CIITA promoter I CARD-deficient mice express functional MHC class II genes in myeloid and lymphoid compartments.

Three distinct promoters control the master regulator of major histocompatibility complex (MHC) class II expression, class II transactivator (CIITA), in a cell type-specific manner. Promoter I (pI) CIITA, expressed primarily by dendritic cells (DCs) and macrophages, expresses a unique isoform that contains a caspase-recruitment domain (CARD). The activity and function of this isoform are not understood, but are believed to enhance the function of CIITA in antigen-presenting cells. To determine whether isoform I of CIITA has specific functions, CIITA mutant mice were created in which isoform I was replaced with isoform III sequences. Mice in which pI and the CARD-encoding exon were deleted were also created. No defect in the formation of CD4 T cells, the ability to respond to a model antigen or bacterial or viral challenge was observed in mice lacking CIITA isoform I. Although CIITA and MHC-II expression was decreased in splenic DCs, pI knockout animals expressed CIITA from downstream promoters, suggesting that control of pI activity is mediated by unknown distal elements that could act at pIII, the B-cell promoter. Thus, no critical function is linked to the CARD domain of CIITA isoform I with respect to basic immune system development, function and challenge.

Antimicrobial properties of porphyrins.

A large number of natural and synthetic porphyrins of diverse chemical compositions and characteristics can be isolated from nature or synthesised in the laboratory. Antimicrobial and antiviral activities of porphyrins are based on their ability to catalyse peroxidase and oxidase reactions, absorb photons and generate reactive oxygen species (ROS) and partition into lipids of bacterial membranes. Light-dependent, photodynamic activity of natural and synthetic porphyrins and pthalocyanines against Gram-positive and Gram-negative bacteria has been well demonstrated. Some non-iron metalloporphyrins (MPs) possess a powerful light-independent antimicrobial activity that is based on the ability of these compounds to increase the sensitivity of bacteria to ROS or directly produce ROS. MPs mimic haem in their molecular structure and are actively accumulated by bacteria via high affinity haem-uptake systems. The same uptake systems can be used to deliver antibiotic-porphyrin and antibacterial peptide-porphyrin conjugates. Haemin, the most well known natural porphyrin, possesses a significant antibacterial activity that is augmented by the presence of physiological concentrations of hydrogen peroxide or a reducing agent. Natural and synthetic porphyrins have relatively low toxicity in vitro and in vivo. The ability for numerous chemical modifications and the large number of different mechanisms by which porphyrins affect microbial and viral pathogens place porphyrins into a group of compounds with an outstanding potential for discovery of novel agents, procedures and materials active against pathogenic microorganisms.

DO11.10 and OT-II T cells recognize a C-terminal ovalbumin 323-339 epitope.

The OVA323-339 epitope recognized by DO11.10 (H-2d) and OT-II (H-2b) T cells was investigated using amino- and carboxy-terminal truncations to locate the approximate ends of the epitopes and single amino acid substitutions of OVA323-339 to identify critical TCR contact residues of the OVA323-339 peptide. DO11.10 and OT-II T cells are both specific for a C-terminal epitope whose core encompasses amino acids 329-337. Amino acid 333 was identified as the primary TCR contact residue for both cells, and amino acid 331 was found to be an important secondary TCR contact residue; however, the importance of other secondary TCR contact residues and peptide flanking residues differ between the cells. Additional OVA323-339-specific clones were generated that recognized epitopes found in the N-terminal end or in the center of the peptide. These findings indicate that OVA323-339 can be presented by I-Ad in at least three binding registers. This study highlights some of the complexities of peptide Ags such as OVA323-339, which contain a nested set of overlapping T cell epitopes and MHC binding registers.

Cutting edge: dueling TCRs: peptide antagonism of CD4+ T cells with dual antigen specificities.

T cells expressing two different TCRs were generated by interbreeding 3A9 and AND CD4+ TCR transgenic mice specific for the hen egg lysozyme (HEL) peptide 48-62:I-Ak and moth cytochrome c (MCC) peptide 88-103:I-Ek peptide:MHC ligands, respectively. Peripheral T cells in the offspring express two TCR V beta-chains and respond to HEL and MCC. We observed minimal or no additive effects upon simultaneous suboptimal stimulation with both agonist peptides; however, an antagonist peptide for the 3A9 TCR was able to inhibit the response of the dual receptor T cells to MCC, the AND TCR agonist. This HEL antagonist peptide did not affect AND single transgenic T cells, indicating that the antagonism observed in the dual TCR cells is dependent on the presence of the HEL-specific 3A9 TCR. In contrast, anti-TCR Abs mediate receptor-specific antagonism. These results demonstrate that peptide antagonism exerts a dominant effect.

Persistence of peptide-induced CD4+ T cell anergy in vitro.

Clonal T cell unresponsiveness, or anergy, has been proposed as a mechanism of peripheral tolerance in vivo, and as a potential means of curbing unwanted T cell responses. In this study, anergy was induced in a T helper cell (Th) clone reactive to hemoglobin (Hb) peptide 64-76 by coculture of the T cells with live antigen-presenting cells (APCs) and 74L, a peptide analog of Hb(64-76) that contains a single amino acid substitution of leucine for glycine at position 74, or with a low concentration of the agonist ligand. The anergic state was characterized by blunted proliferation and interleukin (IL) 2 production upon restimulation with Hb(64-76), and was not the result of impaired TCR/CD3 downmodulation. The addition of exogenous IL-12 transiently restored proliferation of the anergic lines, but removal of IL-12 from culture returned the T cells to their nonproliferative state. Interestingly, persistence of the anergic phenotype was observed despite biweekly restimulation with antigen, APCs, and IL-2. Thus, T cell unresponsiveness induced by a peptide produced a stable, persistent anergic state in a Th0 clone that was not reversible by stimulation with IL-2 or -12.

Enhanced dissociation of HLA-DR-bound peptides in the presence of HLA-DM.

Human leukocyte antigen (HLA)-DM is a critical participant in antigen presentation that catalyzes the release of class II-associated invariant chain-derived peptides (CLIP) from newly synthesized class II histocompatibility molecules, freeing the peptide-binding site for acquisition of antigenic peptides. The mechanism for the selective release of CLIP but not other peptides is unknown. DM was found to enhance the rate of peptide dissociation to an extent directly proportional to the intrinsic rate of peptide dissociation from HLA-DR, regardless of peptide sequence. Thus, CLIP is rapidly released in the presence of DM, because its intrinsic rate of dissociation is relatively high. In antigen presentation, DM has the potential to markedly enhance the rate of peptide exchange, favoring the presentation of peptides with slower intrinsic rates of dissociation.

Specific T cell recognition of minimally homologous peptides: evidence for multiple endogenous ligands.

The T cell receptor (TCR) can interact with a spectrum of peptides as part of its ligand, including the immunogenic peptide, variants of this peptide,and apparently unrelated peptides. The basis of this broad specificity for ligand was investigated by substitution analysis of a peptide antigen and functional testing using a B cell apoptosis assay. A peptide containing as few as 1 aa in common with this peptide could stimulate a specific T cell response. Two endogenous ligands, an agonist and a partial agonist, were readily identified from a search of the SwissProt database, indicating that multiple endogenous ligands likely exist for a given T cell. These findings strongly support the concept that one TCR has the ability to interact productively with multiple different ligands, and provide evidence that such ligands exist in the endogenous peptide repertoire.

Modulation of T cell development by an endogenous altered peptide ligand.

T cells potentially encounter numerous endogenous peptides during selection in the thymus and in the periphery. We examined the impact of an endogenous peptide on in vivo T cell development, using a TCR transgenic mouse model based on a hemoglobin-specific T cell clone. In these mice, the transgenic beta chains paired with endogenous alpha chains. This led to a serendipitous primary reactivity to Ser69 peptide, an altered peptide ligand of the Hbd (64-76) epitope of the parent clone. Two Ser69-reactive T cell populations were identified. A smaller population of the Ser69-reactive T cells responded both to Ser69 and Hbd (64-76). A majority reacted only to Ser69, and not to Hbd(64-76); in fact, Hbd(64-76) was a specific TCR antagonist for these Ser69-only-reactive T cells. Thus, in this unique experimental system, Ser69 became an agonist, and Hbd (64-76) was an antagonist. Endogenous presentation of the antagonist ligand in the thymus selectively eliminated the high-avidity cells, while sparing low-avidity cells in the Ser69-reactive T cell repertoire. These results highlight how specificity guides developing T cells through a network of ligands and indicate that the endogenous peptide pool has a profound effect on T cell development and repertoire.

Th2 cell clonal anergy as a consequence of partial activation.

We have demonstrated Th2 clonal anergy as a consequence of partial T cell activation by immunogenic peptide and chemically fixed APC, as well as by altered peptide ligand and live antigen-presenting cells (APC). Either stimulation resulted in a profound inability of the T cells to proliferate upon restimulation with antigen and functional APC, a similar phenomenon to that found with Th1 cells. The anergic state was long lasting and was restricted to proliferation, since the T cells retained the ability to produce cytokines upon restimulation, albeit at slightly reduced levels. Th2 anergy induction was inhibited by cyclosporine A, but not by provision of exogenous costimulation or growth factors. The data presented unify Th1 and Th2 cells with regard to anergy and suggest that the fundamental control during anergy for both subsets is prevention of clonal expansion, thus blocking amplification of the immune response.

Regulation of the costimulator B7, not class II major histocompatibility complex, restricts the ability of murine kidney tubule cells to stimulate CD4+ T cells.

The proximal segment of murine kidney tubule cells (KTC) constitutively expresses low levels of class II major histocompatibility complex (MHC) that are upregulated during local and systemic inflammation. It is not known if KTC also express the costimulator molecules necessary for them to productively participate in immune responses and stimulate T cells. To answer this question, we studied the ability of KTC to present antigens to four Th1 clones. KTC did not induce T cell proliferation to specific antigen, superantigen, or concanavalin A. However, T cell receptors did engage the peptide/MHC ligand presented by KTC, as indicated by T cell enlargement and upregulation of interleukin-2 receptor expression. Importantly, KTC failed to express the Th1 costimulator, B7, as detected by fluorescence cytometry and reverse transcription polymerase chain reaction. We directly demonstrated that lack of B7 expression accounted for at least part of the KTC presentation defect, in that a KTC line transfected with the cDNA for B7 stimulated T cell proliferation to antigen. Our results suggest that epithelial cells expressing class II MHC have developed mechanisms to prevent costimulator expression and limit parenchymal tissue destruction. Failure of class II-expressing epithelial cells to limit costimulator expression may be an important component of organ-specific autoimmunity.

Antagonism of superantigen-stimulated helper T-cell clones and hybridomas by altered peptide ligand.

T-cell activation by an immunogenic peptide can be antagonized by nonstimulatory analogs of that peptide. We investigated this T-cell receptor antagonism by using staphylococcal enterotoxin superantigen to stimulate hemoglobin-specific helper T (Th) cells because its activation pathway may differ from that of conventional antigen. Interestingly, superantigen activation of these Th cells was antagonized by hemoglobin peptide analogs even though agonist (superantigen) and antagonist (analog peptide) bind at different sites on the major histocompatibility complex-encoded molecule and the T-cell receptor. The antagonism appeared to be a fundamental block in T-cell activation, as phosphoinositol generation, cytokine production, and proliferation were reduced in Th1 clones, and, similarly, proliferative and cytokine responses were inhibited in Th2 cells. Even T-cell hybridoma activation (cytokine production and apoptosis) was inhibited by peptide antagonists. Furthermore, analog peptides that functioned as partial agonists for these Th cells also antagonized superantigen-induced proliferation and thus were a subset of the peptide antagonists. In summary, our results demonstrate that analogs of immunogenic peptide are potent antagonists for Th cell responses induced by superantigen as well as immunogenic peptide.

Tickling the TCR: selective T-cell functions stimulated by altered peptide ligands.

Recent observations of T-cell responses following T-cell receptor (TCR) interaction with altered peptide ligands have highlighted the complexity of this signalling system. The indications are that the TCR responds to minor changes in ligand with gradations of T-cell activation and effector functions. Brian Evavold, Joanne Sloan-Lancaster and Paul Allen review these studies and present a model in which partial T-cell activation and TCR antagonism are related events in a continuum of signalling through the TCR.

Induction of T-cell anergy by altered T-cell-receptor ligand on live antigen-presenting cells.

Activation of CD4+ T helper cells results from the occupancy of the T-cell receptor (TCR) by immunogenic peptide bound to a class II major histocompatibility complex (MHC) molecule, together with a co-stimulatory signal from the antigen-presenting cell (APC). This activation leads to proliferation, cytokine production (Th1 or Th2 profile) and cytolysis. Engagement of the TCR in the absence of co-stimulation causes Th1 cells to become unresponsive to subsequent antigenic stimulation. We have previously demonstrated that analogues of an immunogenic peptide could stimulate Th1 and Th2 cells to carry out some effector functions without inducing proliferation, a phenomenon we term partial activation. Here we study the consequences of such partial activation through the TCR of two Th1 clones using peptide analogues presented by a live APC. A peptide analogue that is unable to stimulate clonal proliferation or production of cytokine or inositol phosphate can induce the T cells to become profoundly unresponsive to subsequent stimulation with the immunogenic peptide. Thus, altering the ligand of the TCR by using a peptide analogue on a functional APC sends a signal to Th1 clones that results in anergy.

Separation of T helper 1 clone cytolysis from proliferation and lymphokine production using analog peptides.

In this report, we investigate the activation of Th1 clones using altered TCR ligand. By changing the immunogenic peptide, cytolytic function can be separated from proliferative and lymphokine responses. These three responses were examined and dissected in two Th1 clones using analogs of the murine hemoglobin [Hb(64-76)] peptide. This analysis was focused on amino acids in the immunogenic peptide that were possible T cell contact residues. Typically, several amino acids were identified as critical contact residues for a Th1 proliferative response. An examination of lymphokine production (IFN-gamma or IL-3) revealed the same pattern of response to the analog peptides indicating that the proliferative and lymphokine responses were directly related. However, for cytolysis, fewer amino acid residues were identified as critical contact residues for effector function. Thus, some altered peptide ligands allowed the disassociation of the cytolytic function from the proliferative and lymphokine responses in Th1 clones. To extend these findings, the activation of T cell hybridomas created from the Th1 clones were similarly examined using the altered TCR ligands. The lymphokine response (IL-2) of the T cell hybridomas identified the same critical amino acids as did the cytolytic response of the Th1 clones. Thus, analog peptides partially activated the Th1 clones such that cytolysis occurred independent from proliferative and lymphokine responses.

Complete dissection of the Hb(64-76) determinant using T helper 1, T helper 2 clones, and T cell hybridomas.

We have generated cloned Th1 cells, Th2 cells, and T cell hybridomas specific for the single immunogenic peptide from the beta-chain of murine hemoglobin (Hb(64-76)). The availability of these various types of T cells provided us an unique opportunity to examine and dissect the T cell response to an immunogenic peptide. A panel of altered Hb peptides was made by replacing each amino acid in the Hb peptide (positions 64-76) with a conservative amino acid substitution or an alanine. Although none of the eleven T cell clones and hybridomas tested exhibited the same pattern of reactivity to the substituted Hb peptides, some general features were identified for all T cell responses. The primary T cell contact residue of Hb(64-76) was shown to be asparagine 72. For every Hb(64-76) specific T cell, no activation was observed using a peptide containing the conservative substitution of a glutamine for the asparagine at position 72. The flanking glutamic acid at position 73 was also required for a proliferative response for all of the Th1 and Th2 clones. The Th subtypes were not grossly unique in their responses to the substituted Hb peptides, but exhibited minor differences in fine specificity with the Th1 cells identifying more critical amino acids then did the Th2 cells. For the Th1 cells and also the T cell hybridomas, the phenylalanine at position 71 was critical for a T cell response. Analysis of peptide affinity for IEk molecules indicated that position 71 played a role in peptide binding to MHC. Secondary T cell contact residues, which were important for many but not all of the T cells, were identified at positions 69, 70, and 76. Overall T cell responses were minimally affected by changes in the amino acid residues at positions 64-68, 74, and 75. We have also demonstrated that cloned Th1 cells, Th2 cells and T hybridomas can be generated against the same Hb(64-76) determinant.

The processing and presentation of the self-antigen hemoglobin. Self-reactivity can be limited by antigen availability and costimulator expression.

APC do not distinguish between self- and foreign proteins. Previous studies from our laboratory demonstrated that most endogenous host APC constitutively processed and presented the self-Ag, hemoglobin (Hb), as detected by the Hb-specific T cell hybridoma, YO1.6. We have now examined APC in organs known to be involved in RBC degradation (liver Kupffer cells and splenic small resting B cells) for the presence of Hb/Ia complexes and for the expression of the costimulation necessary to trigger proliferation of T cell clones. We detected Hb/Ia complexes not only on splenic small resting B cells, but also on liver Kupffer cells. Interestingly, complexes were not present on lymph node small resting B cells. Splenic small resting B cells expressed costimulatory activity and efficiently stimulated the Th2 clones only. The opposite pattern was observed with liver Kupffer cells, which expressed costimulatory activity for Th1 clones only. However, if costimulatory activity was provided for the Th2 clones (IL-1 beta) and Th1 clones (allogenic spleen cells), the clones did proliferate in response to Kupffer cells and small resting B cells, respectively. In this report we have demonstrated that 1) endogenously formed self Hb/Ia complexes are expressed on splenic small resting B cells and liver Kupffer cells but not on lymph node small resting B cells and 2) these APC are also able to limit the expression of costimulatory activity for Th2 and Th1 T cell clones. Thus, endogenous APC not only constitutively process and present the self-Ag Hb, but also limit expression of the costimulatory activity necessary to trigger T cell proliferation against a self-Ag. The constitutive processing and presentation of self-Ag, as well as the regulation of costimulatory activity on APC, is likely an important feature of the maintenance of self-tolerance.

T cell inducing determinants contain a hierarchy of residues contacting the T cell receptor.

T cells through their antigen specific T cell receptor recognize a bi-molecular ligand composed of an antigenic peptide bound to a MHC molecule. Several T cell inducing determinants have been extensively characterized by single amino acid substitutions. In this review, we have summarized our characterization of four immunodominant determinants. Each of these determinants possessed a single amino acid residue which was absolutely critical for the recognition by T cells. From these data we propose a hypothesis that there is a hierarchy in the T cell contact residues of a determinant, composed of a single primary residue, and a few secondary residues.

Separation of IL-4 production from Th cell proliferation by an altered T cell receptor ligand.

In the presence of antigen presenting cells, a murine T helper (Th) cell specific for murine hemoglobin (Hb) responded to its immunogenic peptide by both cytokine (interleukin-4) secretion and proliferation. An altered Hb peptide with a single amino acid substitution induced only cytokine secretion and did not induce proliferation. Interleukin-1 costimulated and restored the Th proliferative response to normal levels. The altered peptide also supported cognate T cell-B cell interactions indicative of T cell helper function. Thus, this result suggests that the T cell receptor has the capacity of differential signaling.

Delayed antigen presentation by epidermal Langerhans cells to cloned T h1 and T h2 cells.

Langerhans (LC) cells require incubation with protein antigen for several days before the cells effectively stimulate proliferation of cloned, H-2 restricted, antigen-specific T h cells. In contrast, splenic antigen-presenting cells are immediately effective. LC are immediately competent, however, if an immunogenic peptide rather than the intact protein is the immunogen, indicating that resident or unchallenged LC have the required class II MHC and can provide the signals necessary for T-cell proliferation but may lack the capacity to internalize or cleave protein antigens. We propose that delayed antigen presentation by LC may be intrinsic and advantageous for promoting early systemic immunity. LC stimulate cloned T h1 and T h2 cells equally well, suggesting that LC may not limit or bias the type of immunity that occurs with cutaneous antigenic challenge.