title: Study of CD8⁺T lymphocyte responses against human herpesviruses
Creators: name: Vargas Cuero, Ana Laura
Publisher: name: The Open University
Year: 2001

description: Human Herpesviruses establish life-long infections in their hosts. They have developed a series of mechanisms to evade immune recognition and destruction and achieve an almost perfect parasitic state, in which they remain latent within the host for its life span. This thesis deals with Human Cytomegalovirus (HCMV) and Herpes Simplex Virus type 2 (HSV-2) and the immune responses they elicit in healthy and immunocompromised individuals. <br><br> HCMV-specific CD8<sup>+</sup> T lymphocyte responses were studied in a cohort of 91 liver allograft recipients. Frequencies of HCMV-specific CD8<sup>+</sup> T lymphocytes were measured, as well as their activation and memory marker expression, using 4-colour FACS analysis and HLA-peptide tetrameric complexes. In parallel, their function was analysed with a series of T cell functional assays such as ELISptit and Intracellular Cytokine Staining. Frequencies of HCMV-specific CD8<sup>+</sup> T lymphocytes were correlated with levels of FK506 (Tacrolimus), the immunosuppressive drug received by some of these patients. Temporal fluctuations in the frequency of HCMV-specific CD8<sup>+</sup> T lymphocytes were observed, which could be explained by undetectable levels of virus stimulating the immune system. In patients with two HLA types susceptible of study there was a good correlation between responses. There was a positive correlation between viraemia and expression of activation markers (CD38 and HLA-DR) but not between frequencies of HCMV-specific CD8<sup>+</sup> T lymphocytes and PCR. There was a negative correlation between high serum levels (&gt;10 ng/ml) of FK506 and low frequencies of HCMV-specific CD8<sup>+</sup> T lymphocytes. Functionally, only a fraction of the HCMV-specific CD8<sup>+</sup> T lymphocytes secreted IFN-<i>ү</i> upon peptide stimulation and little perforin content was found in these cells. <br><br> The phenotypic and functional heterogeneity of HCMV-specific memory T cell subpopulations in healthy seropositive individuals was also studied. The surface expression of a panel of activation and memory markers (CD38, CD45RA, CD45R0, CD27, CD28, CD57, CD62L, HLA-DR), as well as the proliferation marker Ki67, the chemokine receptors CCR5 and CXCR3 and the Vß TCR repertoire of these antiviral memory populations were analysed. There were considerable frequencies of HCMV-specific CD8<sup>+</sup>T lymphocytes in most individuals, all expressing a spectrum of the CD45RA/R0 isoforms. CD62L and CD28 co-segregated with CD45R0<sup>high</sup> HCMV-specific CD8+ T lymphocytes, which might indicate the existence of different subsets of HCMV-specific memory CD8<sup>+</sup> cells. There were large clonal TCR Vß expansions (of up to 54% of total tetramer positive cells) mainly in Vß 2,8,20 and 22, which might imply a preference for these Vp TCRs upon development of the anti-HCMV memory response. These clonal expansions seemed to correlate with CD45R0 surface expression but not with proliferative capacities. Functionally, CD45R0<sup>high</sup> memory T cells secreted IFN-<i>ү</i> more quickly and in higher quantities than CD45R0<sup>low</sup> cells upon peptide stimulation. However, HCMV-specific CD8<sup>+</sup> T lymphocytes showed little killing capacity which correlated with low levels of perforin in these cells. <br><br> Finally, in order to study the CD8<sup>+</sup>T lymphocyte responses against HSV-2,1 cloned the immediate early genes and the VP22 gene of this virus into the pSCl 1 30R vector.