The role of chloroplast movement in C4 photosynthesis: a theoretical analysis using a three-dimensional reaction-diffusion model for maize.

Chloroplasts movement within mesophyll cells in C4 plants is hypothesized to enhance the CO2 concentrating mechanism, but this is difficult to verify experimentally. A three-dimensional (3D) leaf model can help analyse how chloroplast movement influences the operation of the CO2 concentrating mechanism. The first volumetric reaction-diffusion model of C4 photosynthesis that incorporates detailed 3D leaf anatomy, light propagation, ATP and NADPH production, and CO2, O2 and bicarbonate concentration driven by diffusional and assimilation/emission processes was developed. It was implemented for maize leaves to simulate various chloroplast movement scenarios within mesophyll cells: the movement of all mesophyll chloroplasts towards bundle sheath cells (aggregative movement) and movement of only those of interveinal mesophyll cells towards bundle sheath cells (avoidance movement). Light absorbed by bundle sheath chloroplasts relative to mesophyll chloroplasts increased in both cases. Avoidance movement decreased light absorption by mesophyll chloroplasts considerably. Consequently, total ATP and NADPH production and net photosynthetic rate increased for aggregative movement and decreased for avoidance movement compared with the default case of no chloroplast movement at high light intensities. Leakiness increased in both chloroplast movement scenarios due to the imbalance in energy production and demand in mesophyll and bundle sheath cells. These results suggest the need to design strategies for coordinated increases in electron transport and Rubisco activities for an efficient CO2 concentrating mechanism at very high light intensities.

Treatment of sarcoptic and chorioptic mange in an alpaca (Vicugna pacos) herd with a combination of topical amitraz and subcutaneous ivermectin.

Clinical history: An outbreak of intense pruritus and weight loss in a herd of 40 alpacas (Vicugna pacos) in the south-west of France was investigated after the death of 14 adults. One alpaca was referred to a veterinary teaching hospital for diagnosis and treatment but died soon after and one of the dead alpacas was submitted for necropsy. Clinical findings: The remaining alpacas were intensely pruritic with variably severe and extensive alopecia, erythema, lichenification and crusting on the face, ventral abdomen and distal limbs. Superficial skin scrapes from five animals revealed large numbers of Sarcoptes scabiei mites, and less frequent and numerous Chorioptes bovis mites. Coproscopic examinations revealed a median of 1,350 (min 500, max 8800) strongyle epg. The alpaca admitted for treatment was anaemic and hypoalbuminaemic. Skin scrapes revealed copious S. scabiei and C. bovis mites. The two alpacas examined post-mortem had similar skin lesions to those examined on-farm and were cachexic. One had lung lesions attributed to protostrongylid infestation and its liver contained numerous Dicrocoelium spp. adults. Diagnosis: Sarcoptic and chorioptic mange with secondary superficial bacterial skin infection, associated with severe internal parasitism and underfeeding. Treatment and outcome: All 25 alpacas were treated topically with a 3% chlorhexidine shampoo followed by a 0.025% amitraz wash at the initial visit and then 1, 2, 3, 7 and 9 weeks later. A systemic treatment with S/C 500 µg/kg ivermectin was administered at the initial visit and then 2, 7 and 9 weeks later. The alpacas were treated orally with 50 mg/kg praziquantel to control dicrocoeliosis. Nutritional measures, including increased pasture area and supplemental feeding were simultaneously implemented. Pruritus was reduced 1 week after the start of treatment and had resolved after 2 weeks. After 9 weeks, skin lesions were markedly improved. Six months after the initial visit, skin lesions entirely resolved and superficial skin scrapes, taken from half of the animals, were negative for mites. Clinical relevance: This is the first report of the use of two acaricides combined with a chlorhexidine shampoo to successfully treat simultaneous sarcoptic and chorioptic mange in alpacas.

A two-dimensional microscale model of gas exchange during photosynthesis in maize (Zea mays L.) leaves.

CO2 exchange in leaves of maize (Zea mays L.) was examined using a microscale model of combined gas diffusion and C4 photosynthesis kinetics at the leaf tissue level. Based on a generalized scheme of photosynthesis in NADP-malic enzyme type C4 plants, the model accounted for CO2 diffusion in a leaf tissue, CO2 hydration and assimilation in mesophyll cells, CO2 release from decarboxylation of C4 acids, CO2 fixation in bundle sheath cells and CO2 retro-diffusion from bundle sheath cells. The transport equations were solved over a realistic 2-D geometry of the Kranz anatomy obtained from light microscopy images. The predicted responses of photosynthesis rate to changes in ambient CO2 and irradiance compared well with those obtained from gas exchange measurements. A sensitivity analysis showed that the CO2 permeability of the mesophyll-bundle sheath and airspace-mesophyll interfaces strongly affected the rate of photosynthesis and bundle sheath conductance. Carbonic anhydrase influenced the rate of photosynthesis, especially at low intercellular CO2 levels. In addition, the suberin layer at the exposed surface of the bundle sheath cells was found beneficial in reducing the retro-diffusion. The model may serve as a tool to investigate CO2 diffusion further in relation to the Kranz anatomy in C4 plants.

Persistent Antigen and Prolonged AKT-mTORC1 Activation Underlie Memory CD8 T Cell Impairment in the Absence of CD4 T Cells.

Recall responses by memory CD8 T cells are impaired in the absence of CD4 T cells. Although several mechanisms have been proposed, the molecular basis is still largely unknown. Using a local influenza virus infection in the respiratory tract and the lung of CD4(-/-) mice, we show that memory CD8 T cell impairment is limited to the lungs and the lung-draining lymph nodes, where viral Ags are unusually persistent and abundant in these mice. Persistent Ag exposure results in prolonged activation of the AKT-mTORC1 pathway in Ag-specific CD8 T cells, favoring their development into effector memory T cells at the expense of central memory T cells, and inhibition of mTORC1 by rapamycin largely corrects the impairment by promoting central memory T cell development. The findings suggest that the prolonged AKT-mTORC1 activation driven by persistent Ag is a critical mechanism underlying the impaired memory CD8 T cell development and responses in the absence of CD4 T cells.

Remembrance of immunology past: conversations with Herman Eisen.

Herman Eisen and Sondra Schlesinger spent several days together in September 2007 in Woods Hole, Massachusetts, talking about immunology, focusing on his remembrances of the field over the more than 60 years of his involvement. This article is an abridged version of those discussions (the full version is available on the Annual Reviews website). It is both an oral history and a written memory of some important but selected areas of immunology.

Affinity enhancement of antibodies: how low-affinity antibodies produced early in immune responses are followed by high-affinity antibodies later and in memory B-cell responses.

The antibodies produced initially in response to most antigens are high molecular weight (MW) immunoglobulins (IgM) with low affinity for the antigen, while the antibodies produced later are lower MW classes (e.g., IgG and IgA) with, on average, orders of magnitude higher affinity for that antigen. These changes, often termed affinity maturation, take place largely in small B-cell clusters (germinal center; GC) in lymphoid tissues in which proliferating antigen-stimulated B cells express the highly mutagenic cytidine deaminase that mediates immunoglobulin class-switching and sequence diversification of the immunoglobulin variable domains of antigen-binding receptors on B cells (BCR). Of the large library of BCR-mutated B cells thus rapidly generated, a small minority with affinity-enhancing mutations are selected to survive and differentiate into long-lived antibody-secreting plasma cells and memory B cells. BCRs are also endocytic receptors; they internalize and cleave BCR-bound antigen, yielding peptide-MHC complexes that are recognized by follicular helper T cells. Imperfect correlation between BCR affinity for antigen and cognate T-cell engagement may account for the increasing affinity heterogeneity that accompanies the increasing average affinity of antibodies. Conservation of mechanisms underlying mutation and selection of high-affinity antibodies over the ≈200 million years of evolution separating bird and mammal lineages points to the crucial role of antibody affinity enhancement in adaptive immunity.

Koch Institute Symposium on Cancer Immunology and Immunotherapy.

The 12th annual summer symposium of The Koch Institute for Integrative Cancer Research at MIT was held in Cambridge, MA, on June 14th, 1023. The symposium entitled "Cancer Immunology and Immunotherapy" focused on recent advances in preclinical research in basic immunology and biomedical engineering, and their clinical application in cancer therapies. The day-long gathering also provided a forum for discussion and potential collaborations between engineers and clinical investigators. The major topics presented include: (i) enhancement of adoptive cell therapy by engineering to improve the ability and functionality of T-cells against tumor cells; (ii) current therapies using protein and antibody therapeutics to modulate endogenous anti-tumor immunity; and (iii) new technologies to identify molecular targets and assess therapeutic efficacy, and devices to control and target drug delivery more effectively and efficiently.

Promiscuous binding of extracellular peptides to cell surface class I MHC protein.

Algorithms derived from measurements of short-peptide (8-10 mers) binding to class I MHC proteins suggest that the binding groove of a class I MHC protein, such as K(b), can bind well over 1 million different peptides with significant affinity (<500 nM), a level of ligand-binding promiscuity approaching the level of heat shock protein binding of unfolded proteins. MHC proteins can, nevertheless, discriminate between similar peptides and bind many of them with high (nanomolar) affinity. Some insights into this high-promiscuity/high-affinity behavior and its impact on immunodominant peptides in T-cell responses to some infections and vaccination are suggested by results obtained here from testing a model developed to predict the number of cell surface peptide-MHC complexes that form on cells exposed to extracellular (exogenous) peptides.

Interaction of streptavidin-based peptide-MHC oligomers (tetramers) with cell-surface TCRs.

The binding of oligomeric peptide-MHC (pMHC) complexes to cell surface TCR can be considered to approximate TCR-pMHC interactions at cell-cell interfaces. In this study, we analyzed the equilibrium binding of streptavidin-based pMHC oligomers (tetramers) and their dissociation kinetics from CD8(pos) T cells from 2C-TCR transgenic mice and from T cell hybridomas that expressed the 2C TCR or a high-affinity mutant (m33) of this TCR. Our results show that the tetramers did not come close to saturating cell-surface TCR (binding only 10-30% of cell-surface receptors), as is generally assumed in deriving affinity values (K(D)), in part because of dissociative losses from tetramer-stained cells. Guided by a kinetic model, the oligomer dissociation rate and equilibrium constants were seen to depend not only on monovalent association and dissociation rates (k(off) and k(on)), but also on a multivalent association rate (µ) and TCR cell-surface density. Our results suggest that dissociation rates could account for the recently described surprisingly high frequency of tetramer-negative, functionally competent T cells in some T cell responses.

Antigen-bearing dendritic cells regulate the diverse pattern of memory CD8 T-cell development in different tissues.

Memory T cells of the effector type (T(EM)) account for the characteristic rapidity of memory T-cell responses, whereas memory T cells of the central type (T(CM)) account for long-lasting, vigorously proliferating memory T-cell responses. How antigen-stimulated (primed) T cells develop into different memory T-cell subsets with diverse tissue distributions is largely unknown. Here we show that after respiratory tract infection of mice with influenza virus, viral antigen associated with dendritic cells (DCs) was abundant in lung-draining lymph nodes (DLN) and the spleen for more than a week but was scant and transient in nondraining lymph nodes (NDLN). Correspondingly, activated CD8 T cells proliferated extensively in DLN and the spleen but minimally in NDLN. Strikingly, however, although most persisting CD8 T cells in DLN and spleen exhibited the T(EM) phenotype, those persisting in NDLN exhibited the T(CM) phenotype. Reducing antigen exposure by depleting DCs at the peak of primary T-cell responses enhanced the development of T(CM), whereas subjecting primed CD8 T cells from NDLN to additional antigen stimulation inhibited T(CM) development. These findings demonstrate that differences in persistence of antigen-bearing DCs in various tissues regulate the tissue-specific pattern of memory CD8 T-cell development. The findings have significant implications for design of vaccines and immunization strategies.

Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation.

During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.

The impact of TCR-binding properties and antigen presentation format on T cell responsiveness.

TCR interactions with cognate peptide-MHC (pepMHC) ligands are generally low affinity. This feature, together with the requirement for CD8/CD4 participation, has made it difficult to dissect relationships between TCR-binding parameters and T cell activation. Interpretations are further complicated when comparing different pepMHC, because these can vary greatly in stability. To examine the relationships between TCR-binding properties and T cell responses, in this study we characterized the interactions and activities mediated by a panel of TCRs that differed widely in their binding to the same pepMHC. Monovalent binding of soluble TCR was characterized by surface plasmon resonance, and T cell hybridomas that expressed these TCR, with or without CD8 coexpression, were tested for their binding of monomeric and oligomeric forms of the pepMHC and for subsequent responses (IL-2 release). The binding threshold for eliciting this response in the absence of CD8 (K(D) = 600 nM) exhibited a relatively sharp cutoff between full activity and no activity, consistent with a switchlike response to pepMHC on APCs. However, when the pepMHC was immobilized (plate bound), T cells with the lowest affinity TCRs (e.g., K(D) = 30 microM) responded, even in the absence of CD8, indicating that these TCR are signaling competent. Surprisingly, even cells that expressed high-affinity (K(D) = 16 nM) TCRs along with CD8 were unresponsive to oligomers in solution. The findings suggest that to drive downstream T cell responses, pepMHC must be presented in a form that supports formation of appropriate supramolecular clusters.

Rapid tolerization of virus-activated tumor-specific CD8+ T cells in prostate tumors of TRAMP mice.

To study T cell responses to tumors in an autochthonous model, we expressed a CD8 T cell epitope SIYRYYGL (SIY) in the prostate of transgenic adenocarcinoma (TRAMP) mice (referred to as TRP-SIY), which spontaneously develop prostate cancer. Naïve SIY-specific CD8 T cells adoptively transferred into TRP-SIY mice became tolerized in the prostate draining lymph nodes. Vaccination of TRP-SIY mice intranasally with influenza virus that expresses the SIY epitope resulted in generation of SIY-specific effector T cells in the lung-draining lymph nodes. These effector T cells expressed TNFalpha and IFNgamma, eliminated SIY peptide-loaded target cells in vivo, and infiltrated prostate tumors, where they rapidly lost the ability to produce effector cytokines. A population of these T cells persisted in prostate tumors but not in lymphoid organs and could be induced to re-express effector functions following cytokine treatment in vitro. These findings suggest that T cells of a given clone can be activated and tolerized simultaneously in different microenvironments of the same host and that effector T cells are rapidly tolerized in the tumors. Our model provides a system to study T cell-tumor interactions in detail and to test the efficacy of cancer immunotherapeutic strategies.

Stage-dependent reactivity of thymocytes to self-peptide--MHC complexes.

In mice that express a transgene for the 2C T cell antigen-receptor (TCR) and lack a recombinase-activating gene (2C(+)RAG(-/-) mice) most of the peripheral T cells are CD8(+), a few are CD4(+), and a significant fraction are CD4(-)CD8(-) [double negative (DN)]. The DN 2C cells, like DN T cells that are abundant in various other alphabeta TCR-transgenic mice, appear to be derived directly from DN thymocytes that prematurely express the TCR transgene. The DN 2C cells are virtually absent in mice deficient in major histocompatibility complex class II (MHC-II) but more abundant in mice deficient in MHC-I, suggesting that the DN 2C thymocytes are positively selected by self-peptide-MHC-II (pMHC-II) complexes and negatively selected by self-pMHC-I complexes. The pMHC-I complexes, however, positively select CD8(+) 2C T cells in the same mice. The different effects of thymic pMHC-I on DN and CD8(+) thymocytes are consistent with the finding that DN 2C thymocytes are more sensitive than more mature CD4(+)CD8(+) [double positive (DP)] thymocytes to a weak pMHC-I agonist for the 2C TCR. Together with previous evidence that DP thymocytes respond more sensitively than T cells in the periphery to weak pMHC agonists, the findings suggest progressive decreases in responsiveness to self-pMHC-I complexes as thymocytes develop from DN to DP thymocytes and then to mature naïve T cells in the periphery.

Multiple intracellular routes in the cross-presentation of a soluble protein by murine dendritic cells.

Soluble heat shock fusion proteins (Hsfp) stimulate mice to produce CD8+ CTL, indicating that these proteins are cross-presented by dendritic cells (DC) to naive CD8 T cells. We report that cross-presentation of these proteins depends upon their binding to DC receptors, likely belonging to the scavenger receptor superfamily. Hsfp entered DC by receptor-mediated endocytosis that was either inhibitable by cytochalasin D or not inhibitable, depending upon aggregation state and time. Most endocytosed Hsfp was transported to lysosomes, but not the small cross-presented fraction that exited early from the endocytic pathway and required access to proteasomes and TAP. Naive CD8 T cell (2C and OT-I) responses to DC incubated with Hsfp at 1 microM were matched by incubating DC with cognate octapeptides at 1-10 pM, indicating that display of very few class I MHC-peptide complexes per DC can be sufficient for cross-presentation. With an Hsfp (heat shock protein-OVA) having peptide sequences for both CD4+ (OT-II) and CD8+ (OT-I) cells, the CD4 cells responded far more vigorously than the CD8 cells and many more class II MHC-peptide than class I MHC-peptide complexes were displayed.

Poly-beta amino ester-containing microparticles enhance the activity of nonviral genetic vaccines.

Current nonviral genetic vaccine systems are less effective than viral vaccines, particularly in cancer systems where epitopes can be weakly immunogenic and antigen-presenting cell processing and presentation to T cells is down-regulated. A promising nonviral delivery method for genetic vaccines involves microencapsulation of antigen-encoding DNA, because such particles protect plasmid payloads and target them to phagocytic antigen-presenting cells. However, conventional microparticle formulations composed of poly lactic-co-glycolic acid take too long to release encapsulated payload and fail to induce high levels of target gene expression. Here, we describe a microparticle-based DNA delivery system composed of a degradable, pH-sensitive poly-beta amino ester and poly lactic-co-glycolic acid. These formulations generate an increase of 3-5 orders of magnitude in transfection efficiency and are potent activators of dendritic cells in vitro. When used as vaccines in vivo, these microparticle formulations, unlike conventional formulations, induce antigen-specific rejection of transplanted syngenic tumor cells.

DNA damage in lung after oral exposure to diesel exhaust particles in Big Blue rats.

Several chemical mutagens and carcinogens, including polycyclic aromatic hydrocarbons (PAHs) and nitrated PAHs, are adsorbed to the surface of diesel exhaust particles (DEP). DEP can induce formation of reactive oxygen species and cause oxidative DNA damage as well as bulky carcinogen DNA adducts. Lung tissue is a target organ for DEP induced cancer following inhalation. Recent studies have provided evidence that the lung is also a target organ for DNA damage and cancer after oral exposure to other complex mixtures of PAHs. The genotoxic effect of oral administration of DEP was investigated, in terms of markers of DNA damage, mutations and repair, in the lung of Big Blue rats fed a diet with 0, 0.2, 0.8, 2, 8, 20 or 80 mg DEP/kg feed for 21 days. There was no significant increase in the mutation frequency in the cII gene. However, an increase of DNA damage measured as DNA strand breaks (comet assay) and bulky DNA adducts (32P post labeling) was observed. The level of DNA strand breaks increased significantly at all dose levels while the level of DNA adducts increased significantly only at the intermediate dose levels. Similarly, the number of oxidized DNA bases measured as endonuclease III and fapyguanine glycosylase (FPG) sensitive sites increased at the intermediate dose levels. The induction of DNA damage by DEP exposure did not increase the expression of the repair genes OGG1 and ERCC1 at the mRNA level. The present study indicates that the lung is a target organ for primary DNA damage following oral exposure to DEP. DNA damage was induced following exposure to relatively low levels of DEP, but under the conditions used in the present experiment DNA damage did not result in an increased mutation rate.

Molecularly engineered poly(ortho ester) microspheres for enhanced delivery of DNA vaccines.

Genetic vaccination using plasmid DNA presents a unique opportunity for achieving potent immune responses without the potential limitations of many conventional vaccines. Here we report the design of synthetic biodegradable polymers specifically for enhancing DNA vaccine efficacy in vivo. We molecularly engineered poly(ortho ester) microspheres that are non-toxic to cells, protect DNA from degradation, enable uptake by antigen-presenting cells, and release DNA rapidly in response to phagosomal pH. One type of microsphere of poly(ortho esters) that releases DNA vaccines in synchrony with the natural development of adaptive immunity, elicited distinct primary and secondary humoral and cellular immune responses in mice, and suppressed the growth of tumour cells bearing a model antigen. This polymer microparticulate system could, with further study, have implications for advancing the clinical utility of DNA vaccines as well as other nucleic-acid-based therapeutics against viral infections and cancer.

Competition for self-peptide-MHC complexes and cytokines between naive and memory CD8+ T cells expressing the same or different T cell receptors.

To study competition between naïve and memory T cells, we examined proliferation of adoptively transferred naïve CD8(+) T cells in lymphopenic recipients or recipients containing a clonal population of CD8(+) T cells. We find a hierarchy in the extent of T cell proliferation that appears to correlate with the strength of T cell receptor (TCR)-self-peptide-MHC (pepMHC) interactions. CD8(+) T cells also proliferate in recipients containing a full complement of CD8(+) cells with a different TCR if the transferred T cells experience stronger TCR-self-pepMHC interactions than the resident T cells. Furthermore, CD8(+) T cells proliferate in recipients that contain memory CD8(+) cells with a different TCR, but in this case the relative strengths of TCR-self-pepMHC interactions are not as critical. In contrast, CD8(+) T cells do not proliferate significantly in recipients harboring naïve or memory CD8(+) cells that bear the same TCR as the transferred cells. These results suggest that, among naïve T cells and between naïve and memory T cells, CD8(+) cells having the same TCR compete for both self-pepMHC and cytokines, whereas TCR-different CD8(+) cells compete for cytokines. These competitive relationships probably help maintain the size and TCR diversity of naïve and memory T cell populations required for optimal immune responses.

A role for Toll-like receptor 4 in dendritic cell activation and cytolytic CD8+ T cell differentiation in response to a recombinant heat shock fusion protein.

Recombinant heat shock fusion proteins (Hsfp) injected into mice without added adjuvants can stimulate production of CD8 cytolytic T cells. Because initiation of productive immune responses generally requires dendritic cell (DC) activation, the question arises as to whether the Hsfp can activate DC independently of contaminating LPS. Using microarray analyses of DC from LPS-insensitive mice having a point mutation in Toll-like receptor 4 (Tlr4) (C3H/HeJ), or lacking Tlr4 (B10/ScNCr), we show here that unlike a LPS standard, Hsfp activated DC from HeJ mice almost as well as DC from wild-type mice. Consistent with the microarray analysis, the Hsfp's ability to activate DC was not eliminated by polymyxin B but was destroyed by proteinase K. The Hsfp did not, however, stimulate DC from mice lacking Tlr4. In vivo the CD8 T cell response to the Hsfp in mice lacking Tlr4 was impaired: the responding CD8 cells initially proliferated vigorously but their development into cytolytic effector cells was diminished. Overall, the results indicate that this Hsfp can activate DC independently of LPS but still requires Tlr4 for an optimal CD8 T cell response.