Models of immune memory: On the role of cross-reactive stimulation, competition, and homeostasis in maintaining immune memory

There has been much debate on the contribution of processes such as the persistence of antigens, cross-reactive stimulation, homeostasis, competition between different lineages of lymphocytes, and the rate of cell turnover on the duration of immune memory and the maintenance of the immune repertoire. We use simple mathematical models to investigate the contributions of these various processes to the longevity of immune memory (defined as the rate of decline of the population of antigen-specific memory cells). The models we develop incorporate a large repertoire of immune cells, each lineage having distinct antigenic specificities, and describe the dynamics of the individual lineages and total population of cells. Our results suggest that, if homeostatic control regulates the total population of memory cells, then, for a wide range of parameters, immune memory will be long-lived in the absence of persistent antigen (T1/2 > 1 year). We also show that the longevity of memory in this situation will be insensitive to the relative rates of cross-reactive stimulation, the rate of turnover of immune cells, and the functional form of the term for the maintenance of homeostasis.

The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin–serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.

The Yeast Saccharomyces cerevisiae Contains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40–52% identity and 61–76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using β-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins.

Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into β-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by β-strand exchange between the reactive loop of one molecule and β-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin α1-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into β-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma α1-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55–Pro mutation.

Melan-A/MART-151–73 represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4+ T cells

The human Melan-A/MART-1 gene encodes an HLA-A2-restricted peptide epitope recognized by melanoma-reactive CD8+ cytotoxic T lymphocytes. Here we report that this gene also encodes at least one HLA-DR4-presented peptide recognized by CD4+ T cells. The Melan-A/MART-151–73 peptide was able to induce the in vitro expansion of specific CD4+ T cells derived from normal DR4+ donors or from DR4+ patients with melanoma when pulsed onto autologous dendritic cells. CD4+ responder T cells specifically produced IFN-γ in response to, and also lysed, T2.DR4 cells pulsed with the Melan-A/MART-151–73 peptide and DR4+ melanoma target cells naturally expressing the Melan-A/MART-1 gene product. Interestingly, CD4+ T cell immunoreactivity against the Melan-A/MART-151–73 peptide typically coexisted with a high frequency of anti-Melan-A/MART-127–35 reactive CD8+ T cells in freshly isolated blood harvested from HLA-A2+/DR4+ patients with melanoma. Taken together, these data support the use of this Melan-A/MART-1 DR4-restricted melanoma epitope in future immunotherapeutic trials designed to generate, augment, and quantitate specific CD4+ T cell responses against melanoma in vivo.

Reactive oxygen species and nitric oxide mediate plasticity of neuronal calcium signaling

Reactive oxygen species (ROS) and nitric oxide (NO) are important participants in signal transduction that could provide the cellular basis for activity-dependent regulation of neuronal excitability. In young rat cortical brain slices and undifferentiated PC12 cells, paired application of depolarization/agonist stimulation and oxidation induces long-lasting potentiation of subsequent Ca2+ signaling that is reversed by hypoxia. This potentiation critically depends on NO production and involves cellular ROS utilization. The ability to develop the Ca2+ signal potentiation is regulated by the developmental stage of nerve tissue, decreasing markedly in adult rat cortical neurons and differentiated PC12 cells.

Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells

Violet-blue light is toxic to mammalian cells, and this toxicity has been linked with cellular production of H2O2. In this report, we show that violet-blue light, as well as UVA, stimulated H2O2 production in cultured mouse, monkey, and human cells. We found that H2O2 originated in peroxisomes and mitochondria, and it was enhanced in cells overexpressing flavin-containing oxidases. These results support the hypothesis that photoreduction of flavoproteins underlies light-induced production of H2O2 in cells. Because H2O2 and its metabolite, hydroxyl radicals, can cause cellular damage, these reactive oxygen species may contribute to pathologies associated with exposure to UVA, violet, and blue light. They may also contribute to phototoxicity often encountered during light microscopy. Because multiphoton excitation imaging with 1,047-nm wavelength prevented light-induced H2O2 production in cells, possibly by minimizing photoreduction of flavoproteins, this technique may be useful for decreasing phototoxicity during fluorescence microscopy.

Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate

Higher plants share with animals a responsiveness to the Ca2+ mobilizing agents inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular 45Ca2+ flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca2+-releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca2+ with a K1/2 = 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca2+ pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca2+ pathway distinguishes it from the InsP3- and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol/mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca2+ release is insensitive to heparin and 8-NH2-cADPR, specific inhibitors of the InsP3- and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca2+ release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca2+ release pathway is independent of cytosolic free Ca2+ and therefore incapable of operating Ca2+-induced Ca2+ release. In contrast to the sea urchin system, the NAADP-gated Ca2+ release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca2+ mobilization pathways and Ca2+ release sites might contribute to the generation of stimulus-specific Ca2+ signals in plant cells.

Implications of macromolecular crowding for signal transduction and metabolite channeling

The effect of different total enzyme concentrations on the flux through the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in vitro was determined by measuring PTS-mediated carbohydrate phosphorylation at different dilutions of cell-free extract of Escherichia coli. The dependence of the flux on the protein concentration was more than linear but less than quadratic. The combined flux–response coefficient of the four enzymes constituting the glucose PTS decreased slightly from values of ≈1.8 with increasing protein concentrations in the assay. Addition of the macromolecular crowding agents polyethylene glycol (PEG) 6000 and PEG 35000 led to a sharper decrease in the combined flux–response coefficient, in one case to values of ≈1. PEG 6000 stimulated the PTS flux at lower protein concentrations and inhibited the flux at higher protein concentrations, with the transition depending on the PEG 6000 concentration. This suggests that macromolecular crowding decreases the dissociation rate constants of enzyme complexes. High concentrations of the microsolute glycerol did not affect the combined flux–response coefficient. The data could be explained with a kinetic model of macromolecular crowding in a two-enzyme group-transfer pathway. Our results suggest that, because of the crowded environment in the cell, the different PTS enzymes form complexes that live long on the time-scale of their turnover. The implications for the metabolic behavior and control properties of the PTS, and for the effect of macromolecular crowding on nonequilibrium processes, are discussed.

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry

The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor–urotensin–sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of ≈1 μM). An accurate Mr value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L29–R30 and R30–A31 under our assay conditions; some Mas-DH is also oxidized, apparently at M2 and M11. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H2O2 or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.

Tobacco smoke is a source of toxic reactive glycation products

Smokers have a significantly higher risk for developing coronary and cerebrovascular disease than nonsmokers. Advanced glycation end products (AGEs) are reactive, cross-linking moieties that form from the reaction of reducing sugars and the amino groups of proteins, lipids, and nucleic acids. AGEs circulate in high concentrations in the plasma of patients with diabetes or renal insufficiency and have been linked to the accelerated vasculopathy seen in patients with these diseases. Because the curing of tobacco takes place under conditions that could lead to the formation of glycation products, we examined whether tobacco and tobacco smoke could generate these reactive species that would increase AGE formation in vivo. Our findings show that reactive glycation products are present in aqueous extracts of tobacco and in tobacco smoke in a form that can rapidly react with proteins to form AGEs. This reaction can be inhibited by aminoguanidine, a known inhibitor of AGE formation. We have named these glycation products “glycotoxins.” Like other known reducing sugars and reactive glycation products, glycotoxins form smoke, react with protein, exhibit a specific fluorescence when cross-linked to proteins, and are mutagenic. Glycotoxins are transferred to the serum proteins of human smokers. AGE-apolipoprotein B and serum AGE levels in cigarette smokers were significantly higher than those in nonsmokers. These results suggest that increased glycotoxin exposure may contribute to the increased incidence of atherosclerosis and high prevalence of cancer in smokers.

The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes

Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.

CD8+ T cells control the TH phenotype of MBP-reactive CD4+ T cells in EAE mice

Trimolecular interactions between the T cell antigen receptor and MHC/peptide complexes, together with costimulatory molecules and cytokines, control the initial activation of naïve T cells and determine whether the helper precursor cell differentiates into either T helper (TH)1 or TH2 effector cells. We now present evidence that regulatory CD8+ T cells provide another level of control of TH phenotype during further evolution of immune responses. These regulatory CD8+ T cells are induced by antigen-triggered CD4+ TH1 cells during T cell vaccination and, in vitro, distinguish mature TH1 from TH2 cells in a T cell antigen receptor Vβ-specific and Qa-1-restricted manner. In vivo, protection from experimental autoimmune encephalomyelitis (EAE) induced by T cell vaccination depends on CD8+ T cells, and myelin basic protein-reactive TH1 Vβ8+ clones, but not TH2 Vβ8+ clones, used as vaccine T cells, protect animals from subsequent induction of EAE. Moreover, in vivo depletion of CD8+ T cells during the first episode of EAE results in skewing of the TH phenotype toward TH1 upon secondary myelin basic protein stimulation. These data provide evidence that CD8+ T cells control autoimmune responses, in part, by regulating the TH phenotype of self-reactive CD4+ T cells.