T cell responses specific for Respiratory Syncytial Virus : Aspects of antigen presentation

Respiratory Syncytial Virus is a major cause of childhood lower respiratory tract infections. Seventy percent of all children are infected in their first year of life, and nearly all children by age three. Although the majority of patients suffer a relatively mild upper respiratory tract infection, 3% of infected children are hospitalized and require oxygen support. Risk factors for a severe RSV infection are pre-maturity, congenital heart disease and bronchopulmonary dysplasia, although genetic predisposition also seems to play a role. Unfortunately however, the precise mechanism(s) of severe lower respiratory tract disease remain unknown. T cells play an important role in clearing the virus from the infected lung. However, T cells may also be involved in immune pathology that can be associated with respiratory disease. The scope of this thesis was to investigate the mechanism of the initiation of the cellular immune response after RSV infection. New insights of the interaction of the virus with the immune system can provide new directions for the rational design of a vaccine. In the first part of this thesis we focused on characterization of T cell epitopes. One CD4 T cell epitope was found to be HLA-DP4 restricted. The HLA-DP4 restricted epitope is an interesting finding since HLA-DP4 is the most prevalent HLA class II molecule, and epitopes presented by this HLA allele can therefore be used as a tool to measure RSV specific T cell responses in a large population. Furthermore five CD8 T cell epitopes were identified. All epitopes were proven to be processed and presented by RSV infected antigen presenting cells. The information obtained from these studies allowed us to monitor virus specific CD4+ and CD8+ T cell responses and extend the knowledge on the interplay of RSV with the adaptive and innate immune systems. Furthermore, we addressed the question whether mutations in the NS proteins of natural RSV isolates would lead to altered cytotoxic T cell responses, because the NS proteins interfere with the host capacity to induce type I interferon production and thus interfere with the capacity of the cell to suppress viral replication. We found three novel amino acid changes in NS proteins in virus isolates. The mutations lead to altered CD8 T cell responses. Dendritic cells are considered professional antigen presenting cells crucial for the initiation of immune responses. They have the ability to direct and polarize T cells. Since DC are abundantly present in the airways, the effect of RSV infection on DC and how RSV infection impacted on the ability of DC to initiate T cell activation was studied in the second part of this thesis. We have found that RSV-infected myeloid DC secreted an a as yet un-identified soluble suppressive factor that was responsible for the inefficient expansion of naive T cells and an impaired cytokine production by the cells that did expand. In contrast to the inhibitory effect on proliferation by both naive and memory T cells RSV-DC were not impaired in the ability to induce IFN- in memory CD4 and CD8 T cells. Moreover, antigen processing and presentation via the HLA class I and II route were intact in RSV infected DCs. We observed that exposure of DC to inactivated RSV did not lead to the inhibitory phenotype of DC. These DC were able to present viral peptides via both HLA class I and II, which demonstrated that DC could internalize inactivated virus and stimulate CD8 T cells by cross-presentation

Neonatal brain oxygenation during thoracoscopic correction of esophageal atresia

Background. Little is known about the effects of carbon dioxide (CO2) insufflation on cerebral oxygenation during thoracoscopy in neonates. Near-infrared spectroscopy can measure perioperative brain oxygenation [regional cerebral oxygen saturation (rScO2)]. Aims. To evaluate the effects of CO2 insufflation on rScO2 during thoracoscopic esophageal atresia (EA) repair. Methods. This is an observational study during thoracoscopic EA repair with 5 mmHg CO2 insufflation pressure. Mean arterial blood pressure (MABP), arterial oxygen saturation (SaO2), partial pressure of arterial carbon dioxide (paCO2), pH, and rScO2 were monitored in 15 neonates at seven time points: baseline (T0), after anesthesia induction (T1), after CO2-insufflation (T2), before CO2-exsufflation (T3), and postoperatively at 6 (T4), 12 (T5), and 24 h (T6). Results. MABP remained stable. SaO2 decreased from T0 to T2 [97 ± 3–90 ± 6 % (p < 0.01)]. PaCO2 increased from T0 to T2 [41 ± 6–54 ± 15 mmHg (p < 0.01)]. pH decreased from T0 to T2 [7.33 ± 0.04–7.25 ± 0.11 (p < 0.05)]. All parameters recovered during the surgical course. Mean rScO2 was significantly higher at T1 compared to T2 [77 ± 10–73 ± 7 % (p < 0.05)]. Mean rScO2 levels never dropped below a safety threshold of 55 %. Conclusion. The impact of neonatal thoracoscopic repair of EA with insufflation of CO2 at 5 mmHg was studied. Intrathoracic CO2 insufflation caused a reversible decrease in SaO2 and pH and an increase in paCO2. The rScO2 was higher at anesthesia induction but remained stable and within normal limits during and after the CO2 pneumothorax, which suggest no hampering of cerebral oxygenation by the thoracoscopic intervention. Future studies will focus on the long-term effects of this surgery on the developing brain

Characterization of the CD8+ T cell responses directed against respiratory syncytial virus during primary and secondary infection in C57BL/6 mice.

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4+ and CD8+ T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/ 6 background, we characterized the specificity, level and functional capabilities of CD8+ T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes were recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8+ T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice. © 2006 Elsevier Inc. All rights reserved. Keywords: Respiratory syncytial virus; C57BL/6; CD8+ T cells; Inactivation; Tetrame