RESUMEN
A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8+ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4+ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1+ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8+ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.
Asunto(s)
Inmunohistoquímica/métodos , Melanoma/inmunología , Melanoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/inmunología , Citometría de Flujo , Humanos , Ipilimumab/inmunología , Ipilimumab/uso terapéutico , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor , Macrófagos/metabolismo , Melanoma/tratamiento farmacológico , Metastasectomía , Persona de Mediana Edad , Estudios Prospectivos , Estadísticas no Paramétricas , Linfocitos T Reguladores/inmunología , Escape del TumorRESUMEN
Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.
Asunto(s)
Sinapsis Inmunológicas/inmunología , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Complejo CD3 , Adhesión Celular , Muerte Celular , Línea Celular Tumoral , Biología Computacional , Citocinas/metabolismo , Dineínas/química , Ligandos , Activación de Linfocitos , Antígeno-1 Asociado a Función de Linfocito/inmunología , Ratones , Microtúbulos/metabolismo , Transducción de SeñalRESUMEN
The anti-tumor efficacy of adoptively transferred T cells requires their in vivo persistence and memory polarization. It is unknown if human chimeric antigen receptor (CAR)-expressing T cells can also undergo memory polarization. We examined the functional status of CAR CD8(+) T cells, re-directed to Lewis Y antigen (LeY-T), throughout a period of ex vivo expansion. Immediately before culture CD8(+) T cells comprised a mixture of phenotypes including naive (CD45RA(+)/CCR7(+)/CD27(+)/CD28(+)/perforin-), central memory (CM, CD45RA(-)/CCR7(lo)/CD27(+)/CD28(+)/perforin(lo)), effector memory (EM, CD45RA(-)/CCR7(-)/CD27(+)/CD28(+)/perforin(mod)) and effector (Eff, CD45RA(+)/CCR7(-)/CD27(-)/CD28(-)/perforin(hi)) cells. After transduction and expansion culture of peripheral blood mononuclear cells from normal donors or multiple myeloma patients, CD8(+) LeY-T cells polarized to EM- and CM-like phenotype. CD8(+) LeY-T cells differed from starting CD8(+) CM and EM T cells in that CD27, but not CD28, was downregulated. In addition, CD8(+) LeY-T cells expressed high levels of perforin, similar to starting CD8(+) Eff. CD8(+) LeY-T cells also showed hallmarks of both memory and Eff function, underwent homeostatic proliferation in response to interleukin (IL)-15, and showed interferon (IFN)-γ production and cytotoxicity in response to Le-Y antigen on OVCAR-3 (human ovarian adenocarcinoma) cells. This study confirms CD8(+) LeY-T cells have a CM- and EM-like phenotype and heterogeneous function consistent with potential to persist in vivo after adoptive transfer.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Memoria Inmunológica , Receptores de Antígenos/genética , Antígenos CD28/inmunología , Proliferación Celular , Humanos , Interferón gamma/metabolismo , Antígenos Comunes de Leucocito/inmunología , Fenotipo , Receptores de Antígenos/inmunología , Receptores de Antígenos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
We have evaluated the carbohydrate antigen Lewis(Y) (Le(Y)) as a potential target for T-cell immunotherapy of hematological neoplasias. Analysis of 81 primary bone marrow samples revealed moderate Le(Y) expression on plasma cells of myeloma patients and myeloblasts of patients with acute myeloid leukemia (AML) (52 and 46% of cases, respectively). We developed a retroviral vector construct encoding a chimeric T-cell receptor that recognizes the Le(Y) antigen in a major histocompatibility complex-independent manner and delivers co-stimulatory signals to achieve T-cell activation. We have shown efficient transduction of peripheral blood-derived T cells with this construct, resulting in antigen-restricted interferon-gamma secretion and cell lysis of Le(Y)-expressing tumor cells. In vivo activity of gene-modified T cells was demonstrated in the delayed growth of myeloma xenografts in NOD/SCID mice, which prolonged survival. Therefore, targeting Le(Y)-positive malignant cells with T cells expressing a chimeric receptor recognizing Le(Y) was effective both in vitro and in a myeloma mouse model. Consequently, we plan to use T cells manufactured under Good Manufacturing Practice conditions in a phase I immunotherapy study for patients with Le(Y)-positive myeloma or AML.
Asunto(s)
Terapia Genética/métodos , Inmunoterapia Adoptiva/métodos , Leucemia Mieloide Aguda/terapia , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Mieloma Múltiple/terapia , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/trasplante , Animales , Femenino , Vectores Genéticos , Humanos , Interferón gamma/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de Antígenos de Linfocitos T/inmunología , Retroviridae/genética , Linfocitos T/inmunología , Transducción GenéticaRESUMEN
There is considerable concern regarding the transforming potential of retroviral vectors currently used for gene therapy, with evidence that retroviral integration can lead to leukemia in recipients of gene-modified stem cells. However, it is not clear whether retroviral-mediated transduction of T cells can lead to malignancy. We transduced mouse T cells with a Moloney murine retroviral gene construct and transferred them into congenic mice, which were preconditioned to enhance the engraftment of transferred T cells. Recipients were then observed long-term for evidence of cancer. Transferred T cells persisted in mice throughout life at levels up to 17% with gene copy numbers up to 5.89 x 10(5) per million splenocytes. Mice receiving gene-modified T cells developed tumors at a similar rate as control mice that did not receive T cells, and tumors in both groups of mice were of a similar range of histologies. Hematological malignancies comprised approximately 60% of cancers, and the remaining cancers consisted largely of carcinomas. Importantly, the incidence of lymphomas was similar in both groups of mice, and no lymphomas were found to be of donor T-cell origin. This study indicates that the use of retroviral vectors to transduce T cells does not lead to malignant transformation.
Asunto(s)
Traslado Adoptivo , Terapia Genética/efectos adversos , Vectores Genéticos/administración & dosificación , Virus de la Leucemia Murina de Moloney/fisiología , Linfocitos T/virología , Animales , Transformación Celular Viral , Leucemia/virología , Linfoma/virología , Ratones , Ratones SCID , Virus de la Leucemia Murina de Moloney/genética , Neoplasias/inmunología , Neoplasias/patología , Linfocitos T/trasplante , Tiempo , Transducción Genética/métodos , TransgenesRESUMEN
Dendritic cells (DC) perform an important role in the initiation of the immune response through the local secretion of inflammatory mediators within diseased tissue in response to Toll-like receptor (TLR) ligation. However, DC vaccine strategies fail to make use of this capability against cancer. To harness the TLR response capability of DC against cancer, we tested a series of recombinant genes for their ability to redirect DC function specifically against a tumor-associated antigen. Each gene encoded a cell surface chimeric protein made up of extracellular single-chain immunoglobulin anti-erbB2 linked to an intracellular TLR-signaling component composed of either myeloid differentiation factor 88, interleukin-1 receptor-associated kinase-1 (IRAK-1) or the cytoplasmic domain of TLR4. Each gene was expressed in the DC line, JAWS II, to a similar degree following retroviral transduction. However, only the chimera containing IRAK-1 was able to mediate interleukin-12 and tumor necrosis factor-alpha secretion. Since TLR engagement can also activate DC and enhance their ability to stimulate T cells, we ligated the chimeric anti-erbB2-IRAK-1 receptor and determined the effect on the stimulation of T cells. We found that JAWS II cells triggered through chimeric anti-erbB2-IRAK-1 displayed an enhanced ability to stimulate ovalbumin-specific OT-II CD4(+) T cells. This first description of the generation of tumor-reactive DC may lead to the development of new cell-based vaccines that can act at both the tumor site to induce danger and at the lymph node to stimulate a specific T-cell response.
Asunto(s)
Células Dendríticas/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal/genética , Receptores Toll-Like/metabolismo , Animales , Línea Celular Tumoral , Células Dendríticas/inmunología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Activación de Linfocitos , RatonesRESUMEN
Activation and expansion of T cells are important in disease resolution, but tumors do not usually satisfy these immune requirements. Therefore, we employed a novel strategy whereby dual-specific T cells were generated that could respond to both tumor and influenza virus, reasoning that immunization with influenza virus would activate and expand tumor-specific cells, and inhibit tumor growth. Dual-specific T cells were generated by gene modification of influenza virus-specific mouse T cells with a chimeric gene-encoding reactivity against the erbB2 tumor-associated antigen. Dual-specific T cells were demonstrated to respond against both tumor and influenza in vitro, and expanded in vitro in response to influenza to a much greater degree than in response to tumor cells. Following adoptive transfer and immunization of tumor-bearing mice with influenza virus, dual-specific T cells expanded greatly in numbers in the peritoneal cavity and spleen. This resulted in a significant increase in time of survival of mice. However, tumors were not eradicated, which may have been due to the observed poor penetration of tumor by T cells. This is the first demonstration that the potent immunogenic nature of an infectious agent can be utilized to directly impact on T-cell expansion and activity against tumor in vivo.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Neoplasias Mamarias Animales/terapia , Orthomyxoviridae/inmunología , Receptor ErbB-2/antagonistas & inhibidores , Linfocitos T/trasplante , Animales , Antígenos de Neoplasias/inmunología , Ratones , Ratones Endogámicos , Receptor ErbB-2/inmunología , Linfocitos T/inmunologíaRESUMEN
Genetic modification of human lymphocytes is being employed in strategies to correct enzyme deficiencies, encode cytokines and to redirect lymphocytes to antigenic targets other than those encoded by their endogenous T cell receptor. However, expression of transgenes in primary lymphocytes is generally low. Reasoning that vector modification may lead to increased transgene expression and subsequent increases in function, we have performed two retroviral vector modifications and report their effect on the functional expression in primary lymphocytes. A chimeric receptor specific for the colon carcinoma-associated antigen, EGP40, was initially incorporated into the retroviral vector LXSN. In this vector, receptor expression is driven by the Moloney murine leukemia virus LTR, and neomycin phosphotransferase expression driven by the SV40 promoter. Replacement of SV40 with an internal ribosomal entry site (IRES) increased the transgene activity of a mouse T cell line and human PBL as judged by increased cytokine release in response to antigen positive target cells. A further increase in transgene function was generated by the additional incorporation of a splice acceptor motif into the construct. Human PBL transduced with vector incorporating both IRES and intron were consistently more effective at lysing antigen positive colorectal carcinoma cells.
Asunto(s)
Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Neoplasias del Colon/patología , Terapia Genética/métodos , Vectores Genéticos/genética , Inmunoterapia/métodos , Adulto , Animales , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica , Molécula de Adhesión Celular Epitelial , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Inmunofenotipificación , Interferón gamma/biosíntesis , Ratones , Virus de la Leucemia Murina de Moloney/genética , Sitios de Empalme de ARN , Transducción Genética , Transgenes/genética , Células Tumorales CultivadasRESUMEN
Endogenous retroviral gene products have been found in some human tumors, and therefore, may serve as antigens for immunotherapy approaches. The murine colorectal carcinoma CT26 and melanoma B16 have recently been found to express the endogenous retroviral gene products gp70 and p15E, respectively, that can serve as antigens recognized by T cells. To date, though, there has been no demonstration of tumor treatment using an endogenous retroviral protein. In this study, we demonstrate that mice immunized with recombinant vaccinia encoding the gp70 H2-L(d)-restricted minimal determinant were protected from CT26 tumor challenge. Splenocytes from mice immunized with vaccinia gp70 specifically secreted IFN-gamma in response to gp70 peptide-pulsed stimulators. Although this strategy could protect against subsequent tumor challenge, it was ineffective against established tumors. Therefore, to investigate the treatment of established CT26 or B16 lung metastases, mice were treated with cultured dendritic cells (DCs) pulsed with gp70 or p15E peptide. Significant inhibition of established lung metastases required immunization with peptide-pulsed DCs pretreated with CD40 ligand that has been demonstrated to increase the T-cell stimulatory activity of DCs. The ability to immunize against endogenous retroviral tumor antigens may have relevance in the induction of antitumor immunity for some human cancers.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígenos Virales/inmunología , Vacunas contra el Cáncer/inmunología , Proteínas Oncogénicas de Retroviridae/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Antígenos de Neoplasias/genética , Antígenos Virales/genética , Ligando de CD40/inmunología , Vacunas contra el Cáncer/genética , Neoplasias del Colon/terapia , Células Dendríticas/inmunología , Inmunoterapia Adoptiva , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Melanoma Experimental/secundario , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos BALB C , Proteínas Oncogénicas de Retroviridae/genética , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
The destruction of newly forming tumor vasculature is a promising approach to inhibit tumor growth. The goal of the present study was to investigate whether human lymphocytes gene modified to express a chimeric receptor specific for the angiogenic endothelial cell receptor, KDR, could react against KDR(+) cells. Gene-modified lymphocytes specifically lysed KDR(+) cells and secreted cytokines in response to KDR(+) target cells including human umbilical vein endothelial cells (HUVECs). Anti-KDR lymphocytes induced HUVECs to secrete the chemokine interleukin 8 and upregulate the adhesion molecules VCAM and E-selectin, which may be important in the recruitment of further immune effector cells to tumor. These KDR-specific lymphocytes may be useful in the adoptive immunotherapy of a broad range of cancers by inducing immune-mediated destruction of tumor neovasculature.
Asunto(s)
Neoplasias/irrigación sanguínea , Neovascularización Patológica , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Linfocitos T/metabolismo , Antígenos CD28/genética , Adhesión Celular , Línea Celular , Células Cultivadas , Citocinas/biosíntesis , Selectina E/metabolismo , Endotelio Vascular/citología , Citometría de Flujo , Técnicas de Transferencia de Gen , Humanos , Interleucina-8/biosíntesis , Estructura Terciaria de Proteína , Receptores Fc/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Transducción Genética , Venas Umbilicales/citología , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Transduction with chimeric T-cell receptor genes can be used to redirect primary T lymphocytes to recognize specific antigens (Ags), including ovarian and breast cancer Ags. To extend this approach to colon cancer we report here redirection of T cells using a chimeric receptor recognizing the colon cancer-associated Ag EGP40. Chimeric T cell receptors were constructed by ligating single-chain genes of either of two EGP40-specific monoclonal antibodies (CO17.1 A, GA733) to the Fc receptor gamma-signaling chain. Retroviral vectors incorporating these constructs were used to transduce a murine T-cell line and human peripheral blood lymphocytes. These modified T cells were analyzed for specific recognition of colon cancer lines by measuring cytokine release and lytic activity against tumor targets. Murine lymphocytes transduced with the chimeric receptor based on GA733, but not CO17.1A, released cytokine specifically in response to EGP40-expressing colon cancer cell lines. Recognition of colon cancer targets by murine lymphocytes was blocked by the addition of GA733 antibody or soluble EGP40 Ag, confirming that colon cancer recognition is based on specific chimeric receptor-Ag interaction. Human lymphocytes transduced with chimeric GA733 specifically recognized colon carcinoma cells in cytokine release assays and lysed EGP40-expressing tumor cells. Genetic modification of T cells can be used to redirect T cells against EGP40-expressing tumor cells. The expression of chimeric GA733 in the autologous lymphocytes of patients may provide a source of tumor-reactive cells with therapeutic application against colon cancer.
Asunto(s)
Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transfección/inmunología , Células 3T3 , Adulto , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Neoplasias del Colon/genética , Neoplasias del Colon/terapia , Molécula de Adhesión Celular Epitelial , Humanos , Células K562 , Ratones , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/virología , Células Tumorales CultivadasRESUMEN
A chimeric receptor, consisting of the single-chain variable (scFv) domains of an anti-erbB-2 mAb linked via a CD8 membrane-proximal hinge to the Fc receptor gamma chain, was expressed in the mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, MD45. This cell line was grafted with the additional specificity to recognise and bind erbB-2-expressing breast carcinoma target cells T47D, MCF-7 and BT-20 in a non-MHC-restricted manner. Tumour cell lysis was antigen-specific since erbB-2-negative tumours were insensitive to lysis by MD45-scFv-anti-erbB-2-gamma clones, and lysis of erbB-2+ tumour targets was inhibited in the presence of an anti-erbB-2 mAb. Furthermore, target cell death correlated with the level of chimeric receptor expression on the effector MD45 subclones. Redirected MD45 CTL utilised Fas ligand to induce target cell death since soluble Fas-Fc fusion protein completely inhibited cytolysis. The sensitivity of tumour target cells to Fas ligand was further enhanced by treating them with interferon-gamma, a regulator of Fas and downstream signalling components of the Fas pathway. Overall, this study has demonstrated the requirement for successful activation of Fas ligand function in conjunction with cytokine treatment for effective lysis of breast carcinoma target cells mediated by redirected CTL.
Asunto(s)
Antígenos de Superficie/metabolismo , Citotoxicidad Inmunológica/inmunología , Glicoproteínas de Membrana/metabolismo , Neoplasias/inmunología , Receptor ErbB-2/inmunología , Linfocitos T Citotóxicos/inmunología , Receptor fas/metabolismo , Animales , Células COS , Proteína Ligando Fas , Humanos , Ratones , Células Tumorales CultivadasRESUMEN
The popliteal lymph node cells of immunocompetent mice generated a strong in vitro cytotoxic response to footpad injection of several human tumor cell lines and the resulting mouse effector cells predominantly used a perforin-mediated cytotoxic mechanism. A relatively minor FasL-dependent cytotoxic response to CEM-CCRF and Jurkat leukemias, but not colon carcinoma COLO 205 cells, was also detected in immunized perforin-deficient mice. In vitro depletion of CD3+ CD8+ T cells, but not CD4+ T or NK1.1+ cells, completely inhibited lysis of human tumor cells, suggesting that CD3+ CD8+ T cells were effectors of perforin-mediated xenospecific cytotoxicity. Xenospecific cytotoxic T cells from wild-type mice were extremely efficient at rejecting tumor when adoptively transferred into scid mice bearing established COLO 205, CEM-CCRF, or Jurkat tumor xenografts. By contrast, cytotoxic T lymphocytes of perforin-deficient mice had no effect on the growth of established tumor xenografts. These data indicate that perforin, and hence direct cytotoxicity, plays a key role in the ability of adoptively transferred CD8+ cytotoxic T lymphocytes to eradicate established xenografts.
Asunto(s)
Traslado Adoptivo , Rechazo de Injerto/etiología , Glicoproteínas de Membrana/inmunología , Linfocitos T Citotóxicos/inmunología , Trasplante Heterólogo/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Proteína Ligando Fas , Femenino , Rechazo de Injerto/inmunología , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones SCID , Trasplante de Neoplasias , Perforina , Proteínas Citotóxicas Formadoras de Poros , Células Tumorales CultivadasRESUMEN
We recently demonstrated that IFI 16, a human member of a family of interferon-inducible nuclear proteins, can function as a potent repressor of transcription. All members of this family are found in the nucleus and contain 1 or 2 copies of a conserved 200 amino acid repeat domain. IFI 16 migrates on SDS-PAGE as three distinct protein species (IFI 16A, 16B, 16C) clustered at 85-95 kDa, and we therefore set out to determine the molecular mechanisms underpinning the production of these different isoforms. In the present report, we have used thermal cycling amplification of reverse-transcribed mRNA (RT-PCR) and Southern blotting of genomic DNA to show that the three protein isoforms result from translation of three separate mRNA species produced by differential mRNA splicing. This differential splicing gives rise to variability in the central ("hinge") domain of the molecule which separates the two 200 amino acid repeats. The longest mRNA (approximately 2.7 kb) encodes an open reading frame of 2355 bp and generates the IFI 16A isoform of 785 amino acids. It contains sequences from 11 exons, including a newly identified exon (7a) which appears to have arisen by tandem duplication of exon 7. The second isoform (IFI 16B, corresponding to the form reported previously) is the most abundantly expressed, and results from deletion of exon 7a (168 bp) to encode a protein of 729 amino acids. The smallest mRNA encodes the IFI 16C isoform (2019 bp), has deleted both exon 7 and exon 7a, and shortens the protein by a further 56 amino acids. Culture of IFI 16-expressing cells with tunicamycin and incubation of cellular lysates with endoglycosidase H suggested that neither IFI 16A nor IFI 16B is glycosylated; however, some IFI 16C molecules showed a minor degree of complex carbohydrate addition. Furthermore, immunoprecipitation and Western blotting indicated that all three IFI 16 isoforms are phosphorylated on serine and threonine residues, but not on tyrosine. Thus, the three IFI 16 protein isoforms arise due to alternative RNA splicing and not due to differential glycosylation or phosphorylation. Finally, IFI 16 isoforms can homo- and heterodimerize, and we have mapped the dimerization domain to the amino terminus which contains an imperfect leucine zipper domain.
Asunto(s)
Empalme Alternativo , Interferones/fisiología , Proteínas Nucleares , Fosfoproteínas , Proteínas/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Dimerización , Exones/genética , Dosificación de Gen , Glicosilación , Células HeLa , Humanos , Isomerismo , Datos de Secuencia Molecular , Fosforilación , Proteínas/metabolismo , ARN Mensajero/genética , Proteínas Represoras/metabolismo , Células Tumorales CultivadasRESUMEN
Antibodies have found application in the diagnosis and therapy of cancer. These antitumor antibodies are confined to isotypes of IgG and little is known of the potential usefulness of other classes of immunoglobulin. In order to determine a possible antitumor effect of IgE antibody a tumor-specific mouse monoclonal IgE antibody was constructed. This antibody was derived from the mouse monoclonal antibody 30.6 that detects an antigenic determinant expressed on the surface of colorectal adenocarcinoma cells, including COLO 205. Mouse IgE 30.6 inhibited the growth of established COLO 205 tumor growing subcutaneously in scid mice. This effect was transient with tumor growth returning to pretreatment levels after 48 h. By contrast, a mouse IgG 30.6 and a chimeric human/mouse IgE 30.6 were without antitumor effect. This isotype-specific antitumor effect was not attributable to differences in antibody affinity, tumor localization, or serum half-life as these were essentially the same for all three isotypes of antibody. In addition, none of the 30.6 monoclonal antibodies inhibited the growth of COLO 205 cells in vitro. As little as 1 microgram per mouse of the tumor-specific mouse IgE antibody was sufficient to inhibit COLO 205 tumor growth, which is in contrast to previous results in which the comparatively weak antitumor effect of a chimeric human/mouse IgG1 required an optimum dose of 4 x 250 micrograms per mouse. This antitumor effect of mouse IgE 30.6 was specifically abrogated by prior administration of a nonspecific mouse IgE. Given this potency, and the fact that mouse Fc epsilon RI binds mouse IgE, but not human IgE, a role for Fc epsilon receptor bearing effector cells in the observed antitumor effect is discussed.
Asunto(s)
Especificidad de Anticuerpos , Carcinoma/inmunología , Carcinoma/patología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Inhibidores de Crecimiento/inmunología , Inmunoglobulina E/fisiología , Trasplante Heterólogo , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/sangre , Afinidad de Anticuerpos , Carcinoma/metabolismo , Neoplasias Colorrectales/metabolismo , Semivida , Humanos , Inmunoglobulina E/biosíntesis , Inmunoglobulina E/sangre , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Trasplante de Neoplasias , Receptores de IgE/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Controversy exists as to whether natural killer (NK)1.1+ cells additionally support cytotoxic T lymphocyte (CTL) generation. We have previously demonstrated that mice generate a strong in vitro xenospecific CTL response in local popliteal lymph nodes (LN) to footpad immunizations with large numbers of human tumor cells. METHODS: In vivo depletion of various LN subsets using cytotoxic monoclonal antibodies was used to determine their relative importance in stimulating xenospecific CD8+ CTL responses to human Jurkat tumor cells. Depletion of functional NK cells in vivo was evidenced by the relative lack of NK1.1+ cells and NK activity in the spleens and LN of anti-NK1.1 monoclonal antibody-treated mice. CONCLUSION: Depletion of LN subsets indicated that CD4+ T cells were critical in generating an effective xenospecific CD8+ CTL response, but also suggested that NK1.1+ cells play a significant additional accessory role in the development of mouse anti-human xenospecific CTL.
Asunto(s)
Antígenos Heterófilos/análisis , Linfocitos T CD8-positivos/inmunología , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Trasplante Heterólogo/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , División Celular/fisiología , Humanos , Células Jurkat/inmunología , Células Asesinas Naturales/inmunología , RatonesRESUMEN
In the MD45 mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, we have expressed a chimeric receptor, consisting of the single-chain variable domains (scFv) of anti-carcinoma embryonic antigen (CEA) mAb linked to Fcgamma receptor (FcgammaR) chain via a CD8 hinge. Transfected MD45 subclones lysed CEA-positive human colon carcinoma cell lines in an antigen-specific and FasL-dependent manner. The degree of lysis correlated with the level of chimeric receptor expressed on transduced MD45 subclones. The requirement for an intact Y65TGL motif in the signaling gamma chain suggested that interaction of the chimeric receptor with target cell CEA induced the cytotoxicity of MD45-scFv subclones. However, MD45 expressing a Y65F mutant chimera still displayed minor levels of lysis following PMA stimulation, suggesting that PMA could bypass gamma chain induction of functional FasL. Pretreatment of Fas-resistant CEA-positive colon carcinoma target cells with IFN-gamma increased their sensitivity of MD45-scFv subclones and FasL-mediated lysis. This study has demonstrated the successful activation of FasL function via a chimeric receptor introduced into lymphocytes and the susceptibility of human colon carcinoma to combined cytokine and CTL treatment.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Antígeno Carcinoembrionario/inmunología , Carcinoma/inmunología , Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica , Glicoproteínas de Membrana/farmacología , Linfocitos T Citotóxicos/inmunología , Receptor fas/metabolismo , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Células COS , Antígeno Carcinoembrionario/fisiología , Células Clonales , Citotoxicidad Inmunológica/efectos de los fármacos , Citotoxicidad Inmunológica/genética , Proteína Ligando Fas , Expresión Génica/inmunología , Células HT29 , Humanos , Hibridomas , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/farmacología , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/farmacología , Interferón gamma/farmacología , Células Jurkat , Ligandos , Ratones , Receptores de IgG/biosíntesis , Receptores de IgG/genética , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T Citotóxicos/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba/inmunologíaRESUMEN
As little is known about the molecular mechanisms responsible for lymphocyte-mediated rejection of xenografts, we have studied the relative contribution of perforin and Fas pathways in cytotoxic lymphocytes generated in mice transplanted with human cell lines. Responder lymphocytes generated in immunocompetent mice displayed significant lysis of human target cells, which suggests that mice can generate a strong lymphocytotoxic response to human cells. Effector cells generated in immunocompetent and gld (Fas ligand mutant) mice predominantly use a perforin-mediated cytotoxic mechanism. By contrast, a Fas-mediated pathway could be stimulated in perforin-deficient or beta2-microglobulin-deficient mice, providing the human target cells were sensitive to Fas-mediated lysis. In vitro depletion of effector CD3+ CD8+ T cells, but not CD4+ T or NK1.1+ cells, completely inhibited lysis of human target cells. This suggests that CD3+ CD8+ T cells were responsible for perforin-mediated xenospecific cytotoxicity. In vivo depletion of NK1.1+ cells and CD4+ T cells before the final immunization abrogated the capacity of lymph node cells to generate xenospecific CD8+ cytotoxic T lymphocytes. By contrast, in vitro depletion of CD4+ T cells was most effective in abrogating the xenospecific Fas-mediated cytotoxicity of perforin-deficient effector cells. Xenospecific cytotoxic T cells were also capable of mediating tumor rejection when adoptively transferred into scid/scid mice bearing established human COLO 205 xenografts. Overall, these data suggested that xenospecific cytotoxic T lymphocytes can lyse target cells via either perforin- or Fas-mediated pathways and that these cells can provide protective and specific immunity against tumor xenografts in the absence of an intact humoral immune system.
Asunto(s)
Trasplante Heterólogo/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/metabolismo , Proteína Ligando Fas , Rechazo de Injerto/inmunología , Humanos , Inmunidad Celular , Células Jurkat , Células Asesinas Naturales/fisiología , Leucemia-Linfoma de Células T del Adulto , Glicoproteínas de Membrana/farmacología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Perforina , Fenotipo , Proteínas Citotóxicas Formadoras de Poros , Linfocitos T Citotóxicos/trasplante , Células Tumorales CultivadasRESUMEN
Chimeric receptors that redirect effector cell function to tumor cells or virus-infected cells have received much attention. Given the high affinity of Fc(epsilon)RI for immunoglobulin E (IgE) and low serum IgE levels, redirection of effector cells using Fc(epsilon) receptor may provide a novel, versatile, and effective anti-tumor strategy. We have used a mouse perforin 5'-promoter to express a single-chain human Fc(epsilon) receptor in the mouse cytotoxic T lymphocyte cell line, CTLL-R8. Upon ligation of the chimeric Fc(epsilon) receptors by IgE, a signal for effector function is transmitted via the intracellular domain of CD3zeta. Selection in G418-containing medium produced CTLLR8 transfectant clones that: (1) expressed chimeric Fc(epsilon) receptor as determined by flow cytometry; (2) bound human IgE antibodies with high affinity as determined by Scatchard analysis; (3) specifically rosetted IgE-coated SRBC; (4) lysed target cells in IgE-mediated ADCC and reverse ADCC assays; and (5) retarded tumor growth in a Winn assay. Therefore these chimeric Fc(epsilon) receptors can effectively redirect cytotoxicity to tumor cells. Future efforts will assess the versatility and efficacy of these IgE-binding chimeric receptors to redirect killer cell function in animal tumor models.
Asunto(s)
Neoplasias Experimentales/terapia , Receptores de IgE/química , Linfocitos T Citotóxicos/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Complejo CD3/química , Citotoxicidad Inmunológica , Relación Dosis-Respuesta Inmunológica , Humanos , Inmunoglobulina E/inmunología , Inmunoterapia , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores de IgE/inmunología , Proteínas Recombinantes de Fusión , TransfecciónRESUMEN
Lymphocyte-mediated cytotoxicity represents one defense mechanism that contributes to transplant rejection. Comparatively little is known about the molecular mechanisms responsible for cell-mediated rejection of xenografts. Herein, we have investigated the relative contribution of perforin- and Fas- pathways in lymphocyte-mediated cytotoxicity generated in response to a variety of human cell lines and peripheral blood mononuclear cells transplanted into mice. Responder lymphocytes generated in immunocompetent mice displayed significant lysis of human targets, suggesting that normally mice can generate a strong lymphocytotoxic response to human cells. Effector cells from mice immunized with one human cell line were also cytotoxic to other human cells, indicating a cross-reactive mouse antihuman response. Effector cells generated in gld mice that have a mutated Fas ligand displayed apparently normal levels of cytotoxicity against human target cells, suggesting a predominantly perforin-based cytotoxic mechanism. This was confirmed by the low cytotoxic activity of xenospecific lymphocytes from perforin-deficient mice. The residual cytotoxicity in perforin-deficient mice responding to xenografted human cells was completely inhibited by anti-Fas mAb, suggesting that a Fas-mediated pathway can be stimulated in the absence of perforin. The detection of Fas-mediated cytotoxicity correlated with the sensitivity of human target cells to Fas-mediated lysis. Depletion of effector CD8+ T, but not CD4+ T or NK1.1+, cells almost completely inhibited lysis of human target cells, suggesting that CD8+ T cells were responsible for perforin-mediated xenospecific cytotoxicity. Overall, these data suggested that xenospecific cytotoxic T lymphocytes can lyse target cells via either perforin- or Fas-mediated pathways.