Antibody-dependent cell-mediated cytolysis of human colon carcinoma cells induced by specific antisera against carcinoembryonic antigen.

Antibody-dependent lymphocyte cytotoxicity against human colon carcinoma cells grown in vitro was demonstrated with rabbit anti-carcinoembryonic antigen (CEA) antisera and normal human lymphocytes. The same antisera produced no tumor cell lysis in a complement-dependent cytotoxicity test. The specificity of the reaction was demonstrated by the inhibition of antibody-dependent lymphocyte cytotoxicity after the addition of increasing amounts of purified CEA to the antiserum and by the fact that only tumor cell lines expressing CEA on their surface were lysed. Antibody-dependent lymphocyte cytotoxicity was also observed against two colon carcinoma cell lines that expressed Blood Group A antigen, using a human serum containing anti-Blood Group A antibodies of the immunoglobulin G class. This reaction was specifically inhibited by absorption with Blood Group A red cells, whereas the anti-CEA-dependent cytotoxicity was not inhibited by absorption with red cells of different blood groups.

Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex.

Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-1(26-35) multimers at frequencies that are in the range of 10(-4) CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.

Modulation of proteasomal activity required for the generation of a cytotoxic T lymphocyte-defined peptide derived from the tumor antigen MAGE-3.

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201-associated tumor peptide antigen MAGE-3271-279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271-279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271-279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271-279-specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymaticactivities.

Monitoring tumor antigen specific T-cell responses in cancer patients and phase I clinical trials of peptide-based vaccination.

Numerous phase I and II clinical trials testing the safety and immunogenicity of various peptide vaccine formulations based on CTL-defined tumor antigens in cancer patients have been reported during the last 7 years. While specific T-cell responses can be detected in a variable fraction of immunized patients, an even smaller but significant fraction of these patients have objective tumor responses. Efficient therapeutic vaccination should aim at boosting naturally occurring antitumor T- and B-cell responses and at sustaining a large number of tumor antigen specific and fully functional effector T cells at tumor sites. Recent progress in our ability to quantitatively and qualitatively monitor tumor antigen specific CD8 T-cell responses will greatly help in making rapid progress in this field.

Detection of colorectal carcinoma by emission-computerized tomography after injection of 123I-labeled Fab or F(ab')2 fragments from monoclonal anti-carcinoembryonic antigen antibodies.

This clinical study was based on experimental results obtained in nude mice grafted with human colon carcinoma, showing that injected 131I-labeled F(ab')2 and Fab fragments from high affinity anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) gave markedly higher ratios of tumor to normal tissue localization than intact MAb. 31 patients with known colorectal carcinoma, including 10 primary tumors, 13 local tumor recurrences, and 21 metastatic involvements, were injected with 123I-labeled F(ab')2 (n = 14) or Fab (n = 17) fragments from MAb anti-CEA. The patients were examined by emission-computerized tomography (ECT) at 6, 24, and sometimes 48 h after injection using a rotating dual head scintillation camera. All 23 primary tumors and local recurrences except one were clearly visualized on at least two sections of different tomographic planes. Interestingly, nine of these patients had almost normal circulating CEA levels, and three of the visualized tumors weighed only 3-5 g. Among 19 known metastatic tumor involvements, 14 were correctly localized by ECT. Two additional liver and several bone metastases were discovered by immunoscintigraphy. Altogether, 86% of the tumor sites were detected, 82% with F(ab')2 and 89% with Fab fragments. The contrast of the tumor images obtained with Fab fragments suggests that this improved method of immunoscintigraphy has the potential to detect early tumor recurrences and thus to increase the survival of patients. The results of this retrospective study, however, should be confirmed in a prospective study before this method can be recommended for the routine diagnosis of cancer.

Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors.

In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.

HLA-B7-Restricted EBV-Specific CD8+ T Cells Are Dysregulated in Multiple Sclerosis.

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.

Refining abacavir hypersensitivity diagnoses using a structured clinical assessment and genetic testing in the Swiss HIV Cohort Study.

BACKGROUND: We aimed to assess the value of a structured clinical assessment and genetic testing for refining the diagnosis of abacavir hypersensitivity reactions (ABC-HSRs) in a routine clinical setting. METHODS: We performed a diagnostic reassessment using a structured patient chart review in individuals who had stopped ABC because of suspected HSR. Two HIV physicians blinded to the human leukocyte antigen (HLA) typing results independently classified these individuals on a scale between 3 (ABC-HSR highly likely) and -3 (ABC-HSR highly unlikely). Scoring was based on symptoms, onset of symptoms and comedication use. Patients were classified as clinically likely (mean score > or =2), uncertain (mean score > or = -1 and < or = 1) and unlikely (mean score < or = -2). HLA typing was performed using sequence-based methods. RESULTS: From 131 reassessed individuals, 27 (21%) were classified as likely, 43 (33%) as unlikely and 61 (47%) as uncertain ABC-HSR. Of the 131 individuals with suspected ABC-HSR, 31% were HLA-B*5701-positive compared with 1% of 140 ABC-tolerant controls (P < 0.001). HLA-B*5701 carriage rate was higher in individuals with likely ABC-HSR compared with those with uncertain or unlikely ABC-HSR (78%, 30% and 5%, respectively, P < 0.001). Only six (7%) HLA-B*5701-negative individuals were classified as likely HSR after reassessment. CONCLUSIONS: HLA-B*5701 carriage is highly predictive of clinically diagnosed ABC-HSR. The high proportion of HLA-B*5701-negative individuals with minor symptoms among individuals with suspected HSR indicates overdiagnosis of ABC-HSR in the era preceding genetic screening. A structured clinical assessment and genetic testing could reduce the rate of inappropriate ABC discontinuation and identify individuals at high risk for ABC-HSR.

A natural structural variant of the mouse TCR beta-chain displays intrinsic receptor function and antigen specificity.

The Cbeta0 alternate cassette exon is located between the Jbeta1 and Cbeta1 genes in the mouse TCR beta-locus. In T cells with a VDJbeta1 rearrangement, the Cbeta0 exon may be included in TCRbeta transcripts (herein called TCRbeta-Cbeta0 transcripts), potentially inserting an additional 24 aa between the V and C domains of the TCR beta-chain. These TCRbeta splice isoforms may be differentially regulated after Ag activation, because we detected TCRbeta-Cbeta0 transcripts in a high proportion (>60%) of immature and mature T cells having VDJbeta1 rearrangements but found a substantially reduced frequency (<35%) of TCRbeta-Cbeta0 expression among CD8 T cells selected by Ag in vivo. To study the potential activity of the TCRbeta-Cbeta0 splice variant, we cloned full-length TCR cDNAs by single-cell RT-PCR into retroviral expression vectors. We found that the TCRbeta-Cbeta0 splice isoform can function during an early stage of T cell development normally dependent on TCR beta-chain expression. We also demonstrate that T hybridoma-derived cells expressing a TCRbeta-Cbeta0 isoform together with the clonally associated TCR alpha-chain recognize the same cognate peptide-MHC ligand as the corresponding normal alphabetaTCR. This maintenance of receptor function and specificity upon insertion of the Cbeta0 peptide cassette signifies a remarkable adaptability for the TCR beta-chain, and our findings open the possibility that this splice isoform may function in vivo.

Daytime sleepiness with and without cataplexy in Chinese-Taiwanese patients.

BACKGROUND AND PURPOSE: Investigation of Chinese-Taiwanese patients with excessive sleepiness, but no association with other sleep disorders, and with the presence or absence of cataplexy. PATIENTS AND METHODS: Thirty-five patients, successively referred between 2002 and 2004, underwent polysomnography (PSG), repeat multiple sleep latency test (MSLT), and human leukocyte antigen (HLA) typing. Three patients without cataplexy also had cerebrospinal fluid (CSF) hypocretin measurements. RESULTS: DQB1*0602 was associated with cataplexy in over 90% of Chinese-Taiwanese cases. Absence of cataplexy and <2 sleep-onset REM periods (SOREMPs) was seen in only two subjects, but presence of two SOREMPs did not dissociate DQB1*0602 positive and negative or cataplexy positive and negative subjects. As a group, narcoleptics with cataplexy had a higher number of SOREMPs, and the mean sleep latency was much shorter in narcoleptics with cataplexy than in the non-cataplectic patients, independent of the number of SOREMPs. CONCLUSIONS: Chinese-Taiwanese patients with cataplexy present with similar HLA findings as Black and Caucasian patients, but the presence of two or more SOREMPs in Chinese-Taiwanese patients is not a sufficient diagnostic tool to identify narcolepsy. When cataplexy is not present, description of PSG nd HLA findings may be a better approach than using a label with little scientific significance, allowing for better collection of patients' phenotype.

Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells.

Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell-mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.

Gas-filled microbubble-mediated delivery of antigen and the induction of immune responses.

The use of well characterized recombinant or purified protein antigens (Ag) for vaccination is of interest for safety reasons and in the case where inactivated pathogens are not available (cancer, allergy). However it requires the addition of adjuvants such as Ag carrier or immune stimulators to potentiate their immunogenicity. In this study, we demonstrated that gas-filled microbubbles (MB) can serve as an efficient Ag delivery system to promote phagocytosis of the model Ag ovalbumin (OVA) without the need of ultrasound application. Once internalized by DC, OVA was processed and presented to both CD4 and CD8 T cells in vitro; such observations were coupled with the capacity of MB to activate DC. In vivo administration of MB-associated OVA in naïve wild-type Balb/c mice resulted in the induction of OVA-specific antibody and T cell responses. Detailed characterization of the generated immune response demonstrated the production of both IgG1 and IgG2a serum antibodies, as well as the secretion of IFN-γ and IL-10 by splenocytes. Interestingly, similar results were obtained with human DC in regards of Ag delivery and cell activation. Therefore, the data presented here settle the proof of principle for the further evaluation of MB-based immunomodulation studies.