Viable Antigen Research Articles

Abstract 1337: Characterization of anti-AXL CAR-NK cell therapy for the treatment of melanoma

Abstract AXL is a receptor tyrosine kinase (RTK) that is commonly expressed in metastatic melanoma and several other forms of cancer. Activation of AXL signaling is linked to tumor cell growth, invasion, metastatic transformation, drug resistance, and immune response regulation. However, treatment of metastatic melanoma continues to present clinical challenges due to its capacity to rapidly develop drug resistance, which manifests under the influence of AXL signaling. As such, AXL may be a viable antigen target for immunotherapies. Despite their successes in hematological malignancies, chimeric antigen receptor (CAR)-T cell therapies remain limited by their susceptibility to causing off-target toxicity. In contrast, natural killer (NK) cells are not associated with such risks, which makes them clinically appealing as an alternative to T cells. However, studies on the application of CAR-NK cells to treat solid tumors remain acutely scarce. To explore the clinical viability of CAR-NK cells for the treatment of metastatic melanoma, AXL-specific CAR-NK cells were generated by transfecting NK cells obtained from NK cell lines. Preliminary data from a cytotoxicity assay indicated that the cytotoxicity of CAR-NK92-MI cells against AXL-positive melanoma cell lines was significantly stronger (up to a 10-fold difference) than the corresponding parental cells. Further in vitro characterization of this CAR-NK cell line and other CAR-NK cell lines, as well as in vivo evaluation of their efficacy in a mouse xenograft model of melanoma metastasis, is underway. Citation Format: Winston Hibler, Glenn Merlino, Yan Lin Yu. Characterization of anti-AXL CAR-NK cell therapy for the treatment of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1337.

Targeting the activated microenvironment with endosialin (CD248)-directed CAR-T cells ablates perivascular cells to impair tumor growth and metastasis

BackgroundTargeting of solid cancers with chimeric antigen receptor (CAR)-T cells is limited by the lack of suitable tumor-specific antigens and the immunosuppressive, desmoplastic tumor microenvironment that impedes CAR-T cell infiltration,...

EP02.04-002 Synergy of Local Treatment with Pulsed Electric Fields and Anti-PD1 Checkpoint Blockade

Pulsed Electric Fields (PEFs) are a focal ablation treatment that induces cell death without damaging the extracellular matrix, sparing sensitive structures. Its mechanism induces inflammatory signaling and affords viable antigen presentation within the tumor microenvironment. This preclinical study determines the immunogenic impact of PEF and whether its inclusion with anti-PD1 is beneficial.

Irreversible electroporation stimulates a pro-inflammatory tumor microenvironment that facilitate antigen presentation in pancreatic cancer

Abstract Pancreatic cancer is now the third leading cause of cancer-related death and is projected to turn out to be the second by 2030. Pancreatic cancer is often late-diagnosed with few treatment options with extremely low survival rates. Surgical candidacy is only 15% of total diagnosed patients. Irreversible electroporation (IRE), a novel form of tumor non-thermal ablation applies high-voltage microsecond pulses to permeabilize the cell membrane that is irreversible and leads to apoptotic cell death. IRE has had strong success in clinical trials for pancreatic cancer patients. Nevertheless, little is known on the effect of IRE beyond the initiation of cell death in the tumor tissue. We hypothesize that IRE can induce a pro-inflammatory type of cell death in pancreatic cancer that can promote inflammation and anti-tumor immune system responses. We have used in vitro, ex vivo, and immunocompetent in vivo murine models to investigate alterations to pancreatic cancer and its microenvironment. Our findings reveal programmed necrotic cell death, promotion of pro-inflammatory cytokine production, and shifts in immune cell populations from a pro-tumor (such as MDSCs and T regulatory cells) to a proinflammatory types containing cytotoxic lymphocytes and neutrophils after IRE treatment. Progression-free survival was significantly longer for the treated mice. Intriguingly, IRE treatment increases IFNγ signaling and generates viable antigens for presentation to the adaptive immune system but also promotes PD-L1 expression which in turn causes immunological stalemate. Our results suggest possible therapeutic targets for use in combination with IRE, such as gemcitabine or PD-1/PD-L1 inhibitors, to improve patient survival.

Irreversible electroporation can alter immune cell composition and enhance antigen presentation potential in pancreatic cancer

Abstract Pancreatic cancer has a devastating survival rate of only 9%. A major treatment limitation is the immunosuppressive nature of pancreatic tumors created by high amounts of tumor-associate stromal cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). These form a barrier that inhibits anti-tumor immunity and has yet to be overcome by current immunotherapy options. Irreversible electroporation (IRE), an ablative technology that utilizes short electrical pulses to disrupt tumor cell membranes, shows evidence of inducing proinflammatory cell death in pancreatic tumors, but its effect on innate and adaptive immune responses, vital for long-term survival, has not been investigated. We hypothesize that IRE treatment for pancreatic cancer can reduce pro-tumor immune cells, recruit anti-tumor immune cells such as cytotoxic T-cells, and increase tumor-specific antigen presentation potential. We utilized in vivo immunocompetent Pan02 murine pancreatic cancer models to assess progression-free survival and tumor immune cell populations via flow cytometry, as well as an ex vivo antigen presentation model with murine bone marrow-derived dendritic cells and splenic T-cells to determine the ability of IRE to generate viable antigens. Our findings show a temporal increase in anti-tumor immune cell populations, reduction in tumor-associated immune cell populations, and strong antigen presentation potential. However, we also observed an increase in Pdl-1, normally down-regulated in pancreatic cancer, in response to increased immune signaling. This may make IRE a candidate for cotherapy options such as anti-Pdl-1 antibodies or other immunotherapies that could increase patient survival.

Abstract 1436: Development of a chimeric antigen receptor against glycosylated annexin A2 on ovarian tumors

Abstract Chimeric antigen receptors (CARs) have been a significant focus of cancer therapeutic investigation in recent years, due to the remarkably effective responses observed in hematological cancers. However, the utility of CAR T cells has yet to be fully exhibited in solid tumours. One of the key challenges facing the treatment of solid tumors with CAR T cells is the dearth of viable antigen targets, which limits the expansion of this therapeutic space. Here, we describe how the anti-glycan monoclonal antibody 2448, which target annexin A2 on a number of ovarian and breast cancer cell lines, has been successfully reformatted as a CAR T cell construct. Messenger RNA (mRNA) encoding CAR(2448) was transfected into activated T cells, resulting in cytotoxicity and cytokine release upon co-incubation with target cells expressing annexin A2. While CAR(2448) variants with differing spacer chain lengths all mediated cytotoxicity against target cells compared to untransfected T cells, real-time target cell growth monitoring revealed that the long spacer CAR(2448) mediated higher levels of cytotoxicity, consistent with the membrane-proximal location of the annexin A2 target epitope. This optimization of CAR spacer length in the in vitro setting, using mRNA transfected T cells and real-time target cell growth monitoring, suggests that several aspects of CAR construction can be optimized in vitro, thereby lowering the threshold for CAR T cell development. These results suggest the potential for anti-glycan antibodies to be repurposed as CARs, and the development of CAR(2448) is currently being extended to lentiviral vectors for the purpose of developing long-term expressing CAR(2448) T cells, in anticipation of in vivo studies in murine models. This project was funded by the Agency for Science, Technology, and Research (A*STAR), Singapore and the A*STAR Graduate Academy (A*GA). Citation Format: Leonard W. Leong, Simeon Cua, Heng Liang Tan, Andre B. Choo. Development of a chimeric antigen receptor against glycosylated annexin A2 on ovarian tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1436.

Proteomics analysis of faecal proteins in the tick Haemaphysalis flava

BackgroundTicks and tick-borne diseases are of major public health concern. Currently, development of vaccines against ticks is considered crucial for their control. A critical step in this process is the screening of viable antigens. Faeces are byproducts of digestion and blood meal utilization, and partly reflect the vitality and vector potential of ticks. However, an integrated analysis of proteins in tick faeces is lacking. The present study explored the protein components in the faeces of the tick Haemaphysalis flava, by liquid chromatography–tandem mass spectrometry (LC/MS-MS) to identify potential protein antigens for vaccine development against ticks.MethodsFaeces from adult H. flava engorged females were collected. Proteins were extracted from faeces, and the trypsin-digested peptides were analyzed by LC/MS-MS. High confidence proteins were identified based on unique peptides revealed by MS. Potential faecal protein genes, as well as their sources, were also characterized by searching previous transcriptome datasets from the salivary glands and midgut of H. flava.ResultsIn total, 21 were recognized with confidence. Amongst these, 18 were of likely tick origin, while three proteins (serum albumin, haemoglobin α and β subunits) were likely from hosts. Seventeen unigenes corresponding to these proteins were retrieved by searching our previous H. flava salivary glands and midgut transcriptomic datasets. Some proteins were reported to prevent blood clotting, play a role in immunity and antibiosis, and formation of musculature. The functions of the remaining proteins are unknown.ConclusionsIdentifying antigens for tick vaccine development is feasible by analyzing the faecal proteome as well as the transcriptomes of salivary glands and midguts. The vast number of proteins detected in tick faeces highlights the complexity of blood digestion in ticks, a field that needs more investigation.

An overview of FDA-approved vaccines & their innovators

ABSTRACTIntroduction: A survey of FDA-approved biologicals focused upon the development of immunotherapies over time to gain insight on the challenges and trends of vaccine development today.Areas covered: A total of 135 different immune-based therapies were broadly divided into passive or active immunotherapies. Whereas just over half of passive immunotherapies targeted infectious diseases, the vast majority of active immunotherapy products (vaccines) were directed against a handful of viral and bacterial pathogens. We also analyze changes in vaccine strategy, including the use of viable antigens and subunit approaches.Expert commentary: An analysis of vaccine innovators revealed an ever-increasing presence of the private sector and a relatively diminishing role for the public sector . Whereas North American companies have contributed to the approval of two-thirds of vaccines, European companies have regained parity in terms of hosting innovators of vaccine research and development.

Scalable high throughput selection from phage-displayed synthetic antibody libraries.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius.

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.

A3.14 PD-1 Signalling and Low RORC mRNA Content Influence the TREG/TH17 Balance in Human Cord Blood

<h3>Background and Objectives</h3> In neonates, the immune system is less responsive than in adults. Explanations for this involve differences in T helper (Th) 1 versus Th2 cell numbers and altered cytokine profiles after stimulation. We hypothesised that neonatal immune deviation may also involve FOXP3⁺ regulatory T cells (Treg) and Th17 cells. <h3>Materials and Methods</h3> In order to investigate this, we compared CD4⁺CD25^–CD45RO^– naïve T cells from human cord blood (CB) with cells from adult peripheral blood (APB). Cells were activated in vitro by anti-CD3 in the presence of viable antigen presenting cells (APB). The induction of regulatory T cells and Th17 cells was analysed by flow cytometry. Cytokine production was measured by multiplex immunoassays and RORC mRNA content by PCR. Specific interactions between APC and T cells were blocked with monoclonal antibodies during cell culture. <h3>Results</h3> CB cells show lower numbers of Treg ex vivo than APB. However, upon activation, high percentages of functionally suppressive FOXP3⁺ Treg are induced in CB. Replacement of viable APC by irradiated APC or anti-CD28 abrogated this effect, suggesting that live APC from CB in particular may imprint a Treg phenotype. Indeed, CB APC were able to induce high numbers of FOXP3⁺ cells in alloreactive immune reactions with APB T cells as well. Addition of blocking antibodies against CD80, CD86 or CTLA-4 to CB cultures did not affect the number of Tregs induced. Blockade of the PD-1/PD-Ligand (PD-L)1 interaction however showed a marked decrease in FOXP3⁺ cell numbers. CB cells showed a consistently reduced protein kinase B (PKB)/c-Akt phosphorylation upon activation. On top of that, in contrast to APB, low concentrations of APC derived pro-inflammatory cytokines were detected in CB cultures and no Th17 cells were induced. Addition of Th17 inducing cytokines reduced FOXP3⁺ cell numbers in CB, but did not induce IL-17 production. PCR of the Th17 defining transcription factor RORC showed a significantly reduced concentration of this protein in CB. <h3>Conclusions</h3> Human cord blood cells have an increased propensity to become FOXP3⁺ Treg as compared to APB. This effect is due to low production of inflammatory cytokines and PD-1/PD-L1 interaction. Inability to induce Th17 cells in CB could not be abrogated by Th17 inducing cytokines, but was due to reduced RORC mRNA content. This study shows mechanisms involving the human Th17/Treg induction switch that can also be important in inflammation and auto-immune disorders.

Evaluation of a non-viral vaccine in smallpox-vaccinated individuals and immunized HLA-transgenic mice

The current poxvirus vaccine is associated with rare, but serious adverse events. Therefore, we investigated a non-replicating approach to vaccine design. Peptides encoding potential HLA-binding motifs were derived from the orthopoxvirus genes, D8L, A27L, and C12L (the IL-18-binding protein [vIL18BP105]), all of which are preserved among poxviruses that infect humans, and which may be a target of host immunity. The peptides were tested with poxvirus-vaccinated human PBMC and serum for eliciting memory responses, as well as with splenocytes and serum from peptide-immunized, human HLA-DR04 transgenic (HLA tg) mice. vIL18BP105 induced 5-fold proliferation of vaccinated-donor PBMC over non-vaccinated (P<0.001), including IL-2-producing CD8+ cells. Serum IgG recognizing vIL18BP105 was detected (P<0.002 vs non-vaccinated) by ELISA. Viral peptides were conjugated to the HLA-targeting mAb, L243, for immunization of HLA tg mice. Splenocytes from vIL18BP105-L243-immunized mice proliferated upon exposure to vIL18BP105 (P<0.001). Proliferating splenocytes were interferon-γ-producing CD4(+)CD45RA(neg). vIL18BP105-L243-immunized mice generated IgG more rapidly than free-peptide-immunized mice. Peptide-specific antibody was also detected when different L243-peptide conjugates were combined. vIL18BP, by eliciting human memory responses, is a viable antigen for inclusion in a virus-free vaccine. The immunogenicity of peptides was boosted by conjugation to L243, whether administered alone or combined.

Efficient display of scFv antibodies on bacteriophage lambda

In the present work we demonstrate the efficient display of functional scFv antibodies on the bacteriophage lambda capsid. A single-chain (scFv) anti-CEA antibody gene was cloned in two different vectors to obtain fusion of the scFv antibody to the N- or C-terminus of the bacteriophage lambda capsid protein D (gpD). Lambda bacteriophage assembly occurs in the reducing environment of the cytoplasm; despite this the lambda-displayed anti-CEA antibody fragments retain the capacity to recognize the antigen, indicating correct single-chain antibody folding. Efficient production of functional scFv exposed on lambda capsid with viable antigen binding specificity allowed us to study and compare the capacity of display, the stability of recombinant antibody expression and the assembly efficiency of bacteriophage particles decorated with recombinant antibody fused to the amino- or carboxy-terminus of lambda D protein.

Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination

Viable antigen (Ag) delivery systems expressing defined pathogen-derived proteins represent powerful candidates for future vaccination strategies. Here, recombinant (r) Salmonella typhimurium aroA strains secreting listeriolysin (Hly) of Listeria monocytogenes in haemolytic or non-haemolytic form were constructed to direct these carriers into cytosolic or phagosomal host cell compartments, respectively. Oral and intravenous (i.v.) vaccination of mice with either construct induced ‘transporter associated with antigen processing’-dependent protection against the intracellular bacterial pathogen L. monocytogenes. Comparison of oral immunization with both r Salmonella constructs revealed superior vaccine efficacy of the haemolytic r S. typhimurium Hlys construct as compared to the non-haemolytic r Salmonella Hlys 492 strain. In contrast, efficacy of i.v. vaccination with either r Salmonella strain did not significantly differ. Therefore, r Salmonella strains secreting biologically active Hly represent valuable delivery systems for heterologous rAg or DNA which should be exploited for future mucosal vaccination strategies.

Immunogenicity of glycerol-preserved human cadaver skin in vitro.

Donor allograft skin preserved in 85% glycerol is used as a temporary coverage for large burn wounds. Glycerol treatment does not affect the structural integrity of the skin; cells are well preserved but dead. However, cells expressing major histocompatibility class II molecules can still be observed. In this study we investigated the mechanism underlying the clinical observation that glycerol-treated alloskin will be destroyed but after a prolonged period. We compared the in vitro immunogenicity of untreated and 85% glycerol-treated human skin cells. Human purified blood T cells did not proliferate when cultured with allogeneic treated skin cells, whereas untreated cells induced a distinct response. A moderate response was measured after adding T cells and viable antigen presenting cells, such as monocytes, to the allogeneic treated skin cells. However, the response on untreated skin cells was much higher. These results favor the suggestion that after transplantation of glycerol preserved skin is performed, an inflammatory process mediated by infiltrating host monocytes occurs rather than a rejection process mediated by T cells.

Characterization of the N-terminal part of the neutralizing antigenic site I of coxsackievirus B4 by mutation analysis of antigen chimeras

Coxsackievirus B3 (CVB3) as a potential RNA virus vector for the presentation of foreign antigenic epitopes was further characterized. Insertion mutagenesis of infectious CVB3 cDNA yielded viable antigen chimeras containing variant BC loops of VP1 of coxsackievirus B4 (CVB4). Analysis of three antigen chimeras allowed the mapping of the N-terminal part of the neutralizing antigenic site l (N-Agl) of CVB4 which is located in the BC loop of the structural protein VP1. A significant neutralization of a viable chimera with the deletion of CVB4-specific amino acid Ser-83 at the amino terminus of the VP1 BC loop was obtained with CVB4 serotype-specific polyclonal antisera. This neutralization was reduced after further deletion of the adjacent Ala-84, suggesting that this amino acid either constitutes the beginning of N-Agl of CVB4 or is essential for the conformation of the adjacent epitope. In contrast, exchange of amino acid Ser-86 to alanine, in the middle of the BC loop, led to complete loss of reactivity with CVB4-specific antibodies, demonstrating the importance of this residue for binding of CVB4 neutralizing antisera. Furthermore, we observed that manipulations of the VP1 BC loop resulted in increased thermolability of the viable chimeras in comparison to CVB3, although replication efficiencies were similar.

Peptide binding to surface class II molecules is the major pathway of formation of immunogenic class II-peptide complexes for viable antigen presenting cells.

Although studies on fixed APCs have demonstrated that peptide can bind to cell surface class II molecules, the mechanisms by which peptide-class II complexes are formed in viable cells is largely unexplored. To explore the possibility that peptide loading of class II molecules was occurring after endocytosis of peptides as well as by surface binding, we utilized an immunogenic hemagglutinin peptide (HAP 128-145) from the influenza strain A/Japan/57, and studied the appearance of surface complexes of HAP 128-145 bound to HLA-DRw11 molecules on human B-lymphoblastoid cells (BLCLs). Detection of the bound peptide was made possible by a rabbit anti-serum (alpha HAP) raised against HAP 128-145, which recognizes both the free peptide as well as peptide bound to DRw11 on living APCs. Pretreatment of the BLCLs with a variety of inhibitors of protein synthesis and intracellular trafficking failed to decrease the levels of HAP 128-145/DRw11 surface complexes. However, significant inhibition in the appearance of these complexes was caused by a decrease in the temperature at which the cells were incubated with peptide. Temperature-specific inhibition was also observed for fixed DRw11-positive APCs and purified DRw11 molecules indicating that the effect of temperature was directly on the class II molecules. We conclude that surface binding of peptide by class II molecules on human B cells is a major pathway of formation of immunogenic class II-peptide complexes for at least some soluble antigenic peptides, and that endocytosis of soluble peptides with subsequent binding of peptide by intracellular class II molecules plays little if any role in the formation of such complexes. Moreover, class II molecules have evolved to stably bind peptide optimally at physiologic temperatures, independent of cell metabolism.

Delivery of pseudorabies virus envelope antigens enclosed in immunostimulating complexes (ISCOMs): elicitation of neutralizing antibody and lymphoproliferative responses in swine and protection in mice

An experimental subunit vaccine that consisted of pseudorabies virus (PRV) envelope glycoproteins enclosed into immunostimulating complexes (PRVenv/ISCOM) was constructed, and evaluated in DBA/2 mice and inbred swine of the SLA haplotype c/c. Two to three weeks after the first vaccine dose, specific anti-PRV antibodies could be demonstrated by ELISA or virus neutralization (VN) assays. Booster PRVenv/ISCOM vaccinations resulted in rapid and significant increases in antibody titres in both mice and swine. In addition, a week after receiving the third PRVenv/ISCOM vaccine dose swine peripheral blood mononuclear cells exhibited significant proliferation in response to stimulation with PRV virion antigen. Moreover, two doses of vaccine sufficed to protect mice fully against lethal virus challenge. Therefore, the data presented here support the ISCOM as a viable antigen delivery system for subunit PRV vaccines.

Immunity to intracellular bacteria.

Intracellular bacteria are endowed with the capacity to survive and replicate inside mononuclear phagocytes (MP) and, sometimes, within certain other host cells. MP are potent effectors cells that are able to engulf and kill many bacterial invaders. Therefore, intracellular bacteria had to exploit potent evasion mechanisms that allow their survival in this hostile environment. At the early phase, natural killer cells activate antibacterial defense mechanisms. During intracellular persistence, microbial proteins are processed and presented, thus initiating T cell activation. By secreting interleukins, CD4 alpha/beta TH1 cells activate MP, converting them from a habitat to a potent effector cell; TH2-dependent activities seem to be of minor importance. Cytolytic CD8 T cells represent a further element of protection. In the case of Listeria monocytogenes, the gene products responsible for virulence and for the introduction of antigens into the MHC class I pathway are being characterized. Increasing evidence points to a role of gamma/delta T lymphocytes in antibacterial immunity, although their precise function remains to be elucidated. Protection in the host is a local event focussed on granulomatous lesions. MP accumulate at the site of microbial growth and become activated through the CD4 T cell-interleukin-MP axis. Lysis of incapacitated MP and other host cells by CD8 T cells allows release and subsequent uptake by more efficient phagocytes, thus contributing to host protection. At the same time, lysis of host cells promotes microbial dissemination and causes tissue injury, which represent pathogenic aspects of the same mechanism. Research on the immune response against intracellular bacteria not only helps us to better understand how the immune system deals with "viable antigens" in constant trans-mutation, it also forms the basis for the rational design of control measures for major health problems.

An autoantigenic T cell epitope forms unstable complexes with class II MHC: a novel route for escape from tolerance induction.

The peptide rAc1-11 represents the dominant T cell epitope of rat myelin basic protein (MBP) in mice of the H-2u haplotype. Residue 4 has been shown previously to govern binding of the peptide to the class II molecule, I-Au. We have constructed peptide analogues bearing amino acid substitutions at position 4 and have assessed their ability to stimulate an antigen-specific T cell hybridoma when presented by viable antigen presenting cells (APC). Complexes between I-Au and one such analogue, rAc1-11[4A], were rapidly lost from the surface of live APC displaying a half-life (t 1/2) of approximately 10 min. Neither shedding of intact complexes from the cell surface, nor their internalization and recycling through an acidic intracellular compartment were found to account for their loss. The possible dissociation of rAc1-11[4A] from the peptide binding cleft was therefore addressed by comparing the t 1/2 of complexes between I-Au and peptide analogues of higher affinity. The tyrosine-substituted analogue, rAc1-11[4Y], remained stably bound to I-Au for at least 4 h, thereby displaying a t 1/2 far in excess of that evident for rAc1-11[4A]. Significantly, the wild type peptide, rAc1-11, bound so transiently that functional complexes could not be detected on the surface of peptide-pulsed APC. The physiological relevance of these findings was confirmed by extending our studies to an analysis of the homologous epitope of murine MBP; evidence that this epitope likewise displays minimal affinity for I-Au suggests a novel strategy for the escape from tolerance induction by encephalitogenic T cells.