Martin Hage Larsen

Evidence of premature immune aging in patients thymectomized during early childhood.

While the thymus is known to be essential for the initial production of T cells during early life, its contribution to immune development remains a matter of debate. In fact, during cardiac surgery in newborns, the thymus is completely resected to enable better access to the heart to correct congenital heart defects, suggesting that it may be dispensable during childhood and adulthood. Here, we show that young adults thymectomized during early childhood exhibit an altered T cell compartment. Specifically, absolute CD4+ and CD8+ T cell counts were decreased, and these T cell populations showed substantial loss of naive cells and accumulation of oligoclonal memory cells. A subgroup of these young patients (22 years old) exhibited a particularly altered T cell profile that is usually seen in elderly individuals (more than 75 years old). This condition was directly related to CMV infection and the induction of strong CMV-specific T cell responses, which may exhaust the naive T cell pool in the absence of adequate T cell renewal from the thymus. Together, these marked immunological alterations are reminiscent of the immune risk phenotype, which is defined by a cluster of immune markers predictive of increased mortality in the elderly. Overall, our data highlight the importance of the thymus in maintaining the integrity of T cell immunity during adult life.

Pd-1 expression on human Cd8 T cells depends on both state of differentiation and activation status

Objective and design:PD-1 expression on HIV-specific CD8 T cells was recently reported to reflect functional exhaustion, resulting in uncontrolled HIV-1 replication. Assessing PD-1 expression on T cells may be highly relevant in T-cell immunology and vaccine monitoring. However, this requires us to gain further insights into the significance of PD-1 expression on CD8 T cells in humans. Methods:We performed a detailed analysis of PD-1 expression pattern on various CD8 T cell subsets from healthy or HIV infected donors. Results:PD-1 expression has two facets in vivo. On the one hand, it is linked to T-cell differentiation: PD-1 is up-regulated on early/intermediate differentiated subsets, which include HIV and Epstein–Barr virus-specific CD8 T-cell populations, but is down-regulated during late stages of differentiation. On the other hand, it is linked to T-cell activation: on PD-1 positive cells, PD-1 over-expression occurs along with the up-regulation of activation markers such as CD38 or HLA-DR. Conclusions:PD-1 expression on CD8 T cells, including those specific for HIV, can be related both to their differentiation stage and their activation status. It is important to consider these findings when assessing the expression of PD-1 on T cells.

Functional improvement of antibody fragments using a novel phage coat protein III fusion system.

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.

Distinct differentiation profiles of HIV-Gag and Nef-specific central memory CD8+ T cells associated with HLA-B57/5801 and virus control.

Objectives:A superior capacity of controlling HIV has been attributed to CD8+ T cells directed against HIV-Gag compared to Nef, particularly in the context of some protective human leukocyte antigen (HLA) alleles. To further elucidate this protective effect, we compared the multifunctional and differentiation characteristics of CD8+ T cells specific for HIV-Gag and Nef in HLA-B57/5801-positive and negative nonprogressors. Methods:A head-to-head comparison of CD8+ T cells specific for HIV-Gag and Nef frequencies, cytokine production and differentiation was conducted, in 11 HLA-B57/5801+ and 11 HLA-B57/5801− HIV-infected individuals selected from a cohort of 53 nonprogressors by using IFN-γ-ELISpot assay and flow cytometry analysis of intracellular cytokine production and differentiation profile. Correlations with HIV parameters were studied. Results:Frequencies of Gag-specific but not of Nef-specific CD8+ T cells correlated with peripheral blood mononuclear cell (PBMC)-associated HIV-DNA. The HIV-Gag and Nef-specific CD8+ T cells did not differ for IL-2 production in either HLA-B57/5801+ or HLA-B57/5801− individuals. The IFN-γ-producing Gag-specific CD8+ T cells in HLA-B57/5801+ individuals significantly differed from their Nef-specific counterparts by displaying higher proportions of central memory CD45RA-CCR7+ cells positive for CD27. This differentiation pattern was not observed in HLA-B57/5801− individuals. Only these HLA-B57/5801-positive Gag-specific CD27+ central memory CD8+ T cells, but not their Nef-specific counterparts, negatively correlated with cell-associated HIV-DNA. Conclusion:HLA-B57/5801 drives a preferential CD27+ differentiation of central memory CD8+ T cells directed against HIV-Gag but not Nef that may contribute to the ability of Gag-specific CD8+ T cells to better control HIV in HLA-B57/5801+ nonprogressors.

Upregulation of Interleukin 7 Receptor Alpha and Programmed Death 1 Marks an Epitope-Specific CD8+ T-Cell Response That Disappears following Primary Epstein-Barr Virus Infection

ABSTRACT In immunocompetent individuals, the stability of the herpesvirus-host balance limits opportunities to study the disappearance of a virus-specific CD8+ T-cell response. However, we noticed that in HLA-A*0201-positive infectious mononucleosis (IM) patients undergoing primary Epstein-Barr virus (EBV) infection, the initial CD8 response targets three EBV lytic antigen-derived epitopes, YVLDHLIVV (YVL), GLCTLVAML (GLC), and TLDYKPLSV (TLD), but only the YVL and GLC reactivities persist long-term; the TLD response disappears within 10 to 27 months. While present, TLD-specific cells remained largely indistinguishable from YVL and GLC reactivities in many phenotypic and functional respects but showed unique temporal changes in two markers of T-cell fate, interleukin 7 receptor alpha (IL-7Rα; CD127) and programmed death 1 (PD-1). Thus, following the antigen-driven downregulation of IL-7Rα seen on all populations in acute IM, in every case, the TLD-specific population recovered expression unusually quickly post-IM. As well, in four of six patients studied, TLD-specific cells showed very strong PD-1 upregulation in the last blood sample obtained before the cells’ disappearance. Our data suggest that the disappearance of this individual epitope reactivity from an otherwise stable EBV-specific response (i) reflects a selective loss of cognate antigen restimulation (rather than of IL-7-dependent signals) and (ii) is immediately preceded, and perhaps mediated, by PD-1 upregulation to unprecedented levels.

Modifying antibody specificity by chain shuffling of V / V between antibodies with related specificities

Histo‐blood group antigens are important markers of developmental stages and as such also often of tumours. Generation of antibodies towards these carbohydrate structures is still a challenging task as they may lack specificity, affinity or are only of the IgM class. We have examined four own antibodies to Lewis Y/H type 2 for their fine specificities using a large panel of mono‐ and oligosaccharides. Sequence alignment to other antibodies with similar specificity revealed an overall limited variation, and that our antibodies constitute a novel set. Based on produced and analysed chimeric mouse–human antibodies, extensive chain shuffling experiments were performed in order to analyse influences of the respective H and L chains on the specificity of the antibodies, and to generate modified antibodies with improved properties. One chIgG1 out of the shuffled antibodies revealed improved specificity and markedly enhanced functional affinity to Lewis Y compared to the parental chIgG1 antibodies. Therefore, the combinatorial approach of chain shuffling provides a platform to improve specificity and/or affinity of anti‐carbohydrate antibodies.

Enhancement of DNA vaccine potency through linkage of antigen to filamentous bacteriophage coat protein III domain I.

Although DNA‐based cancer vaccines have been successfully tested in mouse models, a major drawback of cancer vaccination still remains, namely that tumour antigens are weak and fail to generate a vigorous immune response in tumour‐bearing patients. Genetic technology offers strategies for promoting immune pathways by adding immune‐activating genes to the tumour antigen sequence. In this work, we converted a model non‐immunogenic antigen into a vaccine by fusing it to domain I of the filamentous bacteriophage coat protein III gene. Vaccination with a DNA construct encoding the domain I fusion generated antigen‐specific T helper 1‐type cellular immune responses. These results demonstrate that the incorporation of protein III into a DNA vaccine formulation can modulate the gene‐mediated immune response and may thus provide a strategy for improving its therapeutic effect.

Stress and food deprivation: linking physiological state to migration success in a teleost fish

ABSTRACT Food deprivation is a naturally occurring stressor that is thought to influence the ultimate life-history strategy of individuals. Little is known about how food deprivation interacts with other stressors to influence migration success. European populations of brown trout (Salmo trutta) exhibit partial migration, whereby a portion of the population smoltifies and migrates to the ocean, and the rest remain in their natal stream. This distinct, natural dichotomy of life-history strategies provides an excellent opportunity to explore the roles of energetic state (as affected by food deprivation) and activation of the glucocorticoid stress response in determining life-history strategy and survival of a migratory species. Using an experimental approach, the relative influences of short-term food deprivation and experimental cortisol elevation (i.e. intra-coelomic injection of cortisol suspended in cocoa butter) on migratory status, survival and growth of juvenile brown trout relative to a control were evaluated. Fewer fish migrated in both the food deprivation and cortisol treatments; however, migration of fish in cortisol and control treatments occurred at the same time while that of fish in the food deprivation treatment was delayed for approximately 1 week. A significantly greater proportion of trout in the food deprivation treatment remained in their natal stream, but unlike the cortisol treatment, there were no long-term negative effects of food deprivation on growth, relative to the control. Overall survival rates were comparable between the food deprivation and control treatments, but significantly lower for fish in the cortisol treatment. Food availability and individual energetic state appear to dictate the future life-history strategy (migrate or remain resident) of juvenile salmonids while experimental elevation of the stress hormone cortisol causes impaired growth and reduced survival of both resident and migratory individuals. Summary: Individual energetic state appears to dictate future life-history strategy whereas an artificial stressor impairs growth and reduces survival regardless of life-history strategy.

An experimental field evaluation of winter carryover effects in semi-anadromous brown trout (Salmo trutta)

For semi-anadromous brown trout, the decision whether or not to smoltify and migrate to the sea is believed to be made at the end of the preceding summer in response to both local environmental conditions and individual physiological status. Stressors experienced during the fall may therefore influence their propensity to migrate as well as carry over into the winter resulting in mortality when fish face challenging environmental conditions. To evaluate this possibility, we artificially elevated cortisol levels in juvenile trout (via intracoelomic injection of cortisol in the fall) and used passive integrated transponder tags to compare their overwinter and spring survival, growth, and migration success relative to a control group. Results suggest that overwinter mortality is high for individuals in this population regardless of treatment. However, survival rates were 2.5 times lower for cortisol-treated fish and they experienced significantly greater loss in mass. In addition, less than half as many cortisol-treated individuals made it downstream to a stationary antenna over the winter and also during the spring migration compared to the control treatment. These results suggest that a fall stressor can reduce overwinter survival of juvenile brown trout, negatively impact growth of individuals that survive, and ultimately result in a reduction in the number of migratory trout. Carryover effects such as those documented here reveal the cryptic manner in which natural and anthropogenic stressors can influence fish populations. J. Exp. Zool. 323A: 645-654, 2015.

Short-term and long-term effects of transient exogenous cortisol manipulation on oxidative stress in juvenile brown trout

ABSTRACT In the wild, animals are exposed to a growing number of stressors with increasing frequency and intensity, as a result of human activities and human-induced environmental change. To fully understand how wild organisms are affected by stressors, it is crucial to understand the physiology that underlies an organisms response to a stressor. Prolonged levels of elevated glucocorticoids are associated with a state of chronic stress and decreased fitness. Exogenous glucocorticoid manipulation reduces an individuals ability to forage, avoid predators and grow, thereby limiting the resources available for physiological functions like defence against oxidative stress. Using brown trout (Salmo trutta), we evaluated the short-term (2 weeks) and long-term (4 months over winter) effects of exogenous cortisol manipulations (versus relevant shams and controls) on the oxidative status of wild juveniles. Cortisol caused an increase in glutathione over a 2 week period and appeared to reduce glutathione over winter. Cortisol treatment did not affect oxidative stress levels or low molecular weight antioxidants. Cortisol caused a significant decrease in growth rates but did not affect predation risk. Over-winter survival in the stream was associated with low levels of oxidative stress and glutathione. Thus, oxidative stress may be a mechanism by which elevated cortisol causes negative physiological effects. Summary: Transient exogenous cortisol administration to brown trout causes an increase in glutathione in the short term, but this increase is not maintained in the long term; overwinter survival is associated with low levels of oxidative stress and glutathione.