Relationship between CD4(+)/CD8(+) T cell ratio and T cell activation in multiple myeloma: reference to IL-16

We found that the ratio of CD4(+) to CD8(+) T cells (CD4/CD8 ratio) was decreased in patients with multiple myeloma (MM) and that this decrease was significantly related to an increase of human leukocyte antigen (HLA)-DR expression by CD8(+) (but not CD4(+)) T cells (P<0.005). In addition, the serum level of interleukin (IL)-16 was significantly higher in stage III MM patients than in healthy controls (P<0.001). The decrease of CD4(+) T cells in MM may be mediated by activation of CD8(+) T cells derived cytokine IL-16. In addition, these T cell phenotypic changes and the IL-16 level appear to be useful indicators of disease activity.     

Maximal T cell-mediated antitumor responses rely upon CCR5 expression in both CD4(+) and CD8(+) T cells

Immune responses against cancer rely upon leukocyte trafficking patterns that are coordinated by chemokines. CCR5, the receptor for chemotactic chemokines MIP1alpha, MIP1beta, and RANTES (CCL3, CCL4, CCL5), exerts major regulatory effects on CD4(+)- and CD8(+) T cell-mediated immunity. Although CCR5 and its ligands participate in the response to various pathogens, its relevance to tumoral immune control has been debated. Here, we report that CCR5 has a specific, ligand-dependent role in optimizing antitumor responses. In adoptive transfer studies, efficient tumor rejection required CCR5 expression by both CD4(+) and CD8(+) T cells. CCR5 activation in CD4(+) cells resulted in CD40L upregulation, leading to full maturation of antigen-presenting cells and enhanced CD8(+) T-cell crosspriming and tumor infiltration. CCR5 reduced chemical-induced fibrosarcoma incidence and growth, but did not affect the onset or progression of spontaneous breast cancers in tolerogenic Tg(MMTV-neu) mice. However, CCR5 was required for TLR9-mediated reactivation of antineu responses in these mice. Our results indicate that CCR5 boosts T-cell responses to tumors by modulating helper-dependent CD8(+) T-cell activation.     

Human ovarian carcinoma cells generate CD4(+)CD25(+) regulatory T cells from peripheral CD4(+)CD25(-) T cells through secreting TGF-beta

Increased CD4(+)CD25(+) regulatory T cells predicted poor prognosis in ovarian carcinoma patients. This study aimed to define whether soluble substances secreted by ovarian carcinoma could up-regulate the proportion of CD4(+)CD25(+) regulatory T cells. Similar to TGF-beta, the low MWF (<50kDa) of supernatant derived from SKOV3 could convert part of freshly isolated CD4(+)CD25(-) T cells into CD25(+) population with similar characters as natural CD4(+)CD25(+) regulatory T cells. To deplete TGF-beta in the low MWF by neutralizing anti-TGF-beta would eliminate this transformation phenomenon. These results indicate that TGF-beta secreted by ovarian carcinoma cells owns vital function in the process of converting peripheral CD4(+)CD25(-) T cells into CD4(+)CD25(+) regulatory T cells, which may provide one immunotherapeutic target for ovarian cancer.     

Attenuation of CD8(+) T-cell function by CD4(+)CD25(+) regulatory T cells in B-cell non-Hodgkin's lymphoma

The underlying mechanisms by which tumor cells are resistant to CTL-mediated apoptosis are not clear. Using a human model of B-cell non-Hodgkin's lymphoma (B-cell NHL), we show that intratumoral T(reg) cells inhibit the proliferation and granule production of activated autologous infiltrating CD8(+) T cells. Our results also show that degranulation and subsequent cytotoxic activity of infiltrating CD8(+) T cells exposed to lymphoma B cells is completely attenuated by the presence of intratumoral T(reg) cells. Furthermore, we show that increased numbers of intratumoral T(reg) cells correlates with the number of CD8(+) T cells in biopsy specimens from patients with B-cell NHL, supporting the in vitro findings that intratumoral T(reg) cells inhibit proliferation of infiltrating CD8(+) T cells. Taken together, these data indicate that human lymphoma B cells are sensitive to autologous CTL-mediated cell death but are protected by the inhibitory function of intratumoral T(reg) cells.     

CD8+ T cell-mediated control of distant tumours following local photodynamic therapy is independent of CD4+ T cells and dependent on natural killer cells

Cancer survival rates decrease in the presence of disseminated disease. However, there are few therapies that are effective at eliminating the primary tumour while providing control of distant stage disease. Photodynamic therapy (PDT) is an FDA-approved modality that rapidly eliminates local tumours, resulting in cure of early disease and palliation of advanced disease. Numerous pre-clinical studies have shown that local PDT treatment of tumours enhances anti-tumour immunity. We hypothesised that enhancement of a systemic anti-tumour immune response might control the growth of tumours present outside the treatment field. To test this hypothesis we delivered PDT to subcutaneous (s.c.) tumours of mice bearing both s.c. and lung tumours and monitored the growth of the untreated lung tumours. Our results demonstrate that PDT of murine tumours provided durable inhibition of the growth of untreated lung tumours. The inhibition of the growth of tumours outside the treatment field was tumour-specific and dependent on the presence of CD8(+) T cells. This inhibition was accompanied by an increase in splenic anti-tumour cytolytic activity and by an increase in CD8(+) T cell infiltration into untreated tumours. Local PDT treatment led to enhanced anti-tumour immune memory that was evident 40 days after tumour treatment and was independent of CD4(+) T cells. CD8(+) T cell control of the growth of lung tumours present outside the treatment field following PDT was dependent upon the presence of natural killer (NK) cells. These results suggest that local PDT treatment of tumours lead to induction of an anti-tumour immune response capable of controlling the growth of tumours outside the treatment field and indicate that this modality has potential in the treatment of distant stage disease.     

Concurrent infiltration by CD8+ T cells and CD4+ T cells is a favourable prognostic factor in non-small-cell lung carcinoma

The purpose of this study was to clarify the relationship between the number of tumour-infiltrating T lymphocytes and the clinicopathological features and clinical outcome in patients with non-small-cell lung cancer (NSCLC). Tissue specimens from 109 patients who underwent surgical resection for NSCLC were immunohistochemically analysed for CD4 and CD8 expression. Patients were classified into two groups according to whether their tumours exhibited a 'high' or 'low' level of CD8(+) or CD4(+) lymphocyte infiltration. Although the level of infiltration by CD8(+) T cells alone had no prognostic significance, the survival rate for patients with both 'high' CD8(+) and 'high' CD4(+) T-cell infiltration was significantly higher than that for the other groups (log-rank test, P=0.006). Multivariate analysis indicated that concomitant high CD8(+) and high CD4(+) T-cell infiltration was an independent favourable prognostic factor (P=0.0092). In conclusion, the presence of high levels of both CD8(+) T cells and CD4(+) T cells is a significant indicator of a better prognosis for patients with NSCLC, and cooperation between these cell populations may allow a significantly more potent antitumour response than either population alone.     

Natural killer receptors on CD8 T cells and natural killer cells from different HLA-C phenotypes in melanoma patients.

PURPOSE: Because immune mechanisms involved in cutaneous melanoma have not been fully elucidated, efforts have been made to achieve prognosis markers and potential targets for immune therapies, but they have not been entirely fruitful thus far. Therefore, the goal of this study was to investigate the involvement of early changes in CD8 T cells and CD56 natural killer (NK) cells expressing NK receptors in different HLA-C dimorphism groups of melanoma patients. EXPERIMENTAL DESIGN: CD8 T cells and CD56 NK cells were analyzed in 41 patients and 39 sex- and age-matched controls with different HLA-C genotypes by flow cytometry. HLA-C dimorphism at position 80 was tested by PCR sequence-specific primers and PCR sequence-specific oligonucleotide to examine whether it could mediate in the emergence of cells expressing killer cell immunoglobulin-like receptors. RESULTS: Thirty-five of 41 patients had benign sentinel node, and showed an imbalance in the absolute number of CD8(+)DR(+) or CD8(+)CD161(+) peripheral blood T cells according to the CD28 coexpression compared with controls. CD8(+)CD28(-)CD158a(+) T and CD56(+)CD158a(+) NK cells were significantly increased in HLA-C(Lys80) homozygous nonmetastatic patients, whereas only CD56(+)CD158a(+) NK cells increased in heterozygous ones. An up-regulation of the CD158a KIR receptor was also seen on NK cells but not in T cells of patients at advanced disease stages. CONCLUSIONS: This work provides, for the first time, evidence of immune activation in early stages of cutaneous melanoma, together with an increase of cells expressing CD158a in patients bearing the corresponding HLA-C ligand, which may be important to evaluate the disease progression and to use individualized immune therapeutic approaches.     

Transfer of IFNgamma-depleted CD4(+) T cells together with CD8(+) T cells leads to rejection of murine kidney sarcoma in mice

In the murine kidney sarcoma, vaccination with the tumor-specific large T antigen induces protective immunity against the tumor. Immunity is dependent both on CD8(+) cytotoxic T cells and on CD4(+) T-helper cells. We analyzed whether the cytokine phenotype of induced CD4(+) T-effector cells might determine whether or not the tumor is successfully rejected. By intracytoplasmic staining of CD4(+) cells, IFNgamma-producing (Th1), IL-4-producing (Th2), and IL-10-expressing cells could be identified in vaccinated and non-vaccinated animals responding to tumor growth. Vaccinated mice rejecting the tumor showed an increase in the percentage of IL-4-producing (Th2) cells. In contrast, in non-vaccinated mice succumbing to the tumor, the immunosuppressive IL-10-producing cells became more abundant and the frequency of IFNgamma-expressing cells dropped at later time points. Yet, dominance by either a Th1 or a Th2 response could not be observed. To further clarify the relevance of these subsets, Th1 cells were enriched by cell sorting according to IFNgamma surface expression. Enriched Th1 and depleted cells, mainly consisting of the Th2 phenotype, were transferred together with CD8(+) T cells. Surprisingly, immunity could be transferred either with Th1 or Th2 cells, but Th2 cells were slightly more efficient. This suggests that, at least in the effector phase, a Th1 phenotype is not crucial for the rejection. Our findings support the view that the Th1/Th2 dichotomy is not central in T-cell-mediated tumor rejection.     

Sustained antigen-specific antitumor recall response mediated by gene-modified CD4+ T helper-1 and CD8+ T cells

Given that specific subsets of T helper 1 (Th1) and T helper 2 (Th2) CD4(+) T cells have been shown to play key roles in tumor rejection models, we wanted to assess the contribution of either Th1 or Th2 CD4(+) cell subtypes for redirected T-cell immunotherapy. In this study, we have developed a novel method involving retroviral transduction and in vitro T-cell polarization to generate gene-engineered mouse CD4(+) Th1 and Th2 cells or T helper intermediate (Thi) cells expressing an anti-erbB2-CD28-zeta chimeric receptor. Gene-modified Th1 and Th2 polarized CD4(+) cells were characterized by the preferential secretion of IFN-gamma and interleukin-4, respectively, whereas Thi cells secreted both cytokines following receptor ligation. In adoptive transfer studies using an erbB2(+) lung metastasis model, complete survival of mice was observed when transduced Th1, Th2, or Thi CD4(+) cells were transferred in combination with an equivalent number of transduced CD8(+) T cells. Tumor rejection was consistently associated with transduced T cells at the tumor site and interleukin-2 secretion. However, the surviving mice treated with gene-modified Th1 CD4(+) cells were significantly more resistant to a subsequent challenge with a different erbB2(+) tumor (4T1.2) implanted s.c. This result correlated with both increased expansion of Th1 CD4(+) and CD8(+) T cells in the blood and a greater number of these cells localizing to the tumor site following rechallenge. These data support the use of gene-modified CD4(+) Th1 and CD8(+) T cells for mediating a sustained antitumor response.     

HER-2/neu-derived peptide epitopes are also recognized by cytotoxic CD3(+)CD56(+) (natural killer T) lymphocytes

The human HER-2/neu gene encodes a 185 kDa transmembrane glycoprotein recognized by MHC class I-restricted CTLs. Here, we report that HER-2/neu peptide CTL epitopes can also be recognized by cytotoxic NK-T lymphocytes. Unfractionated peptides derived from HLA-A2(+), HER-2/neu(+) tumor cells acid cell extract (ACE), collected from patients with metastatic ovarian cancer, were used as antigen to generate in vitro cytotoxic effectors. ACE was able to elicit from cancer patients' PBMCs both alphabetaTCR(+)CD3(+)CD56(-) and alphaTCR(+)CD3(+)CD56(+) (NK-T) CTLs that lysed ACE-sensitized T2 cells in an HLA-A2-restricted manner. The same CTL lines also recognized T2 cells pulsed with HER-2/neu-derived CTL peptide epitopes, a HER-2/neu-transfected HLA-A2(+) cell line and autologous tumor cells. alphaTCR(+)CD3(+)CD56(+) CTL lines also exhibited NK-like cytotoxicity against autologous tumor cells. CTL clones were isolated from alphaTCR(+)CD3(+)CD56(+) bulk cultures displaying both MHC- and non-MHC-restricted cytotoxicity, thus confirming the dual cytolytic function of such cells. Our data demonstrate that ACE from metastatic ovarian tumors can be used as multiepitope vaccines for generating in vitro, besides classical CTLs, NK-T cells exerting efficient MHC- and non-MHC-restricted cytotoxicity against autologous tumor targets. Such NK-T cells expressing dual cytotoxic activity may prove advantageous in cancer immunotherapy.     

Ex vivo expansion of CD8+CD56+ and CD8+CD56- natural killer T cells specific for MUC1 mucin

Prostate cancers express MUC1, but nearly all metastatic cells lack HLA class I molecules. Thus, a lymphocyte population that can sense its antigenic environment, while also able to react to stimuli of natural killer (NK) cells, may be a more versatile effector cell population for antitumor immune responses. Herein, we report that tumor-specific MUC1 peptide, interleukin 2, and interleukin 12 act synergistically to stimulate the ex vivo expansion of CD8(+)CD56(-) T cells and CD8(+)CD56(+) natural killer T (NKT) cells from the peripheral blood mononuclear cells of prostate cancer patients, as well as healthy male and female donors. Both the CD56(+) NKT cells and CD56(-) T cells lysed allogeneic mucin-bearing target cells, as well as NK target cells, but not lymphokine-activated killer target cells. However, the CD56(+) NKT cells displayed a 2-fold greater cytolytic activity than the CD56(-) T cells. The mucin-specific cytolytic activity and NK cytolytic activities for both lymphocyte populations were independent of HLA class I and CD1 molecules. The CD56(-) T cells up-regulated CD56 with continued antigenic stimulation in the presence of interleukin 12, suggesting that CD8(+)CD56(-) T cells are NKT cells. However, CD56(+) NKT cells expand poorly to continued stimulation. All mucin-stimulated NKT cells exhibited the activated/memory CD45RO phenotype. The NKT cell lines express the alpha/beta T-cell receptor (TCR). The TCR repertoire was limited and varied with cell line, but was not the V alpha 24V beta 11 TCR typically associated with NKT cells. Whereas CD161 is generally considered a marker of NKT cells, the mucin-stimulated NKT cells did not express this marker. Thus, we have described two phenotypically distinct NKT types that do not display a biased TCR repertoire, but do display specificity for a tumor-specific peptide antigen (CTL-like activity), as well as HLA class I-deficient target cells (NK-like activity).     

结直肠癌患者外周血中CD4+CD45RA+ T和CD4+CD45RO+ T细胞的变化及其意义

目的 探讨外周血中CD4 CD45RA T细胞和CD4 CD45RO T细胞在结直肠癌中的变化及其临床意义。方法 采用流式细胞术检测60例结直肠癌患者手术前、术后1个月和3个月时,外周血的CD4 T细胞、CD4 CD45RA T细胞和CD4 CD45RO T细胞的比例。选取健康查体人群10例作为对照。结果 结直肠癌患者的CD4 T细胞与健康人群相比无差异。CD4 CD45RO T细胞比例明显增高,术后有显著下降,DukesA、B期患者尤其明显;而CD4 CD45RA T细胞比例正好相反。结论 CD4 CD45RA T细胞和CD4 CD45RO T细胞在肿瘤免疫中起重要作用,其表达同结直肠癌的分期和预后有密切关系。     

Epstein-Barr virus nuclear antigen 1-specific CD4+ T cells directly kill Epstein-Barr virus-carrying natural killer and T cells

Epstein–Barr virus (EBV) nuclear antigen (EBNA)1 is expressed in every EBV-infected cell, regardless of the state of EBV infection. Although EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies, it is less clear whether EBNA1-specific CD4⁺ T cells can act as direct effectors. Herein, we investigated the ability of CD4⁺ T-cell clones induced with overlapping peptides covering the C-terminal region of EBNA1, and identified minimal epitopes and their restricted major histocompatibility complex class II molecules. Of these, a novel epitope, EYHQEGGPD, was found to be presented by DRB1*0401, 0403 and 0406. Five CD4⁺ T-cell clones recognized endogenously processed and presented antigens on EBV-transformed lymphoblastoid cell lines (LCL) and one example proved capable of killing EBV-carrying natural killer (NK) and T-cell lines derived from patients with chronic active EBV infection (CAEBV). Identification of minimal epitopes facilitates design of peptide-based vaccines and our data suggest that EBNA1-specific CD4⁺ T cells may play roles as direct effectors for immunotherapy targeting EBV-carrying NK and T-cell malignancies. ( Cancer Sci 2008; 99: 1633–1642)     

Trafficking of 'immune' CD4(+)/CD8(+)T-lymphocytes into the RENCA tumour microcirculation in vivo in mice

RENCA-IL-2 (Murine Renal Cell Carcinoma transfected with murine IL-2 gene) cells were rejected by immunocompetent (but not T-cell deficient) Balb/c mice, which developed 'immunity' to subsequent parental RENCA tumour cell challenge. Splenocytes adoptively transferred this immunity. CD4(+)and CD8(+)T-lymphocytes prepared from the spleens of 'tumour immune' mice were evaluated for their ability to traffic into the tumour environment using an in vivo model that enables visualization of events within the microvasculature. RENCA cells were implanted into the mouse cremaster muscle and the trafficking of syngeneic lymphocyte subpopulations, derived from naive and 'immune' animals, into both the RENCA tumour and the surrounding normal cremaster muscle microcirculation was measured by in vivo microscopy. Fluorescently labelled CD4(+)and CD8(+)T lymphocytes taken from the spleens of naive mice or mice previously immunized with RENCA-IL-2 were injected systemically into tumour-bearer mice. Naive effector cells migrated to, and flowed through both the tumour and the normal microcirculation, with negligible adhesion. However we observed the selective recruitment, localization and arrest of immune CD4(+)and CD8(+)T lymphocytes (P< 0.05) into the tumour microcirculation, and in some instances the subsequent extravasation of cells into the tumour interstitium. Lymphocyte rolling by 'immune' CD4(+)and CD8(+)T-cells in the tumour microcirculation was greatly reduced, suggesting impaired adhesion molecule expression on the tumour endothelium. This study clearly demonstrates, by direct in vivo microscopy assessment, the localization of effector cells, CD4(+)and CD8(+)lymphocytes into tumours.     

CD1d expression level in tumor cells is an important determinant for anti-tumor immunity by natural killer T cells

Invariant natural killer T (iNKT) cells are thought to regulate anti-tumor immunity. Human iNKT (i.e. Valpha24+ NKT) cells have been reported to recognize CD1d on target cells and show cytotoxicity directly on the target cells in vitro. However, the anti-tumor effect of mouse iNKT (i.e. Valpha14+ NKT) cells has been repeatedly reported to be dependent on the activity of natural killer (NK) cells via interferon-gamma, with no evidence of direct cytotoxicity. In the present study, we report that in vitro cytolysis of EL-4 mouse lymphoblastic lymphoma cells by Valpha24+ NKT cells and in vivo eradication of these cells are both dependent on the level of CD1d expression on the tumor cell surface. These observations possibly suggest that direct cytotoxicity of tumor cells by iNKT cells is common to both humans and mice, and that the high expression level of CD1d may be a predictor whether the tumor is a good target of iNKT cells.     

Induction of myeloma-specific cytotoxic T lymphocytes responses by natural killer cells stimulated-dendritic cells in patients with multiple myeloma

The interaction between dendritic cells (DCs) and natural killer (NK) cells plays a key role in inducing DC maturation for subsequent T-cell priming. We investigated to generate potent DCs by stimulated with NK cells to induce myeloma-specific cytotoxic T lymphocytes (CTLs). NK cells-stimulated-DCs exhibited high expression of costimulatory molecules and high production of IL-12p70. These DCs induce high potency of Th1 polarization and exhibit a high ability to generate myeloma-specific CTLs responses. These results suggest that functionally potent DCs can be generated by stimulation with NK cells and may provide an effective source of DC-based immunotherapy in multiple myeloma.     

Cure of established GL261 mouse gliomas after combined immunotherapy with GM-CSF and IFNgamma is mediated by both CD8+ and CD4+ T-cells

We were the first to demonstrate that combined immunotherapy with GM-CSF producing GL261 cells and recombinant IFNgamma of preestablished GL261 gliomas could cure 90% of immunized mice. To extend these findings and to uncover the underlying mechanisms, the ensuing experiments were undertaken. We hypothesized that immunizations combining both GM-CSF and IFNgamma systemically would increase the number of immature myeloid cells, which then would mature and differentiate into dendritic cells (DCs) and macrophages, thereby augmenting tumor antigen presentation and T-cell activation. Indeed, the combined therapy induced a systemic increase of both immature and mature myeloid cells but also an increase in T regulatory cells (T-regs). Cytotoxic anti-tumor responses, mirrored by an increase in Granzyme B-positive cells as well as IFNgamma-producing T-cells, were augmented after immunizations with GM-CSF and IFNgamma. We also show that the combined therapy induced a long-term memory with rejection of intracerebral (i.c.) rechallenges. Depletion of T-cells showed that both CD4+ and CD8+ T-cells were essential for the combined GM-CSF and IFNgamma effect. Finally, when immunizations were delayed until day 5 after tumor inoculation, only mice receiving immunotherapy with both GM-CSF and IFNgamma survived. We conclude that the addition of recombinant IFNgamma to immunizations with GM-CSF producing tumor cells increased the number of activated tumoricidal T-cells, which could eradicate established intracerebral tumors. These results clearly demonstrate that the combination of cytokines in immunotherapy of brain tumors have synergistic effects that have implications for clinical immunotherapy of human malignant brain tumors.     

Depletion of CD25(+) CD4(+) T cells and treatment with tyrosinase-related protein 2-transduced dendritic cells enhance the interferon alpha-induced, CD8(+) T-cell-dependent immune defense of B16 melanoma.

Transduction of B16 melanoma cells with IFN alpha (B16-IFN alpha) enhances CD8(+) T-cell-dependent tumor immunity in mice, resulting in delayed outgrowth in vivo. Here we provide evidence that CD4(+) T cells down-regulate the IFN alpha-induced tumor immune defense. Importantly, depletion of regulatory CD25(+) CD4(+) T cells prevented growth of B16-IFN alpha in most mice and promoted long-lasting protective tumor immunity. Rejection of B16-IFN alpha could also be achieved with therapeutic injections of dendritic cells genetically engineered to express the melanoma antigen tyrosinase-related protein 2. These results support the development of novel strategies for the immunotherapy of melanoma using IFN alpha in combination with elimination of regulatory T cells or antigen-specific immunization.