A parathyroid-hormone-related-protein (PTH-rP)-specific cytotoxic T cell response induced by in vitro stimulation of tumour-infiltrating lymphocytes derived from prostate cancer metastases, with epitope peptide-loaded autologous dendritic cells and low-dose IL-2.

Bone metastases are one of the most common events in patients with prostate carcinoma. PTH-rP, a protein produced by prostate carcinoma and other epithelial cancers, is a key agent for the development of bone metastases. A PTH-rP-derived peptide, designated PTR-4 was identified, which is capable to bind HLA-A2.1 molecules and to generate PTH-rP-specific cytotoxic T cell (CTL) lines from healthy HLA-A2.1(+) individual peripheral-blood-mononuclear-cells (PBMC). In this model, we investigated the in vitro possibility of generating an efficient PTH-rP specific CTL response by cyclical stimulations with IL-2 and PTR-4 peptide-pulsed autologous dendritic cells (DC), of HLA-A2.1(+) tumour infiltrating lymphocytes (TIL) derived from a patient with metastatic prostate carcinoma. A T cell line generated in this way (called TM-PTR-4) had a CD3(+), CD5(+), CD4(-), CD8(+), CD45(Ro+), CD56(-) immunophenotype and a HLA-A2.1 restricted cytotoxic activity to PTR-4-peptide pulsed CIR-A2 (HLA-A2.1(+)) target cells, PTH-rP(+)/HLA-A2.1(+) CIR-A2 transfected with PTH-rP gene, prostate carcinoma LNCaP cells, and autologous metastatic prostate cancer cells (M-CaP). These lymphocytes were not cytotoxic to HLA-A2.1(+) targets not producing PTH-rP, such as peptide-unpulsed CIR-A2 and colon carcinoma SW-1463, cell lines. Our results provide evidence that PTR-4 peptide-pulsed autologous DC may break the tolerance of human TIL against the autologous tumour by inducing a PTH-rP-specific CTL immune reaction. In conclusion PTR-4 peptide-pulsed autologous DC may be a promising approach for vaccine-therapy and antigen-specific CTL adoptive immunotherapy of hormone-resistant prostrate cancer.     

Expression of cyclooxygenase-2 (COX-2) in tumour and stroma compartments in cervical cancer: clinical implications

This study aims at investigating the relationship between cyclooxygenase-2 expression in tumour vs stroma inflammatory compartment and its possible clinical role. The study included 99 stage IB-IV cervical cancer patients: immunostaining of tumour tissue sections was performed with rabbit antiserum against cyclooxygenase-2. CD3, CD4, CD8, CD25, Mast Cell Tryptase monoclonal antibodies were used to characterise stroma inflammatory cells in nine cervical tumours. An inverse relation was found between cyclooxygenase-2 levels (cyclooxygenase-2 IDV) of tumour vs stroma compartment (r=-0.44, P<0.0001). The percentage of cases showing high tumour/stromal cyclooxygenase-2 IDV ratio was significantly higher in patients who did not respond to treatment (93.3%) with respect to patients with partial (60.5%), and complete (43.7%) response (P= 0.009). Cases with a high tumour/stroma cyclooxygenase-2 IDV ratio had a shorter overall survival rate than cases with a low tumour/stroma cyclooxygenase-2 IDV (P<0.0001). In the multivariate analysis advanced stage and the status of tumour/stroma cyclooxygenase-2 IDV ratio retained an independent negative prognostic role. The proportion of CD3(+), CD4(+), and CD25(+) cells was significantly lower in tumours with high tumour/stroma cyclooxygenase-2 IDV ratio, while a higher percentage of mast cells was detected in tumours showing high tumour/stroma cyclooxygenase-2 IDV ratio. Our study showed the usefulness of assessing cyclooxygenase-2 status both in tumour and stroma compartment in order to identify cervical cancer patients endowed with a very poor chance of response to neoadjuvant therapy and unfavourable prognosis.     

The phenotype of ascitic fluid lymphocytes in patients with ovarian carcinoma and other primaries.

BACKGROUND: Ascitic tumor-infiltrating lymphocytes (TIL) are a potential source of effectors for adoptive immunotherapy. PATIENTS AND METHODS: The TIL phenotype was examined by two-color flow cytometry in malignancy-related ascites of 49 patients with different primaries. Interleukin-10 (IL-10) and neopterin were determined in ascitic fluid by enzyme-linked immunoassay. RESULTS: Malignant melanoma patients had significantly higher CD3(+), CD3(+)CD8(+) and CD3(+)CD95(+), and lower CD3(+)CD4(+) lymphocyte numbers than patients with other primaries. Ovarian cancer patients had higher CD3(+)CD45RO(+), CD8(+)CD28(+), CD19(+)CD86(+), CD19(+) and CD19(+)CD86(+) lymphocyte numbers, and lower NK cell numbers than patients with gastrointestinal and pancreatic primaries. Pretreated patients had significantly lower concentrations of IL-10, lower CD8(+)CD28(+), CD3(+)CD45RA(+), and higher CD3(+)CD80(+) numbers than chemotherapy-na?ve patients. Patients with hepatic metastases had lower CD3(+), CD3(+)CD4(+) and CD3(+)CD45RO(+), and higher CD3(+)CD25(+) and NK cell numbers than patients without liver metastases. A substantial number of cells exhibited dendritic cell phenotype. Significant correlations were observed between neopterin and IL-10 concentrations, and numbers of CD8(+)CD28(+) and CD3(+)CD80(+) lymphocytes. CONCLUSIONS: Some parameters of TIL phenotype differ depending on primary, previous treatment, or the presence of liver metastases. A negative correlation was observed between IL-10 and neopterin, and an opposing effect of local concentrations of IL-10 and neopterin on the numbers of CD8(+)CD28(+) and CD3(+)CD80(+) was noted.     

Tumor-infiltrating cells as a prognostic factor in Hodgkin's lymphoma: a quantitative tissue microarray study in a large retrospective cohort of 267 patients

The present study aimed to describe the general tissular composition of the immune infiltrate observed in Hodgkin's lymphoma (HL) and its possible relationship with clinical and survival prognostic factors. In this retrospective study of 267 HL patients, the relative proportions of infiltrating T lymphocytes (CD4 + , CD8 + ), natural killer cells (CD56 + , CD57 + ), cytotoxic cells (Granzyme B + , TIA-1 + ) and dendritic cells (CD21 + , S-100 + ) were quantified immunohistochemically with tissue microarray technology. Our results confirm the predominance of CD4 + T lymphocytes in the background of tumoral cells, in addition to a high number of cytotoxic lymphocytes (CD8, CD57 and TIA-1). Patients with low numbers of infiltrating CD8, CD56, CD57 + cells and high numbers of Granzyme B and TIA-1 + cells presented a significantly unfavourable clinical course (presence of leukocytosis, B symptoms, advanced clinical stage (III/IV), non-responding patients). A reduced infiltration of CD4 + T lymphocytes was related with the presence of Epstein - Barr virus. Significantly longer survival times were observed in patients with a high level of infiltrating CD57, as well as a low level of Granzyme B and TIA-1 + cells (log-rank test). When evaluated in a multivariate model, high levels of infiltrating TIA-1 and Granzyme B + cells were shown to be independent prognostic factors that negatively influenced overall survival. The presence of TIA-1 + cells was found to be the only unfavorable prognostic factor of event-free survival and disease-free survival. The overall detection of tumor-infiltrating cells in HL confirms the importance of cytotoxic T lymphocyte infiltration (Granzyme B and TIA-1 + cells) in these patients. Independently of the classical clinical and pathological features, these cells appear to be an unfavourable prognostic factor in HL and, more particularly, the presence of cytotoxic TIA-1 + cells.     

Clinical significance of immune cell infiltration within gallbladder cancer

To investigate the pathophysiological significance of infiltrating antitumour immune cells, we evaluated the quantity of immune cell intratumoral infiltration in 110 surgically resected gallbladder specimens by immunohistochemistry. We examined 45 cases of gallbladder cancer and 65 cases of benign gallbladder diseases for CD4(+) T cells, CD8(+) T cells, natural killer cells (NKCs), and dendritic cells (DCs). High levels of CD4(+) T cell, CD8(+) T cell, NKC, and DC infiltration were recognised in 51.1% (23 out of 45), 37.8% (17 out of 45), 33.3% (15 out of 45), and 48.9% (22 out of 45) of cancer specimens, respectively. High numbers of infiltrating CD4(+) and CD8(+) T cells correlated with decreasing tumour invasion, and high numbers of infiltrating DCs correlated with decreasing lymph-node tumour metastasis. Furthermore, increased infiltration of CD4(+) and CD8(+) T cells and DCs exhibited a significant correlation with prolonged survival. NKC infiltration, however, did not correlate with any of the clinicopathological factors examined. Additionally, high levels of infiltration were not identified in specimens from benign diseases, consistent with the cancer-specific activity of CD4(+) and CD8(+) T cells and DCs. In this study, we demonstrate that CD4(+) and CD8(+) tumour-infiltrating lymphocyte and DCs, but not NKCs, are important factors in the accurate prognosis of survival after surgical removal of gallbladder adenocarcinoma.     

Prognostic value of intra‐tumoral CD8+/FoxP3+ lymphocyte ratio in patients with resected colorectal cancer liver metastasis

Background and Objectives

Patients with isolated colorectal‐cancer‐liver‐metastases (CRCLM) frequently undergo metastatectomy. Tumor‐infiltrating‐lymphocytes (TILs) have prognostic potential in the setting of primary colorectal cancer, however, their role in CRCLM is less studied. We aimed to study the spatial distribution and prognostic role of tumor‐infiltrating CD8⁺ cytotoxic T‐cells and FoxP3⁺ regulatory T‐cells at the metastatic site of CRCLM patients.

Methods

TILs were isolated from fresh metastatic tissues of 47 patients with CRCLM. Archived paraffin‐embedded tissue, from the same patients, was retrieved. CD8⁺ and FoxP3⁺ cells, both in the intra‐tumoral and the peri‐tumoral compartments, were measured by immunohistochemistry on full tissue sections. Proportions of cytotoxic T‐cells (CD8⁺) and regulatory T‐cells (CD4⁺CD25⁺FoxP3⁺), within CD45⁺TILs, were measured by flow‐cytometry.

Results

By immunohistochemistry, individual densities of intra‐tumoral or peri‐tumoral CD8⁺ and FoxP3⁺ cells were not prognostic of survival. However, the intra‐tumoral, but not the peri‐tumoral, CD8⁺/FoxP3⁺ ratio was an independent predictor of survival (HR 0.43, 95%CI 0.19‐0.95, P = 0.032). By flow cytometry, the intra‐tumoral CD8⁺/regulatory T‐cell ratio was also an independent predictor of survival (HR 0.45, 95%CI 0.20‐0.99, P = 0.044).

Conclusions

The ratio of cytotoxic (CD8⁺) to regulatory (FoxP3⁺) T‐cells, in the intra‐tumoral compartment, but not in the peri‐tumoral compartment, can predict survival after resection of CRCLM.     

Nitric oxide is a key component in inflammation-accelerated tumorigenesis

Nitric oxide (NO(*)), an important signaling molecule and a component of inflammatory response, is involved in tumorigenesis. However, the quantity of NO(*) and the cellular microenvironment influences the role of NO(*) in tumor development. We used a genetic strategy to test the hypothesis that an inflammatory microenvironment with an enhanced level of NO(*) accelerates spontaneous tumor development. C. parvum-induced inflammation and increased NO(*) synthase-2 (NOS2) expression coincided with accelerated spontaneous tumor development, mostly lymphomas, in p53-/-NOS2+/+ C57BL6 mice when compared with the controls (P = 0.001). However, p53-/-NOS2-/- mice did not show any difference in tumor latency between C. parvum-treated and control groups. In C. parvum-treated p53-/-NOS2+/+ mice, tumor development was preceded by a higher expression of NOS2 and phosphorylated Akt-Ser(473) (pAkt-Ser473) in spleen, increased cell proliferation measured by Ki-67 IHC in spleen and thymus, and a lower apoptotic index and CD95-L expression in spleen and thymus. C. parvum-treated p53-/-NOS2+/+ mice showed an increase in the number of Foxp3(+) T-reg cells, dendritic cells (DC), as well as increased CD80(+), CD86(+), CD40(+), and CD83(+) on DC in the spleen. Regulatory T-cells (T-reg) and the maturation of DC may modulate tumorigenesis. An increase in the FoxP3(+)T-reg cells in C. parvum-treated p53-/-NOS2+/+ mice indicates a role of NO(*) in the regulation of T-reg cells that may contribute to a protumor shift of the immune environment favoring an accelerated tumor development. These data provide genetic and mechanistic evidence that an inflammatory microenvironment and an increased level of NO(*) can accelerate tumor development.     

The clinical impact of tumour‐infiltrating lymphocytes in colorectal cancer differs by anatomical subsite: A cohort study

Accumulating evidence demonstrates an association between dense infiltration of lymphocytes and prognosis in colorectal cancer (CRC), but whether this prognostic impact differs by tumour location remains unknown. This study investigated the prognostic impact of cytotoxic and regulatory T cells in CRC, with particular reference to the anatomical subsite of the primary tumour. The density of CD3⁺, CD8⁺ and FoxP3⁺ tumour‐infiltrating T cells was calculated in tissue microarrays with tumours from 557 incident CRC cases from a prospective population‐based cohort. Kaplan–Meier and Cox regression analyses were applied to determine the impact of high and low lymphocyte density on 5‐year overall survival, in subgroup analysis of right colon, left colon and rectum. High CD8⁺ cell density was a favourable prognostic factor for patients with right‐sided colon tumours (hazard ratio [HR]=0.53, 95% confidence interval [CI] 0.29–0.95), independent of age, sex, TNM stage, differentiation grade and vascular invasion, with a significant prognostic interaction between CD8⁺ cells and right‐sidedness (p = 0.031). High FoxP3⁺ cell density was an independent favourable prognostic factor only in patients with rectal tumours (HR = 0.54, 95% CI 0.30‐0.99), and CD3⁺ cell density was an independent favourable prognostic factor for tumours in the right colon and rectum, but there was no significant prognostic interaction between CD3⁺ or FoxP3⁺ cells and sidedness. These results demonstrate that the prognostic impact of tumour‐infiltrating lymphocytes in CRC differs by primary tumour site, further indicating that tumour location may be an important factor to take into consideration in therapeutic decisions, including eligibility for immunotherapy.     

Enhanced tumor growth in UV-irradiated skin is associated with an influx of inflammatory cells into the epidermis

UV radiation causes a number of cellular changes within the skin which play a role in tumor outgrowth, including immunosuppression and production of growth-enhancing cytokines. Both of these enable tumors to grow but their relative importance in carcinogenesis is poorly defined. In this study, C3H/HeN mice were exposed to a single inflammatory dose of 410 mJ/cm(2) UVB radiation (plus 100 mJ/cm(2) UVA radiation) followed by the inoculation of a regressor squamous cell carcinoma into or the painting of oxazolone onto the treated skin. Tumors transplanted 2 or 3 but not 4 days after irradiation had a significantly higher growth rate than tumors inoculated into unirradiated control mice. In contrast, mice failed to respond to hapten when it was applied 2, 3 or 4 days after irradiation. Cytofluorimetric analysis demonstrated that the number of F4/80(+) Langerhans cells was not significantly reduced until 4 days after irradiation, while the number of dendritic epidermal T cells was significantly lower at all time points observed after UV-irradiation. Furthermore, a large cellular infiltration of CD11b(+), Gr-1(+), CD45(+) MHC class II(+) and CD45(+) MHC class II(-) cells into the epidermis was observed 2 and 3 days after irradiation, which corresponded with the enhanced tumor growth. To a lesser extent tumor growth was also associated with CD45(+) MHC class II(hi) cells, possibly the previously described UV-induced macrophage. In contrast, suppression of contact hypersensitivity corresponded with the reduction in dendritic epidermal T cells but not with other cell changes. The results suggest that, in this model, where immunosuppression did not appear to be responsible for enhanced tumor growth, inflammatory infiltrates may contribute to the promotion of skin tumor growth within UV-irradiated skin.     

T regulatory cell markers in oral squamous cell carcinoma: Relationship with survival and tumor aggressiveness

Tumor-infiltrating lymphocytes (TILs) are a heterogeneous cell family which plays an important role in tumor-associated immune response. Of these, T regulatory (Treg) cells have also been shown to inhibit anti-tumor response. We aimed to evaluate the expression of T regulatory cell markers (CD4, CD25, CTLA-4 and FoxP3) in samples of oral cavity squamous cell carcinoma (OCSCC) and lip SCC (LSCC) by immunohistochemistry. The relationship of Treg markers with survival data and the proliferative index were also evaluated. We observed similar numbers of CD4-, CD25- and FoxP3(+) cells in OCSCC and LSCC. On the other hand, numbers of CTLA-4(+) cells were significantly lower in OCSCC than in LSCC. OCSCC samples with high numbers of CD4 exhibited a high proliferative index, while samples with high CTLA-4 counts demonstrated a low tumoral proliferative index. A log-rank test showed that patients with OCSCC that presented high counts of CD4 showed a significantly decreased survival compared with patients with low cell counts. In contrast, high CD25(+) cell counts were associated with increased survival. Our results suggest an association of CD4 with poor prognosis, while CD25 expression is related with favorable prognosis. These findings result from the heterogeneity of TIL subsets that display an antagonistic role in tumor immune cell response.     

Characterisation of tumour-infiltrating macrophages: impact on response and survival in patients receiving primary chemotherapy for breast cancer

The role of the tumour microenvironment and complex cellular interactions has attracted interest in responses to primary chemotherapy. Of particular interest are tumour-infiltrating T cells and tumour-infiltrating macrophages (TIMs). We evaluated TIMs and their key activation markers in patients with breast cancer undergoing primary chemotherapy related to response and survival. One hundred and ninety nine patients with large or locally advanced breast cancers received primary chemotherapy. Clinical data, histopathological responses to chemotherapy and survival were examined related to infiltrating cells in tumour microenvironments: cluster of differentiation (CD)3 (pan T cell); CD4 (helper T cells); CD8 (cytotoxic T cells); CD25 (activated T cells); CD68, suppressor of cytokine signalling (SOCS)1, SOCS3 (macrophages); and CD11c and CD205 (dendritic). In tumours demonstrating better responses to chemotherapy, there were significantly fewer CD4(+) T-helper cells than a poorer response (p?相似文献   

Induction of tumour-specific CD8(+) cytotoxic T lymphocytes by tumour lysate-pulsed autologous dendritic cells in patients with uterine serous papillary cancer

Uterine serous papillary carcinoma is a highly aggressive variant of endometrial cancer histologically similar to high grade ovarian cancer. Unlike ovarian cancer, however, it is a chemoresistant disease from onset, with responses to combined cisplatinum-based chemotherapy in the order of 20% and an extremely poor prognosis. In this study, we demonstrate that tumour lysate-pulsed autologous dendritic cells can elicit a specific CD8(+) cytotoxic T lymphocyte response against autologous tumour target cells in three patients with uterine serous papillary cancer. CTL from patients 1 and 2 expressed strong cytolytic activity against autologous tumour cells, did not lyse autologous lymphoblasts or autologous EBV-transformed cell lines, and were variably cytotoxic against the NK-sensitive cell line K-562. Patient 3 CD8(+) T cells expressed a modest but reproducible cytotoxicity against autologous tumour cells only at the time of the first priming. Further priming attempts with PBL collected from patient 3 after tumour progression in the lumboaortic lymph nodes were unsuccessful. Cytotoxicity against autologous tumour cells could be significantly inhibited by anti-HLA class I (W6/32) and anti-LFA-1 MAbs. Highly cytotoxic CD8(+) T cells from patients 1 and 2 showed a heterogeneous CD56 expression while CD56 was not expressed by non-cytotoxic CD8(+) T cells from patient 3. Using two colour flow cytometric analysis of intracellular cytokine expression at the single cell level, a striking dominance of IFN-gamma expressors was detectable in CTL populations of patients 1 and 2 while in patient 3 a dominant population of CD8(+) T cells expressing IL-4 and IL-10 was consistently detected. Taken together, these data demonstrate that tumour lysate-pulsed DC can be an effective tool in inducing uterine serous papillary cancer-specific CD8(+) CTL able to kill autologous tumour cells in vitro. However, high levels of tumour specific tolerance in some patients may impose a significant barrier to therapeutic vaccination. These results may have important implications for the treatment in the adjuvant setting of uterine serous papillary cancer patients with active or adoptive immunotherapy.     

Anti‐inflammatory M2 type macrophages characterize metastasized and tyrosine kinase inhibitor‐treated gastrointestinal stromal tumors

We have made a detailed inventory of the immune infiltrate of gastrointestinal stromal tumors (GISTs), which originate from mesenchymal cells in the intestinal tract. These sarcomas are heavily infiltrated with macrophages and T cells, while immune cells of other lineages were much less abundant. Dissecting the functional subtypes of T cells with multicolor fluorescent microscopy revealed substantial populations of cytotoxic T cells, helper T cells and FoxP3⁺ regulatory T cells. The balance of cytotoxic T cells and FoxP3⁺ T cells was toward immune suppression. Analysis of the macrophage population also showed a dominance of anti‐inflammatory cells, as the M2 type scavenger receptor CD163 was abundantly present. Other subsets of macrophages (CD14⁺CD163⁻) were occasionally detected. M2 type CD163⁺ macrophages were associated with the number of infiltrating FoxP3⁺ regulatory T cells and twice as many macrophages were found in metastatic GIST compared to primary lesions. Most metastatic GISTs had been treated with the tyrosine kinase inhibitors imatinib and sunitinib, but the high macrophage infiltrate was not related to this treatment. However, imatinib and sunitinib did induce secretion of anti‐inflammatory IL‐10 in macrophage cultures, indicating that treatment with these inhibitors might contribute to an immune suppressive microenvironment in GIST. Overall, our data reveal a picture of GIST as an active site of tumor‐immune interaction in which suppressive mechanisms overrule potential antitumor responses. Tyrosine kinase inhibitors might promote this negative balance.     

Malignant and non-malignant lung tissue areas are differentially populated by natural killer cells and regulatory T cells in non-small cell lung cancer

Even though the lung represents a special immune compartment with the capacity of a high inflammatory response, ineffective anti-tumour immunity is common in lung-associated malignancies. We asked whether a differential composition of the immune cell infiltrate in malignant (MLTAs) and non-malignant lung tissue areas (N-MLTAs) exists and might potentially contribute to this effect. We performed a comparative analysis of immune cells residing in MLTAs and N-MLTAs of non-small cell lung cancer (NSCLC) patients. To this end, we used immunophenotyping and functional analyses on directly isolated immune cells and tissue arrays on archived paraffin-embedded specimens. A strong T cell infiltration was prominent in both tissue compartments whereas CD4(+)CD25(+)CD127(-) T regulatory cells were present in MLTAs only. Nonetheless, concurrent functional ex vivo T cell analyses revealed no significant difference between T cells of MLTA and N-MLTA, suggesting that tumour-infiltrating T cells were not functionally impaired. Interestingly, T cell infiltration was less pronounced in specimens with a high neutrophilic infiltrate. NK cell infiltration was strikingly heterogenous between MLTA and N-MLTA. While NK cells were almost absent in the malignant tissue regions, non-malignant counterparts were selectively populated by NK cells and those NK cells showed strong cytotoxic activity ex vivo. We report that malignant and non-malignant tissue areas in NSCLC are selectively infiltrated by certain immune cell types with NK cells being displaced from the tumour tissue. These phenomena have important implications for tumour immunology of NSCLC and should be considered for the development of future immunologic intervention therapies.     

Inhibition of melanoma growth after treatment with dendritic cells in a Tyr-SV40E murine model requires CD4+ T cells but not CD8+ T cells

Melanomas are promising targets for immunotherapy, as they express a number of tissue-specific antigens against which immune responses can be elicited. We have previously described transgenic mice in which malignant cutaneous melanomas are produced. The 1042 melanoma cell line, derived from a primary melanoma in one of these mice, was used here to generate tumours by subcutaneous inoculation in syngeneic animals. All mice injected with 1 x 10(6) cells of the 1042 cell line developed a tumour. CD4+ T cells, CD8+ T cells and macrophages infiltrated the tumours. Treatment with dendritic cells pulsed with peptides from melanogenic proteins slowed tumour growth and resulted in increased numbers of infiltrating lymphocytes and macrophages, expansion of CD4+ T cells specific for 1042 cell antigens, and increased levels of 1042-specific immunoglobulin G1 (IgG1) and IgM in serum. The frequency of cytotoxic T lymphocytes (CTLs) specific for the MART-1 melanocytic antigen did not increase after dendritic cell treatment. Indeed, the presence of CD8+ T cells was apparently not required for the anti-tumour effects: slowing of tumour growth was not abrogated in animals depleted of CD8+ T cells using antibodies, or in syngeneic CD8-/- animals. In contrast, treatment with dendritic cells + peptides was ineffective after depletion of CD4+ T cells and in syngeneic CD4-/- mice. This experimental system therefore provides an opportunity to investigate CD4-dependent anti-tumour effector mechanisms, and for studies designed to activate the quiescent CTLs which infiltrate melanomas.     

Dendritic cell density and activation status of tumour-infiltrating lymphocytes in metastatic human melanoma: possible implications for sentinel node metastases

Nodal deposits of melanoma may present many years after resection of the primary tumour, implying initial suppression of tumour growth with subsequent immune escape. Using immunocytochemical techniques on frozen sections, the cellular types and activation status of infiltrating cells within a series of 19 clinically apparent nodal metastases of melanoma were studied. Infiltrating cells were assessed using a semiquantitative grading system. Macrophages (CD68+) and T-lymphocytes (CD3+) (including both CD8+ and probably also CD4+ T-cells) were the predominant cells infiltrating the tumours. B-lymphocytes (CD20+) were generally present in low numbers. CD1a+ putative dendritic cell density and expression of the early lymphocyte activation markers interleukin-2 receptor alpha (IL2Ralpha) and CD69 was low. However, greater evidence of intermediate lymphocyte activation (CD38) was identified. Expression of interleukin-2 (IL2) by tumour-infiltrating cells was not detected. The paucity of staining for IL2 and IL2Ralpha, with greater expression of CD38 by infiltrating cells, suggests that the usual pathways of lymphocyte activation via IL2 were bypassed or impaired within the lymph node metastases. Low numbers of CD1a+ putative dendritic cells may result in reduced effector cell activation. These findings provide evidence to support the hypothesis that antitumour immune responses within clinically involved lymph nodes are reduced in metastatic melanoma. This also has possible implications for micrometastases to the sentinel lymph node.     

Prognostic value of arginase‐II expression and regulatory T‐cell infiltration in head and neck squamous cell carcinoma

Tumor‐infiltrating lymphocytes are present in a variety of tumors and play a central role in antitumor immune responses. Nevertheless, most cancers progress probably because tumors are only weakly immunogenic and develop multiple immunosuppressive mechanisms. In the present study, on head and neck squamous cell carcinoma, we found high intraepithelial infiltration of regulatory FOXP3⁺ T cells, and relatively high levels of BDCA2⁺ and FOXP3⁺ cells in stromal (peripheral) regions of the tumors. Tumor‐infiltrating (intraepithelial) FOXP3⁺ T cells were significantly more frequent in patients with oropharynx and oral cavity squamous cell carcinoma and in patients without lymph node metastasis. Furthermore, arginase‐II (ARG2) was expressed by 60%, inducible nitric oxide synthetase by 9%, cyclooxygenase‐2 by 43%, and B‐cell lymphoma 2 (BCL2) by 26% of tumors. Interestingly, the absence of ARG2 expression, enhanced stromal infiltration of CD11c⁺ myeloid dendritic cells, and high numbers of FOXP3⁺ T cells were each significantly associated with prolonged overall survival, and the latter two parameters were also confirmed by multivariate analysis. For disease‐free survival, multivariate analysis revealed significant negative correlations with BCL2 and ARG2 expression by tumor cells. These findings shed new light on mechanisms of cancer progression, and provide rationales for therapeutic inhibition of immunosuppressive mechanisms in head and neck squamous cell carcinoma.     

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.     

Stromal infiltration of CD8 T cells is associated with improved clinical outcome in HPV-positive oropharyngeal squamous carcinoma

Background:

Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) have a better prognosis than those with HPV-negative tumours. There is interest in de-escalating their treatment but strategies are needed for risk stratification to identify subsets with a poor prognosis. This study investigated tumour-infiltrating lymphocytes (TILs) in relation to HPV tumour status and patient survival.

Methods:

Biopsies from 218 patients diagnosed with OPSCC between 2002 and 2011, who underwent chemo/radiotherapy were analysed for HPV by PCR, in-situ hybridisation and p16 immunohistochemistry (IHC). One hundred and thirty-nine samples with concordant HPV detection were analysed for CD3, CD4, CD8 and FoxP3 expression in tumour and stromal regions using multiplexIHC and multispectral image analysis. Labelling of smooth muscle actin (SMA) identified activated stroma.

Results:

Human papillomavirus-positive compared with HPV-negative OPSCC had higher infiltration in both tumour and stromal areas of CD4 and CD8 T cells but not FoxP3 T regulatory cells. Only CD3+CD8+ stromal and not tumour area infiltration was associated with increased survival (P=0.02). There was significantly higher SMA expression in HPV-positive compared with -negative tumours, which did not correlate with survival.

Conclusions:

Studies of TILs for risk stratification in OPSCC should assess stromal infiltration.     

Intratumoral CD8+ T lymphocytes as a prognostic factor of survival in endometrial carcinoma.

PURPOSE: CTLs are a prominent immune component infiltrating many solid tumors. These cells are considered to be a manifestation of host-immune response to the tumor; however, their prognostic significance remains a subject of considerable debate. The objective of this study was to evaluate the distribution pattern and prognostic value of CD8(+) T cells in endometrial carcinoma. EXPERIMENTAL DESIGN: We studied 90 cases of endometrial carcinoma, including 75 endometrioid and 15 papillary serous carcinomas. Immunohistochemical staining for CD8 and granzyme B was performed on paraffin-embedded sections. The number of immunohistochemically staining CD8(+) T cells was enumerated in the following four regions: lymphocytes infiltrating the tumor epithelium at the invasive border, within the underlying tumor stroma, within the superficial tumor epithelium, and in the perivascular areas of the myometrium. RESULTS: Patients with >10 CD8(+) T lymphocytes/high-power field within the tumor epithelium at the invasive border displayed improved overall survival compared with patients with fewer intraepithelial CD8(+) T lymphocytes (87 and 50%, respectively; P = 0.027). Multivariate analysis revealed that stage, vascular invasion, grade, and the number of intraepithelial CD8(+) T lymphocytes at the invasive border were the only independent predictors of survival (P < 0.0001, P = 0.001, P = 0.011, and P = 0.025, respectively). Granzyme B(+) cytoplasmatic granules were detected in a high proportion of CTLs, confirming their activated cytotoxic phenotype. CONCLUSIONS: Our study demonstrates for the first time that increased numbers of CTLs at the invasive border may be a reliable independent prognostic factor of survival in patients with endometrial carcinoma.