Targeted regulation of a lymphocyte-endothelial-interleukin-6 axis by thermal stress

Immune protection from microbial invaders or malignant progression is dependent on the ability of lymphocytes to efficiently traffic across morphologically and biochemically distinct vascular sites throughout the body. Lymphocyte trafficking to target tissues is orchestrated by adhesion molecules and chemokines that stabilize dynamic interactions between circulating lymphocytes and endothelial cells lining blood vessels. While the molecular mechanisms that regulate the efficient migration of lymphocytes across specialized high endothelial venules (HEVs) in secondary lymphoid organs have been extensively characterized, there is a paucity of information available regarding the mechanisms that dictate the rate of lymphocyte entry into tumor tissues. This article summarizes recent evidence that inflammatory cues associated with fever-range thermal stress promote lymphocyte extravasation across HEVs of lymphoid organs through a highly regulated lymphocyte–endothelial–interleukin-6 (IL-6) biological axis. The potential for using thermally-based strategies to improve lymphocyte delivery to the tumor microenvironment during T cell-based immunotherapy will also be discussed.     

Cancer cells regulate lymphocyte recruitment and leukocyte-endothelium interactions in the tumor-draining lymph node

The physiologic function of the secondary lymphoid organs to recruit large numbers of na?ve lymphocytes increases the probability that antigens encounter their rare, sometimes unique, specific T lymphocytes and initiate a specific immune response. In peripheral lymph nodes (LNs), this recruitment is a multistep process, initiated predominantly within the high endothelial venules (HEVs), beginning with rolling and chemokine-dependent firm adhesion of the lymphocytes on the venular endothelium surface. We report here that, in C57BL/6 mice, the recruitment of na?ve lymphocytes is impaired in LNs draining a B16 melanoma tumor. Intravital microscopy analysis of the tumor-draining LNs revealed that this effect is associated with an important defect in lymphocyte adhesion in the HEVs and a progressive decrease in the expression of the LN chemokine CCL21. In parallel with these effects, the tumor up-regulated, essentially through a P-selectin-dependent mechanism, the rolling and sticking of circulating polymorphonuclear cells within the LN low-order venules where few rolling and sticking events are usually observed. These effects of the tumor were independent of the presence of metastasis into the LN and occurred as long as the tumor developed. Together, these results indicate that the tumor proximity disturbs the LN physiology by modifying the molecular, spatial, and cellular rules that usually control leukocyte-endothelium interactions into the peripheral LNs. In addition, they emphasize a new role for the low-order venules of the peripheral LNs, which compared with the HEVs, seem to be the preferential port of entry for cells linked to inflammatory processes.     

Neogenesis of Lymphoid Structures and Antibody Responses Occur in Human Melanoma Metastases

Lymphoid neogenesis, or the development of lymphoid structures in nonlymphoid organs, is frequently observed in chronically inflamed tissues, during the course of autoimmune, infectious, and chronic graft rejection diseases, in which a sustained lymphocyte activation occurs in the presence of persistent antigenic stimuli. The presence of such ectopic lymphoid structures has also been reported in primary lung, breast, and germline cancers, but not yet in melanoma. In this study, we observed ectopic lymphoid structures, defined as lymphoid follicles comprising clusters of B lymphocytes and follicular dendritic cells (DC), associated with high endothelial venules (HEV) and clusters of T cells and mature DCs, in 7 of 29 cutaneous metastases from melanoma patients. Some follicles contained germinal centers. In contrast to metastatic lesions, primary melanomas did not host follicles, but many contained HEVs, suggesting an incomplete lymphoid neogenesis. Analysis of the repertoire of rearranged immunoglobulin genes in the B cells of microdissected follicles revealed clonal amplification, somatic mutation and isotype switching, indicating a local antigen-driven B-cell response. Surprisingly, IgA responses were observed despite the nonmucosal location of the follicles. Taken together, our findings show the existence of lymphoid neogenesis in melanoma and suggest that the presence of functional ectopic lymphoid structures in direct contact with the tumor makes the local development of antimelanoma B- and T-cell responses possible. Cancer Res; 72(16); 3997-4007. ?2012 AACR.     

Alterations of high endothelial venules in primary and metastatic tumors are correlated with lymph node metastasis of oral and pharyngeal carcinoma

High endothelial venules (HEVs) are special blood vessels in the paracortical region of lymph nodes (LNs) and govern lymphocyte recruitment. LN metastasis has similarity to circulating lymphocytes homing to LNs, but the role of HEVs in the progression of oral and pharyngeal squamous cell carcinoma (OPSCC) is unclear. In this study, we found that HEVs experienced a series of morphological and functional changes during OPSCC progression and were correlated with LN metastasis. In 9 cases of 73 metastatic LNs, tumor emboli were located adjacent to HEVs or just out of the vessels but not lymphatic channels. Gap junctions of tumor cells close to HEVs decreased or disappeared, and gaps were left at contact points where tumor cells attached to the HEVs. Moreover, the proliferation rate of endothelial cells of HEVs was the highest in metastatic LNs. Finally, L-selectin was detected in both primary and metastatic tumors, and it facilitated tumor cells adhering to LNs. In conclusion, our findings suggest that remodeled HEVs are correlated with LN metastasis of OPSCC and play important role in this process by preparing premetastatic soil for cancer cell metastasis.     

Neogenesis and development of the high endothelial venules that mediate lymphocyte trafficking

Physiological recruitment of lymphocytes from the blood into lymph nodes and Peyer’s patches is mediated by high endothelial venules (HEV), specialized blood vessels found in secondary lymphoid tissues except for the spleen. The HEV are distinguished from other types of blood vessels by their tall and plump endothelial cells, and by their expression of specific chemokines and adhesion molecules, which all contribute to the selective lymphocyte trafficking across these blood vessels. The development of HEV is ontogenically regulated, and they appear perinatally in the mouse. High endothelial venules can appear ectopically, for instance in chronically inflamed tissues. Given that HEV enable the efficient trafficking of lymphocytes into tissues, the induction of HEV at a tumor site could potentiate tumor‐specific immune responses, and the artificial manipulation of HEV neogenesis might thus provide a new tool for cancer immunotherapy. However, the process of HEV development and the mechanisms by which the unique features of HEV are maintained are incompletely understood. In this review, we discuss the process of HEV neogenesis and development during ontogeny, and their molecular requirements for maintaining their unique characteristics under physiological conditions. (Cancer Sci 2010; 101: 2302–2308)     

Cytokine and adhesion molecule expression in primary human endothelial cells stimulated with fever-range hyperthermia.

Migration of blood-borne lymphocytes into lymphoid tissues and sites of inflammation is initiated by vascular adhesion molecules and proinflammatory cytokines. Previous in vivo studies have shown that febrile temperatures dynamically stimulate adhesion in differentiated high endothelial venules (HEV), which are portals for lymphocyte extravasation. This report examines the direct effect of fever-range hyperthermia on the expression of adhesion molecules and cytokines by primary cultured endothelial cells. In both macrovascular (HUVEC) and microvascular (HMVEC) endothelial cells, fever-range hyperthermia (40 degrees C for 6-12 h) did not affect expression of adhesion molecules (ICAM-1, E-selectin, VCAM-1, P-selectin, PECAM-1, PNAd, MAdCAM-1), cytokine release (IL-1beta, TNF-alpha, IFN-gamma, IL-6, IL-11, IL-12, IL-13), or chemokine secretion (IL-8, RANTES, MCP-1, MIP-1beta, MIG). This is in contrast to the stimulatory effects of TNF-alpha or 43 degrees C heat shock. However, a novel role for fever-range hyperthermia was identified in augmenting actin polymerization in cultured endothelial cells and enhancing the ability of endothelial-derived factors to transactivate the alpha4beta7 integrin lymphocyte homing receptor. These findings provide insight into the tightly regulated effects of fever-range hyperthermia that exclude induction of adhesion in non-activated endothelium of normal blood vessels. Through these mechanisms, it is proposed that febrile temperatures associated with infection or clinical hyperthermia avoid the unproductive exodus of lymphocytes to non-involved extralymphoid tissues while simultaneously promoting lymphocyte delivery to sites of immune activation.     

Studies of the mechanisms for the induction of in vivo tumor immunity. II. Distribution and homing of cytotoxic effector and precursor cells.

Cytotoxic T (thymus)-lymphocytes (CTL) with specific cytotoxicity against the leukemia-associated antigens of FBL-3, a syngeneic Friend virus-induced leukemia in C57BL/6 mice, could be adoptively transferred to sublethally X-irradiated (350 R) syngeneic hosts and could be induced by adoptive transfer of either normal or presensitized lymphocytes obtained from immunocompetent hosts. The CTL and their precursor cells were systemically distributed in peripheral lymph nodes and spleen, although they had the tendency of homing to the lymphoid tissue of the same origin. Direct cytotoxicity was obtained with the lymphocytes from these lymphoid tissues, and cells obtained from these lymphoid tissues could produce secondary cytotoxic responses by the mixed lymphocyte tumor cell culture reactions 40--60 days after adoptive transfer. In addition, lymph node and spleen cells had a synergistic effect on the induction of cytotoxicity. These findings indicated that tumor immunity was widely distributed and that various populations of lymphocytes were involved in the generation of efficient cell-mediated cytotoxic responses.     

Cytokine and adhesion molecule expression in primary human endothelial cells stimulated with fever-range hyperthermia

Migration of blood-borne lymphocytes into lymphoid tissues and sites of inflammation is initiated by vascular adhesion molecules and proinflammatory cytokines. Previous in vivo studies have shown that febrile temperatures dynamically stimulate adhesion in differentiated high endothelial venules (HEV), which are portals for lymphocyte extravasation. This report examines the direct effect of fever-range hyperthermia on the expression of adhesion molecules and cytokines by primary cultured endothelial cells. In both macrovascular (HUVEC) and microvascular (HMVEC) endothelial cells, fever-range hyperthermia (40°C for 6-12h) did not affect expression of adhesion molecules (ICAM-1, E-selectin, VCAM-1, P-selectin, PECAM-1, PNAd, MAdCAM-1), cytokine release (IL-1 &#103 , TNF- &#102 , IFN- &#110 , IL-6, IL-11, IL-12, IL-13), or chemokine secretion (IL-8, RANTES, MCP-1, MIP-1 &#103 , MIG). This is in contrast to the stimulatory effects of TNF- &#102 or 43°C heat shock. However, a novel role for fever-range hyperthermia was identified in augmenting actin polymerization in cultured endothelial cells and enhancing the ability of endothelial-derived factors to transactivate the &#102 4 &#103 7 integrin lymphocyte homing receptor. These findings provide insight into the tightly regulated effects of fever-range hyperthermia that exclude induction of adhesion in non-activated endothelium of normal blood vessels. Through these mechanisms, it is proposed that febrile temperatures associated with infection or clinical hyperthermia avoid the unproductive exodus of lymphocytes to non-involved extralymphoid tissues while simultaneously promoting lymphocyte delivery to sites of immune activation.     

Extravasation of antitumor effector cells.

Development of effective local immune responses depends on the ability of lymphocytes to extravasate and migrate into nonlymphoid solid tissues. Different lymphocyte subpopulations seem to vary in their abilities to extravasate. In this review, recent advances made in understanding lymphocyte extravasation, interactions between lymphocytes and vascular endothelial cells, receptors on lymphocytes which are involved in their movement through the extracellular matrix, and cytokines, which regulate lymphocyte mobility through tissue, are described. In pathologic conditions, lymphocyte extravasation may be compromised, and delivery of immune effector cells to the site of injury is an important therapeutic end point. Adoptive therapy of solid tumors with antitumor effector cells depends on their successful delivery to the tumor. Although parameters which determine this delivery are not yet defined, considerable progress has been made in studies of the mechanisms involved in lymphocyte movement through tissues.     

Principles of tumor immunosurveillance and implications for immunotherapy

Although antigen loss variants, major histocompatibility (MHC) class I down-regulation, or the expression of inhibitory molecules may explain the failure of immunosurveillance against some tumors, this seems not to apply for many other solid peripheral or lymphohematopoietic tumors. Why then is immunosurveillance so ineffective and can it be improved? This review focuses on one important aspect of tumor immunity, namely the relevance of antigen dose and localization. Immune responses in vivo are induced in organized lymphoid tissues, i.e., in lymph nodes and spleen. The antigen dose that reaches secondary lymphoid organs over time is a crucial parameter that drives antiviral and antitumoral immune responses. Tumors use various strategies to prevent efficient presentation of their antigens in lymphoid organs. A major obstacle to the induction of an endogenous tumor-specific cytotoxic T lymphocyte (CTL) response is the inefficient presentation of tumor antigen on MHC class I molecules of professional antigen-presenting cells. Peripheral solid tumors that develop outside lymphoid organs are, therefore, often ignored by the immune system. In other situations, tumors - especially of lymphohematopoietic origin - may tolerize specific CTLs. Understanding tumor immunosurveillance is key to the design of efficient antitumor vaccines. Attempts to improve immunity to tumors include vaccination strategies to (a) provide the tumor antigen to secondary lymphoid organs using recombinant viruses or dendritic cells as carriers, (b) express costimulatory signals on tumor cells, or (c) improve the efficiency of cross-priming.     

Thermal regulation of lymphocyte trafficking: hot spots of the immune response.

Lymphocytes use extensive vascular networks to traffic to various destinations in the body, including lymphoid organs and extra-lymphoid tissues. This discussion will focus on the emerging evidence that thermal stress regulates the traffic signals that direct the exit of lymphocytes from the vascular freeway. This issue is particularly relevant to T cell-based cancer immunotherapy where delivery of immune effector lymphocytes to neoplastic lesions depends on their extravasation across tumour micro-vessels. Although tumours are frequently highly vascularized by vessels that are competent to support blood flow, the tumour micro-environment has been characterized as non-permissive to lymphocyte extravasation. This may lead to a scenario where limited leukocyte infiltration at tumour sites correlates with a poor prognosis. These observations support the thesis that adjuvant strategies that promote trafficking of tumour-reactive cytolytic leukocytes to tumour sites have the potential to improve the efficacy of immune-based cancer therapy.     

Thermal regulation of lymphocyte trafficking: Hot spots of the immune response

Lymphocytes use extensive vascular networks to traffic to various destinations in the body, including lymphoid organs and extra-lymphoid tissues. This discussion will focus on the emerging evidence that thermal stress regulates the traffic signals that direct the exit of lymphocytes from the vascular freeway. This issue is particularly relevant to T cell-based cancer immunotherapy where delivery of immune effector lymphocytes to neoplastic lesions depends on their extravasation across tumour micro-vessels. Although tumours are frequently highly vascularized by vessels that are competent to support blood flow, the tumour micro-environment has been characterized as non-permissive to lymphocyte extravasation. This may lead to a scenario where limited leukocyte infiltration at tumour sites correlates with a poor prognosis. These observations support the thesis that adjuvant strategies that promote trafficking of tumour-reactive cytolytic leukocytes to tumour sites have the potential to improve the efficacy of immune-based cancer therapy.     

Uridine kinase activities in normal and neoplastic lymphoid cells.

Both adult (I) and embryonic (II) forms of uridine kinase have been identified in the transplantable EL-4 leukemia of C57BL/6 mice and in the P815Y mastocytoma of DBA/2 mice. Only Species I is found in primary tumor cells of lymphoid orgin (virus-induced feline lymphosarcoma, human acute and chronic lymphocytic leukemia) and in normal calf thymocytes and porcine peripheral blood lymphocytes; Species I was induced 4-fold upon stimulation of the normal blood lymphocytes with phytohemagglutinin. The level of uridine kinase activity in the feline lymphosarcoma of thymus-dependent lymphocyte orgin and childhood lymphocytic leukemia of possible thymus-dependent lymphocyte or null-cell origin was similar to the induced level in phytohemagglutinin-stimulated normal lymphocytes, i.e., thymus-dependent lymphocytes. In contrast lymphocytes of a patient with chronic lymphocytic leukemia of thymus-independent lymphocyte origin had a level of uridine kinase activity comparable to that of the unstimulated normal lymphocytes or thymocytes. The uridine kinase activity in the EL-4 tumor cells was repressed by acute treatment of the mice with 5-azacytidine.     

趋化因子CCL20与恶性肿瘤之间的关系

趋化因子是一类控制细胞向炎性部位迁移的细胞因子,在调控免疫细胞分化、发育及定向迁移过程中起重要作用。CCL20是趋化因子家族中的重要成员之一,属于CC亚族,其受体为CCR6。CCL20在被激活的单核细胞、T细胞、树突状细胞及内皮细胞中表达;CCR6主要在肝、肺及淋巴组织中表达。CCL20的表达可被肿瘤坏死因子α(tumor necrosis factorα,TNFα)、白细胞介素1β(interleukin1β,IL-1β)、IL-17、CD40配体和干扰素γ(interferonγ,IFN-γ)等细胞因子诱导,在恶性肿瘤的生长及促进肿瘤细胞的侵袭和转移(主要是肝内转移)中发挥重要作用。     

Immunohistochemical detection of high endothelial venules in extranodal B-cell lymphomas

The hypothesis that extranodal malignant lymphomas, such as thyroid, stomach, rectum, ileum and uterus, eventuate from long-standing inflammation is essentially tested using a monoclonal antibody (HECA-452) to an endothelial-associated antigen selectively expressed by the high endothelim of post-capillary venules (HEVs) normally present in lymph nodes, tonsils and mucosa-associated lymphoid tissues. The presence of HEVs was examined on freshly prepared specimens from 16 cases of extranodal lymphoma of the B-cell type (eight thyroid, three stomach, two rectum, one ileum, one uterus, one tonsil). Control cases included reactive lymph nodes, nodal lymphoma of T-cell type, epithelial neoplasia, bone and soft tissue sarcomas, glioblastoma, thyroid affected by chronic lymphocytic thyroiditis (CLTH), thyrotoxicosis (Grave's disease) or nodular goiter, and salivary gland affected by Sjogren's syndrome (SS). HECA+ HEVs were present in reactive lymph nodes, nodal lymphoma, and all but one case of thyroid, all cases of stomach and all cases of rectal lymphoma, together with all CLTH and SS, both known to be autoimmune diseases. No thyroid, ileal or uterine lymphoma cases had any HECA positive venules. HECA+ venules were present in extranodal lymphomas, either adjoining the lymphoid follicles observed in several cases or in the diffusely proliferating tumor cells. Previous studies have shown that HEVs appeared in association with long-standing inflammation. The present study therefore, suggested that extranodal lymphomas of the thyroid, stomach and rectum evolve in long-standing inflammation, forming HEVs.     

Systemic distribution and tumor localization of adoptively transferred lymphocytes in mice: comparison with physiologically based pharmacokinetic model

The mechanisms by which tumors are able to evade cellular immune responses are still largely unknown. It is likely, however, that the initial recruitment of lymphocytes to tumor vessels is limited by cell retention in normal tissue, which results in a low flux of these cells into the tumor vasculature. We grew MCaIV (mouse mammary carcinoma) tumors in the leg of SCID mice and injected 111In-oxine-labeled, primed T lymphocytes directed against the tumor intravenously. The systemic distribution of cells in normal organs was similar between mice injected with primed and control lymphocyte populations, except for a delayed clearance of primed lymphocytes from the lungs. Kinetics of lymphocyte localization to the tumor were identical between the primed and control lymphocyte populations. Splenectomy before the injection of primed lymphocytes increased delivery of cells to the lungs and liver after 1 hour with no significant improvement in tumor localization. Within 24 to 168 hours after injection, localization of cells in the liver of splenectomized mice was higher than in the control group. However, no significant difference in tumor localization was observed between groups. A physiologically based compartmental model of lymphocyte distribution predicted the compartmental sequestration and identified model parameters critical for experimental planning and therapeutic optimization.     

A study of surface markers in non-Hodgkin's lymphoma by using anti-T and anti-B lymphocyte sera

Cell surface markers of 44 cases of non-Hodgkin's lymphoma (NHL) were studied with various surface markers, especially by using antihuman B lymphocyte serum (ABS), antihuman thymocyte serum (ATS-T), and antihuman peripheral T lymphocyte serum (ALS-T), which were rendered specific for human B lymphocytes, human thymocytes, and human peripheral T lymphocytes, respectively. An immunofluorescent study with ABS, ATS-T, and ALS-T enabled us to demonstrate the histologic localization of normal or neoplastic B and T cells in preserving the original structure of lymphoid organs or tumor tissues. The proportion of cell types in NHL was B cell type 59%, T1 (ATS-T reactive) type 7%, T2 (ALS-T reactive) type 23%, and Null (non T, non B) type 11%. The relationships among cell types, histologic findings, and clinical characteristics were also investigated. Patients with T1-NHL had mediastinal tumors, which were histologically classified into "Lymphoblastic lymphoma." These facts suggest that T1-NHL may have originated in the thymus. Patients with T2-NHL showed a high incidence of skin lesions. Median survival of ten patients with T1- and T2-NHL was seven months, which was much shorter than that of B- or Null-NHL.     

Lymphocyte homing receptors and adhesion molecules in intravascular malignant lymphomatosis

Intravascular malignant lymphomatosis (IML) is a highly malignant, recently recognized form of lymphoma. It is characterized by multifocal proliferation of malignant lymphocytes within small blood vessels, primarily in the central nervous system and skin, frequently resulting in circulatory disturbances. The cause of the impaired capability of the malignant lymphocytes to extravasate has remained unclear. We analyzed the presence of immunoreactivity for certain homing receptor and adhesion molecules associated with lymphocyte extravasation in 3 patients with this disease. Compared with non-neoplastic leukocytes, large malignant lymphocytes appeared either negative or only weakly positive for the leukocyte surface glycoprotein, CD18 that is the beta chain of the CDIIa/CD18 complex (lymphocyte-function associated antigen-I, LFA-I), which mediates cell-to-cell adhesion of lymphocytes. On the other hand, antibody to one of the proposed ligands for this complex, intercellular adhesion molecule-I, gave positive reactivity both on lymphocytes and on endothelial cells. Further, the malignant lymphoid cells stained positively with Hermes-3 antibody, which recognizes a common structure of CD44 class of molecules involved in lymphocyte homing. It was also shown that HECA-452 antigen, a marker of high endothelial venules (HEV) supporting lymphocyte extravasation, can be synthesized by an IML patient even at the site of inflammation but it is not prerequisite for extravasation of inflammatory lymphocytes. Our results suggest that the deficiency or absence of the adhesion molecule CDIIa/CD18 may contribute to the inability of the malignant lymphoid cells to extravasate in IML, and perhaps also to the high malignancy of this form of lymphoma.     

Lymphocytes and leukemia viruses: tropism and transtropism of murine leukemia virus.

The tropism of naturally occurring murine leukemia virus (MuLV) was investigated in short-term lymphocyte cultures. The tropism of MuLV was readily defined in fibroblast cultures, but not in lymphocyte cultures. Lymphocytes free of infectious MuLV could be infected across the tropism barrier by partially purified MuLv or by in vitro contact with MuLV-producing lymphocytes. Stimulation of lymphocytes was not required for this cross-infection and replication of MuLV. When cross-infected lymphocytes and was specifically associated with lymphocytes were stimulated in vitro by allogeneic cells, they facilitated MuLV infection of ordinarily non-permissive fibroblasts. This phenomenon (transtropism) required antigenically stimulated lymphocytes and was specifically associated with infection of the lymphocyte by MuLV across the tropism barrier. Thus in contrast with the resting lymphocyte, the transformed lymphocyte acquired the ability to disseminate infectious MuLV to nonpermissive cells. These findings suggest a novel relationship between lymphocytes and leukemia viruses. They indicate one mechanism whereby antigenic stimulation may enhance the development of virus-induced lymphoid neoplasms.