Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

NLRC5, at the Heart of Antigen Presentation.

Nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs) are intracellular proteins mainly involved in pathogen recognition, inflammatory responses, and cell death. Until recently, the function of the family member NLR caspase recruitment domain (CARD) containing 5 (NLRC5) has been a matter of debate. It is now clear that NLRC5 acts as a transcriptional regulator of the major-histocompatibility complex class I. In this review we detail the development of our understanding of NLRC5 function, discussing both the accepted and the controversial aspects of NLRC5 activity. We give insight into the molecular mechanisms, and the potential implications, of NLRC5 function in health and disease.

Inflammatory role of ASC in antigen-induced arthritis is independent of caspase-1, NALP-3, and IPAF.

Because IL-1beta plays an important role in inflammation in human and murine arthritis, we investigated the contribution of the inflammasome components ASC, NALP-3, IPAF, and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC(-/-) mice showed reduced severity of AIA, decreased levels of synovial IL-1beta, and diminished serum amyloid A levels. In contrast, mice deficient in NALP-3, IPAF, or caspase-1 did not show any alteration of joint inflammation, thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes, we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro, as was the production of IFN-gamma, whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC(-/-) T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC(-/-) mice, but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion, these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation.

In vivo localisation of radiolabelled antibodies to carcinoembryonic antigen in human colon carcinoma grafted into nude mice.

SEVERAL attempts have been made to show the specific localisation in vivo of anti-tumour antibodies. Most of these studies, however, either in experimental animals1,2 or in humans3 were performed with antibodies obtained by adsorption and elution from poorly characterised crude tumour fractions.

Sandwich enzyme immunoassay using three monoclonal antibodies against different epitopes of carcinoembryonic antigen (CEA).

Purified monoclonal antibodies (Mab) produced by 3 hybridomas and reacting with 3 different epitopes of carcinoembryonic antigen (CEA) were used in a solid phase enzyme immunoassay. Two Mabs were physically adsorbed to polystyrene balls and the third Mab was coupled to alkaline phosphatase using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. During a first incubation, CEA from heat-extracted serum samples was immunoadsorbed to the antibody coated balls. After washing of the balls, bound CEA was detected by a second incubation with the enzyme coupled Mab. The sensitivity of the assay was 0.6 ng per ml of serum. A total of 196 serum samples from patients with various types of carcinoma, with liver cirrhosis, or from healthy blood donors with or without smoking habits, were tested. The results obtained with the monoclonal enzyme immunoassay (M-EIA) were compared with those obtained with perchloric acid extracts of the same serum samples tested by an inhibition radioimmunoassay using conventional goat anti-CEA antiserum. There was an excellent correlation between the two assays. In particular, the new M-EIA gave good results for the detection of tumor recurrences in the follow-up of colon carcinoma patients. However, despite the use of exclusively monoclonal antibodies the new assay detected a similar percentage of slightly elevated CEA values as the conventional assay in patients with non-malignant disease, suggesting that the CEA associated with non-malignant diseases is immunologically identical to the CEA released by colon carcinoma.

MM-GBSA binding free energy decomposition and T cell receptor engineering.

Recognition by the T-cell receptor (TCR) of immunogenic peptides (p) presented by class I major histocompatibility complexes (MHC) is the key event in the immune response against virus infected cells or tumor cells. The major determinant of T cell activation is the affinity of the TCR for the peptide-MHC complex, though kinetic parameters are also important. A study of the 2C TCR/SIYR/H-2Kb system using a binding free energy decomposition (BFED) based on the MM-GBSA approach had been performed to assess the performance of the approach on this system. The results showed that the TCR-p-MHC BFED including entropic terms provides a detailed and reliable description of the energetics of the interaction (Zoete and Michielin, 2007). Based on these results, we have developed a new approach to design sequence modifications for a TCR recognizing the human leukocyte antigen (HLA)-A2 restricted tumor epitope NY-ESO-1. NY-ESO-1 is a cancer testis antigen expressed not only in melanoma, but also on several other types of cancers. It has been observed at high frequencies in melanoma patients with unusually positive clinical outcome and, therefore, represents an interesting target for adoptive transfer with modified TCR. Sequence modifications of TCR potentially increasing the affinity for this epitope have been proposed and tested in vitro. T cells expressing some of the proposed TCR mutants showed better T cell functionality, with improved killing of peptide-loaded T2 cells and better proliferative capacity compared to the wild type TCR expressing cells. These results open the door of rational TCR design for adoptive transfer cancer therapy.

gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells.

Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.

Soluble major histocompatibility complex-peptide octamers with impaired CD8 binding selectively induce Fas-dependent apoptosis.

Fluorescence-labeled soluble major histocompatibility complex class I-peptide "tetramers" constitute a powerful tool to detect and isolate antigen-specific CD8(+) T cells by flow cytometry. Conventional "tetramers" are prepared by refolding of heavy and light chains with a specific peptide, enzymatic biotinylation at an added C-terminal biotinylation sequence, and "tetramerization" by reaction with phycoerythrin- or allophycocyanin-labeled avidin derivatives. We show here that such preparations are heterogeneous and describe a new procedure that allows the preparation of homogeneous tetra- or octameric major histocompatibility complex-peptide complexes. These compounds were tested on T1 cytotoxic T lymphocytes (CTLs), which recognize the Plasmodium berghei circumsporzoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on Lys(259) in the context of H-2K(d). We report that mutation of the CD8 binding site of K(d) greatly impairs the binding of tetrameric but not octameric or multimeric K(d)-PbCS(ABA) complexes to CTLs. This mutation abolishes the ability of the octamer to elicit significant phosphorylation of CD3, intracellular calcium mobilization, and CTL degranulation. Remarkably, however, this octamer efficiently activates CTLs for Fas (CD95)-dependent apoptosis.

Mutation of the BAFF furin cleavage site impairs B-cell homeostasis and antibody responses.

B-cell-activating factor of the TNF family (BAFF)/BLyS contributes to B-cell homeostasis and function in the periphery. BAFF is expressed as a membrane-bound protein or released by proteolytic cleavage, but the functional importance of this processing event is poorly understood. Mice expressing BAFF with a mutated furin consensus cleavage site, i.e. furin-mutant BAFF (fmBAFF), were not different from BAFF-deficient mice with regard to their B-cell populations and responses to immunization. It is however noteworthy that an alternative processing event releases some soluble BAFF in fmBAFF mice. Mild overexpression (∼ 5-fold) of fmBAFF alone generated intermediate levels of B cells without improving humoral responses to immunization. Processed BAFF was however important for B-cell homeostasis, as peripheral B-cell populations and antibody responses were readily restored by administration of soluble BAFF trimers in BAFF-deficient mice. However, the rescue of CD23 expression in B cells of BAFF-deficient mice required both soluble BAFF trimers and fmBAFF, or a polymeric form of soluble BAFF (BAFF 60-mer). These results point to a predominant role of processed BAFF for B-cell homeostasis and function, and indicate possible accessory roles for membrane-bound BAFF.

CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes.

Thymocytes and class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes express predominantly heterodimeric alpha/beta CD8. By interacting with non-polymorphic regions of MHC class I molecules CD8 can mediate adhesion or by binding the same MHC molecules that interact with the T-cell antigen receptor (TCR) function as coreceptor in TCR-ligand binding and T-cell activation. Using TCR photoaffinity labelling with a soluble, monomeric photoreactive H-2Kd-peptide derivative complex, we report here that the avidity of TCR-ligand interactions on cloned cytotoxic T cells is very greatly strengthened by CD8. This is primarily explained by coordinate binding of ligand molecules by CD8 and TCR, because substitution of Asp 227 of Kd with Lys severely impaired the TCR-ligand binding on CD8+, but not CD8- cells. Kinetic studies on CD8+ and CD8- cells further showed that CD8 imposes distinct dynamics and a remarkable temperature dependence on TCR-ligand interactions. We propose that the ability of CD8 to act as coreceptor can be modulated by CD8-TCR interactions.

Evaluation of the specific IgG- and IgA-antibody response against immundominant Aspergillus fumigatus antigens as diagnostic markers of invasive aspergillosis

An improvement in the serological diagnostic toolbox of invasive aspergillosis (IA) is necessary. So far, most laboratories do not perform antibody detection assays at all to diagnose IA, as commercial test systems are based on crude and undefined antigen mixtures of A. fumigatus. Utilizing the A. fumigatus protein mitogillin, we could demonstrate that the use of selected characterized immunodominant antigens can improve the serodiagnosis of Aspergillus-related diseases. In an animal model we were able to identify additional 36 immunodominant antigens of a cDNA library of A. fumigatus germlings. Five selected antigens were expressed recombinantly in E. coli, purified and used for Westernblot und ELISA analyses to study the kinetics of the specific antibody response in rabbits that were infected systemically with A. fumigatus. Subsequently, the specific IgG- and IgA-antibody responses against these antigens were studied in patients suffering from proven IA and compared to healthy blood donors and patients with other forms of pneumonia. Furthermore, we examined how total IgG- and IgA-levels influence the diagnostic value of antibody detection in IA patients.

Low levels of human leukocyte antigen donor-specific antibodies detected by solid phase assay before transplantation are frequently clinically irrelevant.

Since new technologies based on solid phase assays (SPA) have been routinely incorporated in the transplant immunology laboratory, the presence of pretransplantation donor-specific antibodies (DSA) against human leukocyte antigen (HLA) molecules has generally been considered as a risk factor for acute rejection (AR) and, in particular, for acute humoral rejection (AHR). We retrospectively studied 113 kidney transplant recipients who had negative prospective T-cell and B-cell complement-dependent cytotoxicity (CDC) crossmatches at the time of transplant. Pretransplantation sera were screened for the presence of circulating anti-HLA antibody and DSA by using highly sensitive and HLA-specific Luminex assay, and the results were correlated with AR and AHR posttransplantation. We found that approximately half of our patient population (55/113, 48.7%) had circulating anti-HLA antibody pretransplantation. Of 113 patients, 11 (9.7%) had HLA-DSA. Of 11 rejection episodes post-transplant, only two patients had pretransplantation DSA, of whom one had a severe AHR (C4d positive). One-year allograft survival was similar between the pretransplantation DSA-positive and -negative groups. Number, class, and intensity of pretransplantation DSA, as well as presensitizing events, could not predict AR. We conclude that, based on the presence of pretransplantation DSA, post-transplantation acute rejections episodes could not have been predicted. The only AHR episode occurred in a recipient with pretransplantation DSA. More work should be performed to better delineate the precise clinical significance of detecting low titers of DSA before transplantation.

Detection of serum antibodies against Leishmania 94 kDa antigen in visceral and cutaneous leishmaniosis due to Leishmania infantum.

Leishmania promastigotes polypeptides are analyzed by immunoblotting with sera from patients infected with different Leishmania species and presenting visceral or cutaneous infections. These sera recognize Leishmania polypeptides in several molecular masses. The major findings of this study are as follow. 1) The Leishmania 94 kDa antigen, which is specifically recognized by all sera from L. infantum-infected patients with visceral infection, is recognized by some sera from L. infantum-infected patients presenting cutaneous infection. 2) All patients with cutaneous infections due to L. tropica, L. amazonensis, or L. guyanensis do not develop anti-94 kDa antibodies, whatever the Leishmania species used as antigens. 3) Difference in electrophoretic mobilities is seen between the 94 kDa antigen identified by sera from Leishmania infantum-infected patients, and the antigen both recognized by the Concavalin A lectin and a rabbit antiserum raised against deglycosylated Promastigote Surface Protease.

Probing the interaction between feline immunodeficiency virus and CD134 by using the novel monoclonal antibody 7D6 and the CD134 (Ox40) ligand.

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumor necrosis factor receptor superfamily, and all primary viral strains tested to date use CD134 for infection. We examined the expression of CD134 in the cat using a novel anti-feline CD134 monoclonal antibody (MAb), 7D6, and showed that as in rats and humans, CD134 expression is restricted tightly to CD4+, and not CD8+, T cells, consistent with the selective targeting of these cells by FIV. However, FIV is also macrophage tropic, and in chronic infection the viral tropism broadens to include B cells and CD8+ T cells. Using 7D6, we revealed CD134 expression on a B220-positive (B-cell) population and on cultured macrophages but not peripheral blood monocytes. Moreover, macrophage CD134 expression and FIV infection were enhanced by activation in response to bacterial lipopolysaccharide. Consistent with CD134 expression on human and murine T cells, feline CD134 was abundant on mitogen-stimulated CD4+ T cells, with weaker expression on CD8+ T cells, concordant with the expansion of FIV into CD8+ T cells with progression of the infection. The interaction between FIV and CD134 was probed using MAb 7D6 and soluble CD134 ligand (CD134L), revealing strain-specific differences in sensitivity to both 7D6 and CD134L. Infection with isolates such as PPR and B2542 was inhibited well by both 7D6 and CD134L, suggesting a lower affinity of interaction. In contrast, GL8, CPG, and NCSU were relatively refractory to inhibition by both 7D6 and CD134L and, accordingly, may have a higher-affinity interaction with CD134, permitting infection of cells where CD134 levels are limiting.

Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation.

Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes.