A long, naturally presented immunodominant epitope from NY-ESO-1 tumor antigen: implications for cancer vaccine design.

The tumor antigen NY-ESO-1 is a promising cancer vaccine target. We describe here a novel HLA-B7-restricted NY-ESO-1 epitope, encompassing amino acids 60-72 (APRGPHGGAASGL), which is naturally presented by melanoma cells. The tumor epitope bound to HLA-B7 by bulging outward from the peptide-binding cleft. This bulged epitope was not an impediment to T-cell recognition, however, because four of six HLA-B7(+) melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX vaccine generated a potent T-cell response to this determinant. Moreover, the response to this epitope was immunodominant in three of these patients and, unlike the T-cell responses to bulged HLA class I viral epitopes, the responding T cells possessed a remarkably broad TCR repertoire. Interestingly, HLA-B7(+) melanoma patients who did not receive the NY-ESO-1 ISCOMATRIX vaccine rarely generated a spontaneous T-cell response to this cryptic epitope, suggesting a lack of priming of such T cells in the natural anti-NY-ESO-1 response, which may be corrected by vaccination. Together, our results reveal several surprising aspects of antitumor immunity and have implications for cancer vaccine design.

Differential requirement for CD4 help in the development of an antigen-specific CD8+ T cell response depending on the route of immunization.

Previous studies in our laboratory have shown that DBA/2 mice injected i.p. with syngeneic P815 tumor cells transfected with the HLA-CW3 gene (P815-CW3) showed a dramatic expansion of activated CD8+CD62L- T cells expressing exclusively the Vbeta10 segment. We have used this model to study the regulatory mechanisms involved in the development of the CW3-specific CD8+ response, with respect to different routes of immunization. Whereas both intradermal (i.d.) and i.p. immunization of DBA/2 mice with P815-CW3 cells led to a strong expansion of CD8+CD62L-Vbeta10+ cells, only the i.d. route allowed this expansion after immunization with P815 cells transfected with a minigene coding for the antigenic epitope CW3 170-179 (P815 miniCW3). Furthermore, depletion of CD4+ T cells in vivo completely abolished the specific response of CD8+CD62L-Vbeta10+ cells and prevented the rejection of P815-CW3 tumor cells injected i.p., whereas it did not affect CD8S+CD62L-Vbeta10+ cell expansion after i.d. immunization with either P815-CW3 or P815 miniCW3. Finally, the CW3-specific CD8+ memory response was identical whether or not CD4+ T cells were depleted during the primary response. Collectively, these results suggest that the CD8+ T cell response to P815-CW3 tumor cells injected i.p. is strictly dependent upon recognition of a helper epitope by CD4+ T cells, whereas no such requirement is observed for i.d. injection.

Characterization of tumor antigen specific CD8+ T cells and semi-invariant Vα24/Vβ11 NKT cells in tumor invaded liver

Summary: Detailed knowledge on tumor antigen expression and specific immune cells is required for a rational design of immunotherapy for patients with tumor invaded liver. In this study, we confirmed that Cancer/Testis (CT) tumor-associated antigens are frequently expressed in hepatocellular carcinoma (HCC) and searched for the presence of CD8+ T cells specific for these antigens. In 2/10 HLA-A2+ patients with HCC, we found that MAGE-A10 and/or SSX-2 specific CD8+ T cells naturally responded to the disease, since they were enriched in tumor lesions but not in non-tumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, suggesting that these CTL were selected in vivo for high avidity antigen recognition, providing the rational for specific immunotherapy of HCC, based on immunization with CT antigens such as MAGE-Al 0 and SSX-2. Type 1 NKT cells express an invariant TCR α chain (Vα24.1α18, paired with Vβ11 in human) and share a specific reactivity to αGalactosylceramide (αGC) presented by CD1d. These cells can display paradoxical immuno-regulatory properties including strong anti-tumor effects upon αGC administration in murine models. To understand why NKT cells were not sufficiently protective against tumor development in patients with tumor invaded liver, we characterized the diversity of Vα24/Vβ11 NKT cells in healthy donors (HD) and cancer patients: NKT cells from HD and patients were generally diverse in terms of TCR β chain (Vβ11) variability and NKT cells from HD showed a variable recognition of αGC loaded CD 1 d multimers. Vα24/ Vβ11 NKT cells can be divided in 3 populations, the CD4, DN (CD4-/CD8-) and CD8 NKT cell subsets that show distinct ability of cytokine production. In addition, our functional analysis revealed that DN and CD8 subsets displayed a higher cytolytic potential and a weaker IFNγ release than the CD4 NKT cell subset. NKT cell subsets were variably represented in the blood of HD and cancer patients. However, HD with high NKT cell frequencies displayed an enrichment of the DN and CD8 subsets, and few of them were suggestive of an oligoclonal expansion in vivo. Comparable NKT cell frequencies were found between blood, non-tumoral liver and tumor of patients. In contrast, we identified a gradual enrichment of CD4 NKT cells from blood to the liver and to the tumor, together with a decrease of DN and CD8 NKT cell subsets. Most patient derived NKT cells were unresponsive upon αGalactosylceramide stimulation ex vivo; NKT cells from few patients displayed a weak responsiveness with different cytokine polarization. The NKT cell repertoire was thus different in tumor tissue, suggesting that CD4 NKT cells infiltrating tumors may be detrimental for protection against tumors and instead may favour the tumor growth/recurrence as recently reported in mice. Résumé en français scientifique : Afin de développer le traitement des patients porteurs d'une tumeur dans le foie par immunothérapie, de nouvelles connaissances sont requises concernant l'expression d'antigènes par les tumeurs et les cellules immunitaires spécifiques de ces antigènes. Nous avons vérifié que des antigènes associés aux tumeurs, tels que les antigènes « Cancer-Testis » (CT), sont fréquemment exprimés par le carcinome hepatocéllulaire (CHC). La recherche de lymphocytes T CD8+ spécifiques (CTL) de ces antigènes a révélé que des CTL spécifiques de MAGE-A10 et/ou SSX-2 ont répondu naturellement à la tumeur chez 2/10 patients étudiés. Ces cellules étaient présentes dans les lésions tumorales mais pas dans le foie adjacent. De plus, ces CTL ont démontré une activité cytolytique forte et spécifique contre les cellules tumorales in vitro, ce qui suggère que ces CTL ont été sélectionnés pour une haute avidité de reconnaissance de l'antigène in vivo. Ces données fournissent une base pour l'immunothérapie spécifique du CHC, en proposant de cibler les antigènes CT tels que MAGE-A10 ou SSX-2. Les cellules NKT de type 1 ont une chaîne α de TCR qui est invariante (chez l'homme, Vα24Jα18, apparié avec Vβ11) et reconnaissent spécifiquement l'αGalactosylceramide (αGC) présenté par CD1d. Ces cellules ont des propriétés immuno¬régulatrices qui peuvent être parfois contradictoires et leur activation par l'αGC induit une forte protection anti-tumorale chez la souris: Afin de comprendre pourquoi ces cellules ne sont pas assez protectrices contre le développement des tumeurs dans le foie chez l'homme, nous avons étudié la diversité des cellules NKT Vα24/Vβ11 d'individus sains (IS) et de patients cancéreux. Les cellules NKT peuvent être sous-divisées en 3 populations : Les CD4, DN (CD4- /CD8-) ou CDS, qui ont la capacité de produire des cytokines différentes. Nos analyses fonctionnelles ont aussi révélé que les sous-populations DN et CD8 ont un potentiel cytolytique plus élevé et une production d'IFNγ plus faible que la sous-population CD4. Ces sous-populations sont représentées de manière variable dans le sang des IS ou des patients. Cependant, les IS avec un taux élevé de cellules NKT ont un enrichissement des sous- populations DN ou CDS, et certains suggèrent qu'il s'agit d'une expansion oligo-clonale in vivo. Les patients avaient des fréquences comparables de cellules NKT entre le sang, le foie et la tumeur. Par contre, la sous-population CD4 était progressivement enrichie du sang vers le foie et la tumeur, tandis que les sous-populations DN ou CD8 était perdues. La plupart des cellules NKT des patients ne réagissaient pas lors de stimulation avec l'αGC ex vivo et les cellules NKT de quelques patients répondaient faiblement et avec des polarisations de cytokines différentes. Ces données suggèrent que les cellules NKT CD4, prédominantes dans les tumeurs, sont inefficaces pour la lutte anti-tumorale et pourraient même favoriser la croissance ou la récurrence tumorale. Donc, une mobilisation spécifique des cellules NKT CD4 négatives par immunothérapie pourrait favoriser l'immunité contre des tumeurs chez l'homme. Résumé en français pour un large public Au sein des globules blancs, les lymphocytes T expriment un récepteur (le TCR), qui est propre à chacun d'entre eux et leur permet d'accrocher de manière très spécifique une molécule appelée antigène. Ce TCR est employé par les lymphocytes pour inspecter les antigènes associés avec des molécules présentatrices à la surface des autres cellules. Les lymphocytes T CD8 reconnaissent un fragment de protéine (ou peptide), qui est présenté par une des molécules du Complexe Majeur d'Histocompatibilité de classe I et tuent la cellule qui présente ce peptide. Ils sont ainsi bien adaptés pour éliminer les cellules qui présentent un peptide issu d'un virus quand la cellule est infectée. D'autres cellules T CD8 reconnaissent des peptides comme les antigènes CT, qui sont produits anormalement par les cellules cancéreuses. Nous avons confirmé que les antigènes CT sont fréquemment exprimés par le cancer du foie. Nous avons également identifié des cellules T CD8 spécifiques d'antigènes CT dans la tumeur, mais pas dans le foie normal de 2 patients sur 10. Cela signifie que ces lymphocytes peuvent être naturellement activés contre la tumeur et sont capables de la trouver. De plus les lymphocytes issus d'un patient ont démontré une forte sensibilité pour reconnaître l'antigène et tuent spécifiquement les cellules tumorales. Les antigènes CT représentent donc des cibles intéressantes qui pourront être intégrés dans des vaccins thérapeutiques du cancer du foie. De cette manière, les cellules T CD8 du patient lui-même pourront être induites à détruire de manière spécifique les cellules cancéreuses. Un nouveau type de lymphocytes T a été récemment découvert: les lymphocytes NKT. Quand ils reconnaissent un glycolipide présenté par la molécule CD1d, ils sont capables, de manière encore incomprise, d'initier, d'augmenter, ou à l'inverse d'inhiber la défense immunitaire. Ces cellules NKT ont démontré qu'elles jouent un rôle important dans la défense contre les tumeurs et particulièrement dans le foie des souris. Nous avons étudié les cellules NKT de patients atteints d'une tumeur dans le foie, afin de comprendre pourquoi elles ne sont pas assez protectrice chez l'homme. Les lymphocytes NKT peuvent être sous-divisés en 3 populations: Les CD4, les DN (CD4-/CD8-) et les CD8. Ces 3 classes de NKT peuvent produire différents signaux chimiques appelés cytokines. Contrairement aux cellules NKT DN ou CDS, seules les cellules NKT CD4 sont capables de produire des cytokines qui sont défavorables pour la défense anti-tumorale. Par ailleurs nous avons trouvé que les cellules NKT CD4 tuent moins bien les cellules cancéreuses que les cellules NKT DN ou CD8. L'analyse des cellules NKT, fraîchement extraites du sang, du foie et de la tumeur de patients a révélé que les cellules NKT CD4 sont progressivement enrichies du sang vers le foie et la tumeur. La large prédominance des NKT CD4 à l'intérieur des tumeurs suggère que, chez l'homme, ces cellules sont inappropriées pour la lutte anti-tumorale. Par ailleurs, la plupart des cellules NKT de patients n'étaient pas capables de produire des cytokines après stimulation avec un antigène. Cela explique également pourquoi ces cellules ne protègent pas contre les tumeurs dans le foie.

Adjuvant radioimmunotherapy trial with iodine-131-labeled anti-carcinoembryonic antigen monoclonal antibody F6 F(ab')2 after resection of liver metastases from colorectal cancer.

PURPOSE: To evaluate the feasibility of radioimmunotherapy (RIT) with radiolabeled anti-carcinoembryonic antigen antibodies after complete resection of liver metastases (LM) from colorectal cancer. Patients and Methods: Twenty-two patients planned for surgery of one to four LM received a preoperative diagnostic dose of a 131I-F(ab')2-labeled anti-carcinoembryonic antigen monoclonal antibody F6 (8-10 mCi/5 mg). 131I-F(ab')2 uptake was analyzed using direct radioactivity counting, and tumor-to-normal liver ratios were recorded. Ten patients with tumor-to-normal liver ratios of >5 and three others were treated with a therapeutic injection [180-200 mCi 131I/50 mg F(ab')2] 30 to 64 days after surgery. RESULTS: Median 131I-F(ab')2 immunoreactivity in patient serum remained at 91% of initial values for up to 96 hours after injection. The main and dose-limiting-toxicity was hematologic, with 92% and 85% grades 3 to 4 neutropenia and thrombocytopenia, respectively. Complete spontaneous recovery occurred in all patients. No human anti-mouse antibody response was observed after the diagnosis dose; however, 10 of the 13 treated patients developed human anti-mouse antibody approximately 3 months later. Two treated patients presented extrahepatic metastases at the time of RIT (one bone and one abdominal node) and two relapsed within 3 months of RIT (one in the lung and the other in the liver). Two patients are still alive, and one of these is disease-free at 93 months after resection. At a median follow-up of 127 months, the median disease-free survival is 12 months and the median overall survival is 50 months. CONCLUSION: RIT is feasible in an adjuvant setting after complete resection of LM from colorectal cancer and should be considered for future trials, possibly in combination with chemotherapy, because of the generally poor prognosis of these patients.

High antigen concentration inhibits T cell proliferation but not interleukin 2 production: examination of limiting dilution microcultures and T cell clones.

The effect of high antigen dose on the activation of cytochrome c peptide-primed lymph node cells was determined in several strains of mice by a limiting dilution analysis. It was found that proliferation of cytochrome c peptide-specific T cells was completely inhibited at high antigen concentration in C57BL/6 but only partially in DBA mice and had no effect in SJL mice. Clones derived from DBA mice showed a differential capacity to be inhibited by high antigen dose. On the other hand, interleukin 2 production by these clones was not impaired regardless of the antigen concentrations used.

Plasmodium falciparum merozoite surface protein 2: epitope mapping and fine specificity of human antibody response against non-polymorphic domains.

BACKGROUND: Two long synthetic peptides representing the dimorphic and constant C-terminal domains of the two allelic families of Plasmodium falciparum merozoite surface proteins 2 are considered promising malaria vaccine candidates. The aim of the current study is to characterize the immune response (epitope mapping) in naturally exposed individuals and relate immune responses to the risk of clinical malaria. METHODS: To optimize their construction, the fine specificity of human serum antibodies from donors of different age, sex and living in four distinct endemic regions was determined in ELISA by using overlapping 20 mer peptides covering the two domains. Immune purified antibodies were used in Western blot and immunofluorescence assay to recognize native parasite derivate proteins. RESULTS: Immunodominant epitopes were characterized, and their distribution was similar irrespective of geographic origin, age group and gender. Acquisition of a 3D7 family and constant region-specific immune response and antibody avidity maturation occur early in life while a longer period is needed for the corresponding FC27 family response. In addition, the antibody response to individual epitopes within the 3D7 family-specific region contributes to protection from malaria infection with different statistical weight. It is also illustrated that affinity-purified antibodies against the dimorphic or constant regions recognized homologous and heterologous parasites in immunofluorescence and homologous and heterologous MSP2 and other polypeptides in Western blot. CONCLUSION: Data from this current study may contribute to a development of MSP2 vaccine candidates based on conserved and dimorphic regions thus bypassing the complexity of vaccine development related to the polymorphism of full-length MSP2.

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component.

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.

Protection from radiation-induced colitis requires MHC class II antigen expression by cells of hemopoietic origin.

Ulcerative colitis, an inflammatory bowel disease, is believed to result from a breakdown of dominant tolerance mechanisms that normally control intestinal immunity. Although CD4+ T lymphocyte subpopulations and expression of MHC class II molecules have been shown to play a role in the pathogenesis of the disease, the nature of the responsible mechanisms remains unclear. In this paper we describe a novel mouse model for inflammatory bowel disease, radiation-induced colitis, that occurs with complete penetrance 6-8 wk postinduction. A combination of high dose gamma-irradiation and lack of MHC class II expression on cells of hemopoietic origin results in development of colitis in C57BL/6 mice. Because of its versatility (due to susceptibility of mice of the widely genetically manipulated C57BL/6 background), high reproducibility, and 100% penetrance, radiation-induced colitis will be a useful mouse model for colitis and a significant tool to study dominant immunological tolerance mechanisms. Moreover, our data imply that tolerization to enteric Ags requires MHC class II mediated presentation by APC of hemopoietic origin.

Immune response mechanisms and protection against "Plasmodium falciparum" and "Plasmodium berghei"

Malaria is one of the most important tropical and infectious diseases causing many deaths and enormous social and economic consequences, particularly in the developing countries. Despite of widely use of anti-malaria drugs and insecticide, the development of successful vaccines constitutes one of the main strategies to control malaria transmission. Several proteins expressed from blood stage such as merozoite surface proteins (MSP] or liver stage as circumsporozoite protein (CSP) are shown to be the targets of immune responses in humans and in animals. Thus, several studies have illustrated that natural infection and laboratory immunizations of humans and animals with Plasmodium sporozoite (SPZ) and its derivate-proteins (peptides) can elicit protection and control of parasite infection. However, a clear understanding of immune response against defined Plasmodium proteins should be the prerequisite conditions before any development of appropriate vaccines. In this order, our study focused on the immune responses to MSP2 (dimorphic and C-terminal fragments) in human and mice; and the mechanisms by which mouse infected hepatocytes present Plasmodium antigens to CD8+ T-cells to induce protective immunity in mice.¦The first part of this work shows that infected hepatocytes can present Plasmodium antigens to PbCSP-specific CD8+ T-cells and induce a protective immunity in mice. Here, this was addressed in vivo and showed that the infected hepatocytes were able of stimulating of primed-and naive-CD8+ T-cell clones and induced fully protective immunity against SPZ challenge. The role of infected hepatocytes in antigen presentation was illustrated here by their graft into immuno-deficient mice and depletion of cosspresenting dentritic cells (DCs) that are known to have key role in the activation of CD8+ T-cells during the liver cycle stage of Plasmodium.¦The second part of this project concerned the fine specificity of Ab responses regarding D and C regions of the two allelic families of MSP2 (3D7 and FC27). Covering of the two regions by overlapping-20 mers led to delineate the epitopes in the different endemic areas and different age groups of donors. The major epitopes characterizing D or C regions were conserved in different endemic areas (P12/P13 and P15/P16 for the 3D7-D, P23/24 and P25/26 for the FC27-D; P29/P30 for the C region). This offers thus, the possibility of a multi-epitope vaccine design including the major epitopes from the two domains of the two allelic MSP2 families. On the other, the 20 mers, particularly some major epitopes of the 3D7-Dregion (P12, P13 and P16) belonged to the epitopes that presented a high probability to be associated with protection in the children group [1 to 5 year-old). In addition, D and C LSP purified Abs (pAbs) recognized merozoite derived polypeptides and native proteins. A crossreactivity activity of homologous pAbs against the heterologous was also illustrated between the two allelic MSP2 parasites. Finally, the functional analysis of D regions pAbs showed an inhibition of Plasmodium falciparum growth suggesting the functional biological activity of the D region pAbs in the control of malaria.¦The last part of this project aimed the evaluation of the immunogenicity of the D and C region LSPs of the two allelic MSP2 families in the presence of adjuvants for the possible use in clinical trial study in humans. The MSP2 LSP mixture showed that D and C were immunogenic and defined limited epitopes (whose intensity of immune responses) depending on the adjuvants and mouse strain for the D regions. The major epitopes characterizing the C region were usually conserved in different strains of mouse and adjuvants used. Furthermore, the single region (either with D or C) immunization of mice confirmed the immunogenicity and the presence of their limited epitopes. We concluded that the possibility to finely delineate in animals the immune responses to antigens might help to select optimal antigen/adjuvant combinations to be tested later in clinical trials. Thus, formulation of glucopyranosyl-lipid A stable emulsion, GLA-SE (toll like receptor (TLR) 4 agonist) and its different combination (CpG: TLR9 agonist and GDQ: LR7 agonist) with MSP2 LSP was better than with alum, montanide ISA 720 (Mt) and virosome. Immunization of mice with allelic LSP did not show a crossreactivity between the two allelic MSP2 parasites unlike as humans, suggesting that the crossreactivity could be acquired during natural infection of the population who are usually exposed to both allelic parasite forms (3D7 and FC27).¦Nevertheless, similar epitope of D (P12, P13 and P25) and C (P29) regions have been found both in mice and human. This offers an opportunity to compare their epitopes in naïve immunized donors with LSPs and naturally infected populations in the endemic areas.

Basal and antigen-induced exposure of the proline-rich sequence in CD3ε.

The CD3ε cytoplasmic tail contains a conserved proline-rich sequence (PRS) that influences TCR-CD3 expression and signaling. Although the PRS can bind the SH3.1 domain of the cytosolic adapter Nck, whether the PRS is constitutively available for Nck binding or instead represents a cryptic motif that is exposed via conformational change upon TCR-CD3 engagement (CD3Δc) is currently unresolved. Furthermore, the extent to which a cis-acting CD3ε basic amino acid-rich stretch (BRS), with its unique phosphoinositide-binding capability, might impact PRS accessibility is not clear. In this study, we found that freshly harvested primary thymocytes expressed low to moderate basal levels of Nck-accessible PRS ("open-CD3"), although most TCR-CD3 complexes were inaccessible to Nck ("closed-CD3"). Ag presentation in vivo induced open-CD3, accounting for half of the basal level found in thymocytes from MHC(+) mice. Additional stimulation with either anti-CD3 Abs or peptide-MHC ligands further elevated open-CD3 above basal levels, consistent with a model wherein antigenic engagement induces maximum PRS exposure. We also found that the open-CD3 conformation induced by APCs outlasted the time of ligand occupancy, marking receptors that had been engaged. Finally, CD3ε BRS-phosphoinositide interactions played no role in either adoption of the initial closed-CD3 conformation or induction of open-CD3 by Ab stimulation. Thus, a basal level of open-CD3 is succeeded by a higher, induced level upon TCR-CD3 engagement, involving CD3Δc and prolonged accessibility of the CD3ε PRS to Nck.

IL-12 controls cytotoxicity of a novel subset of self-antigen-specific human CD28+ cytolytic T cells.

Activated CD8 T cells develop cytotoxicity against autologous cells bearing foreign Ags and self/tumor Ags. However, self-specific cytolysis needs to be kept under control to avoid overwhelming immunopathology. After peptide vaccination of melanoma patients, we studied molecular and functional properties of T cell subsets specific for the self/tumor Ag Melan-A/MART-1. Ex vivo analysis revealed three Ag-specific effector memory (EM) populations, as follows: CD28-negative EM (EM28(-)) T cells strongly expressing granzyme/perforin, and two EM28(+) subsets, one with high and the other with low level expression of these cytotoxic proteins. For further functional characterization, we generated 117 stable CD8 T cell clones by ex vivo flow cytometry-based sorting of these subsets. All EM28(-)-derived clones lysed target cells with high efficacy. In contrast, EM28(+)-derived clones were heterogenous, and could be classified in two groups, one with high and the other with low killing capacity, correlating with granzyme/perforin expression. High and low killer phenotypes remained surprisingly stable for several months. However, strongly increased granzyme expression and cytotoxicity were observed after exposure to IL-12. Thus, the data reveal a newly identified subset of CD28(+) conditional killer T cells. Because CD28 can mediate strong costimulatory signals, tight cytotoxicity control, as shown in this study through IL-12, may be particularly important for subsets of T cells expressing CD28.

An exogenous mouse mammary tumor virus with properties of Mls-1a (Mtv-7).

The classical minor lymphocyte stimulating (Mls) antigens, which induce a strong primary T cell response in vitro, are closely linked to endogenous copies of mouse mammary tumor viruses (MMTV). Expression of Mls genes leads to clonal deletion of T cell subsets expressing specific T cell receptor (TCR) V beta chains. We describe the isolation and characterization of a new exogenous (infectious) MMTV with biological properties similar to the Mls antigen Mls-1a. In vivo administration of either Mls-1a-expressing B cells or the infectious MMTV (SW) led to an increase of T cells expressing V beta 6 followed by their deletion. Surprisingly, different kinetics of deletion were observed with the exogenous virus depending upon the route of infection. Infection through the mucosa led to a slow deletion of V beta 6+ T cells, whereas deletion was rapid after subcutaneous infection. Sequence analysis of the open reading frames in the 3' long terminal repeat of both this exogenous MMTV (SW) and of Mtv-7 (which is closely linked to Mls-1a) revealed striking similarities, particularly in the COOH terminus, which has been implicated in TCR V beta recognition. The identification of an infectious MMTV with the properties of a strong Mls antigen provides a new, powerful tool to study immunity and tolerance in vivo.

Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein.

Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) beta chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (orf) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism of action of the orf gene product is unknown. Whereas predicted orf amino acid sequences show strong overall homology, the 20-30 COOH-terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR V beta specificity. We have generated monoclonal antibodies to a synthetic peptide encompassing the 19 COOH-terminal amino acid residues of Mtv-7 orf, which encodes the Mls-1a determinant. We show here that these antibodies block Mls responses in vitro and can interfere specifically with thymic clonal deletion of Mls-1a reactive V beta 6+ T cells in neonatal mice. Furthermore, the antibodies can inhibit V beta 6+ T cell responses in vivo to an infectious MMTV that shares orf sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the orf COOH terminus and imply that the orf proteins of both endogenous and exogenous MMTV interact directly with TCR V beta.

Tumor localization in patients by radiolabeled monoclonal antibodies against colon carcinoma.

A radiolabeled monoclonal antibody (MAb) that has been shown to react specifically in vitro and ex vivo to human colorectal carcinoma and to inhibit growth of human carcinomas grafted in nude mice was administered to 52 colorectal carcinoma patients and 15 patients with other types of cancer. Of 63 colorectal carcinoma tumor sites studied, 34 showed significant accumulation of antibody by external photoscanning and tomoscintigraphy, whereas none of the 20 sites of other cancer types gave positive results. One-third of the patients received F(ab')2 fragments of the MAb, which gave a slightly higher percentage (61%) of positive results than did intact MAbs (51%). A few patients scheduled for tumor resection were given injections simultaneously of 131I-labeled MAb and 125I-labeled normal immunoglobulin G. Antibody concentration in resected tumors was 3.6 to 6.3 times higher than the average antibody concentration in adjacent normal tissues (1.5, 3.4, and 9.4 as compared with normal mucosa, serosa, and fat, respectively), and the specificity indices, calculated by differential radioactivity analysis, ranged from 2.1 to 5.1. The results show the potential value and limitations of this particular MAb for tumor detection by immunoscintigraphy.