Intratumoral regulatory T cells alone or in combination with cytotoxic T cells predict prognosis of hepatocellular carcinoma after resection

Tumor-infiltrating lymphocytes (TILs) represent the host immune response to cancer. CD8(+) cytotoxic T cells (CTLs) have a central role in the elimination of tumors, while regulatory T cells (Tregs) can suppress the immune reaction. The aim of this study was to investigate the prognostic value of TILs, especially Tregs and CTLs, in hepatocellular carcinoma (HCC) patients after resection. CD3(+), CD4(+), CD8(+), and FoxP3(+) TILs were assessed by immunohistochemistry in tumor tissue from 141 randomly selected HCC patients. Prognostic effects of low- or high-density TIL subsets were evaluated by Kaplan-Meier and Cox regression analysis using the median values as cutoff. The density of intratumoral Tregs (P = 0.040) and peritumoral CTLs (P = 0.004) were an independent factor for overall survival (OS), but not for disease-free survival (DFS). The density of CD3(+) and CD4(+) TILs, and the prevalence of Tregs and CTLs were associated with neither OS nor DFS. The presence of low intratumoral Tregs with high intratumoral CTLs was a negative independent prognostic factor for OS (P = 0.001), while that of low intratumoral Tregs and low peritumoral CTLs independently correlated with improved DFS (P = 0.008). Moreover, the combined analysis of Tregs and CTLs displayed better prognostic performances than any of them alone. Additionally, higher density of intratumoral Tregs correlated with both the presence of liver cirrhosis (P = 0.025) and increased tumor size (P = 0.050). Tregs within tumor environment are promising prognostic parameters for HCC patients, and their combination with CTLs can predict prognosis more effectively.     

Associations Between Infiltrating Lymphocyte Subsets and Hepatocellular Carcinoma

Aims: We aimed to analyze the phenotype of tumor-infiltrating lymphocytes (TILs) and non-tumor infiltrating lymphocytes (NILs) in HCC and non-tumor tissues, and evaluate relationships between changes in these cells and the prognosis of HCC. Methods: Lymphocytes were isolated from HCC and corresponding non-tumor tissues and tested by flow cytometry. For comparison, clinical parameters were analyzed. Results: Compared with the non-tumor tissue, tumor tissue had a lower intensity of NK, NKT andCD8+T cell infiltration. TILs had higher intensity of CD4+CD25+Foxp3+regulatory T cell (Treg cells) infiltration compared with that in NILs. The prevalence of Treg cells was associated with fewer CD8 + T lymphocytes in the HCC immune microenvironment. The frequencies of NK cells and CD8+T cells in TILs of HCC patients with metastasis less than 12 months were lower than those without metastasis. However, the frequency of Treg cells was higher than those without metastasis. Conclusion: These results suggest that the frequencies of CD8+T, NK and NKT cells as well as Treg cells in the tumor tissue of HCC are significantly associated with patient survival, and could be applied as predictive indicators for HCC prognosis.     

肝细胞性肝癌组织浸润淋巴细胞表型与分布研究

目的:肝细胞性肝癌组织浸润淋巴细胞与外周血T 细胞表型可能与肿瘤进展及预后相关,本研究检测肝癌患者组织及外周血T 细胞表型与分布,分析淋巴细胞表型变化与预后的关系。方法:分析2007年10月至12月中山医院147 例肝癌及癌旁组织浸润淋巴细胞表型（T 细胞或B 细胞表面标志物:CD3、CD8、CD4、CD20、CD19、Foxp 3）,表型与临床病理特征及预后的关系;检测26例肝癌外周血CD3、CD8、CD4 +T细胞数量并其比例变化。结果:癌巢内肿瘤浸润细胞明显少于癌周组织（P < 0.01）,癌周淋巴细胞主要分布于癌旁正常肝组织、汇管区,其与患者肝炎病史及肝硬化相关,表型以CD3 +T细胞为主,其中又以CD8 + 细胞毒性T 细胞为主;CD4 染色在多数病例为阴性,Foxp 3 仅在个别病例（15/ 109）呈阳性。肿瘤浸润淋巴细胞B 细胞标志CD20、CD19均为阴性。肿瘤组织内CD8 +T细胞浸润数量与预后正相关,而癌周浸润淋巴细胞数目与患者转移及复发无显著关系。结论:肝癌肿瘤浸润细胞在癌巢内明显少于癌周组织,肿瘤及癌周浸润细胞以CD8 + 细胞毒性T 细胞为主。肿瘤组织内CD8 +T细胞浸润数量与预后相关,而癌周浸润淋巴细胞数量与患者转移及复发无显著关系。      

Up-regulation of inhibitory natural killer receptors CD94/NKG2A with suppressed intracellular perforin expression of tumor-infiltrating CD8+ T lymphocytes in human cervical carcinoma

Inhibitory signals that govern the cytolytic functions of CD8(+) T lymphocytes have been linked to the expression of natural killer cell receptors (NKRs) on CTLs. There is limited knowledge about the induction of inhibitory NKR (iNKR) expression in vivo. Up-regulation of iNKRs has been linked to the modulation of the virus- and/or tumor-specific immune responses in animal models. In the present study, we directly examined the expression of various NKRs on tumor-infiltrating lymphocytes (TILs) derived from human cervical cancer. We found that in human cervical cancer, the percentage expression of immunoglobulin-like NKR(+)CD8(+) T lymphocytes were similar in gated CD8(+)-autologous TILs and peripheral blood mononuclear cells. On the contrary, cervical cancer-infiltrating CD8(+) T lymphocytes expressed up-regulated C-type lectin NKRs CD94/NKG2A compared with either peripheral blood CD8(+) T cells or normal cervix-infiltrating CD8(+) T lymphocytes. Dual NKR coexpression analyses showed that CD94 and NKG2A were mainly expressed on CD56(-)CD161(-)CD8(+) TILs within the cancer milieu. Immunohistochemical study showed that cervical cancer cells expressed abundant interleukin 15 (IL-15) and transforming growth factor-beta (TGF-beta). In kinetic coculture assay, cervical cancer cells can promote the expression of CD94/NKG2A on CD8(+) T lymphocytes. The cancer-derived effects can be reversed by addition of rIL-15Ralpha/Fc and anti-TGF-beta antibody. Functional analyses illustrated that intracellular perforin expression of CD8(+) T cells was minimal upon up-regulation of CD94/NKG2A. Kinetic cytotoxicity assays showed that up-regulated expressions of CD94/NKG2A restrain CD8(+) T lymphocyte cytotoxicity. Our study strongly indicated that cervical cancer cells could promote the expression of iNKRs via an IL-15- and possibly TGF-beta-mediated mechanism and abrogate the antitumor cytotoxicity of TILs.     

Expression of Fas antigen (CD95) in peripheral blood lymphocytes and in liver-infiltrating, cytotoxic lymphocytes in patients with hepatocellular carcinoma

BACKGROUND: Fas-expressing cytotoxic T lymphocytes (CTLs) are important antitumor immune effector cells in patients with hepatocellular carcinoma (HCC). The role of transforming growth factor beta 1 (TGF-beta1) in modulating the expression of Fas by CTLs is not known in HCC. The objectives of this study were to characterize the expression of Fas by CTLs and natural killer (NK) cells among peripheral blood lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs) in patients with HCC and to correlate the association, if any, with serum TGF-beta1 levels. METHODS: PBLs from 18 patients with HCC and TILs from 5 HCC liver specimens were isolated, and Fas expression was analyzed by three-color flow cytometry. The results were compared with results from normal control volunteers (n = 19 individuals). Serum TGF-beta1 levels in patients with HCC were measured by enzyme-linked immunosorbent assay. RESULTS: The median percentage of Fas expression by CD3 positive T cells was significantly higher in patients with HCC compared with normal controls (54.37% vs. 32.03%, respectively; P = 0.0036), and this was attributable solely to Fas expression by CD4 positive PBLs (54.46% vs. 34.90%, respectively; P = 0.0234). In contrast, Fas expression was significantly higher in all the subtypes of TILs (CD3 positive, CD4 positive, CD8 positive, NK cells, and natural T cells) compared with controls (all P values were < 0.001). Tumor size was inversely proportional to the TGF-beta1 levels (correlation coefficient [r] = -0.725; P < 0.0001), which were correlated inversely with Fas expression by CD4 positive PBLs (r = -0.516; P = 0.01). CONCLUSIONS: In patients with HCC, TILs exhibit significantly increased expression of Fas compared with PBLs that may enhance their susceptibility to apoptotic mechanisms. Larger tumors were associated with lower serum TGFbeta1 levels, and this was correlated with greater Fas expression by CD4 positive PBLs.     

CD8+ lymphocyte infiltration and apoptosis in hepatocellular carcinoma.

AIMS: Tumour-infiltrating lymphocytes (TILs) play a role in local anti-tumour immunity. Tumour cells may escape from immune surveillance by expressing RCAS1, a receptor-binding cancer antigen expressed on SiSo cells, which inhibits T cell growth. In this study, the correlation between the density of CD8+ TILs, tumour cell apoptosis, and tumour RCAS1 expression was investigated in hepatocellular carcinoma (HCC). METHODS: We obtained tissues from 60 patients with surgically resected HCCs. CD8+ TILS, apoptotic cancer cells, and RCAS1 expressing cancer cells were identified by immunohistochemistry. RESULTS: The density of CD8+ T cells in tumours (mean: 9.5/HPF, HPF: high power field) was significantly less than in non-cancerous hepatic lobules (17.8/HPF, p<0.001) and in relation to the progression of tumour stage. The density of CD8+ T cells in tumours positively correlated with the occurrence of tumour cell apoptosis, but did not correlate with RCAS1 protein expression. CONCLUSIONS: CD8+ TILs may play a role in the occurrence of tumour cell apoptosis in HCC, but CD8+ TILs may not be controlled by RCAS1 in HCC.     

Characterization of T-cell memory phenotype after in vitro expansion of tumor-infiltrating lymphocytes from melanoma patients

Memory T-cell populations in human antitumor tumor-infiltrating lymphocytes (TILs) for adoptive cell transfer have not been fully characterized. Our studies demonstrated that CD62L, CD27 and CD28 positive effector memory T-cells were present in the TIL samples from the tumor tissues of melanoma patients and T-cell expansion led to the significant loss of memory T-cells. CD27- and CD28-positive T-cells had high levels of CD44 expression. T-Cell expansion resulted in significant down-regulation of CD44 expression. Interleukin-2 (IL-2) and anti-CD3 antibody stimulation may be responsible for CD44 down-regulation on CD8(+) T-cells during expansion. Furthermore, CD44 down-regulation using small interfering RNA (siRNA) on TILs dramatically reduced interferon-gamma and IL-2 release upon tumor stimulation. These results suggest that the regulation of CD44 expression in TILs may play an important role in memory T-cell maintenance and antitumor immune response.     

Regression of engineered myeloma cells secreting interferon-gamma-inducing factor is mediated by both CD4(+)/CD8(+) T and natural killer cells

IL-18 is a novel cytokine that stimulates T and NK cell activity and has potent antitumor effects. In this study, a mouse IL-18 gene was transfected into the mouse myeloma cell line J558. Our data demonstrated that (i) inoculation of 0.5x10(6) engineered tumor cells J558/IL-18 into syngeneic mice induced a Th1 dominant immune response and resulted in tumor regression in all 8/8 mice; (ii) the IL-18 antitumor effect was significantly decreased in mice depleted of either the CD4(+), or CD8(+), or NK cell subset, respectively but was completely abrogated in mice depleted of both CD4(+) and CD8(+) T cells; (iii) in vivo neutralization of IFN-gamma was accompanied by the growth of J558/IL-18 tumor in all the mice; and (iv) the J558/IL-18 tumor regression further induced protective immunity against a subsequent challenge by the parental J558 tumor, which is mediated by CD8(+) T cells as examined in the cytotoxicity assay in vitro and in the animal study in vivo. Taken together, our findings indicate that: (i) IL-18 can induce antitumor immune responses mediated by both CD4(+)/CD8(+) T cells and NK cells; and (ii) it is associated with IFN-gamma production. This study thus highlights the potential utility of IL-18 as an antitumor agent, a role that it can fulfil alone or in combination with other immunomodulatory cytokines such as IL-12.     

Administration of 6-gingerol greatly enhances the number of tumor-infiltrating lymphocytes in murine tumors

Tumor-infiltrating lymphocytes (TILs) play critical roles in host antitumor immune responses. It is known that cancer patients with tumor-reactive lymphocyte infiltration in their tumors have better prognoses, while patients with tumors infiltrated by immunosuppressive cells have worse prognoses. We found that administration of 6-gingerol, which is a component of ginger, inhibited tumor growth in several types of murine tumors, such as B16F1 melanomas, Renca renal cell carcinomas and CT26 colon carcinomas, which were established by inoculating tumor cells on the flanks of mice. However, administration of 6-gingerol did not lead to complete eradication of the tumors. 6-Gingerol treatment of tumor-bearing mice caused massive infiltration of CD4 and CD8 T-cells and B220(+) B-cells, but reduced the number of CD4(+) Foxp3(+) regulatory T-cells. The CD8 tumor-infiltrating T lymphocytes in 6-gingerol-treated mice strongly expressed IFN-γ, a marker of activation of cytotoxic T lymphocytes (CTL) CD107a and chemokine receptors that are expressed on T(H) 1 cells, such as CXCR3 and CCR5. To test whether 6-gingerol could promote infiltration of tumor antigen-specific CD8 T-cells into tumors, we adoptively transferred CFSE-labeled OT-1 CD8 T-cells into EG7 tumor-bearing mice. We found that CD8 T cells isolated from 6-gingerol pretreated OT-1 mice, but not from control OT-1 mice, massively infiltrated tumors and tumor draining lymph nodes and divided several times. Our results strongly suggest that 6-gingerol can be used in tumor immunotherapy to increase the number of TILs.     

Immunization with tumor-derived ER chaperone grp170 elicits tumor-specific CD8+ T-cell responses and reduces pulmonary metastatic disease

Glucose-regulated protein (grp) 170 is a molecular chaperone localized in endoplasmic reticulum (ER), which has been demonstrated to interact with the peptides translocated by transporter associated with antigen presentation (TAP). In our study, we have evaluated the therapeutic efficacy of tumor-derived grp170 against the highly metastatic and poorly immunogenic murine melanoma B16F10. Immunization of mice with grp170 preparations from autologous tumor significantly delayed progression of the primary cancer and reduced established pulmonary metastases, which was associated with the prolonged survival of metastases-bearing mice. However, grp170 from normal liver or antigenically distinct tumor failed to exhibit therapeutic effect. In addition, tumor-derived grp170 elicited a potent cytotoxic T-lymphocyte response specific for B16F10 tumor, which correlates with in vivo protective effects. Adoptive transfer of splenocytes obtained from B16F10-grp170-primed animals remarkably suppressed pulmonary metastases. Depletion of either CD4(+) or CD8(+) T cells in priming phase significantly abrogated the tumor immunity induced by the B16F10-grp170. However, the vaccine activity was intact when CD4(+), not CD8(+), T cells were depleted in effector phase. It suggests that CD4(+) T helper cells play an important role in the generation of effective antitumor response, whereas CD8(+) T cells are predominantly involved in direct killing of tumor cells. These observations have strong clinical implications for using tumor-derived grp170 as a therapeutic vaccine against metastatic diseases.     

Increased populations of regulatory T cells in peripheral blood of patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a poor prognosis and one for which immunotherapy remains a viable option. Experimental tumor models have shown that regulatory T cells, a functionally unique subset of T cells, can suppress effective antitumor immune responses. This suppression might explain the poor outcome of some of the immunotherapy protocols currently being used. A better understanding of the role of regulatory T cells in HCC is important for design of future immunotherapy-based clinical protocols. We have studied regulatory T cells from 84 patients with HCC and 74 controls, including healthy donors, patients with chronic hepatitis B virus and hepatitis C virus infection and nonviral liver cirrhosis. Regulatory T cells were identified by fluorescence-activated cell sorting using a panel of antibodies and by real-time PCR analysis for Foxp3 expression. Functional studies were done to analyze their inhibitory role. Finally, regulatory T cells were analyzed in tumors and ascites from patients with HCC. Patients with HCC have increased numbers of CD4+CD25+ regulatory T cells in their peripheral blood, which express high levels of HLA-DR, GITR, and low or no CD45RA. These cells were anergic toward T-cell receptor stimulation and, when cocultured with activated CD4+CD25- cells, potently suppressed their proliferation and cytokine secretion. There were also high numbers of regulatory T cells in tumor-infiltrating lymphocytes of HCC patients comparable with the increase in their peripheral blood. Our data suggest that the increase in frequency of regulatory T cells might play a role in modulation of the immune response against HCC and could be important in design of immunotherapeutic approaches.     

Prognostic implications of type and density of tumour-infiltrating lymphocytes in gastric cancer

The study aims to determine whether type and density of tumour-infiltrating lymphocytes (TILs) can predict the clinical course in gastric cancer. Gastric carcinomas (n=220) were immunostained for CD3, CD8, CD20, and CD45RO and evaluated for clinicopathologic characteristics. Number of TILs that immunostained positively for each marker were counted using NIH ImageJ software. Tumours were grouped into low- and high-density groups for each marker (CD3, CD8, CD45RO). The densities of CD3(+), CD8(+), and CD45RO(+) TILs were found to be independent predictors of lymph node metastasis by multivariate analysis with odds ratios (95% CI) of 0.425 (0.204-0.885), 0.325 (0.150-0.707), and 0.402 (0.190-0.850), respectively. Kaplan-Meier survival analysis revealed that patients in the high-density groups for CD3, CD8, and C45RO had a significantly longer survival time than the patients in the corresponding low-density groups, respectively. In multivariate survival analysis, the densities of CD3(+), CD8(+), and CD45RO(+) TILs remained independent prognostic factors with hazard ratios (95% CI) of 0.549 (0.317-0.951), 0.574 (0.347-0.949), and 0.507 (0.298-0.862), respectively. In conclusion, density of TILs was found to be independently predictive of regional lymph node metastasis and patient survival in gastric cancer.     

CD4+ T cells in cancer stroma, not CD8+ T cells in cancer cell nests, are associated with favorable prognosis in human non-small cell lung cancers

We investigated intratumoral tumor-infiltrating lymphocytes (TILs), including CD4(+) and CD8(+) T cells, in non-small cell lung cancers (NSCLCs) and their relationships with clinicopathological variables and post-operative survival. Tumor specimens from 178 NSCLCs were consecutively obtained by surgery at the Hokkaido University Medical Hospital between 1976 and 1994. CD8(+) T cells, CD4(+) T cells and Ki-67/CD8(+) T cells were visualized immunohistochemically, and counted within cancer cell nests and in cancer stroma. CD8(+) T cells and CD4(+) T cells were observed at higher frequencies within cancer cell nests in moderately and poorly differentiated tumors compared with well differentiated tumors (P < 0.01), and in tumors with high Ki-67 expression compared with low Ki-67 expression (P < 0.01), that showed severe cellular atypia and a higher growth rate. Patients with higher numbers of CD8(+) T cells within cancer cell nests showed significantly shorter survival times compared to those with lower numbers of CD8(+) T cells within cancer cell nests (5-year survival rates, 47% and 60%, respectively; P = 0.03). Moreover, patients with higher labeling index of Ki-67/CD8(+) T cells showed significantly shorter survival than those with lower labeling index of Ki-67/CD8(+) T cells within cancer cell nests (5-year survival rates, 41% and 69%, respectively; P = 0.02), and the labeling index of Ki-67/CD8(+) T cells within cancer cell nests was found to be a significant and independent unfavorable prognostic factor by multivariate analysis (P = 0.01). On the other hand, higher numbers of CD4(+) T cells in cancer stroma, but not within cancer cell nests, were correlated with longer survival times in patients with NSCLC (5-year survival rates, 64% and 43%, respectively; P = 0.04). CD4(+) T cells in cancer stroma might reflect immune responses against cancer cells, while CD8(+) T cells do not appear to work as effectors in tumor tissues of NSCLC. Moreover, the higher labeling index of Ki-67/CD8(+) T cells within cancer cell nests is a strong indicator of unfavorable clinical outcome.     

CD8+ Foxp3+ regulatory T cells mediate immunosuppression in prostate cancer.

PURPOSE: Although elevated proportions of CD4(+)CD25(+) regulatory T (Treg) cells have been shown in several types of cancers, very little is known about the existence and function of CD8(+) Treg cells in prostate cancer. In this study, we investigated prostate tumor-derived CD8(+) Treg cells and their function. EXPERIMENTAL DESIGN: Tumor-infiltrating lymphocytes (TIL) from fresh tumor specimens of patients with prostate cancer were generated and subjected to phenotypic and suppressive function analyses. In particular, we investigated the role and function CD8(+) Treg cells in prostate cancer. RESULTS: We show that high percentages of CD4(+)CD25(+) T cells are probably present in the majority (70%) of prostate TILs. Remarkably, both CD4(+) and CD8(+) T-cell subpopulations possessed potent suppressive activity. T-cell cloning and fluorescence-activated cell sorting analyses showed the presence of CD8(+)CD25(+) Treg cell clones that expressed FoxP3 and suppressed na?ve T-cell proliferation, in addition to the previously known CD4(+)CD25(+) Treg cells. These CD8(+) Treg cells suppressed na?ve T-cell proliferation mainly through a cell contact-dependent mechanism. Importantly, the suppressive function of CD8(+) Treg cells could be reversed by human Toll-like receptor 8 (TLR8) signaling. CONCLUSION: Our study shows that like CD4(+)CD25(+) Treg cells, CD8(+) Foxp3(+) Treg cells present in prostate tumor-derived TILs suppress immune responses and that their suppressive function can be regulated by TLR8 ligands, raising the possibility that the manipulation of Treg cell function by TLR8 ligands could improve the efficacy of immunotherapy for prostate cancer patients.     

The spontaneous CD8+ T-cell response to HLA-A2-restricted NY-ESO-1b peptide in hepatocellular carcinoma patients.

PURPOSE: Hepatocellular carcinoma (HCC) can express various cancer-testis antigens including NY-ESO-1, members of the SSX family, members of the MAGE family, SCP-1, and CTP11. Immunotherapy directed against these antigens is a potential alternative treatment for HCC. To date, it remains unclear whether HCC patients have spontaneous immune responses to these tumor antigens. The objectives of this study were to measure immune responses to NY-ESO-1, a promising cancer vaccine candidate, in HCC patients using the HLA-A2-restricted NY-ESO-1b peptide (p157-165) to measure cellular responses and whole protein to measure antibody responses. EXPERIMENTAL DESIGN: In HLA-A2(+) patients with NY-ESO-1(+) HCC, we analyzed T-cell antigen-dependent interferon (IFN)-gamma and/or Granzyme B release by enzyme-linked immunospot (ELISPOT) assay and IFN-gamma-producing intracellular cytokine flow cytometry (CytoSpot). As an assay independent of T-cell function, we performed tetramer staining. Antibodies to whole NY-ESO-1 were assayed by enzyme-linked immunosorbent assay. RESULTS: The frequency of specific CD8(+) T-cell responses to NY-ESO-1b in 28 NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients was 35.7% (10 of 28). The average magnitude of effector CD8(+) T cells was 0.3% (89 +/- 59 per 2.5 x 10(4) CD8(+) cells) and 1.2% as measured by IFN-gamma release ELISPOT and CytoSpot assays, respectively. These in vitro induced NY-ESO-1b-specific CD8(+) T cells can also recognize HepG2 cells transfected with pcDNA3.1-NY-ESO-1 in both IFN-gamma and Granzyme B ELISPOT assays. Frequencies of NY-ESO-1b-specific T cells in several patients were confirmed by tetramer staining. Nonfunctional tetramer(+)CD8(+) T cells were also present. The CD8(+) T-cell response was apparently increased in patients with late-stage HCC. A discordance between antibody and CD8(+) T-cell responses in HCC patients was observed. CONCLUSIONS: The elevated frequency of specific CD8(+) T-cell responses to NY-ESO-1b in NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients suggests that NY-ESO-1 is appropriate for use in the immunotherapy of HCC patients.     

Activation of tumor-associated macrophages by the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid induces an effective CD8+ T-cell-mediated antitumor immune response in murine models of lung cancer and mesothelioma

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a small molecule in the flavanoid class that has antitumor activity thought to be due to ability to induce high local levels of tumor necrosis factor (TNF)-alpha that disrupt established blood vessels within tumors. The drug has completed phase 1 testing in humans and is currently in phase 2 trials in combination with chemotherapy. Although characterized as a "vascular disrupting agent," there are some studies suggesting that DMXAA also has effects on the immune system that are important for its efficacy. The goal of this study was to carefully define the immune effects of DMXAA in a series of murine lung cancer and mesothelioma cell lines with varying immunologic characteristics. We show that DMXAA efficiently activated tumor-associated macrophages to release a variety of immunostimulatory cytokines and chemokines, including TNF-alpha; IFN-inducible protein-10; interleukin-6; macrophage inflammatory protein-2; monocyte chemotactic protein-1; and regulated on activation, normal T-cell expressed, and secreted. DMXAA treatment was highly effective in both small and large flank tumors. Animals cured of tumors by DMXAA generated a systemic memory response and were resistant to tumor cell rechallenge. DMXAA treatment led to initial tumor infiltration with macrophages that was followed by an influx of CD8(+) T cells. These CD8(+) T cells were required for antitumor efficacy because tumor inhibitory activity was lost in nude mice, mice depleted of CD8(+) T cells, and perforin knockout mice, but not in CD4(+) T-cell-depleted mice. These data show that activation of tumor-associated macrophages by DMXAA is an efficient way to generate a CD8(+) T-cell-dependent antitumor immune response even in animals with relatively nonimmunogenic tumors. Given these properties, DMXAA might also be useful in boosting other forms of immunotherapy.     

Proliferative activity of intratumoral CD8(+) T-lymphocytes as a prognostic factor in human renal cell carcinoma: clinicopathologic demonstration of antitumor immunity.

Tumor-infiltrating lymphocytes, particularly CD8(+) T cells, could be a manifestation of antitumor immunity. We clinicopathologically analyzed the biological significance of tumor-infiltrating lymphocytes in 221 patients with renal cell carcinoma without preoperative treatments. More abundant infiltration of tumor tissue not only by CD8(+) but also CD4(+) T cells was associated with shorter survival of the patients, because of the positive correlation between the number of lymphocytes and representative tumor grade factors. This suggests that immune cell reactions are more pronounced as the tumor grade/biological malignancy progresses, probably because of increased antigenicity of tumor cells. We next analyzed the proliferative activity of CD8(+) T cells that infiltrated in tumor cell nests, which could also reflect antitumor immunity. Higher labeling index of Ki-67, a proliferation-associated antigen, among CD8(+) T cells in contact to tumor cells was associated with a longer survival by both uni- and multivariate analyses. Our data in human renal cell carcinoma suggest that infiltration of tumor tissue by T cells itself does not denote the efficacy of antitumor immunity because of its dependence on the biological malignancy of tumor cells, but infiltration of tumor tissue by CD8(+) T cells bearing more pronounced proliferative activity could reflect effective antitumor immunity. This concept would be important for future immunotherapy of human cancer.     

Disturbed patterns of immunocompetent cells in usual-type vulvar intraepithelial neoplasia

Genital infection with human papillomavirus (HPV) is usually transient, as the immune system is capable of eliminating the virus. When immunity "fails" and the infection persists, vulvar intraepithelial neoplasia (VIN) may develop. In this study, we examined the distribution of inflammatory cells in 51 patients with HPV-associated usual-type VIN and in 19 healthy controls. Frozen vulvar tissue samples were tested for the presence of HPV-DNA, and immunohistochemical staining for the markers CD1a, CD207, CD208, CD123/CD11c, CD94, CD4, CD8, and CD25/HLA-DR was performed. Cells were counted in both the epidermis and dermis over at least 2 mm of basal membrane length. In the epidermis of VIN patients, CD1a(+) and CD207(+) (Langerin) dendritic cells (DC) and CD8(+) T cells were significantly lower than in controls, whereas the number of CD123(+)/CD11c(-) plasmacytoid DCs (pDC) was significantly increased. No significant changes were observed for CD208(+) DCs, CD94(+) natural killer (NK) cells, CD4(+) T cells, and CD25(+)/HLA-DR(+) regulatory T cells. In the dermis of VIN patients, elevated numbers of CD208(+), CD123(+)/CD11c(-), CD94(+), CD4(+), CD8(+), and CD25(+)/HLA-DR(+) cells were observed when compared with healthy controls. The numbers of CD1a(+) and CD207(+) DCs were not different between groups. In summary, high-risk HPV-related usual-type VIN lesions are characterized by an immunosuppressive state in the epidermis, showing a reduction of immature myeloid DCs (mDC) and CD8(+) T cells. In the dermis, inflammatory activation is reflected by the influx of mature mDCs and pDCs, NK cells, and T cells, suggesting that the cellular immune response on viral HPV infection occurs in the dermis of VIN patients.     

Soluble type II transforming growth factor-beta receptor inhibits established murine malignant mesothelioma tumor growth by augmenting host antitumor immunity.

PURPOSE: Transforming growth factor (TGF)-beta blockade has been proposed as an anticancer therapy; however, understanding which tumor patients might benefit most from such therapy is crucial. An ideal target of such inhibitory therapy might be malignant mesothelioma (MM), a highly lethal, treatment-resistant malignancy of mesothelial cells of the pleura and peritoneum that produces large amounts of TGF-beta. The purpose of this study was to explore the possible therapeutic utility of TGF-beta blockade on MM. EXPERIMENTAL DESIGN: To evaluate this hypothesis, we tested the effects of a soluble TGF-beta type II receptor (sTGF-beta R) that specifically inhibits TGF-beta1 and TGF-beta 3 in three different murine MM tumor models, AB12 and AC29 (which produce large amounts of TGF-beta) and AB1 (which does not produce TGF-beta). RESULTS: Tumor growth of both established AB12 and AC29 tumors was inhibited by sTGF-beta R. In contrast, AB1 tumors showed little response to sTGF-beta R. The mechanism of these antitumor effects was evaluated and determined to be primarily dependent on immune-mediated responses because (a) the antitumor effects were markedly diminished in severe combined immunodeficient mice or mice depleted of CD8(+) T cells and (b) CD8(+) T cells isolated from spleens of mice treated with sTGF-beta R showed strong antitumor cytolytic effects, whereas CD8(+) T cells isolated from spleens of tumor-bearing mice treated with of control IgG2a showed no antitumor cytolytic effects. CONCLUSIONS: Our data suggest that TGF-beta blockade of established TGF-beta-secreting MM should be explored as a promising strategy to treat patients with MM and other tumors that produce TGF-beta.