Inflammation Control and Immunotherapeutic Strategies in Comprehensive Cancer Treatment.

Tumor growth and expansion are determined by the immunological tumor microenvironment (TME). Typically, early tumorigenic stages are characterized by the immune system not responding or weakly responding to the tumor. However, subsequent tumorigenic stages witness the tumor promoting its growth and metastasis by stimulating tumor-protective (pro-tumor) inflammation to suppress anti-tumor immune responses. Here, we propose the pivotal role of inflammation control in a successful anti-cancer immunotherapy strategy, implying that available and novel immunotherapeutic modalities such as inflammation modulation, antibody (Ab)-based immunostimulation, drug-mediated immunomodulation, cancer vaccination as well as adoptive cell immunotherapy and donor leucocyte transfusion could be applied in cancer patients in a synergistic manner to amplify each other's clinical effects and achieve robust anti-tumor immune reactivity. In addition, the anti-tumor effects of immunotherapy could be enhanced by thermal and/or oxygen therapy. Herein, combined immune-based therapy could prove to be beneficial for patients with advanced cancers, as aiming to provide long-term tumor cell/mass dormancy by restraining compensatory proliferation of surviving cancer cells observed after traditional anti-cancer interventions such as surgery, radiotherapy, and metronomic (low-dose) chemotherapy. We propose the Inflammatory Prognostic Score based on the blood levels of C-reactive protein and lactate dehydrogenase as well as the neutrophil-to-lymphocyte ratio to effectively monitor the effectiveness of comprehensive anti-cancer treatment.

Therapeutic Stimulation of Glycolytic ATP Production for Treating ROS-Mediated Cellular Senescence.

Cellular senescence is conditioned through two interrelated processes, i.e., a reduction in adenosine triphosphate (ATP) and the enhancement of reactive oxygen species (ROS) production levels in mitochondria. ATP shortages primarily influence the energy-intensive synthesis of large biomolecules, such as deoxyribonucleic acid (DNA). In addition, as compared to small biomolecules, large biomolecules are more prone to ROS-mediated damaging effects. Based on the available evidence, we suggest that the stimulation of anaerobic glycolytic ROS-independent ATP production could restrain cellular senescence. Consistent with this notion, non-drug related intermittent hypoxia (IH)-based therapy could be effectively applied in sports medicine, as well as for supporting the physical activity of elderly patients and prophylactics of various age-related disorders. Moreover, drug therapy aiming to achieve the partial blockade of respiratory chain and downstream compensatory glycolysis enhancement could prove to be useful for treating cardiovascular, neurological and hormonal diseases. We maintain that non-drug/drug-related therapeutic interventions applied in combination over the entire lifespan could significantly rejuvenate and prolong a high quality of life for individuals.

Oxygen therapy in traditional and immunotherapeutic treatment protocols of cancer patients: current reality and future prospects.

INTRODUCTION: The metabolic environment in ischemic and hypoxic tumors is known to contribute to cancer progression. Importantly, peculiar metabolic changes occurring in malignant cells (the increased glycolysis and the hampered Krebs cycle) may contribute to decreased antioxidant-dependent defense in ischemic and hypoxic tumors. AREAS COVERED: In the clinic, oxygen saturation of tumors is usually achieved by the application of water-soluble ozone and hyperbaric oxygen therapy. Tumor oxygenation has been shown to inhibit tumor growth and potentiate anti-tumor effects of chemoradiotherapy in animal experiments and the clinical setting. Tumor oxygenation could enhance anti-tumor effects achieved by tumor blood vessel occlusion or angiostatic therapy. EXPERT OPINION: Owing to a profound influence of ROS on both the innate and adaptive immunity, oxygen therapy, when combined simultaneously or sequentially with immunotherapeutic interventions (such as immune checkpoint inhibition, drug-induced immunostimulation, adoptive cell therapy, hyperthermia, etc.), could be considered as a novel highly-effective clinical biological approach to cancer treatment.

Clinically feasible and prospective immunotherapeutic interventions in multidirectional comprehensive treatment of cancer.

INTRODUCTION: The immune system is able to exert both tumor-destructive and tumor-protective functions. Immunotherapeutic technologies aim to enhance immune-based anti-tumor activity and (or) weaken tumor-protective immunity. AREAS COVERED: Cancer vaccination, antibody (Ab)-mediated cytotoxicity, Ab-based checkpoint molecule inhibition, Ab-based immunostimulation, cytokine therapy, oncoviral therapy, drug-mediated immunostimulation, exovesicular therapy, anti-inflammatory therapy, neurohormonal immunorehabilitation, metabolic therapy, as well as adoptive cell immunotherapy, could be coherently used to synergize and amplify each other in achieving robust anti-cancer responses in cancer patients. Tumor-specific immunotherapy applied at early stages is capable of eliminating remaining tumor cells after surgery, thus preventing the development of minimal residual disease. Patients with advanced disease stages could benefit from combined immunotherapy, which would be aimed at providing tumor cell/mass dormancy. Traditional therapeutic anti-cancer interventions (chemoradiotherapy, hyperthermia, anti-hormonal therapy) could significantly enhance tumor sensitivity to anti-cancer immunotherapy. It is important that lower-dose (metronomic) chemotherapy regimens, which are well-tolerated by normal cells, could advance immune-mediated control over tumor growth. EXPERT OPINION: We envisage that combined immunotherapy regimens in the context of traditional treatment could become the mainstream modality for treating cancers in all phases of the tumorigenesis. The effectiveness of the anti-cancer treatment could be monitored by the following blood parameters: C-reactive protein, lactate dehydrogenase, and neutrophil-to-lymphocyte ratio.

Immune memory limits human longevity: the role of memory СD4+ T cells in age-related immune abnormalities.

Introduction: With age, the proportion of memory T cells increases, while the proportion and number of naive T cell decreases. Memory T cells are more sensitive to antigenic stimulation and less dependent on co-stimulation signals, as compared to naïve T cells. Differentiation of naïve T cells into memory T cells is accompanied by an increase in T cell reactivity to self-peptide/MHC complexes, which allowed for positive selection of their naïve precursors in the thymus.Areas covered: We envisage that in geriatric age memory Th1-type autoreactivity leads to age-associated immune hyporeactivity, atherosclerosis, and degenerative neuropathology, such as Alzheimer's and Parkinson's diseases, whereas autoreactive memory Th2 cells could promote tumorigenesis.Expert option: Stimulation of adaptive immunoregulatory mechanisms by polyclonal T-cell vaccination could constrain the development of age-related T-cell autoreactivity surplus. Another approach to immune system 'rejuvenation' could involve adoptive cell transfer of naïve T cells with a view to restrain the expansion of pathological memory T cells and support immune responsiveness to novel antigenic challenges. The proposed concept conjectures the occurrence of a hard-wired immunological clock that could determine the duration of life, which theoretically could be subject to immune-based therapy.

Immunotherapy for treating metastatic colorectal cancer.

BACKGROUND: Colorectal cancer remains one of the leading causes of death in the world. Surgery still remains the mainstay of treatment for primary and metastatic colorectal cancer. Immunotherapy used as an adjunct to surgery can play an important role in controlling the spread of tumour. METHODS: The online databases PubMed, Medline, Scirus and Medscape Oncology were used to identify articles of relevance. Keywords included; "Immunotherapy", "Cellular Immunotherapy", "Metastatic Colorectal Cancer", "Monoclonal Antibody" "Tumour Vaccines" and "Adoptive Cell Therapy". The databases search was from the period of June 1995 until May 2010 inclusive. RESULTS: Our understanding of tumour immunology has allowed the development of some successful therapies. Immunotherapy through the use of monoclonal antibodies is an effective adjunct to chemotherapy for metastatic colorectal cancer. Other modalities that are in the stages of development are cellular and conjugated vaccines. However, these vaccines are being experimented in advanced stages of colorectal tumours. CONCLUSION: Colorectal cancer vaccines are being developed for advanced stages of colorectal tumour. However, their use as an early adjunct could potentially limit the spread of tumour or even result in cure. Further trials are required to ensure the safety and efficacy of cellular vaccines against colorectal tumours to allow their use on patients early in their disease presentation.

Invariant natural killer T (iNKT) cell deficiency in chronic mucocutaneous candidiasis--a consequence or a cause?

Chronic mucocutaneous candidiasis (CMC) is a group of heterogeneous disorders characterised by primary selective susceptibility to chronic, recurrent Candida infections. The genetic defect of one subgroup of CMC patients have been identified as mutations of the autoimmune regulator (AIRE) gene. Recent data implicated the AIRE gene in iNKT cell development, raising the possibility that iNKT cells may be important in defending against Candida infections. In this study, we enumerated the circulating iNKT frequency in 22 CMC patients (9 with AIRE gene mutations) and 25 healthy controls. We also examined the effect of Candida stimulation on iNKT cells in vitro. Our data demonstrated that peripheral iNKT cell frequency is significantly reduced in CMC patients compared to healthy controls, regardless of their AIRE gene mutation status. Direct stimulation with heat-inactivated whole Candida did not induce iNKT cell proliferation. Furthermore, circulating iNKT cell frequencies in some healthy controls were comparable to CMC patients. These observations suggest that iNKT cell deficiency is part of the CMC disease phenotype irrespective of the presence of AIRE gene mutations but does not appear to confer susceptibility to chronic Candida infections. We postulate that the reduced circulating iNKT cell frequency in CMC is a consequence rather than a cause of chronic Candida infections.

Impaired T(H)17 responses in patients with chronic mucocutaneous candidiasis with and without autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.

BACKGROUND: Accumulating evidence implicates T(H)17 cytokines in protection against Candida species infections, but the clinical relevance is not clear. Chronic mucocutaneous candidiasis (CMC) is a heterogeneous syndrome with the unifying feature of selective susceptibility to chronic candidiasis. Different subgroups with distinct clinical features are recognized, including autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), CMC with hypothyroidism, and isolated CMC. Understanding immune defects in patients with CMC will define cellular and molecular mechanisms crucial for protection against Candida species in human subjects. OBJECTIVES: We sought to determine whether impaired T(H)17 responses underlie susceptibility to Candida species infections and whether the same defect is present in different CMC subgroups. METHODS: We assessed T(H)17 responses of PBMCs to Candida and non-Candida species stimuli by measuring IL-17, IL-22, IL-21, IL-6, IL-23, and IFN-γ cytokine production using cytokine arrays and intracellular cytokine-producing cell numbers and proliferation with flow cytometry. PBMCs from healthy subjects and unaffected family members served as controls. RESULTS: In patients with CMC with hypothyroidism, T(H)17 cells demonstrated decreased proliferation and IL-17 production in response to Candida species. In contrast, in patients with APECED, T(H)17 cell proliferation and IL-17 production were normal unless exposed to APECED plasma, which inhibited both functions in both APECED and normal PBMCs. Candida species-stimulated IL-22 production was impaired in all patients with CMC, whereas IL-6 and IL-23 responses were unaltered. CONCLUSION: An impaired T(H)17 response to Candida species, although mediated by different mechanisms, was present in all CMC subgroups studied and might be a common factor predisposing to chronic candidiasis.

Therapeutic effect of tolerogenic dendritic cells in established collagen-induced arthritis is associated with a reduction in Th17 responses.

OBJECTIVE: Tolerogenic dendritic cells (DCs) are antigen-presenting cells with an immunosuppressive function. They are a promising immunotherapeutic tool for the attenuation of pathogenic T cell responses in autoimmune arthritis. The aims of this study were to determine the therapeutic action of tolerogenic DCs in a type II collagen-induced arthritis model and to investigate their effects on Th17 cells and other T cell subsets in mice with established arthritis. METHODS: Tolerogenic DCs were generated by treating bone marrow-derived DCs with dexamethasone and vitamin D(3) during lipopolysaccharide-induced maturation. Mice with established arthritis received 3 intravenous injections of tolerogenic DCs, mature DCs, or saline. Arthritis severity was monitored for up to 4 weeks after treatment. Fluorescence-labeled tolerogenic DCs were used for in vivo trafficking studies. The in vivo effect of tolerogenic DCs on splenic T cell populations was determined by intracellular cytokine staining and flow cytometry. RESULTS: Tolerogenic DCs displayed a semi-mature phenotype, produced low levels of inflammatory cytokines, and exhibited low T cell stimulatory capacity. Upon intravenous injection into arthritic mice, tolerogenic DCs migrated to the spleen, liver, lung, feet, and draining lymph nodes. Treatment of arthritic mice with type II collagen-pulsed tolerogenic DCs, but not unpulsed tolerogenic DCs or mature DCs, significantly inhibited disease severity and progression. This improvement coincided with a significant decrease in the number of Th17 cells and an increase in the number of interleukin-10-producing CD4+ T cells, whereas tolerogenic DC treatment had no detectable effect on Th1 cells or interleukin-17-producing γ/δ T cells. CONCLUSION: Treatment with type II collagen-pulsed tolerogenic DCs decreases the proportion of Th17 cells in arthritic mice and simultaneously reduces the severity and progression of arthritis.

Profound invariant natural killer T-cell deficiency in inflammatory arthritis.

OBJECTIVES: Data from rodent models indicate that invariant natural killer T (iNKT) cells are key regulators of many immune responses including autoimmune arthritis, but their role in human diseases is unclear. The aims of this study are to determine whether iNKT cell frequency and function are altered in patients with rheumatoid arthritis (RA), and the clinical significance of such iNKT cell abnormalities. METHODS: Peripheral blood iNKT cell frequency and proliferative response to an iNKT cell-specific agonist, α-galactosylceramide were measured in 46 RA patients (including 23 untreated, newly diagnosed patients), 22 healthy controls and 27 patients presenting with recent-onset joint pain. The relationship between iNKT cell frequency and clinical characteristics and the effects of immunosuppressive treatment was examined. RESULTS: Compared with healthy controls, RA patients had a decreased frequency of peripheral blood iNKT cells (median 0.001% vs 0.021%, p<0.001) and the proliferative response of this subset to α-galactosylceramide was also diminished in the patient group (median fold-expansion 31 vs 121, p=0.037). These abnormalities preceded the initiation of disease-modifying or immunosuppressive therapy, whose effect was to increase the circulating iNKT cell frequency (p=0.037). Furthermore, iNKT cell frequency correlated inversely with the systemic inflammatory marker, C-reactive protein (p=0.008). Finally, in patients presenting with recent-onset joint symptoms, normal peripheral blood iNKT cell frequency predicted a non-inflammatory cause of joint pain. CONCLUSION: iNKT cell deficiency is present in patients with RA and other inflammatory arthropathy. Normal iNKT cell frequency predicts non-inflammatory causes of joint pain.

Response of Th17 cells to a citrullinated arthritogenic aggrecan peptide in patients with rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is considered to be a prototypical autoimmune disease. However, the autoantigens that play an important role in the development of RA remain unclear. The aim of this study was to investigate whether T cells specific for citrullinated epitopes from self proteins are present in patients with RA. METHODS: Peripheral blood mononuclear cells (PBMCs) from 28 RA patients and 18 healthy controls were stimulated with citrullinated or noncitrullinated aggrecan peptide Agg(84-103), and proliferative and cytokine responses were assessed using (3)H-thymidine incorporation assay, enzyme-linked immunosorbent assay, and intracellular cytokine analysis. RESULTS: A proliferative response to the citrullinated aggrecan peptide was detected in >60% of RA patients but not in healthy controls. Furthermore, citrullinated aggrecan peptide-stimulated PBMCs from RA patients produced high levels of the proinflammatory cytokine interleukin-17 (IL-17), accompanied by an induction of IL-17+CD4+ T cells. In contrast, PBMCs from RA patients and healthy controls exhibited no response to stimulation with the noncitrullinated aggrecan peptide. CONCLUSION: Proinflammatory T cell responses to stimulation with a citrullinated arthritogenic aggrecan peptide were detected in RA patients but not in healthy individuals, suggesting a role for these autoantigen-specific T cells in the pathogenesis of RA. Our results suggest that the lack of response to the noncitrullinated analog peptide not only implicates the citrulline residue in T cell recognition but also highlights the potential value of citrullinated aggrecan peptide-specific responses as biomarkers of RA. To our knowledge, this is the first study to demonstrate the presence of citrullinated antigen-specific T cells in human RA.

LPS activation is required for migratory activity and antigen presentation by tolerogenic dendritic cells.

Autoimmune pathologies are caused by a breakdown in self-tolerance. Tolerogenic dendritic cells (tolDC) are a promising immunotherapeutic tool for restoring self-tolerance in an antigen-specific manner. Studies about tolDC have focused largely on generating stable maturation-resistant DC, but few have fully addressed questions about the antigen-presenting and migratory capacities of these cells, prerequisites for successful immunotherapy. Here, we investigated whether human tolDC, generated with dexamethasone and the active form of vitamin D3, maintained their tolerogenic function upon activation with LPS (LPS-tolDC), while acquiring the ability to present exogenous autoantigen and to migrate in response to the CCR7 ligand CCL19. LPS activation led to important changes in the tolDC phenotype and function. LPS-tolDC, but not tolDC, expressed the chemokine receptor CCR7 and migrated in response to CCL19. Furthermore, LPS-tolDC were superior to tolDC in their ability to present type II collagen, a candidate autoantigen in rheumatoid arthritis. tolDC and LPS-tolDC had low stimulatory capacity for allogeneic, naïve T cells and skewed T cell polarization toward an anti-inflammatory phenotype, although LPS-tolDC induced significantly higher levels of IL-10 production by T cells. Our finding that LPS activation is essential for inducing migratory and antigen-presenting activity in tolDC is important for optimizing their therapeutic potential.

T cell responses to a non-glycosylated epitope predominate in type II collagen-immunised HLA-DRB1*0101 transgenic mice.

AIM: To study collagen-induced arthritis in human leucocyte antigen (HLA)-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules (MHC-II) and to determine T cell specificity against the arthritogenic CII(259-273) epitope of type II collagen either unmodified or post-translationally glycosylated at Lys(264). METHODS: Arthritis was induced by immunisation with human type II collagen in complete Freund's adjuvant and measured by footpad swelling, clinical score and histology. T cell responses were assessed by proliferation of spleen and lymph node cells and in antigen presentation assays, using T cell hybridomas specific for the glycosylated and non-glycosylated CII(259-273) epitope. RESULTS: The incidence of arthritis was 50% in DR1-transgenic mice lacking endogenous MHC-II molecules. Recall T cell responses in draining lymph nodes and spleen were consistently greater against the non-glycosylated epitope than to the glycosylated CII(259-273). Most of the T cell hybridomas generated from CII-immunised mice recognised the non-glycosylated CII epitope and this form of the epitope was also presented with 100-fold higher efficiency and 1 h faster kinetics by both macrophages and dendritic cells. CONCLUSION: This study shows that T cell responses to the non-glycosylated epitope of heterologous (human) CII are dominant in HLA-DR1 transgenic mice lacking MHC-II, which could contribute to the pathogenicity of autoimmune arthritis.

Mechanisms of major histocompatibility complex class II-restricted processing and presentation of the V antigen of Yersinia pestis.

We mapped mouse CD4 T-cell epitopes located in three structurally distinct regions of the V antigen of Yersinia pestis. T-cell hybridomas specific for epitopes from each region were generated to study the mechanisms of processing and presentation of V antigen by bone-marrow-derived macrophages. All three epitopes required uptake and/or processing from V antigen as well as presentation to T cells by newly synthesized major histocompatibility complex (MHC) class II molecules over a time period of 3-4 hr. Sensitivity to inhibitors showed a dependence on low pH and cysteine, serine and metalloproteinase, but not aspartic proteinase, activity. The data indicate that immunodominant epitopes from all three structural regions of V antigen were presented preferentially by the classical MHC class II-restricted presentation pathway. The requirement for processing by the co-ordinated activity of several enzyme families is consistent with the buried location of the epitopes in each region of V antigen. Understanding the structure-function relationship of multiple immunodominant epitopes of candidate subunit vaccines is necessary to inform choice of adjuvants for vaccine delivery. In the case of V antigen, adjuvants designed to target it to lysosomes are likely to induce optimal responses to multiple protective T-cell epitopes.

Inhibition of macropinocytosis blocks antigen presentation of type II collagen in vitro and in vivo in HLA-DR1 transgenic mice.

Professional antigen-presenting cells, such as dendritic cells, macrophages and B cells have been implicated in the pathogenesis of rheumatoid arthritis, constituting a possible target for antigen-specific immunotherapy. We addressed the possibility of blocking antigen presentation of the type II collagen (CII)-derived immunodominant arthritogenic epitope CII259-273 to specific CD4 T cells by inhibition of antigen uptake in HLA-DR1-transgenic mice in vitro and in vivo. Electron microscopy, confocal microscopy, subcellular fractionation and antigen presentation assays were used to establish the mechanisms of uptake, intracellular localization and antigen presentation of CII by dendritic cells and macrophages. We show that CII accumulated in membrane fractions of intermediate density corresponding to late endosomes. Treatment of dendritic cells and macrophages with cytochalasin D or amiloride prevented the intracellular appearance of CII and blocked antigen presentation of CII259-273 to HLA-DR1-restricted T cell hybridomas. The data suggest that CII was taken up by dendritic cells and macrophages predominantly via macropinocytosis. Administration of amiloride in vivo prevented activation of CII-specific polyclonal T cells in the draining popliteal lymph nodes. This study suggests that selective targeting of CII internalization in professional antigen-presenting cells prevents activation of autoimmune T cells, constituting a novel therapeutic strategy for the immunotherapy of rheumatoid arthritis.

The impact of glycosylation on HLA-DR1-restricted T cell recognition of type II collagen in a mouse model.

OBJECTIVE: Type II collagen (CII) is a candidate autoantigen implicated in the pathogenesis of rheumatoid arthritis (RA). Posttranslational glycosylation of CII could alter intracellular antigen processing, leading to the development of autoimmune T cell responses. To address this possibility, we studied the intracellular processing of CII for presentation of the arthritogenic glycosylated epitope CII(259-273) to CD4 T cells in macrophages from HLA-DR1-transgenic mice. METHODS: HLA-DR1-transgenic mice were generated on a class II major histocompatibility complex-deficient background, and T cell hybridomas specific for the glycosylated and nonglycosylated epitope CII(259-273) were developed. Subcellular fractionation of macrophages was used to localize CII degradation to particular compartments and to identify the catalytic subtype of proteinases involved. RESULTS: We showed that the glycosylated CII(259-273) epitope required more extensive processing than did the nonglycosylated form of the same epitope. Dense fractions containing lysosomes were primarily engaged in the processing of CII for antigen presentation, since these compartments contained 1) enzyme activity that generated antigenic CII fragments bearing the arthritogenic glycosylated epitope, 2) the antigenic CII fragments themselves, 3) CII peptide-receptive HLA-DR1 molecules, and 4) peptide/HLA-DR1 complexes that could directly activate T cell hybridomas. Degradation of CII by dense fractions occurred optimally at pH 4.5 and was abrogated by inhibitors of serine and cysteine proteinases. CONCLUSION: Processing of the arthritogenic glycosylated CII(259-273) epitope, which is implicated in the induction of autoimmune arthritis, is more stringently regulated than is processing of the nonglycosylated form of the same epitope. Mechanisms of intracellular processing of the glycosylated epitope may constitute novel therapeutic targets for the treatment of RA.

Major histocompatibility class II molecules prevent destructive processing of exogenous peptides at the cell surface of macrophages for presentation to CD4 T cells.

We studied factors affecting major histocompatibility complex class II (MHC-II)-restricted presentation of exogenous peptides at the surface of macrophages. We have previously shown that peptide presentation is modulated by surface-associated proteolytic enzymes, and in this report the role of the binding of MHC-II molecules in preventing proteolysis of exogenous synthetic peptides was addressed. Two peptides containing CD4 T-cell epitopes were incubated with fixed macrophages expressing binding and non-binding MHC-II, and supernatants were analysed by high-performance liquid chromatography and mass spectrometry to monitor peptide degradation. The proportion of full-length peptides that were degraded and the number of peptide fragments increased when non-binding macrophages were used, leading to reduction in peptide presentation. When MHC-II molecules expressed on the surface of fixed macrophages were blocked with monoclonal antibody and incubated with peptides and the supernatants were transferred to fixed macrophages, a significant reduction in peptide presentation was observed. Peptide presentation was up-regulated at pH 5.5 compared to neutral pH, and the latter was found to be the pH optimum of the proteolytic activity of the surface enzymes involved in the degradation of exogenous peptides and proteins. The data suggest that MHC-II alleles that bind peptides protect them from degradation at the antigen-presenting cell surface for presentation to CD4 T cells and we argue that this mechanism could be particularly pronounced at sites of inflammation.

Regulation of peptide presentation by major histocompatibility complex class II molecules at the surface of macrophages.

We studied major histocompatibility complex class II-dependent presentation of two T cell epitopes delivered as synthetic peptides by fixed macrophages. Treatment of bone marrow macrophages with inhibitors of proteinases of the metallo-, aspartic and serine proteinase families enhanced presentation of peptides, indicating that several enzyme families participate in destructive antigen processing of exogenous peptides. High performance liquid chromatography and mass spectrometry analysis demonstrated the presence of peptide fragments in macrophage supernatants, and permitted identification of the cleavage sites which confirmed the enzyme families involved. Peptide fragments were shown to be competitive inhibitors of presentation of the full-length peptide to CD4 T cells by fixed and live macrophages. The results indicate that several classes of proteinases can modulate antigen presentation by at least two mechanisms: (1) degradation of extracellular oligopeptides and (2) generation of natural peptide ligands that block antigen presentation to CD4 T cells. The generation of inhibitory natural peptide ligands is a new mechanism of immunoregulation which could operate during the induction of T cell responses in a variety of situations.

Localization of peptide/MHC class II complexes in macrophages following antigen processing of viable Streptococcus pyogenes.

The subcellular localization of peptide/MHC complexes was investigated during processing of the surface M5 protein from Streptococcus pyogenes. Bone marrow-derived macrophages were pulsed with viable S. pyogenes for 20 min followed by various periods of chase. T hybridoma cells detected complexes of one epitope, M5(17-31) with E(d) on the surface of macrophages within 30 min of chase. In contrast, complexes with another epitope, M5(308-319) with A(d) peaked later. Intracellular localization of peptide/MHC-II complexes was studied by subcellular fractionation and detection of complexes in fractions by T hybridoma cells. M5(17-31)/E(d) complexes were detected in light membrane fractions containing plasma membrane and early endosomes by 10-30 min. M5(308-319)/A(d) complexes were detected in these light membranes after 3 h of chase. Thus, the time course of M5(308-319)/A(d) presentation was delayed relative to M5(17-31)/E(d). However, neither type of complex was detected at any time in fractions containing phagosomes. Both species of peptide/MHC complexes localized to endocytic compartments, indicating a role for endosomes in presentation of antigens from phagocytosed bacteria.

The route of bacterial uptake by macrophages influences the repertoire of epitopes presented to CD4 T cells.

We studied MHC class II (MHC-II)-restricted antigen processing of viable Streptococcus pyogenes by murine macrophages for presentation of two CD4 T cell epitopes of the surface M5 protein. We show that presentation of both epitopes was prevented if actin polymerization was inhibited by cytochalasin D, but not if clathrin-dependent receptor-mediated endocytosis was prevented, suggesting uptake of streptococci by phagocytosis or macropinocytosis was required for presentation of the surface M protein. However, treatment of macrophages with amiloride, which selectively blocks membrane ruffling and subsequent macropinocytosis, inhibited the response to one epitope (M5(308-319)), but had no effect on presentation of the other (M5(17-31)). The effect of the inhibitors on uptake of streptococci was analyzed by electron microscopy. Cytochalasin D completely blocked uptake of streptococci, while dimethyl-amiloride only inhibited uptake into spacious compartments. Neither of the inhibitors altered the cell-surface expression of MHC-II and costimulatory molecules analyzed by flow cytometry. The data suggest that distinct epitopes of a protein associated with viable bacteria may be presented optimally following different uptake mechanisms in the same antigen-presenting cells.