CMV-specific Responses Research Articles

Cytomegalovirus-specific responses of CD38+ memory T cells are skewed towards IFN-γ and dissociated from CD154 in HIV-1 infection

Despite the strong correlation of T-cell CD38 expression with HIV disease progression, evidence linking CD38 expression and dysfunction at the single cell level is scant. Since CD38⁺ memory CD4⁺ T cells, especially those from HIV-infected persons, fail to induce CD154 (CD40L) while responding to a superantigen with interferon (IFN)-γ or interleukin (IL)-2, we aimed to determine if recall responses to cytomegalovirus (CMV) were similarly affected in the CD38⁺ memory CD4⁺ T-cell subpopulation. Peripheral blood mononuclear cells from HIV+ patients and healthy controls were incubated 14 h with CMV antigens, the superantigen Staphylococcus aureus enterotoxin B or medium, and labeled for identification of central memory (T(CM)) and effector memory (T(EM)) CD4⁺ T cells, and for the intracellular detection of induced CD154, IFN-γ and/or IL-2 by flow cytometry. Compared with CD38⁻ cells, CD38⁺ T(CM) cells from patients had less CD40L induction after CMV stimulation, and increased IFN-γ response. Patients' CD38⁺ T(EM) cells showed a lower IL-2 response, and tended to have a greater IFN-γ response, in which CD154 induction frequently failed. CMV-specific responses of patients' CD38⁺ T(CM) and T(EM) cells were dominated by IFN-γ, and almost all IL-2⁺ cells co-expressed IFN-γ. IL-2 responses to the polyclonal activator S. aureus enterotoxin B were also significantly less frequent among CD38⁺ T(CM) and T(EM) cells than in CD38⁻ cells. Patients' CD38⁺ memory CD4⁺T-cell responses to CMV favor the effector cytokine IFN-γ over IL-2, in the context of deficient CD154 induction, which may limit co-stimulation, proliferation and survival.

Boosting In Vivo CAR-Redirected Virus-Specific CTLs With Universal-Artificial Antigen Presenting Cells

Virus-specific cytotoxic T lymphocytes (CTLs) targeting EBV and/or CMV and engrafted with chimeric antigen receptors (CARs) can receive appropriate costimulation from professional antigen presenting cells (APCs) that process latent viral antigens and can then target tumor cells through their CAR. In a clinical trial in neuroblastoma patients, CAR-redirected virus specific CTLs persisted for over 6 weeks after infusion and produced complete tumor responses in 3/11 patients. To improve the in vivo expansion and persistence of CAR-redirected virus specific CTLs, we determined whether K562 cells engineered to express viral antigens and either CD40L or OX40L can act as universal artificial APCs (aAPCs) to boost CAR-modified virus-specific CTLs in vivo through a cross-presentation mechanism. Using CMV as the viral antigen model, we found that aAPCs/pp65 produce superior in vitro activation of CMV-specific CTLs from seropositive healthy donors (292±56 IFNγ spot forming cells (SFC)/105 cells) than control non modified K562 (83±25 IFNγ SFC/105 cells). The frequency of CMV-CTLs was further enhanced if the aAPC coexpressed CD40L (aAPCs/pp65/CD40L) (502±104 SFC/105 cells; p<0.001) but not by incorporation of OX40L (aAPCs/pp65/OX40L) (357±40 SFC/105 cells) or the combination CD40L/OX40L (477±91 SFC/105 cells). We then assessed whether these aAPCs can boost in vivo CMV-specific CTL responses in NSG mice engrafted with human cells from CMV-seropositive healthy donors. Spleens isolated from mice that had been vaccinated with either aAPCs/pp65 or aAPCs/pp65/CD40L or aAPCs/pp65/OX40L showed an increased frequency of CMV-specific precursors (57±24 vs. 41±14 vs. 35±17 IFNγ-SFC/105 cells) as compared to mice infused with control aAPCs (27±6) (p=0.005). In contrast to the in vitro experiments, the combination in vivo of aAPCs/CD40L/pp65 and aAPCs/OX40L/pp65 elicited the highest number of CMV-specific precursors (101±21 SFC/105 cells). We then tested if CMV-CTLs retained this response to the aAPC vaccine when the CTLs were grafted with a CAR. As a model we used a CAR targeting the neuroblastoma associated antigen GD2. We found that in vitro stimulation with aAPCs/CD40L/OX40L/pp65 promoted the greatest increase of CMV-specific (925±201 IFNγ SFC/105 cells) and CAR-GD2 responses (2725±585 IFNγ SFC/105 cells). The effector function of CAR+ CMV-CTLs was maintained through both their native TCR and their CAR as indicated by cytotoxicity against both GD2+ target cells (CHLA-255=63±14% at a 20:1 E:T ratio) and pp65-infected cells (59±3%); there was no killing of GD2- targets (Raji = 18%±8%) or of CMV uninfected cells (3%±1%). Sustained persistence of CAR+ CMV-CTLs in response to the aAPCs was also maintained in vivo. Thus, vaccination of NSG mice with aAPCs/CD40L/OX40L/pp65 enhanced the frequency of CAR+ CTLs (range 2.4% - 6.25%) as compared to mice vaccinated with control aAPCs (0.98% - 1.08%). IFNγ ELISpot assays confirmed increased functional frequency of both CAR and pp65 mediated recognition of T cells isolated from mice vaccinated with aAPCs/CD40L/OX40L/pp65 against GD2+ targets (71±24 SFC/105 cells) and pp65-infected targets (85±16) as compared to control mice (23±8 and 41±11, respectively) (p=0.048 and p=0.035). Importantly, vaccination increased the antitumor activity of CAR-CTLs in a xenograft model of neuroblastoma since 48% of the mice vaccinated with aAPCs/CD40L/OX40L/pp65 were tumor free by day 40, while mice vaccinated with control aAPCs uniformly succumbed to the tumor (p≤0.037). In conclusion, we have identified a broadly applicable strategy to stimulate CAR redirected virus-specific CTLs in vivo through their native TCR that lengthens their persistence and thereby increases their antitumor activity. Disclosures:Savoldo:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

PD-1 Expression On Total and CMV-Specific T Cells In Early Post Transplant Is Associated With Donor Source, T Cell Maturation Profile, and Effectiveness Of CMV Control

T cell inhibitory receptors upregulate in parallel with activating receptors to prevent overwhelming immune response. Excessive or persistent inhibitory receptor expression on T cells, however, has been associated with several pathologic states, including chronic viral infection and malignancy. Programmed death-1 (PD-1) is among the most well studied inhibitory receptors, and PD-1 inhibition has resulted in regression of solid tumor malignancies and improved control of HIV infection.To examine the role of PD-1 in T cell function post allotransplant we examined PD-1 expression on total and CMV antigen specific T cells on samples collected from 43 patients between 55 and 85 days post cord blood transplant (CBT) (n=13),matched unrelated donor transplant (MURD) (n=12), matched related donor transplant (MRD) (n=18) and compared results to normal adult peripheral blood controls (PBMCs) (n=22), and cord blood controls (CB) (n=11). PD-1 expression was significantly upregulated (p<0.05) on CD4+ T cells following CBT and MURD transplantation as compared to PBMCs and on all donor sources as compared to normal CB. On CD8+ T cells, PD-1 expression was significantly upregulated (p<0.05) following CB and MURD transplants as compared to PBMC. For each donor source, there was no difference in PD1 expression on CD4+ or CD8+ T cells based on conditioning intensity, GVHD prophylaxis, use of prednisone at the time the sample was drawn, or degree of T cell chimerism. Naïve CD4+ T cells were a statistically smaller proportion of total T cells following CB transplants as compared to PBMC and central memory (CM) cells were a statistically greater proportion of total T cells following CB and MURD transplant compared to PBMC. Effector memory terminally differentiated (EMTD)s were a statistically greater portion of total T cells on MRD transplants as compared to CB.We next assessed PD-1 expression on CMV specific CD8+ T cells from HLA A2 patients using a CMVpp65 peptide (NLVPMVATV) specific A2 tetramer. Among 15 CMV positive patients assessed between 55 and 85 days post-transplant, tetramer+, CD8+ T cells were detectable in 10 patients. In patients who effectively controlled CMV clinically, defined as no reactivation (n=12) or reactivation controlled within 1 week of preemptive therapy (n=2), tetramer+, CD8+ T cell populations consisted primarily of EM/EMTD cells (28.9-98.2% of cells, median 75%, n=8) and PD-1 expression was comparable, though modestly increased, compared to total CD8+ T cell populations. In 2 patients not controlling CMV, tetramer+, CD8+ T cells had a greater proportion of naïve/CM phenotype (45.7-54.6% of cells, median 50%, n=2) and expressed markedly higher PD-1 levels than total CD8+T cells.To investigate the potential for PD-1 pathway blockade to enhance CMV responses, we performed stimulations with and without PD-L1 antibody blockade on 17 patient samples collected between 55 and 85 days post-transplant. Eleven patients demonstrated T cell proliferation in response to CMV. For nine of 11 patients, proliferative responses in CD4+ T cells were enhanced by PD-L1 antibody blockade and for eight of 11 patients proliferative responses in CD8+ T cells were enhanced by PD-L1 antibody blockade.Our observations suggest different patterns of T cell immune reconstitution depending on donor source. Similar patterns of immune reconstitution were demonstrated in the case of CMV specific responses. Our data suggests that PD-1 interactions might be manipulated to affect immune responses following transplantation. Further investigation of PD-1 and its interactions with other inhibitory receptors as well as the potential to manipulate PD-1 to enhance GVT and abrogate GVHD are ongoing. Disclosures:No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia Patients Have a Preserved Cytomegalovirus-Specific Antibody Response despite Progressive Hypogammaglobulinemia

Chronic lymphocytic leukemia (CLL) is characterized by progressive hypogammaglobulinemia predisposing affected patients to a variety of infectious diseases but paradoxically not to cytomegalovirus (CMV) disease. Moreover, we found reactivity of a panel of CLL recombinant antibodies (CLL-rAbs) encoded by a germ-line allele with a single CMV protein, pUL32, despite differing antibody binding motifs. To put these findings into perspective, we studied prospectively relative frequency of viremia, kinetics of total and virus-specific IgG over time, and UL32 genetic variation in a cohort of therapy-naive patients (n=200). CMV-DNA was detected in 3% (6/200) of patients. The decay of total IgG was uniform (mean, 0.03; SD, 0.03) and correlated with that of IgG subclasses 1-4 in the paired samples available (n=64; p<0.001). Total CMV-specific IgG kinetics were more variable (mean, 0,02; SD, 0,06) and mean decay values differed significantly from those of total IgG (p=0.034). Boosts of CMV-specific antibody levels were observed in 49% (22/45) of CMV-seropositive patients. In contrast, VZV- and EBV-specific IgG levels decayed in parallel with total IgG levels (p=0.003 and p=0.001, respectively). VZV-specific IgG even became undetectable in 18% (9/50) of patients whereas CMV-specific ones remained detectable in all seropositive patients. The observed CMV-specific IgG kinetics were predicated upon the highly divergent kinetics of IgG specific for individual antigens - glycoprotein B-specific IgG were boosted in 51% and pUL32-specific IgG in 32% of patients. In conclusion, CLL patients have a preserved CMV-specific antibody response despite progressive decay of total IgG and IgG subclasses. CMV-specific IgG levels are frequently boosted in contrast to that of other herpesviruses indicative of a higher rate of CMV reactivation and antigen-presentation. In contrast to the reactivity of multiple different CLL-rAbs with pUL32, boosts of humoral immunity are triggered apparently by other CMV antigens than pUL32, like glycoprotein B.

Non-Immunogenicity of Overlapping Gag Peptides Pulsed on Autologous Cells after Vaccination of HIV Infected Individuals

BackgroundHIV Gag-specific CD4+ and CD8+ T-cell responses are important for HIV immune control. Pulsing overlapping Gag peptides on autologous lymphocytes (OPAL) has proven immunogenic and effective in reducing viral loads in multiple pigtail macaque studies, warranting clinical evaluation.MethodologyWe performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel therapeutic vaccine approach ‘OPAL-HIV-Gag(c)’. This vaccine is comprised of 120 15mer peptides, overlapping by 11 amino acids, spanning the HIV Gag C clade sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenous reinfusion. Patients with undetectable HIV viral loads (<50 copies/ml plasma) on HAART received four administrations at week 0, 4, 8 and 12, and were followed up for 12 weeks post-treatment. Twenty-three people were enrolled in four groups: 12 mg (n = 6), 24 mg (n = 7), 48 mg (n = 2) or matching placebo (n = 8) with 18 immunologically evaluable. T-cell immunogenicity was assessed by IFNγ ELIspot and intracellular cytokine staining (ICS).ResultsThe OPAL-HIV-Gag(c) peptides were antigenic in vitro in 17/17 subjects. After vaccination with OPAL-HIV-Gag(c), 1/6 subjects at 12 mg and 1/6 subjects at 24 mg dose groups had a 2- and 3-fold increase in ELIspot magnitudes from baseline, respectively, of Gag-specific CD8+ T-cells at week 14, compared to 0/6 subjects in the placebo group. No Gag-specific CD4+ T-cell responses or overall change in Rev, Nef, Tat and CMV specific responses were detected. Marked, transient and self-limiting lymphopenia was observed immediately post-vaccination (4 hours) in OPAL-HIV-Gag(c) but not in placebo recipients, with median fall from 1.72 to 0.67 million lymphocytes/mL for active groups (P<0.001), compared to post-placebo from 1.70 to 1.56 lymphocytes/ml (P = 0.16).Conclusion/SignificanceDespite strong immunogenicity observed in several Macaca nemestrina studies using this approach, OPAL-HIV-Gag(c) was not significantly immunogenic in humans and improved methods of generating high-frequency Gag-specific T-cell responses are required.Name of RegistryClinicalTrials.gov, Registry number: NCT01123915, URL trial registry database: http://www.clinicaltrials.gov/ct2/results?term=OPAL-HIV-1001&Search=Search

Cytomegalovirus Specific Immunity in Paediatric Cardiac Transplantation

Purpose Cytomegalovirus (CMV) disease remains an important risk factor in transplant recipients, particularly in pediatric patients. The role of CMV specific immunity in protecting against viral infection/rebound is yet to be elucidated in the paediatric context. We investigated the relationship between CMV-specific T-cell responses and CMV viral load. We also measured soluble markers of inflammation, such as CX3CL1 (fractalkine), an atypical chemokine with a documented role in the development of numerous inflammatory diseases including atherosclerosis. Methods and Materials Blood samples from 28 heart-transplanted children were taken at 0, 1, 2, 4, and then 4-weekly time points, up to one year. Patients were divided into 3 groups according to CMV exposure (donor or recipient positive) and CMV viral load. Peripheral blood mononuclear cells were assessed for CMV-specific T cell response, and CD57 and Granzyme B was measured. Soluble markers were measured in plasma. Results 7 children had viremia within 12 weeks after transplant. Their CD8+ cells responded to CMV stimulation between 16-32 weeks post-transplant. Patients who had been exposed to CMV, but did not get CMV viremia (n=11), had CMV specific responses within the first 12 weeks post-transplant. CMV specific responses were similar in the CMV exposed groups by one year post transplant. CMV exposed patients showed a slight augmentation in the granzyme B production while those who had viral inactivation/reinfection post-transplant exhibited increased cytotoxic profiles. CMV exposed patients had higher plasma fractalkine levels than CMV negative patients, but patients with viremia appeared to maintain higher levels throughout the first year post-transplant. Conclusions Our data show that CMV specific T cell responses are predictive of CMV viremia. Monitoring of T cell responses can guide immunosuppression and thus may reduce CMV disease post transplant. The demonstration of elevated fractalkine further illustrated the potential of CMV to propagate coronary allograft vasculopathy.

Distinct gene-expression profiles associated with the susceptibility of pathogen-specific CD4 T cells to HIV-1 infection

AbstractIn HIV infection, CD4 responses to opportunistic pathogens such as Candida albicans are lost early, but CMV-specific CD4 response persists. Little is currently known about HIV infection of CD4 T cells of different pathogen/antigen specificities. CFSE-labeled PBMCs were stimulated with CMV, tetanus toxoid (TT), and C albicans antigens and subsequently exposed to HIV. HIV infection was monitored by intracellular p24 in CFSElow population. We found that although TT- and C albicans–specific CD4 T cells were permissive, CMV-specific CD4 T cells were highly resistant to both R5 and X4 HIV. Quantification of HIV DNA in CFSElow cells showed a reduction of strong-stop and full-length DNA in CMV-specific cells compared with TT- and C albicans–specific cells. β-Chemokine neutralization enhanced HIV infection in TT- and C albicans–specific cells, whereas HIV infection in CMV-specific cells remained low despite increased entry by β-chemokine neutralization, suggesting postentry HIV restriction by CMV-specific cells. Microarray analysis (Gene Expression Omnibus accession number: GSE42853) revealed distinct transcriptional profiles that involved selective up-regulation of comprehensive innate antiviral genes in CMV-specific cells, whereas TT- and C albicans–specific cells mainly up-regulated Th17 inflammatory response. Our data suggest a mechanism for the persistence of CMV-specific CD4 response and earlier loss of mucosal Th17-associated TT- and C albicans–specific CD4 response in AIDS.

High-Quality CMV-Specific CD4+ Memory Is Enriched in the Lung Allograft and Is Associated With Mucosal Viral Control

The maintenance of CMV-specific T cell memory in lung transplant recipients (LTRs) is critical for host defense and allograft durability, particularly in donor(+) /recipient(-) (D(+) R(-) ) individuals who demonstrate increased mortality. We studied CD4(+) and CD8(+) CMV-specific memory responses to phosphoprotein 65 (pp65) in a prospective cohort of 18 D(+) R(-) LTRs, from bronchoalveolar lavage (BAL)-obtained lung mononuclear cells (LMNC) and PBMC. Unexpectedly, pp65-specific CD4(+) and CD8(+) IFN-γ memory responses from LMNC were similar, in contrast to persistent CD8(+) predominance in PBMC. Unlike the pulmonary CD8(+) predominance during acute primary infection, compartmental equalization occurred in the CMV-specific CD8(+) memory pool during chronic infection, whereas CMV-specific CD4(+) memory was enriched in the bronchoalveolar space. Moreover, CMV-specific CD4(+) memory T cells with multifunctional production of IFN-γ, TNF-α, IL-2 and MIP-1β were significantly increased in LMNCs, in contrast to similar intercompartmental CD8(+) memory function. Moreover, the absolute number of CMV-specific CD4(+) IFN-γ(+) memory cells in BAL was significantly increased in LTRs exhibiting viral control compared to those with CMV early antigen positivity. Collectively, these data demonstrate both preferential distribution and functional quality of CMV-specific CD4(+) memory in the lung allograft during chronic infection, and show an important association with CMV mucosal immunity and viral control.

Vectored co-delivery of human cytomegalovirus gH and gL proteins elicits potent complement-independent neutralizing antibodies

Human cytomegalovirus (hCMV) is prevalent worldwide with infection generally being asymptomatic. Nevertheless, hCMV infection can lead to significant morbidity and mortality. Primary infection of seronegative women or reactivation/re-infection of seropositive women during pregnancy can result in transmission to the fetus, leading to severe neurological defects. In addition, hCMV is the most common viral infection in immunosuppressed organ transplant recipients and can produce serious complications. Hence, a safe and effective vaccine to prevent hCMV infection is an unmet medical need.Neutralizing antibodies to several hCMV glycoproteins, and complexes thereof, have been identified in individuals following hCMV infection. Interestingly, a portion of the CMV-specific neutralizing antibody responses are directed to epitopes found on glycoprotein complexes but not the individual proteins. Using an alphavirus replicon particle (VRP) vaccine platform, we showed that bicistronic VRPs encoding hCMV gH and gL glycoproteins produce gH/gL complexes in vitro. Furthermore, mice vaccinated with these gH/gL-expressing VRPs produced broadly cross-reactive complement-independent neutralizing antibodies to hCMV. These neutralizing antibody responses were of higher titer than those elicited in mice vaccinated with monocistronic VRPs encoding gH or gL antigens, and they were substantially more potent than those raised by VRPs encoding gB. These findings underscore the utility of co-delivery of glycoprotein components such as gH and gL for eliciting potent, broadly neutralizing immune responses against hCMV, and indicate that the gH/gL complex represents a potential target for future hCMV vaccine development.

BK Virus Reactivation After Double Umbilical Cord Blood Transplantation in Adults Correlates with Tregs and Delayed Reconstitution of CD4+ and CD8+ T Effector Cells

Abstract 4174Umbilical cord blood transplantation (UCBT) in adults is associated with impaired immune function and increased infection-related morbidity and mortality due to lack of antigen experienced cells and delayed immune reconstitution. BK virus (BKV) is a human polyomavirus that remains latent in renal epithelial cells and can be reactivated after hematopoietic stem cell transplantation (HSCT), leading to hemorrhagic cystitis. Data regarding BKV reactivation and its association with immune reconstitution after UCBT is lacking. We evaluated the status, cellular mechanisms, and clinical implications of immune reconstitution on BK viremia in adults with hematologic malignancies undergoing double unit cord blood transplantation. Thirty-two patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion of two sequential UCB grafts and GvHD prophylaxis with tacrolimus and sirolimus. The grafts were at least a 4/6 match with each other and the recipient. The results are based on 27 evaluable patients. Assessments were done prior to transplant and at 1, 2, 3, 6 and 12 months after UCBT. After UCBT, 15 patients had detectable serum BKV DNA, median 2.6×104 copies/ml (range, 2.5×102–7.9×106) with a median time to viremia of 40 days (range, 26–733). The cumulative probability of developing BK viremia by day 100 was 0.52 (95% CI, 0.33–0.71). In 9 of the 15 patients with detectable serum BKV DNA, urinary BKV PCR was also performed. All 9 tested patients had detectable urinary BKV and developed clinical symptoms ranging from dysuria to hemorrhagic cystitis. To determine whether development of BK viremia was related to the immunological status, we analyzed detection of serum BKV DNA in conjunction with parameters of immune reconstitution. At 6 and 12 months after transplantation development of BK viremia displayed a statistically significant inverse correlation with CD4+ and CD8+ T cells (p<0.05). Development of BK viremia at these time intervals also inversely correlated with CD4+CD45RO+ and CD8+CD45RO+ T cells (p<0.05), consistent with a potentially significant role of these effector populations in preventing and/or clearing BKV. Conversely, simultaneoulsy, there was a significant positive correlation of BK viremia with T regulatory cell numbers (p<0.05) suggesting that cellular mechanisms of Treg-mediated immune suppression were directly involved in regulating this clinical outcome. At 3 months after UCBT there was a significant positive correlation (p<0.05) between BK viremia and T cell receptor excision circles (TRECs), which are expressed in recent thymic emigrant T cells. BK viremia was not dependent on any other immune cell populations including CD20+ B cells, CD16+CD56+ NK cells and CD14+ monocytes. Furthermore, prevention and/or clearance of BK viremia was not dependent on naïve cell numbers as determined by lack of correlation between BK viremia -or absence thereof- with CD4+CD45RA+ T cells and CD8+CD45RA+ T cells. These observations were in complete contrast to our previous findings regarding CMV-specific immunity, which revealed that prevention and/or clearance of CMV viremia depend on reconstitution of thymopoiesis and increase of TRECs and naïve CD4+CD45RA+ cells. In addition, we found no correlation between development of CMV viremia and BK viremia in UCBT recipients. Our results indicate that reactivation of BK virus occurs with high frequency in adult UCBT recipients and is related to the inability of TREC positive cells to control BK viremia, the impaired and delayed reconstitution of CD4+ and CD8+ T effector cells, and the suppressive function of Treg. Furthermore, our results indicate that distinct immunological mechanisms govern CMV-specific and BK-specific anti-viral responses after UCBT. Disclosures:No relevant conflicts of interest to declare.

Stimulation of Human EBV- and CMV-Specific Cytolytic Effector Function Using Allogeneic HLA Molecules

Viral infection is a major cause of morbidity and mortality, and there are few therapeutic options available to augment a virus-specific T cell response. Although allo-HLA cross-reactivity from virus-specific memory T cells is common, it is unclear whether priming with specific allogeneic cells could conversely elicit a viral peptide/self-HLA restricted cytotoxic T cell response in humans. First, we used the previously described allo-HLA-B*44:02 cross-reactivity of EBV peptide/HLA-B8 restricted T cells, to determine whether allogeneic HLA stimulation can elicit a cytolytic immune response against EBV. HLA-B8(+) HLA-B44(-) EBV-seropositive PBMCs were stimulated with either HLA-B*44:02(+) or HLA-B*44:03(+) mismatched irradiated PBMCs in a 7-10 d MLR. The allo-HLA stimulated responder cells were then evaluated for cytotoxicity using EBV peptide loaded autologous target cells and unloaded HLA-B8(+) EBV LCL target cells. PBMCs from EBV-seropositive donors gained EBV-specific cytolytic effector function following specific allo-HLA stimulation. Finally, we also elicited cytolytic CMV-specific responses using specific allogeneic cell stimulation, to confirm that this technique can be used to elicit viral peptide/self-HLA restricted responses even from nonpublic TCR responses. Allogeneic cell stimulation used as a cell therapy may be a potential tool to augment an antiviral T cell response in patients with EBV or CMV infection.

Everolimus Treated Renal Transplant Patients Develop a More Robust CMV-Specific CD8 Response Compared to Cyclosporine or Mycophenolate Sodium Treated Patients

Everolimus Treated Renal Transplant Patients Develop a More Robust CMV-Specific CD8 Response Compared to Cyclosporine or Mycophenolate Sodium Treated Patients

Increased Memory Differentiation Is Associated with Decreased Polyfunctionality for HIV but Not for Cytomegalovirus-Specific CD8+ T Cells

The generation of polyfunctional CD8(+) T cells, in response to vaccination or natural infection, has been associated with improved protective immunity. However, it is unclear whether the maintenance of polyfunctionality is related to particular cellular phenotypic characteristics. To determine whether the cytokine expression profile is linked to the memory differentiation stage, we analyzed the degree of polyfunctionality of HIV-specific CD8(+) T cells within different memory subpopulations in 20 antiretroviral therapy-naive HIV-1-infected individuals at ∼34 wk postinfection. These profiles were compared with CMV-specific CD8(+) T cell responses in HIV-uninfected control subjects and in individuals chronically infected with HIV. Our results showed that the polyfunctional abilities of HIV-specific CD8(+) T cells differed according to their memory phenotype. Early-differentiated cells (CD45RO(+)CD27(+)) exhibited a higher proportion of cells positive for three or four functions (p < 0.001), and a lower proportion of monofunctional cells (p < 0.001) compared with terminally differentiated (TD; CD45RO(-)CD27(-)) HIV-specific CD8(+) T cells. The majority of TD HIV-specific CD8(+) T cells were monofunctional (median 69% [interquartile range: 57-83]), producing predominantly CD107a or MIP1β. Moreover, proportions of HIV-specific monofunctional CD8(+) T cells positively associated with proportions of TD HIV-specific CD8(+) T cells (p = 0.019, r = 0.54). In contrast, CMV-specific CD8(+) T cell polyfunctional capacities were similar across all memory subpopulations, with terminally and early-differentiated cells endowed with comparable polyfunctionality. Overall, these data show that the polyfunctional abilities of HIV-specific CD8(+) T cells are influenced by the stage of memory differentiation, which is not the case for CMV-specific responses.

Reconstitution of protective immune responses against CMV and VZV does not require disease development in paediatric recipients of umbilical cord blood transplantation. (126.4)

Abstract Cytomegalovirus (CMV) and varicella-zoster virus (VZV) are significant causes of morbidity and mortality following umbilical cord blood transplant (UCBT). Yet the kinetics of reconstitution and protective potential of antiviral cell-mediated immune responses following UCBT remain poorly characterized. Here, the reconstitution of CMV and VZV-specific T cell responses was assessed from 1 to 36 months post-transplant using IFN-γ ELISpot and MHC-peptide tetramers in 28 children who underwent UCBT to treat haematological or inherited disorders. Barely detectable in the first 3 months post-UCBT, antiviral T cell responses directed against CMV and VZV were observed in 30.4% and 40.3% of subjects after 36 months. Four CMV-seronegative subjects developed IFN-γ responses against CMV, and 4 subjects developed VZV-specific IFN-γ responses without clinical signs of VZV infection. No CMV- or VZV-related events were observed in subjects following the development of CMV- or VZV-specific responses &amp;gt;150 SFU per 10E6 PBMC, consistent with T cell-mediated protection. Famciclovir prophylaxis did not strictly prevent the reconstitution of the VZV-specific T cell repertoire. Lastly, the magnitude of VZV- and CMV-specific responses was correlated with the frequency of naïve CD8+ T cells, underlining the importance of immune reconstitution to achieve antiviral protection. Monitoring of cell-mediated immunity during the post-transplant period could guide the tailoring of antiherpetic prophylaxis.

Maternal Cytomegalovirus-Specific Immune Responses and Symptomatic Postnatal Cytomegalovirus Transmission in Very Low-Birth-Weight Preterm Infants

Transmission of cytomegalovirus (CMV) via breast milk can lead to severe acute illness in very low-birth-weight (VLBW) preterm infants. Although the majority of CMV-seropositive women shed CMV in milk, symptomatic postnatal infection of VLBW infants occurs infrequently, suggesting that virologic or immunologic factors in milk may be associated with the risk and severity of postnatal CMV infection. We investigated the magnitude of CMV-specific cellular and humoral immune responses in milk of 30 seropositive mothers of VLWB preterm infants and assessed their relationship to milk CMV load and symptomatic CMV transmission. Milk immunoglobulin G (IgG) avidity was inversely correlated to milk CMV load (r = -0.47; P = .009). However, milk CMV load and CMV-specific cellular and humoral immune responses were similar in mothers of VLBW infants with and those without symptomatic postnatal CMV infection. Similar immunologic parameters in milk of CMV-seropositive mothers of VLBW infants with and without symptomatic postnatal CMV infection indicate that screening milk by these parameters may not predict disease risk. However, the inverse correlation between milk CMV IgG avidity and CMV load may suggest that enhancement of maternal CMV-specific IgG responses could aid in reduction of CMV shedding into breast milk.

Old age and anti-cytomegalovirus immunity are associated with altered T-cell reconstitution in HIV-1-infected patients

Increasing evidence supports a parallel between HIV-1 infection and immune aging, which is particularly apparent with common changes in naive versus memory T-cell proportions. Here, we aimed at refining the value of common T-cell-associated markers of immunosenescence during HIV disease progression or aging, and at exploring further the impact in this context of old age as well as cytomegalovirus (CMV) co-infection, which is predominant in HIV-1-infected individuals. Frequencies of naive or CD57(+) memory T cells as well as the magnitude of CMV-pp65 T cells were measured in HIV-1-infected patients grouped according to disease progression status, treatment and age. Our results indicate that the decline in naive T-cell levels rather than the accumulation of CD57(+) senescent T cells identifies best the premature development of an immunosenescence phenotype with HIV disease progression. Moreover, advanced age or mounting of strong CMV-specific responses impact independently on CD4(+) T-cell counts and recovery with antiretroviral therapy. The present findings indicate that HIV-1 infection amplifies the effect of age on naive T-cell levels, and highlight the constraint on the capacity of treated patients to reconstitute their CD4(+) T-cell compartment due to age and CMV co-infection.

Immunological insights into the pathogenesis of active CMV infection in non‐immunosuppressed critically ill patients

Dissociation of cytomegalovirus (CMV) DNA loads between the lower respiratory tract and blood, with high levels in the former compartment and low or undetectable levels in the latter, commonly occurs during active CMV infection in critically ill patients despite the presence of high frequencies of CMV-specific IFN-γ-producing CD8(+) and CD4(+) T cells in blood. Data presented in this case report suggest that inter-compartmental differences in interleukin-10 (IL-10) levels may, in part, explain the pathobiology of this phenomenon. In the absence of ganciclovir treatment, a significant correlation was observed between IL-10 levels and CMV DNA loads in lower respiratory tract specimens (P = 0.016), but not in plasma samples (P = 0.46). Comparable data were obtained during the course of active CMV infection episodes that developed in six CMV-seropositive critically ill patients with no canonical immunosuppression. The presence of higher levels of IL-10 in the lower respiratory tract than in plasma may result in increased impairment of CMV-specific T-cell effector responses in the lung compared to the systemic compartment, facilitating local CMV replication.

A Molecular Assay for Sensitive Detection of Pathogen-Specific T-Cells

Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters - monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

IL‐15 preferentially enhances functional properties and antigen‐specific responses of CD4+CD28null compared to CD4+CD28+ T cells

One of the most prominent changes during T-cell aging in humans is the accumulation of CD28(null) T cells, mainly CD8+ and also CD4+ T cells. Enhancing the functional properties of these cells may be important as they provide an antigen-specific defense against chronic infections. Recent studies have shown that IL-15 does in fact play an appreciable role in CD4 memory T cells under physiological conditions. We found that treatment with IL-15 increased the frequency of elderly CD4+CD28(null) T cells by the preferential proliferation of these cells compared to CD4+CD28+ T cells. IL-15 induced an activated phenotype in CD4+CD28(null) T cells. Although the surface expression of IL-15R α-chain was not increased, the transcription factor STAT-5 was preferentially activated. IL-15 augmented the cytotoxic properties of CD4+CD28(null) T cells by increasing both the mRNA transcription and storage of granzyme B and perforin for the cytolytic effector functions. Moreover, pretreatment of CD4+CD28(null) T cells with IL-15 displayed a synergistic effect on the IFN-γ production in CMV-specific responses, which was not observed in CD4+CD28+ T cells. IL-15 could play a role enhancing the effector response of CD4+CD28(null) T cells against their specific chronic antigens.

Primary response against cytomegalovirus during antiviral prophylaxis with valganciclovir, in solid organ transplant recipients

Antiviral prophylaxis has proved successful for prevention of cytomegalovirus (CMV) disease in solid organ transplant (SOT) patients; though emerging data suggest that antiviral agents interfere with immunity, and may inhibit immune priming. In this context, we investigated levels and phenotype of primary CMV-specific immune responses that developed during antiviral prophylaxis in a cohort of CMV seronegative recipients (R(-) ) of a SOT from a seropositive donor (D(+) ). We longitudinally monitored CMV viral load, antibodies and levels of the negative immuno-modulator IL-10. PBMC were stimulated with CMV-specific peptide libraries to measure CD137 activation marker on CMV-specific T-cells and levels of PD-1 receptor, which is over expressed on exhausted T-cells. Unexpectedly, the majority (13/18) of D(+) R(-) patients who developed a primary CMV response showed early post-transplant CMV-specific responses, though levels of PD-1 on CMV-specific T-cells remained elevated throughout prophylaxis. A strong inverse association was found between levels of plasma IL-10 and CMV-specific cellular immune responses. Our study suggests that during prophylaxis, subclinical CMV infection might have occurred in the D(+) R(-) patients, and primary CMV-specific responses were detected early post-transplant when levels of plasma IL-10 were low. Extended prophylaxis or antiviral treatment did not appear to suppress CMV-specific antibodies or T-cells, which, however, showed exhaustion phenotypes.