Rachel Bruton

Cytomegalovirus Seropositivity Drives the CD8 T Cell Repertoire Toward Greater Clonality in Healthy Elderly Individuals

The deterioration in immune function with aging is thought to make a major contribution to the increased morbidity and mortality from infectious disease in old age. One aspect of immune senescence is the reduction in CD8 T cell repertoire as due to the accumulation of oligoclonal, memory T cells and a reduction in the naive T cell pool. CD8 T cell clonal expansions accumulate with age, but their antigenic specificity remains unknown. In this study, we show that in elderly individuals seropositivity for human CMV leads to the development of oligoclonal populations of CMV-specific CTL that can constitute up to one-quarter of the total CD8 T cell population. Furthermore, CMV-specific CTL have a highly polarized membrane phenotype that is typical of effector memory cells (CD28−, CD57+, CCR7−). TCR analyses show that CMV-specific CTL have highly restricted clonality with greater restriction in the larger expansions. Clonal analysis of the total CD8 T cell repertoire was compared between CMV-seropositive and CMV-seronegative donors. Thirty-three percent more clonal expansions were observed in CMV-seropositive donors in comparison with seronegative individuals. These data implicate CMV as a major factor in driving oligoclonal expansions in old age. Such a dramatic accumulation of virus-specific effector CTL might impair the ability to respond to heterologous infection and may underlie the negative influence of CMV seropositivity on survival in the very elderly.

The cytomegalovirus-specific CD4+ T-cell response expands with age and markedly alters the CD4+ T-cell repertoire.

ABSTRACT Immune function in the elderly is associated with a number of phenotypic and functional abnormalities, and this phenomenon of immune senescence is associated with increased susceptibility to infection. The immune response to pathogens frequently declines with age, but the CD8+ T-cell response to cytomegalovirus (CMV) is unusual, as it demonstrates a significant expansion over time. Here we have documented the CD4+ T-cell immune response to CMV in healthy donors of different ages. The magnitude of the CMV-specific CD4+ T-cell immune response increases from a mean of 2.2% of the CD4+ T-cell pool in donors below 50 years of age to 4.7% in donors aged over 65 years. In addition, CMV-specific CD4+ T cells in elderly donors demonstrate decreased production of interleukin-2 and less dependence on costimulation. CMV seropositivity is associated with marked changes in the phenotype of the overall CD4+ T-cell repertoire in healthy aged donors, including an increase in CD57+ expression and a decrease in CD28 and CD27 expression, a phenotypic profile characteristic of immune senescence. This memory inflation of CMV-specific CD4+ T cells contributes to evidence that CMV infection may be damaging to immune function in elderly individuals.

T Cell Recognition Patterns of Immunodominant Cytomegalovirus Antigens in Primary and Persistent Infection

Replication of human cytomegalovirus is controlled by a vigorous CD8 T cell response. The persistent nature of infection is believed to periodically stimulate T cell responses resulting in considerable expansions of virus-specific CD8 T cells over time. In this study, we describe the magnitude and breadth of CD8 T cell responses against the immunodominant viral Ags, IE-1 and pp65, in acute and long-term infection using the IFN-γ ELISPOT assay. Simultaneously, we have identified several novel MHC class I restricted CD8 T cell epitopes. Acute phase responses in immunocompetent donors appear to be extremely focused as early as 1 week post diagnosis with dominant peptide-specific responses observed against both proteins. These dominant responses remain detectable at all later time points over a 4-year follow-up. Interestingly the IE-1 responses show an increase over time whereas the pp65 responses do not, which contrasts with data showing that responses against both Ags are elevated in elderly individuals. We also observe the rapid emergence of an effector memory phenotype for virus-specific CD8 T cells as observed in persistent infection. Over time the revertant CD45RApos effector cell population is also expanded, and this is more evident in the preferentially expanded IE-1 responses. We postulate that periodic low-level virus reactivation after the acute infection phase preferentially stimulates these responses whereas pp65-specific T cell expansions probably occur during the infrequent episodes of lytic viral replication or secondary infection.

Early reconstitution of effector memory CD4+ CMV-specific T cells protects against CMV reactivation following allogeneic SCT.

Reactivation of CMV is a common complication following allogeneic haematopoietic SCT and is associated with significant morbidity and mortality. The relative importance of the CD4+ and CD8+ components of the CMV-specific immune response in protection from reactivation is unclear. The CMV-specific CD4+ and CD8+ immune response was measured at serial time points in 32 patients following allogeneic HSCT. Intracellular cytokine staining following CMV lysate stimulation and HLA-peptide tetramers were used to determine CMV-specific CD4+ and CD8+ responses, respectively. A deficient CMV-specific CD4+ T-cell immune response within the first 30–50 days post transplant was associated with high risk of viral reactivation. Patients with combined impairment of the CD4+ and CD8+ immune response within the first 100 days were susceptible to late viral reactivation. The frequency of CMV-specific CD4+ T cells correlated with CMV-specific CD8+ T cells, comprising 10% of the whole T-cell repertoire. Early CMV-specific CD4+ T-cell reconstitution was dominated by effector memory cells with normal levels of IL-2 resuming 6 months following transplantation. In summary, both CD4 and CD8 CMV-specific immune reconstitution is required for protection from recurrent activation. Measurement of the magnitude of the CMV-specific CD4+ immune response is useful in managing viral reactivation following HSCT.

Purification of cytomegalovirus‐specific CD8 T cells from peripheral blood using HLA–peptide tetramers

Cytomegalovirus (CMV) reactivation and disease remains an important clinical problem for patients after allogeneic stem cell transplantation. Impaired cellular immune control of viral replication is responsible for viral reactivation, and transfer of CMV‐specific T cells from transplant donors can be effective in providing protection. Recent reports have indicated that the frequency of CMV‐specific CD8+ T cells in the peripheral blood of healthy donors is surprisingly high. Here we demonstrate that by using a combination of human leucocyte antigen (HLA) Class I‐peptide tetramers and magnetic selection it is possible to select CMV‐specific T cells from CMV antibody‐positive individuals to high purity. Reliable purification of CMV‐specific T cells up to 99·8% of CD8+ cells was possible within hours, even when starting with a precursor frequency of < 0·1% of peripheral blood CD8+ T cells. CMV‐specific T cells remained functional after the selection process. This novel form of antigen‐specific T‐cell selection should facilitate the selection of T cells for cellular immunotherapy to treat or prevent CMV disease after transplantation. In addition, this technique could potentially be applied to many antigens including against other infective agents and tumour‐specific antigens.

Ability of 5‐HT4 receptor ligands to modulate rat striatal dopamine release in vitro and in vivo

1 The ability of 5‐HT4 (5‐hydroxytryptamine4) receptor ligands to modify dopamine release from rat striatal slices in vitro and in the striatum of freely moving rats was assessed by the microdialysis technique. 2 The release of dopamine from slices of rat striatum continually perfused with Krebs buffer was enhanced by 5‐HT4 receptor agonists; 5‐HT (10 μm), 5‐methoxytryptamine (5‐MeOT; 10 μm), renzapride (10 μm) and (S)‐zacopride (10 μm) maximally increased dopamine release by 133 ± 5, 214 ± 25, 232 ± 29 and 264 ± 69%, respectively (mean ± s.e.mean, n = 3–8). The drug‐induced responses were maximal within the first 2 min of drug application, and subsequently declined. The non‐selective 5‐HT3/5‐HT4 receptor antagonist, SDZ205‐557 (10 μm), failed to modify basal dopamine release from striatal slices but completely antagonized the (S)‐zacopride (10 μm)‐induced increase in dopamine release. 3 To allow faster drug application, the modulation of dopamine release from rat striatal slices in a static release preparation was also investigated. The 5‐HT4 receptor agonist, renzapride (10 μm) also enhanced dopamine release in this preparation (maximal increase = 214 ± 35%, mean ± s.e.mean, n = 14), whilst a lower concentration of renzapride (3 μm) was less effective. The renzapride‐induced response was maximal within the first 2 min of drug application, before declining. In this preparation, the stimulation of dopamine release by renzapride (10 μm), was completely antagonized by the selective 5‐HT4 receptor antagonist, GR113808 (100 nM). In addition, both the Na+ channel blocker, tetrodotoxin (100 nM) and the non‐selective protein kinase A inhibitor, H7 (100 nM) completely prevented the stimulation of dopamine release induced by renzapride (10 μm). 4 In vivo microdialysis studies demonstrated that the 5‐HT4 receptor agonists, 5‐MeOT (10 μm), renzapride (100 μm) and (S)‐zacopride (100 μm) maximally elevated extracellular levels of dopamine in the striatum by 220 ± 20, 161 ± 10 and 189 ± 53%, respectively (mean ± s.e.mean, n = 5–9). A lower concentration of renzapride (10 μm) was less effective. The elevation of extracellular striatal dopamine levels induced by either renzapride (100 μm) or (S)‐zacopride (100 μm) were completely antagonized by the non‐selective 5‐HT3/5‐HT4 receptor antagonist, SDZ205‐557 (100 μm). In addition, the elevation of extracellular levels of dopamine induced by either 5‐MeOT (10 μm) or renzapride (100 μm) was completely prevented by the selective 5‐HT4 receptor antagonist, GR113808 (1 μm) and the renzapride (100 μm)‐induced response was also completely prevented by the non‐selective protein kinase A inhibitor, H7 (1 μm). In this in vivo preparation, both GR113808 (1 μm) and H7 (1 μm), when perfused alone, reduced extracellular levels of dopamine. 5 In conclusion, the present study provides evidence that the 5‐HT4 receptor facilitates rat striatal dopamine release in vitro and in vivo.

Group I mGlu receptor modulation of dopamine release in the rat striatum in vivo

The present study investigated the ability of mGlu (metabotropic glutamate) receptor to modulate dopamine release in the striatum of freely moving rats assessed using the microdialysis technique. The group I and II mGlu receptor agonist (1S,3R)-ACPD (1-amino-cyclopentane-1,3-dicarboxylate; 1-3 mM) increased dopamine release (367% of basal levels) which was prevented by the non-selective mGlu receptor antagonist, (+)-MCPG (alpha-methyl-4-carboxyphenylglycine; 10 mM). The group I mGlu receptor agonist, DHPG (3,5-dihydroxyphenylglycine; 0.3-1 mM), also increased dopamine release (maximum increase 229%) which was also antagonised by (+)-MCPG (10 mM). In contrast, the group II mGlu receptor agonist, DCG-IV (2-(2,3-dicarboxycyclopropyl)glycine; 3-50 microM), induced a more modest increase in dopamine release (156% of basal levels). Combined administration of DHPG (1 mM) and DCG-IV (50 microM) maximally increased dopamine release by 252% of basal levels which was antagonised completely by (+)-MCPG (10 mM). Such findings indicate that group I (and possibly group II) mGlu receptors facilitate rat striatal dopamine release in vivo.

Identification of Cytomegalovirus-Specific Cytotoxic T Lymphocytes In Vitro Is Greatly Enhanced by the Use of Recombinant Virus Lacking the US2 to US11 Region or Modified Vaccinia Virus Ankara Expressing Individual Viral Genes

ABSTRACT Cytomegalovirus (CMV) elicits a potent T-cell response in humans that appears to protect the host from virus-associated disease. Despite facing strong host defense mechanisms, CMV remains as a lifelong infection that may reactivate and cause life-threatening disease in immunocompromised individuals. This persistence is probably assisted by expression of immune subversion proteins of the virus encoded by genes belonging to the US gene family. These proteins modulate major histocompatibility complex expression in infected cells and bias in vitro experiments toward the detection of only certain specificities. We have combined the use of recombinant CMV, lacking the US2 to US11 region genes, and cytoplasmic gamma interferon staining to define a more accurate assessment of CMV-specific responses in vivo. Recombinant CMV stimulation reveals a CD8 response much larger than that of parental virus in all donors tested. In some cases, this represented up to 10-fold increases in the number of cells detected. Responses were directed mainly against pp65, IE-1, and pp50 in the majority of donors. In addition, previously unreported IE-2-specific T-cell responses could be detected in a minority of cases. Furthermore, we observed a less marked increase in the response to mutant CMV by CD4 T cells in some donors. This suggests that a much broader T-cell response to CMV exists in vivo than is revealed by restimulation with wild-type virus and adds to the evidence that the efficacy of immune evasion strategies may not be as absolute as previously believed.

A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection

ABSTRACT E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here, we show a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during Ad infection. E1B-AP5 is recruited to viral replication centers during infection, where it colocalizes with ATR-interacting protein (ATRIP) and the ATR substrate replication protein A 32 (RPA32). Indeed, E1B-AP5 associates with ATRIP and RPA complex component RPA70 in both uninfected and Ad-infected cells. Additionally, glutathione S-transferase pull-downs show that E1B-AP5 associates with RPA components RPA70 and RPA32 directly in vitro. E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9, and Smc1 during infection such that Ad12 promotes a significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implications for the regulation of ATR activity during cellular DNA replication or in response to DNA damage.

The number of cytomegalovirus-specific CD4 + T cells is markedly expanded in patients with B-cell chronic lymphocytic leukemia and determines the total CD4 + T-cell repertoire

B-cell chronic lymphocytic leukemia is associated with immune suppression and an altered T-cell repertoire with expansion of memory cells. Cytomegalovirus (CMV) is a common herpes virus that elicits a strong virus-specific T-cell immune response after infection. We studied the CMV-specific CD4(+) T-cell response in 45 patients and 35 control subjects and demonstrated that it was markedly expanded in the patient group, averaging 11% of the CD4(+) pool compared with 4.7% in controls. The magnitude of the CMV-specific CD4(+) immune response increased with disease stage and was particularly high in patients who received chemotherapy. Within this group, the CMV-specific response comprised over 46% of the CD4(+) T-cell repertoire in some patients. Serial analysis revealed that CMV-specific immunity increased during treatment with chemotherapy and remained stable thereafter. CMV-seropositive patients exhibited a markedly altered CD4(+) T-cell repertoire with increased numbers of CD45R0(+) T cells and a reduction in CD27, CD28, and CCR7 expression. Overall survival was reduced by nearly 4 years in CMV-seropositive patients, although this did not reach statistical significance. CLL patients therefore demonstrate an expansion of the CD4(+) CMV-specific immune response, which is likely to contribute to the immunological and clinical features of this disease.