The ability of cultured human fibroblasts to reorganize and contract three dimensional collagen I gels is regarded as an in vitro model for the reorganization of connective tissue during wound healing. We investigated whether adhesion receptors of the integrin family are involved. It was found that synthesis and transcription of the alpha 2 beta 1 integrin (but not of alpha 1 beta 1 or alpha 3 beta 1) is selectively upregulated when fibroblasts are seeded into type I collagen gels. Time course experiments revealed that high synthetic levels of alpha 2 beta 1 parallel the gel contraction process and return to "baseline" levels after the contraction has subsided. Furthermore, function-blocking mAbs directed to the alpha 2 and beta 1 chain of integrins inhibited gel contraction. Remodelling of connective tissue can be important for tumor cells during invasion and formation of metastases. Therefore, we tested human melanoma cell lines for this function. Five out of nine melanoma lines contracted collagen gels in vitro. Among these, two highly aggressive melanoma cell lines (MV3 and BLM) most efficiently contracted gels almost reaching the rate of normal adult fibroblasts. In these cells, synthesis of alpha 2 beta 1 was also significantly upregulated when seeded into collagen I gels. Moreover, function blocking anti-alpha 2 in conjunction with anti-beta 1 chain mAbs completely inhibited gel contraction for several days. Other melanoma cells (530) with lower metastatic potential which were not able to contract gels, showed no induction of alpha 2 beta 1 synthesis in gel culture. Our results suggest an important role of integrin alpha 2 beta 1 in the contraction of collagen I by normal diploid fibroblasts during wound healing and in the reorganization of collagen matrices by highly aggressive human melanoma cells.
Here, it is established that human peripheral blood lymphocytes (HuPBLs), injected s.c. with a human lung tumor into severe combined immunodeficient (SCID) mice, engraft and display antitumor cytotoxic activity. Initial studies used HuPBLs from normal donors and an allogeneic tumor cell line derived from biopsy tissue of a patient with a squamous cell carcinoma of the lung. Evidence of HuPBL antitumor activity was revealed by a cell dose-dependent suppression of the tumor xenograft. Tumor suppression was shown to be dependent upon both CD8+ T cells and CD56+ natural killer cells in the donor HuPBLs. By titrating the antitumor activity of HuPBLs in SCID mice with and without cytokines, it was established that interleukin (IL)-12 enhanced the HuPBL-mediated tumor suppression and that IL-2 had a synergistic effect upon the IL-12 enhancement of cytotoxicity. Subsequent studies revealed that a lung cancer patient's PBLs also suppress the growth of the patient's (autologous) tumor when coinjected s.c. with the tumor cells into SCID mice. The patient's antitumor immunity was shown to be mediated by CD8+ T cells and CD56+ natural killer cells. The data presented here indicate that the s.c. coengraftment of HuPBLs and tumor into SCID mice represents a viable model with which to study (and to periodically monitor) patients' immune responses to their tumors for extended periods of time and suggest that this SCID/Winn assay could be used to evaluate novel immunotherapeutic approaches, such as bolus injections of cytokines, cytokine gene therapy, or vaccination strategies for the treatment of human cancer.
Abstract Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor responses. However, achieving local expression of cytokines at disseminated sites is challenging and systemic delivery has significant toxicity. We describe a novel approach employing an activatable fusion protein (FP) to reduce the negative side-effects of systemic delivery of the cytokine but which can preferentially affect all tumor sites. This FP consists of a cytokine linked to an inhibitory single chain antibody fragment (scFv) separated by a specific protease cleavage sequence. Importantly, the scFv does not bind to the tumor but rather binds to the cytokine keeping it inactive. However, at the tumor site, the FP can be cleaved by proteases such as Matrix metalloproteases (MMPs) that are over-expressed at tumor sites. This cleavage allows the release of the cytokine from the scFv and its binding to higher affinity receptors on immune cells. We will present data on the construction, expression, and characterization of a panel of murine and human IL-12 FPs. Using phage display, we have identified a panel of 15 scFv that bind to epitopes of murine IL-12 on either the P35 or P40 subunit as well as scFv that bind to human IL-12. Functional and biochemical data on baculovirus expressed IL-12 FPs will be presented. This strategy has the potential to reduce the negative side effects associated with systemic delivery of constitutively active cytokines yet retain their efficacy.
Tumor cells, injected s.c., were maintained until spontaneous metastases to the lungs were established in all of the mice. Mice were then treated with a single dose of cytokine-encapsulated biodegradable microspheres injected directly into primary s.c. tumors to achieve a local and sustained release of interleukin 12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), or a combination of these cytokines to the tumor microenvironment. The s.c. tumors were surgically excised 6 days after microsphere injections, and the mice were monitored for recurrence of the primary tumor, survival, and progression of metastatic disease. Combined neoadjuvant treatment with IL-12 and GM-CSF microspheres was superior to all other treatments in reducing the recurrence of primary tumors, enhancing postoperative survival, and suppressing established metastatic disease. Long-term survival analysis demonstrated that intratumoral injection of IL-12 + GM-CSF-loaded microspheres resulted in the complete cure of disseminated disease in the majority of the animals. The addition of systemic low-dose IL-2 therapy to the treatment protocol resulted in the loss of the antitumor activity induced by IL-12 + GM-CSF treatment. In vivo lymphocyte subset depletions established that both T- and natural killer-cell subsets were required for the suppression of primary and metastatic tumors. Long-term, tumor-specific T-cell activity was demonstrated by immunohistochemical analysis of metastatic lesions, IFN-gamma enzyme-linked immunosorbent spot (ELISPOT) assays and tumor challenge studies. These results establish that neoadjuvant in situ tumor immunotherapy with IL-12 + GM-CSF microspheres induces both innate and adaptive antitumor immune responses resulting in the eradication of disseminated disease.
Abstract A combination rosette-plaque assay was used to demonstrate that most (92%) of the plaque-forming cells (PFC) bound hapten to their surface 5 to 8 days after one injection of 3-nitro-4-hydroxy-5-iodophenyl acetic acid (NIP) coupled to diphtheria toxoid, (NIP-TOX). There was a significant decrease in hapten binding by these cells later in the primary response. Rosette formation was observed when NIP-TOX-primed lymph node cells bound NIP-coupled sheep red blood cells stabilized with pyruvic aldehyde (NIP/PASRBC). This suspension of cells was then plaqued on fresh NIP-coupled sheep red blood cells (NIP/SRBC). A rosetted lymph node cell in the center of a plaque clearly demonstrated simultaneous hapten binding and anti-hapten antibody secretion. The specificity of hapten binding and antibody secretion was established by rosette and plaque inhibition with NIP coupled to bovine serum albumin (NIP-BSA). The specificity of each was further characterized by comparative inhibition with analogues of NIP (also coupled to BSA): 3,5-dinitro-4-hydroxyphenyl acetic acid (NNP-BSA), 3,5-diiodo-4-hydroxyphenyl acetic acid (DIP-BSA) and 3-nitro-4-hydroxyphenyl acetic acid (NP-BSA). The homologous hapten NIP was always the best inhibitor of plaques (97.8% inhibition) and rosettes (96.2% inhibition). NNP was always a better plaque and rosette inhibitor than either DIP or NP. This hierarchy of inhibition, and the correlation between rosette and plaque inhibition demonstrated the similarity of receptor and antibody specificity.
Abstract Regulatory T cells (TR) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ TR that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These TR actively suppress both the proliferation of autologous nodal CD8+CD25− and CD4+CD25− T cells, as well as cytokine production (IFN-γ, TNF-α and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these TR are also capable of suppressing the proliferation of allogeneic CD8+CD25− and CD4+CD25− T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25− and CD4+CD25− T cells, suggesting that FLN TR are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-β signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-β in FLN TR-mediated suppression.
