Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.     

Immune defects in 28-kDa proteasome activator gamma-deficient mice

Protein complexes of the 28-kDa proteasome activator (PA28) family activate the proteasome and may alter proteasome cleavage specificity. Initial investigations have demonstrated a role for the IFN-gamma-inducible PA28alpha/beta complex in Ag processing. Although the noninducible and predominantly nuclear PA28gamma complex has been implicated in affecting proteasome-dependent signaling pathways, such as control of the mitotic cell cycle, there is no previous evidence demonstrating a role for this structure in Ag processing. We therefore generated PA28gamma-deficient mice and investigated their immune function. PA28gamma(-/-) mice display a slight reduction in CD8+ T cell numbers and do not effectively clear a pulmonary fungal infection. However, T cell responses in two viral infection models appear normal in both magnitude and the hierarchy of antigenic epitopes recognized. We conclude that PA28gamma(-/-) mice, like PA28alpha(-/-)/beta(-/-) mice, are deficient in the processing of only specific Ags.     

The structural transition of the prion protein into its pathogenic conformation is induced by unmasking hydrophobic sites

A series of structural intermediates in the putative pathway from the cellular prion protein PrP(C) to the pathogenic form PrP(Sc) was established by systematic variation of low concentrations (<0.1%) of the detergent sodium dodecyl sulfate (SDS) or by the interaction with the bacterial chaperonin GroEL. Most extended studies were carried out with recombinant PrP (90-231) corresponding to the amino acid sequence of hamster prions PrP 27-30. Similar results were obtained with full-length recombinant PrP, hamster PrP 27-30 and PrP(C) isolated from transgenic, non-infected CHO cells. Varying the incubation conditions, i.e. the concentration of SDS, the GroEL and GroEL/ES, but always at neutral pH and room temperature, different conformations could be established. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and to molecular mass, as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation: alpha-helical monomers, soluble alpha-helical dimers, soluble but beta-structured oligomers of a minimal size of 12-14 PrP molecules, and insoluble multimers were observed. A high activation barrier was found between the alpha-helical dimers and beta-structured oligomers. The numbers of SDS-molecules bound to PrP in different conformations were determined: Partially denatured, alpha-helical monomers bind 31 SDS molecules per PrP molecule, alpha-helical dimers 21, beta-structured oligomers 19-20, and beta-structured multimers show very strong binding of five SDS molecules per PrP molecule. Binding of only five molecules of SDS per molecule of PrP leads to fast formation of beta-structures followed by irreversible aggregation. It is discussed that strongest binding of SDS has an effect identical with or similar to the interaction with GroEL thereby inducing identical or very similar transitions. The interaction with GroEL/ES stabilizes the soluble, alpha-helical conformation. The structure and their stabilities and particularly the induction of transitions by interaction of hydrophobic sites of PrP are discussed in respect to their biological relevance.     

In vivo expansion of the residual tumor antigen-specific CD8+ T lymphocytes that survive negative selection in simian virus 40 T-antigen-transgenic mice

Mice that express the viral oncoprotein simian virus 40 (SV40) large T antigen (T-Ag) as a transgene provide useful models for the assessment of the state of the host immune response in the face of spontaneous tumor progression. Line SV11 (H2(b)) mice develop rapidly progressing choroid plexus tumors due to expression of full-length T-Ag from the SV40 promoter. In addition, T-Ag expression in the thymus of SV11 mice results in the deletion of CD8(+) T cells specific for the three H2(b)-restricted immunodominant epitopes of T-Ag. Whether CD8(+) T cells specific for the immunorecessive H2-D(b)-restricted epitope V of T-Ag survive negative selection in SV11 mice has not been determined. Immunization of SV11 mice with rVV-ES-V, a recombinant vaccinia virus expressing epitope V as a minigene, resulted in the induction of weak, but reproducible, epitope V-specific cytotoxic T-lymphocyte (CTL) responses. This weak lytic response corresponded with a decreased frequency of epitope V-specific CTL that could be recruited in SV11 mice. In addition, CTL lines derived from rVV-ES-V-immunized SV11 mice had reduced avidities compared to that seen with CTL derived from healthy mice. Despite this initial weak response, significant numbers of epitope V-specific CD8(+) T cells were detected in SV11 mice ex vivo following a priming-boosting approach and these cells demonstrated high avidity for epitope V. The results suggest that low numbers of tumor-reactive CD8(+) T cells with high avidity for epitope V survive negative selection in SV11 mice but can be expanded by specific boosting approaches in the tumor bearing host.     

Effects of pollution on human growth and development: an introduction

Pollution is a worldwide problem and its potential to influence the physiology of human populations is great. Studies of human growth and development in relation to pollution have increased in number and quality since the mid-twentieth century. Many studies have found that some pollutants have detrimental effects on human growth, particularly prenatal growth. The heavy metal, lead, is commonly found in human populations and is related to smaller size at birth and studies have reported decrements that range up to about 200 grams. Noise stress from transportation sources also is related to reduced prenatal growth with somewhat smaller decrements reported. Studies of humans exposed to polychlorinated biphenyls, one of the persistent organic pollutants, have reduced size at birth, advanced sexual maturation and altered hormone levels related to thyroid regulation. Thus different pollutants exert effects through different physiological pathways. However, some studies have not observed these effects, which indicates that the situation is complex and requires further study with better study designs. Determining the effects of pollutants on human physiology and growth is difficult as it requires fairly large numbers of subjects who are not purposely exposed but for whom exposure can be measured. These effects of pollutants and the mechanisms of effect require further study to understand and, it is hoped, to blunt or block any detrimental effects on human health and well-being.     

Dendritic Cell Migration Limits the Duration of CD8+ T-Cell Priming to Peripheral Viral Antigen

CD8⁺ T cells (T_CD8⁺) play a crucial role in immunity to viruses. Antiviral T_CD8⁺ are initially activated by recognition of major histocompatibility complex (MHC) class I-peptide complexes on the surface of professional antigen-presenting cells (pAPC). Migration of pAPC from the site of infection to secondary lymphoid organs is likely required during a natural infection. Migrating pAPC can be directly infected with virus or may internalize antigen derived from virus-infected cells. The use of experimental virus infections to assess the requirement for pAPC migration in initiation of T_CD8⁺ responses has proven difficult to interpret because injected virus can readily drain to secondary lymphoid organs without the need for cell-mediated transport. To overcome this ambiguity, we examined the generation of antigen-specific T_CD8⁺ after immunization with recombinant adenoviruses that express antigen driven by skin-specific or ubiquitous promoters. We show that the induction of T_CD8⁺ in response to tissue-targeted antigen is less efficient than the response to ubiquitously expressed antigen and that the resulting T_CD8⁺ fail to clear all target cells pulsed with the antigenic peptide. This failure to prime a fully functional T_CD8⁺ response results from a reduced period of priming to peripherally expressed antigen versus ubiquitously expressed antigen and correlated with a brief burst of pAPC migration from the skin, a requirement for induction of the response to peripheral antigen. These results indicate that a reduced duration of pAPC migration after virus infection likely reduces the amplitude of the T_CD8⁺ response, allowing persistence of the peripheral virus.The induction of effector CD8⁺ T cells (T_CD8⁺) is a vital step in the eradication or control of many viral infections. The induction of antiviral T_CD8⁺ requires the presentation of virally derived peptides in complex with major histocompatibility complex (MHC) class I on the surface of specialized professional antigen-presenting cells (pAPC), most commonly a subset of dendritic cells (DC) that bear the CD8α chain (1, 29). The CD8α⁺ DC reside only in secondary lymphoid organs and not in the tissues, implying that cell-mediated transport or drainage of virus particles to a lymph node is required for initiation of a T_CD8⁺ response. Partial inhibition of DC migration from the skin can impair the initiation of a T_CD8⁺ response (2). After influenza infection in the lungs, there is a burst of DC migration, followed by a refractory period in which no DC migration occurs (19). The functional consequences of this refractory period of DC migration have not been explored.A number of viruses, particularly human papillomaviruses, infect the skin and are ignored by the immune response for extended periods of time (31). We sought to explore the possibility that, after a low-level peripheral virus infection of the skin, changes in DC migration may limit the availability of antigen in the draining lymph node and thus the induction of a T_CD8⁺ response. There are a number of confounding factors that make the study of DC migration in the initiation of an antiviral T_CD8⁺ response difficult. Virus particles may directly drain to the lymph node within seconds (11, 13, 25). In addition, many viruses will alter DC functions, including migration, after infection of the DC itself. This may occur via specific viral modulation of DC function (16) or via nonspecific shut down of host protein synthesis (26), both of which will affect migration. Thus, it is often not possible to distinguish between the effects of virus infection upon DC migration, drainage of virus directly to the lymph node, and the natural response that follows migration of DC responding to a peripheral virus infection.There is currently no mouse model of a peripheral virus infection that is confined to the skin, as no natural mouse papillomavirus has ever been isolated. Therefore, to address these issues, we have made use of another small DNA virus, namely, an adenovirus vector that is replication deficient (rAd). These vectors express influenza virus nucleoprotein (NP) under the control of a ubiquitous (cytomegalovirus [CMV] immediate-early) or tissue-targeted promoter (K14, targeted to keratinocytes, the site of papillomavirus replication). Antigen driven by the K14 promoter is expressed only in skin cells, so only uninfected DC can present antigen in this system, removing the need to account for modulation of the function of virus-infected DC.We demonstrate that when antigen is expressed in only keratinocytes in the skin, the efficiency of T_CD8⁺ induction is reduced and the time period for which antigen is available to prime effector cells is reduced dramatically. DC-mediated transport is required for antigen to reach the lymph node where a T_CD8⁺ response is initiated. The reduced time period of antigen presentation is the result of a transient blockade in DC migration from the site of infection. The blockade in DC migration reduced the delivery of viral antigen to the lymph node needed to induce a T_CD8⁺ response. The resulting T_CD8⁺ response to peripheral viral antigen is not capable of clearing all target cells presenting a viral peptide, thus allowing the persistence of peripheral virus-infected cells. These results provide a potential mechanism for the long-term evasion of the immune response by papillomaviruses following natural infection and also have important implications for tissue targeted gene therapy vectors.     

Inflammatory phase of bone healing initiates the regenerative healing cascade

Bone healing commences with an inflammatory reaction which initiates the regenerative healing process leading in the end to reconstitution of bone. An unbalanced immune reaction during this early bone healing phase is hypothesized to disturb the healing cascade in a way that delays bone healing and jeopardizes the successful healing outcome. The immune cell composition and expression pattern of angiogenic factors were investigated in a sheep bone osteotomy model and compared to a mechanically-induced impaired/delayed bone healing group. In the impaired/delayed healing group, significantly higher T cell percentages were present in the bone hematoma and the bone marrow adjacent to the osteotomy gap when compared to the normal healing group. This was mirrored in the higher cytotoxic T cell percentage detected under delayed bone healing conditions indicating longer pro-inflammatory processes. The highly activated periosteum adjourning the osteotomy gap showed lower expression of hematopoietic stem cell markers and angiogenic factors such as heme oxygenase and vascular endothelial growth factor. This indicates a deferred revascularization of the injured area due to ongoing pro-inflammatory processes in the delayed healing group. Results from this study suggest that there are unfavorable immune cells and factors participating in the initial healing phase. In conclusion, identifying beneficial aspects may lead to promising therapeutical approaches that might benefit further by eliminating the unfavorable factors.     

CD8 T cells recruited early in mouse polyomavirus infection undergo exhaustion

Repetitive Ag encounter, coupled with dynamic changes in Ag density and inflammation, imparts phenotypic and functional heterogeneity to memory virus-specific CD8 T cells in persistently infected hosts. For herpesvirus infections, which cycle between latency and reactivation, recent studies demonstrate that virus-specific T cell memory is predominantly derived from naive precursors recruited during acute infection. Whether functional memory T cells to viruses that persist in a nonlatent, low-level infectious state (smoldering infection) originate from acute infection-recruited naive T cells is not known. Using mouse polyomavirus (MPyV) infection, we previously showed that virus-specific CD8 T cells in persistently infected mice are stably maintained and functionally competent; however, a sizeable fraction of these memory T cells are short-lived. Further, we found that naive anti-MPyV CD8 T cells are primed de novo during persistent infection and contribute to maintenance of the virus-specific CD8 T cell population and its phenotypic heterogeneity. Using a new MPyV-specific TCR-transgenic system, we now demonstrate that virus-specific CD8 T cells recruited during persistent infection possess multicytokine effector function, have strong replication potential, express a phenotype profile indicative of authentic memory capability, and are stably maintained. In contrast, CD8 T cells recruited early in MPyV infection express phenotypic and functional attributes of clonal exhaustion, including attrition from the memory pool. These findings indicate that naive virus-specific CD8 T cells recruited during persistent infection contribute to preservation of functional memory against a smoldering viral infection.     

Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.