Memory T cells specific for HBV enumerated by a peptide-based cultured enzyme-linked immunospot assay in healthy HBV-vaccinated subjects.

Hepatitis B vaccine is the most effective strategy to control hepatitis B virus (HBV) infection and disease. It is considered that an anti-HBs (antibodies against HBV surface antigen) titer >10 mIU/ml, measured shortly after a complete vaccination schedule, provides protection against infection. Approximately 4-10% of healthy individuals fail to respond to 3-dose vaccination. Long-term HBV-specific memory T-cell response has not been fully investigated, mainly due to the lack of a suitable assay. We quantified HBV-specific expandable memory T cells by using a cultured IFN-γ enzyme-linked immunospot (ELISPOT) assay. Cultured ELISPOT response to an overlapping peptide pool representing the complete L (large) HBV envelope polypeptide was evaluated in 41 healthy subjects vaccinated 15-20 y earlier and 5 unvaccinated. Plasma samples were tested for anti-HBs. Vaccinated subjects had significantly higher HBV-specific T-cellular response than unvaccinated (p = 0.0002). HBV-specific T-cell response was mainly mediated by CD4+ T cells. No concordance was found between cultured ELISPOT and anti-HBs data in vaccinated subjects. Thirty-one (76%) vaccinated subjects were responders (anti-HBs >10 mIU/ml). Nineteen (46%) vaccinated subjects were considered to be responders in cultured ELISPOT. Twenty-two (54%) vaccinated subjects were considered non-responders in cultured ELISPOT; 5 of them (23%) were also humoral non-responders. About 12% of healthy HBV-vaccinated subjects are both humoral and cellular non-responders. Although the prognostic value of this assay has not been established in terms of predictability for susceptibility to de-novo HBV infection, ELISPOT data suggest that these subjects may be at risk for HBV infection and disease, especially health care workers.

T-lymphocyte subsets in lung transplant recipients: association between nadir CD4 T-cell count and viral infections after transplantation.

BACKGROUND: Little is known about the kinetics of T-cell subsets in lung transplant recipients (LTR) and their association with the occurrence of opportunistic infections (OI). OBJECTIVES: To analyze the kinetics of T-lymphocyte subsets in LTR and the association between nadir CD4 T-cell count and viral infections after transplantation. STUDY DESIGN: Serial measurements of peripheral blood CD4 and CD8 T-cell counts obtained during the first year post-transplantation from 83 consecutive LTR and their correlation with both viral OI and community-acquired infections post-transplantation were retrospectively analyzed. RESULTS: LTR with a nadir CD4 T-cell count <200 cells/µl had consistently lower CD4 and CD8 T-cell counts than LTR with a nadir CD4 T-cell count >200 cells/µl (p<0.001). In LTR with a nadir CD4 T-cell count <200 cells/µl, the cumulative incidence of viral infections detected in peripheral blood and in bronchoalveolar lavage (BAL) samples was higher than in LTR with a nadir CD4 T-cell count >200 cells/µl (p=0.0012 and p=0.0058, respectively). A nadir CD4 T-cell count <200 cells/µl within the first three months post-transplantation predicted a higher frequency of viral infectious episodes in BAL samples within the subsequent six month period (p=0.0066). CONCLUSIONS: Stratification of patients according to nadir CD4 T-cell count may represent a new and simple approach for early identification of patients at risk for subsequent virus infections.

Neutrophil CD64 expression: a reliable diagnostic marker of infection in advanced cancer patients?

Infection and sepsis are major health problems in cancer patients. There is a need for the identification and validation of biomarkers to improve their early diagnosis and treatment. Emerging evidence showed that neutrophil CD64 is a highly sensitive and specific marker for systemic infection and sepsis in critically ill patients with various diseases but data on patients bearing solid tumors are still lacking. Using a dedicated flow cytometric assay we evaluated neutrophil CD64 expression in patients with advanced cancer without active infections to verify if it could be utilized as a reliable biomarker of early infections also in oncologic patients.

Liver fibrosis, microbial translocation and immune activation markers in HIV and HCV infections and in HIV/HCV co-infection.

BACKGROUND: Liver fibrosis is accelerated in patients co-infected with human immunodeficiency virus and hepatitis C viruses. AIMS: We investigated the correlation between liver fibrosis, immune activation and microbial translocation. METHODS: This cross-sectional study included patients with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) mono-infections, HIV/HCV co-infection, and healthy controls (20 subjects/group). Peripheral blood was analysed to determine the levels of Forkhead box 3 (Foxp3) T cells, TGF-ß1, CD14 (soluble and surface isoforms), IL-17 and bacterial translocation products. These measurements were correlated to the severity of liver fibrosis, measured with the FIB-4 score and transient elastography. RESULTS: Foxp3T cell levels were significantly elevated in HIV mono-infected and co-infected groups (p<0.0005). FIB-4 and liver stiffness values inversely correlated with TGF-ß1 (p=0.0155 and p=0.0498). Bacterial DNA differed significantly in the HIV-positive compared to the other groups: HIV/HCV co-infected subjects had significantly higher serum levels of bacterial translocation products, CD14, and IL-17 levels (p<0.001). CONCLUSIONS: Fibrosis stage in HIV/HCV co-infection may be influenced by immune activation due either by viral infections or to bacterial translocation.

Normalizing ELISPOT responses to T-cell counts: a novel approach for quantification of HCMV-specific CD4(+) and CD8(+) T-cell responses in kidney transplant recipients.

BACKGROUND: Human cytomegalovirus (HCMV) is the most common opportunistic virus infection in solid organ transplant recipients. The analysis of HCMV-specific T-cell immunity after organ transplant is of relevant clinical interest. OBJECTIVES: To analyze HCMV-specific CD4(+) and CD8(+) T-cell responses in healthy subjects and kidney transplant recipients (KTR). STUDY DESIGN: HCMV-specific T-cell responses were evaluated by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) using overlapping 15-mer peptide pools of immediate early (IE)-1, IE-2, phosphoprotein 65 (pp65) (for stimulation of both CD4(+) and CD8(+) T-cell responses) and a pool of 34 short peptides (8-12 amino acids in length, for stimulation of CD8(+) T-cell responses). ELISPOT results were normalized to T-cell subset counts and their correlations with a reported dendritic cell (DC)-based assay, which simultaneously quantifies HCMV-specific CD4(+) and CD8(+) T-cell responses, were analyzed. RESULTS: HCMV-seropositive KTR showed higher ELISPOT responses compared to HCMV-seropositive healthy subjects. IE-1 and pp65 ELISPOT responses were mediated mainly by CD8(+) T-cells and, to a lesser extent, CD4(+) T cells; IE-2 peptides appear to stimulate CD56(+) cells (natural killer cells). In HCMV-seropositive healthy subjects, ELISPOT results (expressed either as net spots/million cells or normalized to the corresponding T-cell count) significantly correlated with the DC assay. However, in HMCV-seropositive KTR, only normalized ELISPOT responses to overlapping 15-mer peptide pools significantly correlated with DC-assay responses. CONCLUSIONS: The normalized ELISPOT represents a novel and simple approach for quantifying and monitoring HCMV-specific CD4(+) and CD8(+) T-cell responses in KTR.

Detection of Epstein-Barr virus-specific memory CD4+ T cells using a peptide-based cultured enzyme-linked immunospot assay.

Approaches to evaluate T-cell responses to Epstein-Barr virus (EBV) include enzyme-linked immunospot (ELISPOT), which quantifies cells capable of immediate interferon-γ secretion upon antigen stimulation. However, evaluation of expandable EBV-specific memory T cells in an ELISPOT format has not been described previously. We quantified EBV-specific T-cell precursors with high proliferative capacity by using a peptide-based cultured interferon-γ ELISPOT assay. Standard and cultured ELISPOT responses to overlapping peptide pools (15-mers overlapping by 11 amino acids) covering the lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) EBV proteins were evaluated in 20 healthy subjects with remote EBV infection and, for comparison, in four solid organ transplant recipients. Cultured ELISPOT responses to both lytic and latent EBV antigens were significantly higher than standard ELISPOT responses. The distribution of EBV-specific T-cell responses detected in healthy virus carriers showed more consistent cultured ELISPOT responses compared with standard ELISPOT responses. T-cell responses quantified by cultured ELISPOT were mainly mediated by CD4+ T cells and a marked pattern of immunodominance to latent-phase antigens (EBNA1 > EBNA3 family antigens > LMP2 > LMP1) was shown. Both the magnitude and distribution of EBV-specific T-cell responses were altered in solid organ transplant recipients; in particular, cultured ELISPOT responses were almost undetectable in a lung-transplanted patient with EBV-associated diseases. Analysis of T-cell responses to EBV by ELISPOT assays might provide new insights into the pathogenesis of EBV-related diseases and serve as new tools in the monitoring of EBV infection in immunocompromised patients.

Monitoring of human cytomegalovirus and virus-specific T-cell response in young patients receiving allogeneic hematopoietic stem cell transplantation.

In allogeneic hematopoietic stem-cell transplantation (HSCT) recipients, outcome of human cytomegalovirus (HCMV) infection results from balance between viral load/replication and pathogen-specific T-cell response. Using a cut-off of 30,000 HCMV DNA copies/ml blood for pre-emptive therapy and cut-offs of 1 and 3 virus-specific CD4(+) and CD8(+) T cells/µl blood for T-cell protection, we conducted in 131 young patients a prospective 3-year study aimed at verifying whether achievement of such immunological cut-offs protects from HCMV disease. In the first three months after transplantation, 55/89 (62%) HCMV-seropositive patients had infection and 36/55 (65%) were treated pre-emptively, whereas only 7/42 (17%) HCMV-seronegative patients developed infection and 3/7 (43%) were treated. After 12 months, 76 HCMV-seropositive and 9 HCMV-seronegative patients (cumulative incidence: 90% and 21%, respectively) displayed protective HCMV-specific immunity. Eighty of these 85 (95%) patients showed spontaneous control of HCMV infection without additional treatment. Five patients after reaching protective T-cell levels needed pre-emptive therapy, because they developed graft-versus-host disease (GvHD). HSCT recipients reconstituting protective levels of HCMV-specific T-cells in the absence of GvHD are no longer at risk for HCMV disease, at least within 3 years after transplantation. The decision to treat HCMV infection in young HSCT recipients may be taken by combining virological and immunological findings.

Kinetics of T-lymphocyte subsets and posttransplant opportunistic infections in heart and kidney transplant recipients.

BACKGROUND: The potential use of T-lymphocyte measurements as infection risk markers after solid organ transplant has not been fully investigated. We analyzed the kinetics of T-lymphocyte subsets within the first 8 months posttransplant and their correlation with opportunistic infections (OIs) in solid organ transplant recipients. METHODS: Serial measurement of CD4 and CD8 T cells was performed retrospectively in 48 heart transplant recipients (HTR) and 42 kidney transplant recipients (KTR). Generalized estimating equation models were used to analyze longitudinal data separately for HTR and KTR. RESULTS: An initial CD4 T-cell drop (at months 1 and 2, in HTR and KTR, respectively) coincided with the peak of OIs. HTR with a low nadir CD4 T-cell count (≤ 200/µL) showed poor CD4 T-cell recovery (175 ± 277 cells/µL at baseline vs 242 ± 99 cells/µL at month 8) and their CD8 T cells increased from 153 ± 194 cells/µL at baseline to 601 ± 399 cells/µL at month 8. KTR with a low nadir CD4 T-cell count (≤ 200/µL) showed a modest CD4 T-cell recovery (138 ± 46 cells/µL at baseline vs. 440 ± 448 cells/µL at month 8), and their CD8 T cells increased from 90 ± 41 cells/µL at baseline to 450 ± 242 cells/µL at month 8. HTR developing OIs had lower CD4 (P<0.001) and CD8 T cells (P=0.001) than those without infections, whereas in KTR the risk for OIs seemed restricted to patients with low CD8 T cells. HTR with OIs had a low CD4/CD8 T-cell ratio, whereas KTR had a high CD4/CD8 T-cell ratio. CONCLUSIONS: Determination of T-lymphocyte subsets is a simple and effective parameter to identify patients at risk of developing OIs.

Human cytomegalovirus-specific CD4(+) and CD8(+) T-cell response determination: comparison of short-term (24h) assays vs long-term (7-day) infected dendritic cell assay in the immunocompetent and the immunocompromised host.

Human cytomegalovirus (HCMV)-specific CD4(+) and CD8(+) T-cells were measured in the immunocompetent host as well as in 13 solid-organ transplant recipients (SOTR), and 12 young hematopoietic stem cell transplant recipients (HSCTR) by using a long-term (7-day) assay based on PBMC stimulation by HCMV-infected dendritic cells (iDC), and two short-term (24h) assays, one for CD4(+) stimulation by infected cell lysate (iCL), and the other for CD8(+) stimulation by a pool of 34 epitopic peptides (pep-pool). In the immunocompetent, the number of T-cells activated by either iCL or the pep-pool was significantly reduced with respect to iDC. In both SOTR and HSCTR, the number of T-cells activated by iDC was comparable to that activated by iCL or the pep-pool. A significant correlation between iDC-activated T-cells and T-cells activated by either iCL or the pep-pool was observed. In conclusion, whenever a rapid result is needed, short-term assays may efficiently replace the iDC assay.

Human cytomegalovirus-specific T cell reconstitution in young patients receiving T cell-depleted, allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Reconstitution of human cytomegalovirus (HCMV) T cell-specific immunity is of crucial relevance in patients receiving a hematopoietic stem cell transplant (HSCT). Scarce data on this subject are available for children receiving a T cell-depleted HSCT. METHODS: We investigated HCMV-specific T cell recovery in 48 recipients of a T cell-depleted HSCT from a human leukocyte antigen (HLA)-disparate relative. Autologous HCMV-infected dendritic cells were used to stimulate HCMV-specific CD8+ and CD4+ T cells producing interferon-gamma (IFN). RESULTS: The 1-year cumulative incidence of both HCMV infection and specific T cell reconstitution was 83% among the 23 HCMV-seropositive patients and 4% and 8%, respectively, among the 25 HCMV-seronegative patients (P < .01). HCMV-specific T cell reconstitution was significantly delayed in patients receiving T cell-depleted grafts, compared with patients receiving unmanipulated HSCTs (median time to reconstitution, 75 vs. 47 days, respectively; P < .01). The median time from HCMV infection to immune recovery in recipients of T cell-depleted grafts was 47 days. Detection of HCMV-specific T cells correlated with control of HCMV infection. The number of residual T cells in the graft predicted earlier T cell recovery (P = .02). CONCLUSIONS: Latent HCMV in the recipient was the major cause of HCMV reactivation and also promoted specific T cell reconstitution in patients given a T cell-depleted HSCT from an HLA-disparate relative. Routine immunologic monitoring is valuable in identifying patients with early HCMV-specific T cell reconstitution.

Comparison of a new Light Diagnostics and the CMV Brite to an in-house developed human cytomegalovirus antigenemia assay.

BACKGROUND: Antigenemia, i.e. detection and quantification of human cytomegalovirus (HCMV) peripheral blood pp65-positive leukocytes, is still one of the two major assays available for diagnosis and monitoring of HCMV infections. OBJECTIVES: To evaluate the performance of a new commercial assay under development (Light Diagnostics). STUDY DESIGN: To compare the performance of the new assay with a commercial assay (CMV Brite) already available on 300 blood samples from immunocompromised patients using as a reference the original in-house developed assay. RESULTS: Although 30 blood samples gave discrepant results among the 3 antigenemia assays, the Light Diagnostics detected an overall number of antigenemia-positive blood samples (sensitivity 84%) identical to that detected by CMV Brite (sensitivity 88%) and in-house assay (91/300, 30.3%). Problems of non-specific cytoplasmic staining were encountered with the CMV Brite assay in 219/300 (73%) blood samples. CONCLUSIONS: The Light Diagnostics assay provides results comparable to those of the reference assay both in terms of specificity and sensitivity (number of pp65-positive leukocytes).

Human cytomegalovirus-specific CD4+ and CD8+ T-cell reconstitution in adult allogeneic hematopoietic stem cell transplant recipients and immune control of viral infection.

BACKGROUND: Human cytomegalovirus infection is the most frequent viral complication in patients undergoing hematopoietic stem cell transplantation. We investigated the development of human cytomegalovirus-specific T cells in adult recipients of hematopoietic stem cell transplants. DESIGN AND METHODS: From May 2003 through October 2006 a total of 45 patients were monitored for human cytomegalovirus-specific T-cell reconstitution. Human cytomegalovirus-infected autologous dendritic cells were used as a stimulus to detect interferon-gamma-producing human cytomegalovirus-specific CD8(+) and CD4(+) T cells during the first year after transplantation. Interleukin-2 production by specific T cells was also determined. RESULTS: Human cytomegalovirus infection was detected in the blood of 39/45 patients at a median of 29 days after transplantation. Human cytomegalovirus-specific T-cell reconstitution followed reactivation of latent human cytomegalovirus infection at a median time of about 2 months after transplantation. Only donor human cytomegalovirus-seronegativity and bone marrow as a stem cell source were found to delay specific T-cell reconstitution significantly. Levels of three CD8(+) and one CD4(+) human cytomegalovirus-specific T-cells/microL blood had a positive predictive value of around 80% for identifying patients able to control human cytomegalovirus infection spontaneously. Five patients who received high doses of steroids for treatment of graft-versus-host disease developed human cytomegalovirus infection requiring pre-emptive treatment despite high levels of interferon-gamma-producing T cells in response to human cytomegalovirus. Specific interleukin-2 production was not detected in patients with human cytomegalovirus infection requiring treatment, while 90% of patients who spontaneously controlled human cytomegalovirus infection had T cells that produced interleukin-2 and interferon-gamma. CONCLUSIONS: Pre-transplant human cytomegalovirus infection of the recipient is a major factor driving human cytomegalovirus-specific immune reconstitution. Control of human cytomegalovirus infection likely requires the presence of both interferon-gamma and interleukin-2 producing T cells. Corticosteroid treatment may favor active viral replication even in patients with specific T cells.

Gln145Met/Leu changes in human immunodeficiency virus type 1 reverse transcriptase confer resistance to nucleoside and nonnucleoside analogs and impair virus replication.

The frequencies of multidrug resistance-associated mutations at codons 145, 151, and 69 of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene in strains from a group of 3,595 highly active antiretroviral therapy (HAART)-experienced patients were 0.22, 2.36, and 0.86%, respectively. Several amino acid substitutions different from the recently reported Gln145Met change (S. Paolucci, F. Baldanti, M. Tinelli, G. Maga, and G. Gerna, AIDS 17:924-927, 2003) were detected at position 145. Thus, amino acid substitutions selected at position 145 were introduced into the wild-type HIV type 1 (HIV-1) RT gene by site-directed mutagenesis, and recombinant HIV strains were assayed for their drug susceptibilities. Only Met and Leu substitutions at position 145 of the HIV-1 RT conferred multidrug resistance, while other amino acid changes did not. Lower levels of replication of the Gln145Met recombinant strain compared with those of both Gln151Met and wild-type recombinant strains were observed. In in vitro inhibition assays, expression and purification of the recombinant Gln145Met HIV-1 RT revealed a strong loss of catalytic efficiency of the mutated enzyme, as well as significant resistance to both zidovudine and efavirenz. Specific amino acid substitutions in the HIV RT nucleotide-binding pocket might affect both antiretroviral drug recognition and binding and decrease the level of virus replication, possibly by interfering with the enzyme activity. This finding may explain the lower frequency of Gln145Met/Leu mutations observed compared with the frequencies of Gln151Met/Leu mutations and the insertion at position 69 in HAART-experienced patients.

Monitoring of human cytomegalovirus (HCMV)-specific CD4+ T cell frequency by cytokine flow cytometry as a possible indicator for discontinuation of HCMV secondary prophylaxis in HAART-treated AIDS patients.

OBJECTIVE: Absolute CD4+ T cell count and human cytomegalovirus (HCMV)-specific CD4+ T cell frequency (as determined by cytokine flow cytometry, CFC) were compared for their ability to predict HCMV disease and safe discontinuation of HCMV secondary prophylaxis. STUDY DESIGN: Three groups of AIDS patients with previous nadir CD4+ T cell count <100/microl were studied. Group A included 48 HAART-treated patients with no HCMV disease. Group B included 11 HAART-treated patients with previous HCMV disease who discontinued HCMV prophylaxis. Group C included 23 HAART-treated (n = 16) or -naive (n = 7) patients with previous HCMV disease either continuing or starting HCMV prophylaxis. Patients underwent follow-up for detection of HCMV viremia or disease (groups A and B) and for discontinuation of HCMV secondary prophylaxis on the basis of either HCMV-specific or absolute CD4+ T cell count (group C). RESULTS: During follow-up, while some patients showed a stable HCMV-specific CD4+ T cell response, others had a fluctuating response (unstable responders) or showed no response at all. In detail, 13/48 group A patients were either HCMV non-responders or unstable responders and 2 of them developed HCMV viremia; 3/11 group B patients were unstable responders, none developing either HCMV viremia or disease; finally, 9 group C patients discontinued HCMV prophylaxis based on absolute CD4+ T cell count > 150 cells/microl, but in 2 of them lacking HCMV-specific response HCMV retinitis relapsed. None of the seven group C patients discontinuing HCMV prophylaxis on the basis of CFC showed HCMV disease relapse. CONCLUSIONS: CFC may support absolute CD4+ T cell count for both guiding HCMV prophylaxis discontinuation and better monitoring HCMV infection in AIDS patients with no previous HCMV disease or having discontinued HCMV prophylaxis.