Human Cytomegalovirus-specific Memory CD8+ and CD4+ T Cell Differentiation after Primary Infection

Daniele Lilleri 0 Chiara Fornara 0 Maria Grazia Revello 0 Giuseppe Gerna 0 0 Servizio di Virologia, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo , Pavia , Italy Background. The development of human cytomegalovirus (HCMV)-specific T cell immunity after primary infection and its correlation with virus transmission to the fetus were investigated. Methods. The membrane phenotype (CCR7 and CD45RA expression) of and intracellular cytokine (interferon [IFN]- and interleukin-2) production by HCMV-specific T cells (stimulated with HCMV-infected dendritic cells) were investigated in 21 immunocompetent pregnant women (12 transmitters and 9 nontransmitters) and in 5 nonpregnant subjects during the first year after infection. Results. IFN- -producing CD4 and CD8 T cells were readily detected during the first month, and their levels did not significantly change with time. CCR7 expression was negligible during both the early and the late stage of infection. Among CCR7 cells, those reexpressing CD45RA progressively increased until they reached median levels of 33% (range, 7%-51%) and 51% (range, 22%-76%) for HCMV-specific CD4 and CD8 T cells, respectively, similar to those observed in subjects with remote infection. CD45RA reexpression correlated with HCMV disappearance from blood. The level of HCMV-specific CD45RA T cells during the first months after infection was significantly lower in mothers who were transmitters than in those who were nontransmitters. Conclusions. After primary infection, circulating HCMV-specific effector T cells revert to the CD45RA phenotype, which appears to be associated with control of viremia and vertical transmission. Thus, these cells may represent long-lived true memory lymphocytes in the HCMV-specific pool. - Human cytomegalovirus (HCMV) infection is generally clinically mild or silent [1, 2] in immunocompetent hosts, whereas it can lead to severe disease and mortality in immunocompromised patients [3, 4]. In addition, HCMV is the major cause of congenital viral infection, often resulting in sensory and developmental impairment [5, 6]. Several studies have documented the importance of both the T cellmediated and the humoral immune response for the control of HCMV replication [710] and have shown that women with preconceptional immunity to HCMV only rarely transmit infection to the fetus [5, 6, 11]. Pregnant women who acquire primary HCMV infection and transmit the virus to the fetus show a delayed appearance of peripheral blood T cells that can proliferate in vitro in response to HCMV antigen, compared with nontransmitting women [12, 13]. Nevertheless, little is known about the development of a cell-mediated immune response and the generation of the memory T cell pool after primary HCMV infection in immunocompetent hosts. Therefore, a deeper knowledge of the natural development of HCMVspecific memory T cells is needed both to study and to verify the protective activity of potential vaccine candidates and to investigate how the natural development of the immune response can influence virus transmission to the fetus in pregnant women. Different subpopulations of T cells can be recognized by analyzing surface expression of the chemokine receptor CCR7 (which enables cells to migrate to lymph nodes) and the different isoforms of the tyrosine phosphatase CD45 [14]. Naive T cells (CD45RA CCR7 ), after encountering antigen, switch from the CD45RA isoform to CD45RO, and memory T cells, according to the differential CCR7 expression, can be divided into central memory T cells (TCM cells; CD45RA CCR7 ), which are able to migrate to lymph nodes and display a high proliferation potential, and effector memory T cells (TEM cells; CD45RA CCR7 ), which exert effector functions in peripheral tissues. Furthermore, a proportion of TEM cells specific for some persistent viruses, such as HCMV and Epstein-Barr virus (EBV), can revert to the RA isoform of CD45 (TEMRA cells; CD45RA CCR7 ) after the acute phase of infection [15, 16]. However, the developmental pathways of the different memory T cell lineages are still debated [1719]. To date, only a few studies have analyzed the differentiation pattern of memory T cells during primary HCMV infection in immunocompetent hosts [20, 21], focusing on T cells specific for some HCMV proteins, such as pp65 and IE-1. The present study was designed to investigate this issue by analyzing the cytokine (interferon [IFN] and interleukin [IL]2) production by and membrane phenotype (CCR7 and CD45RA expression) of HCMV-specific T cells stimulated in vitro by HCMV-infected autologous dendritic cells (DCs) [22], in a series of immunocompetent subjects undergoing primary HCMV infection. Subjects (most of whom were pregnant women) were followed up from the acute phase until 1 year after infection, and the correlation between memory T cell differentiation kinetics and virus transmission to the fetus was analyzed. Samples. Sequential blood samples were obtained from 24 pregnant women and 5 nonpregnant subjects experiencing primary HCMV infection. A total of 69 blood samples (1 6 per patient) collected 7 452 days after the onset of infection were tested. In addition, 9 HCMV-seropositive healthy subjects with remote HCMV infection (5 years after onset) were included as control subjects. The study was approved by the local ethics committee, and informed consent was obtained from all subjects enrolled in the study. HCMV-specific T cell determination. HCMV-specific CD4 and CD8 T cells were simultaneously quantified ex vivo by a recently developed method based on the use of autologous, monocyte-derived, HCMV-infected immature DCs, as described elsewhere [22]. Briefly, immature DCs were generated from peripheral blood mononuclear cells (PBMCs) [23] and infected for 24 h with an endotheliotropic and leukotropic strain of HCMV (VR1814), as reported elsewhere [24]. Infected DCs were then cocultured overnight with autologous PBMCs at a ratio of 1:20 in the presence of brefeldin A (Sigma), to prevent cytokine release. Finally, PBMCs were tested for the frequency of HCMV-specific, IFN- producing CD4 and CD8 T cells by cytokine flow cytometry. IL-2 production and differentiation markers of HCMVspecific T cells (CD45RA and CCR7 expression) [14] were also determined. Absolute CD3 CD4 and CD3 CD8 T cell counts were determined in heparinized peripheral blood samples by direct immunofluorescence flow cytometry (Beckman Coulter). The total number of HCMV-specific CD4 and CD8 T cells was calculated by multiplying the percentages of HCMV-specific T cells positive for IFN- by the relevant absolute CD4 and CD8 T cell counts. Flow cytometry analysis. After incubation with HCMVinfected DCs, PBMCs were tested by cytokine flow cytometry for intracellular IFN- and IL-2 production. PBMCs were washed and incubated for 30 min on ice with phycoerythrin (PE) cyanine 5 conjugated anti-CD4 monoclonal antibody (MAb) and allophycocyanin ( (...truncated)