemiluminescence technology.

Results

No difference was observed in the overall expression of TLR4 and TLR9 in the lung tissue and peripheral blood of COPD patients compared to control subjects. However, COPD patients had increased TLR4 and TLR9 expression on lung CD8⁺ T cells. Exposure of CD8⁺ T cells to CSC resulted in an increase of TLR4 and TLR9 protein expression. CSC exposure also caused the activation of CD8⁺ T cells, resulting in the production of IL-1β, IL-6, IL-10, IL-12p70, TNFα and IFNγ. Furthermore, inhibition of TLR4 or TLR9 significantly attenuated the production of TNFα and IL-10.

Conclusions

Our results demonstrate increased expression of TLR4 and TLR9 on lung CD8⁺ T cells in COPD. CD8⁺ T cells exposed to CSC increased TLR4 and TLR9 levels and increased cytokine production. These results provide a new perspective on the role of CD8⁺ T cells in COPD.     

Enhanced and prolonged efficacy of superantigen-induced cytotoxic T lymphocyte activity by interleukin-2 in vivo

The bacterial superantigen, staphylococcal enterotoxin A (SEA) activates T cells with high frequency and directs them to lyse MHC-class-II-expressing cells in superantigen-dependent cell-mediated cytotoxicity (SDCC). Treatment of mice with SEA induced strong CD8⁺ T-cell(CTL)-mediated SDCC, as well as abundant cytokine production from CD4⁺ and CD8⁺ T cells. However, both cytotoxicity and cytokine release were transient. In contrast, combined treatment with SEA and recombinant interleukin-2 (rIL-2) increased peak levels and maintained CTL activity. These effects were concomitant with an increased number of SEA-reactive V11⁺ T cells. Both the CD4⁺ and CD8⁺ populations contained higher frequencies of cells expressing IL-2 receptor (IL-2R) , which suggests that continuous IL-2R signaling preserves its high expression and subsequently prevents loss of growth factor signal necessary for expansion of T cells. Although IL-2R expression was increased among both CD4⁺ and CD8⁺ cells, only the cytotoxic function of CTL, but not cytokine production from either CD4 or CD8, was augmented. These findings demonstrate that treatment with rIL-2 potentiates superantigen-induced cytotoxicity and maintains high CTL activity. rIL-2 might therefore be useful in improving superantigenbased tumor therapy.     

Expression of β-1,4-galactosyltransferase-I affects cellular adhesion in human peripheral blood CD4 T cells

β-1,4-galactosyltransferase-I (β-1,4-GalT-I) has two isoforms that differ only in the length of their cytoplasmic domains. In this study, we found that both the long and short isoforms of β-1,4-GalT-I were expressed in human CD4⁺ T lymphocytes, and localized in the cytoplasm and on the plasma membrane. The expression level of β-1,4-GalT-I was increased in CD4⁺ T cells after stimulation with interleukin (IL)-2, and was further increased after stimulation with IL-2 + IL-12, but decreased after stimulation with IL-2 + IL-4 when compared to stimulation with IL-2 alone. We also demonstrated that the cellular adhesion of CD4⁺ T cells was significantly increased upon cytokine stimulation, and was inhibited by α-lactalbumin, indicating that the increase in adhesion was positively correlated with the expression and activity of long β-1,4-GalT-I. Collectively, the data suggest that β-1,4-GalT-I plays a role in the cellular adhesion of CD4⁺ T cells.     

Priming of naive CD8+ T cells in the presence of IL-12 selectively enhances the survival of CD8<Superscript>+</Superscript>CD62L<Superscript>hi</Superscript> cells and results in superior anti-tumor activity in a tolerogenic murine model

During the antigen-dependant activation process several subsets CD8⁺ T cells appear with different phenotypic and functional characteristics. Recent studies indicate that the state of T cell differentiation radically affects their ability to effectively respond to tumor challenge, with early effector CD8⁺ T (CD62L^high) cells having better anti-tumor activity. Thus strategies aimed at optimizing the generation of such subpopulations could significantly enhance the effectiveness of adoptive cell therapy (ACT) for cancer. In this study, we show that priming of naïve CD8⁺ T cells in the presence of IL-12 selectively rescued early CD8⁺ CD62L^hi from activation induced cell death and resulted in the increased accumulation of this subset of CD8⁺ T cells. Furthermore, we demonstrated that IL-12 directly modulated the expression of CD62L on activated CD8⁺ T cells. When used for ACT, naïve CD8⁺ T cells primed in vitro in the presence of IL-12 showed superior anti-tumor activity toward B16 melanoma. Importantly, using the Pmel-1 model, priming pmel-1 cells in vitro with IL-12 reduced the state of functional tolerance associated with the non-mutated “self” tumor antigen gp100, as demonstrated by significant tumor responses in the absence of vaccination. Together, our results suggest that in vitro conditioning of naïve CD8⁺ T cells with IL-12 prior to ACT could significantly enhance their anti-tumor activity.     

CD8CD25 T cells reduce atherosclerosis in apoE(−/−) mice

Background

It is increasingly evident that CD8⁺ T cells are involved in atherosclerosis but the specific subtypes have yet to be defined. CD8⁺CD25⁺ T cells exert suppressive effects on immune signaling and modulate experimental autoimmune disorders but their role in atherosclerosis remains to be determined. The phenotype and functional role of CD8⁺CD25⁺ T cells in experimental atherosclerosis were investigated in this study.

Methods and results

CD8⁺CD25⁺ T cells were observed in atherosclerotic plaques of apoE(−/−) mice fed hypercholesterolemic diet. Characterization by flow cytometric analysis and functional evaluation using a CFSE-based proliferation assays revealed a suppressive phenotype and function of splenic CD8⁺CD25⁺ T cells from apoE(−/−) mice. Depletion of CD8⁺CD25⁺ from total CD8⁺ T cells rendered higher cytolytic activity of the remaining CD8⁺CD25⁻ T cells. Adoptive transfer of CD8⁺CD25⁺ T cells into apoE(−/−) mice suppressed the proliferation of splenic CD4⁺ T cells and significantly reduced atherosclerosis in recipient mice.

Conclusions

Our study has identified an athero-protective role for CD8⁺CD25⁺ T cells in experimental atherosclerosis.