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Keywords:

  • Cytomegalovirus;
  • lung transplantation;
  • valganciclovir

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Cytomegalovirus (CMV) is a common opportunistic infection after lung transplant. Despite effective antiviral medications to treat CMV, invasive CMV disease contributes to lung allograft dysfunction and worse survival. Efforts to prevent CMV have led to the use of valganciclovir prophylaxis for increasingly longer periods after transplant. A pivotal concern with long-term antiviral prophylaxis is that it may prevent or delay the development of CMV-specific immunity and increase the subsequent risk of late onset disease. To address this issue, we conducted a pilot study to determine if CMV-specific immunity was detectable in lung transplant recipients at risk for CMV while on antiviral prophylaxis. Utilizing polychromatic flow cytometry panels, CMV-specific immunity was determined by peripheral blood CD4 and CD8 T cell expression of cytokines in response to the HLA restricted CMV peptides pp65 and IE-1. We determined CMV seropositive lung transplant recipients on valganciclovir for a median of 6 months from transplant have a detectable polyfunctional CMV-specific T cell response which is comparable to seropositive recipients not on antiviral medications and to healthy seropositive nontransplant controls. Thus, valganciclovir prophylaxis does not appear to impair the development of CMV-specific immunity in lung transplantation.


Abbreviations: 
CMV

Cytomegalovirus

BOS

bronchiolitis obliterans syndrome

HLA

human leukocyte antigen

ICS

intracellular staining

D–/R–

CMV serology status donor negative/recipient negative

D+/R+

CMV serology status donor positive/recipient positive

D–/R+

CMV serology status donor negative/recipient positive

D+/R–

CMV serology status donor positive/recipient negative

FBS

Fetal bovine serum

DMSO

Dimethyl sulfoxide

PBMC

peripheral blood mononuclear cell

EDTA

Ethylenediaminetetraacetic acid

IL-2

Interleukin 2

TNFα

Tumor necrosis factor alpha

IFNγ

Interferon. gamma

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Cytomegalovirus (CMV) is one of the most prevalent opportunistic infections in solid organ transplant recipients. In particular, lung transplant recipients have an exceptionally high burden of CMV infection and disease both by frequency and severity (1,2). This may be due to the intensive immunosuppression utilized after lung transplant and/or the significant amount of lymphatic tissue and thus viral load transplanted with the lung allograft. Regardless, CMV after lung transplant has been associated with acute rejection, long-term allograft dysfunction manifested as bronchiolitis obliterans syndrome (BOS) and poor overall posttransplant survival (3,4). Most lung transplant centers, therefore, employ antiviral prophylaxis, such as intravenous ganciclovir or a highly bioavailable oral ganciclovir formulation, valganciclovir, for a fixed duration after transplant (5).

While prophylaxis generally prevents infection in the early posttransplant period, there is increasing recognition of late onset CMV disease, often defined as occurring after discontinuation of prophylaxis (2–6). Although the reasons for the development of late onset disease are likely complex, it has been hypothesized that extended valganciclovir prophylaxis prevents or delays the recovery of CMV-specific CD4 positive and CD8 positive T cells after transplant (7,8). CMV-specific T cells are critical for limiting viral replication after solid organ transplant (9). Thus, if antiviral prophylaxis impairs CMV-specific immunity, then recipients may be at a higher risk for CMV disease after discontinuation of prophylaxis.

In order to clarify this critical issue, we sought to characterize in detail the CD4 and CD8 T cell cytokine responses to CMV in lung transplant recipients receiving extended antiviral prophylaxis with valganciclovir as compared to recipients not on prophylaxis and healthy nontransplant controls. In this pilot cross-sectional study, we measured concurrent intracellular cytokine production of IFNγ, TNFα and IL-2 in CD4 and CD8 T cell populations in response to a panel of human leukocyte antigen (HLA) restricted CMV pp65 or IE-1 antigens using polychromatic intracellular staining (ICS) flow cytometry on peripheral blood mononuclear cells. This study provides the first description of the polyfunctional T cell response to immunodominant CMV peptides in CMV seropositive lung transplant recipients on and off extended valganciclovir prophylaxis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Recipient cohort, immunosuppression and prophylaxis protocol

Fourteen CMV seropositive lung transplant recipients were recruited from the lung transplant clinic at Duke University Medical Center. Additionally one CMV seronegative recipient who received a seronegative donor was recruited and two CMV seronegative recipients who received seropositive donors and have continuously received antiviral prophylaxis were recruited as negative controls. Healthy, nontransplant subjects with documented CMV positive serology but without active infection were recruited for comparison as positive controls. All subjects signed consent to participate in an IRB approved protocol.

At the time of transplant, all recipients received a CD25 antagonist induction agent (basiliximab) and triple immunosuppression with tacrolimus, azathioprine and corticosteroids. Immunosuppression was adjusted based on white blood cell count, intolerance to side effects, infection and rejection history. Cytomegalovirus prophylaxis was given according to donor/recipient CMV serology (IgG and/or IgM antibody detected). Low risk (donor negative/recipient negative, D–/R–) received acyclovir for 12 weeks, medium risk (donor positive or negative/recipient positive, D+/R+, D–/R+) received ganciclovir initially followed by valganciclovir for up to 1 year posttransplant and high risk (donor positive/recipient negative, D+/R–) received ganciclovir for 12 weeks followed by valganciclovir indefinitely. All recipients received pneumocystis carinii prophylaxis. Between transplant and the blood collection, none of the recipients developed CMV viremia by PCR DNA in the peripheral blood, CMV shell vial positivity in the bronchoalveolar lavage culture or CMV pneumonitis as defined by prospectively stained positive IE-1 immunohistochemistry on transbronchial lung biopsies. At sample collection, none of the lung transplant recipients met criteria for BOS as determined by spirometry.

Sample collection and storage of mononuclear peripheral blood cells

Peripheral blood was obtained by venipuncture and collected in acid-citrate-dextrose tubes (BD Vacutainer, Franklin Lakes, NJ, USA). Mononuclear cells were separated by Ficoll density gradient, washed and counted prior to storage. Cells were resuspended in a 90% FBS (Gemini, West Sacramento, CA, USA) and 10% DMSO (Sigma, St Louis, MO, USA) solution, progressively cooled to –80°C and transferred to liquid nitrogen for storage. Prior studies comparing fresh and cryopreserved peripheral blood mononuclear cells have shown recovery to be consistently above 80% viable lymphocytes and no significant differences in cytokine production (data not shown).

Cell preparation, stimulation and flow cytometry

Cells were thawed, washed and rested overnight in RPMI (Gibco, Carlsbad, CA, USA) with 10% FBS (Cell Generation, Fort Collins, CO, USA) and 1% penicillin/streptomycin/glutamine (Gibco) at 37°C in a 5% CO2 incubator. For each subject, 2 × 106 PBMCs were separately stimulated with CMV pp65 peptide pool (JPT Peptide Technologies, Berlin, Germany) + costimulatory molecules anti-CD28 and anti-CD49d (BD Biosciences, San Jose, CA, USA) and with an IE-1 peptide pool (JPT Peptide Technologies) + costimulatory molecules anti-CD28 and anti-CD49d. CMV peptide pools spanned the entire target region (pp65 or IE-1) in 15 amino acid peptides, overlapping by 11 amino acids and used at a concentration of 1μg/mL. A negative control (costimulatory molecules anti-CD28 and anti-CD49d alone) and a positive control with staphylococcal enterotoxin B (Sigma) + costimulatory molecules anti-CD28 and anti-CD49d was included for each subject. Brefeldin-A (Sigma) at a concentration of 5 μg/mL and Monensin (BD Biosciences) at a concentration of 1 μg/mL were added to the stimulation mixes prior to incubation for 6 h at 37°C then 4°C overnight. The following day, EDTA (FastImmune, BD) was added to the samples. After an incubation period, the cells were washed and then incubated with a cell surface antibody mix of anti-CD3 AmCyan, anti-CD4 PerCP-Cy5.5, anti-CD8 APC-Cy7 and anti-CD14 PacBlue (BD Biosciences) and Live/Dead Fixable Violet Dye (Invitrogen). Following this incubation, the cells were washed, lysed (FACSLysing Solution, BD Biosciences) and permeabilized (FACSPermeabilizing Solution, BD Biosciences) according to the manufacturer's instructions. The cells were then incubated with an intracellular antibody mix of anti-IL-2 APC, anti-TNFα Alexa700 and anti-IFNγ PE-Cy7 (BD Biosciences). After incubation, the cells were washed and cell pellets fixed in 1% formalin. Compensation beads (BD Biosciences) were stained with monoclonal antibodies and used to determine compensation. Data were acquired immediately with a LSRII flow cytometer (BD Biosciences).

Statistical analysis

Flow cytometry data were analyzed by a sequential gating strategy and Boolean gating using FlowJo analysis software (Ashland, OR, USA). Data for each group were expressed as a mean and standard error. Unpaired t-tests were used to compare groups with Welch's correction for unequal variances. Graphs were generated using GraphPad Software (La Jolla, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Recipient cohort

Clinical characteristics and demographics of the medium risk lung transplant recipients are outlined in Table 1. Patients on valganciclovir were similar to those off valganciclovir with regards to age, gender, type of transplant operation and immunosuppression. The native disease distribution was different between the groups with notably more restrictive lung disease in the valganciclovir group. As expected given our center's CMV prophylaxis protocol, all medium risk recipients on valganciclovir at the time of blood draw were within the first year posttransplant (median 174 days, IQR 98–220).

Table 1.  Demographic characteristics of lung transplant recipients with CMV positive serology (medium risk)
CharacteristicValganciclovir N = 10Not on valganciclovir N = 4
Gender, male, n (%)6 (60) 4 (100)
Age at transplant, years, median (IQR)  64 (54, 68)  59 (54, 63)
Bilateral transplant, n (%)5 (50) 4 (100)
Days from Transplant, median (IQR)174 (98.3,  220)1202 (1068,  2344)
Native lung disease, n (%)
 Obstructive2 (20)2 (50)
 Cystic0 (0) 1 (25)
 Pulmonary vascular0 (0) 1 (25)
 Restrictive8 (80)0 (0) 
Immunosuppression at time of sample, n (%)
   Prednisone10 (100) 4 (100)
   Tacrolimus9 (90)3 (75)
   Cyclosporine1 (10)1 (25)
   Azathioprine6 (60)3 (75)
   Mycophenolate mofetil1 (10)0 (0) 

Staphylococcal enterotoxin B (SEB) produces a robust cytokine response in immunosuppressed transplant recipients

We first confirmed that lung transplant recipients on multiple drug immunosuppression could produce a detectable polyfunctional cytokine response to the super antigen, staphylococcal enterotoxin B. With similar percentages of CD4+ and CD8+ T cells, lymphocytes from lung transplant recipients produced at least as effective cytokine response compared to the nontransplant control subject samples (Figure 1). In both T cell subsets, lymphocytes were polyfunctional with production of multiple cytokines concurrently.

image

Figure 1. Representative CD8 + T cell cytokine expression in response to negative control, staphylococcal enterotoxin (SEB), pp65 and IE-1 stimulation in CMV seropositive transplant on valganciclovir (R+ on valganciclovir), off valganciclovir (R+ NO valganciclovir) and in a CMV seropositive nontransplant control. Initial gating was based on CD3+. The positive and negative gates for each cytokine are individually determined by the negative control (costimulatory molecules alone).

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Cellular response to CMV peptides is polyfunctional with predominantly concurrent IFNγ and TNFα expression

A unique aspect of this experimental design was the ability to concurrently measure IFNγ, IL-2 and TNFα expression in individual CD4 and CD8 cells. For both pp65 and IE-1 stimulation, the CD4 and CD8 cellular CMV-specific cytokine response was predominantly polyfunctional with concurrent expression of IFNγ and TNFα. IL-2 cytokine response to CMV peptides was low in both T cell subsets.

D+/R+ and D–/R+ recipients have evidence of CMV-specific immunity by pp65 and IE-1 stimulation regardless of valganciclovir use

CMV seropositive transplant recipients demonstrated IFNγ and TNFα expression but not IL-2 expression in response to both pp65 and IE-1 peptide stimulation (Figure 1 and Table 2). Within the CD8 positive population, the cytokine response to pp65 stimulation was similar between the recipients regardless of antiviral use (Table 2 and Figure 2). Similarly, IE-1 stimulation induced IFNγ and TNFα expression with no difference between the recipients on or off valganciclovir (Table 2 and Figure 2).

Table 2.  Frequency of CD8 cells producing cytokines in response to CMV peptide stimulation. All values are expressed as mean percentage ± standard error
 pp65 stimulation1IE-1 stimulation2
R+ on valganciclovir N = 10R+ NO valganciclovir N = 4Seropositive nontransplant N = 4R+ on valganciclovir N = 10R+ NO valganciclovir N = 4Seropositive nontransplant N = 4
  1. 1pp65 stimulation: p values comparing R+ on valganciclovir versus R+ not on valganciclovir and R+ on valganciclovir versus seropositive nontransplant controls all not significant for individual and multiple cytokines.

  2. 2IE-1 stimulation: p values comparing R+ on valganciclovir versus R+ not on valganciclovir and R+ on valganciclovir versus seropositive nontransplant controls all not significant for individual and multiple cytokines.

IFNγ2.27 ± 0.552.67 ± 0.632.96 ± 0.622.73 ± 1.101.10 ± 0.801.48 ± 0.80
IL-20.24 ± 0.050.10 ± 0.030.19 ± 0.070.34 ± 0.130.16 ± 0.090.12 ± 0.04
TNFα2.43 ± 0.582.49 ± 0.542.97 ± 0.643.09 ± 1.211.29 ± 0.801.56 ± 0.80
IFNγ and TNFα1.97 ± 0.522.27 ± 0.472.47 ± 0.472.50 ± 1.070.91 ± 0.681.26 ± 0.69
IFNγ, IL-2 and TNFα0.11 ± 0.040.04 ± 0.030.16 ± 0.060.10 ± 0.050.09 ± 0.090.07 ± 0.04
image

Figure 2. CMV-specific immunity by intracellular cytokine production in the CD4 and the CD8 population. (A) In the CD8 population, CMV-specific immunity was notable in recipients seropositive for CMV and on valganciclovir (R+ on valganciclovir) by IFNγ total production and by TNFα total production. This CD8 cytokine response is similar to recipients seropositive for CMV not on valganciclovir (R+ NO valganciclovir) and to CMV seropositive nontransplant controls (Seropositive nontransplant). (B) CMV-specific cytokine response in the CD4 population was low (<0.5%). Bars indicate mean T cell percentage with standard error bars.

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Regarding polyfunctional expression, the percentage of CD8+ expressing concurrent IFNγ and TNFα in response to pp65 stimulation was similar between the groups (Table 2 and Figure 3). The percentage of CD8+ expressing concurrent IFNγ and TNFα in response to IE-1 stimulation was similar between the groups (Table 2 and Figure 3). Thus, most CD8 T cells produced both IFNγ and TNFα in response to pp65 or IE-1 stimulation. In contrast, for both pp65 and IE-1 stimulation, there were very low percentages (<0.5%) of CD4 positive cells expressing individual or polyfunctional cytokines (Figure 2).

image

Figure 3. CMV-specific immunity polyfunctional cytokine response in the CD8 population defined by concurrent IFNγ and TNFα expression. (A) Representative subjects demonstrating polyfunctional expression of both IFNγ and TNFα in recipients seropositive for CMV and on valganciclovir (R+ on valganciclovir), off valganciclovir (R+ NO valganciclovir) and CMV seropositive nontransplant controls (Seropositive nontransplant). (B) CMV-specific immunity polyfunctional cytokine response in the each defined group. Bars indicate mean T cell percentage with standard error bars.

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Cytokine responses to CMV peptides are similar between D+/R+ and D–/R+ recipients on valganciclovir and healthy nontransplant subjects

In comparing seropositive CMV transplant recipients on valganciclovir to seropositive nontransplant control subjects, the cytokine expression pattern was similar for both CMV pp65 stimulation and IE-1 stimulation. The predominant cytokines expressed in the CD8 population were IFNγ and TNFα and this was predominantly polyfunctional with IL-2 expression very low (Figure 2). In contrast, for both pp65 and IE-1 stimulation, there were very low percentages (<0.6%) of CD4 positive cells expressing individual or polyfunctional cytokines.

Absence of CMV-specific immunity in D+/R– and D–/R– recipients

D+/R– recipients were maintained on valganciclovir after transplant and did not develop CMV pneumonitis, CMV shell vial culture positive BAL or detectable CMV by DNA PCR. These high-risk recipients, tested at 365 days or 707 days after transplant, did not demonstrate CMV-specific immunity by cytokine response after stimulation with pp65 or IE-1 peptides in vitro (data not shown). This lack of CMV-specific immunity was similar to the undetectable cytokine response seen in a D–/R– recipient tested 1198 days after transplant (data not shown). Thus, the CMV seronegative recipients who have not developed primary posttransplant CMV infection after transplant did not have detectable CMV-specific immunity.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Despite the availability of effective antiviral agents to prevent and treat CMV, the development of invasive CMV disease at any point after lung transplant is a risk factor for poor long-term lung allograft function and survival (4). Recently, a randomized controlled clinical trial of lung transplant recipients demonstrated that 1 year of valganciclovir prophylaxis more effectively prevented CMV disease without significant adverse drug side effects as compared to shorter course therapy (10). However, a major concern with this type of extended prophylaxis is the risk of late onset CMV disease once prophylaxis ends (6). One potential explanation for of the high incidence and severity of late onset disease is that extended prophylaxis might prevent the development or maintenance of CMV-specific immunity. We, therefore, sought to determine if CMV-specific immunity was detectable in lung transplant recipients receiving valganciclovir using polyfunctional analysis of CD4 and CD8 CMV-specific cytokine production. Furthermore we sought to compare the quantity and quality of the anti-CMV response in seropositive lung transplant recipients currently receiving valganciclovir to those off valganciclovir for an extended period of time and to healthy seropositive nontransplant controls. Our findings document that CMV seropositive lung transplant recipients on valganciclovir have detectable CMV-specific immunity in the CD8 population and this response is polyfunctional with predominantly concurrent IFNγ and TNFα cytokine production. Furthermore, the level and pattern of the CMV-specific T cell response is similar to CMV seropositive recipients off antiviral prophylaxis for an extended period of time and to healthy CMV seropositive controls.

Our study includes several novel features. First, to our knowledge, our study is the first to consider polyfunctional cytokine production in response to CMV antigens among CMV seropositive lung transplant recipients on valganciclovir. This adds to the experience previously reported in primary CMV infection after lung transplant where both single cytokine and polyfunctional cytokine responses were measured (11,12). In contrast to most other studies that measured a single cytokine by ELISPOT assays in CMV seropositive recipients, our flow cytometry design measured polyfunctional cytokine responses from CD4 or CD8 T cell populations (13,14). Although the full significance of polyfunctional cytokine production is an area of intense research in virology, polyfunctional T cells might have a greater capacity to limit viral replication (15). For example, a polyfunctional response to GAG proteins in HIV infection is correlated with disease nonprogression (16). Similarly, a polyfunctional cytokine response in the CD8 population after vaccinia vaccination is thought to contribute to the success of the vaccine in protection from smallpox (17). Our results indicate that a polyfunctional response to CMV occurs in solid organ transplantation with predominantly IFNγ and TNFα produced by CD8 T cells in response to CMV antigen stimulation. While IFNγ and TNFα have concurrent expression, IL-2 expression is distinctly different and diminished in comparison to the other cytokines.

Second, we provide the first detailed characterization of CMV-specific immunity in a population of patients receiving extended valganciclovir prophylaxis. Because of the high burden of CMV in lung transplantation and its adverse consequences, prophylaxis regimens are now extended to 6–12 months after transplant in this patient population. Consistent with that approach, patients enrolled in this study received a median of 174 days of valganciclovir prophylaxis prior to undergoing analysis. We demonstrate that the quality and quantity of their CMV-specific immunity is comparable to those off valganciclovir for an extended period of time, and to healthy seropositive nontransplant controls.

Finally, our study considered both the immune response to IE-1 and to pp65. While there were no significant differences in single or multiple cytokine responses to either CMV antigen between the groups, the highest polyfunctional cytokine response to IE-1 was seen in CMV seropositive recipients on valganciclovir (Figures 2 and 3). The clinical significance of this observation is unclear and may simply reflect our sample size. Alternatively, IE-1 response and pp65 response may correspond to different CMV immunity. In normal healthy subjects with prior CMV exposure, there is a higher percentage of CD4 and CD8 positive cells producing cytokines in response to pp65 peptides compared to IE-1 peptides (18). However, at least one study in transplant recipients has suggested that IE-1-specific response in the CD8 positive population correlates to CMV protection more than pp65-specific response (19,20). Other studies in transplant recipients have not been able to confirm that response to one antigen correlates to risk of CMV disease (18). The conflicting reports on effective protection by cytokine response are likely due to older techniques measuring cytokine production (ELISPOT), small sample size and differences in CMV-specific immunity among different types of solid organ transplant.

Our study also has several limitations that should be noted. Most importantly, this was a pilot study with a small sample of patients. Although a larger sample size would enhance our ability to detect subtle differences in CMV-specific immunity among the groups, it would be unlikely to change our overall conclusion that CMV-specific immunity is present in the setting of extended valganciclovir prophylaxis and immunosuppression. Another potential limitation is our use of IE-1 and pp65 peptide pools targeting the known immunodominant regions of the CMV viral protein to determine CMV-specific immunity. It is possible there are other regions of the viral protein that are critical to CMV-specific immunity. In limited studies of transplant recipients, these peptide pools used in vitro appear to underestimate CMV-protective immunity compared to more laborious cell-based approaches (18). Thus, we believe this peptide stimulation approach is conservative in determining CMV-specific immunity. In addition, we only considered a limited range of cytokines in our polyfunctional analysis; further studies are needed to identify other cytokines that might be upregulated as part of a polyfunctional anti-CMV response and may define recipients at risk once prophylaxis ends.

Finally, because of the cross-sectional nature of the study, we cannot determine the extent to which the individual or polyfunctional cytokine response actually corresponds to clinical control of viral replication and predicts sustained protection against CMV development after lung transplantation. A study of predominantly heart transplant recipients suggested a threshold of 0.4% CD8 positive cells producing IFNγ in response to IE-1 peptide stimulation correlated with protection from CMV disease in the first few months (19). In our study, most recipients on valganciclovir had a higher measurable level of immunity. However, two recipients off valganciclovir without any history of CMV, three recipients currently on valganciclovir and 1 normal nontransplant control subject were below this threshold and would be considered at higher risk for CMV. Another study of lung transplant recipients that measured IFNγ in response to a CMV peptide pool that included IE-1 and pp65 stimulation did not find a consistent correlation between T cells expressing IFNγ and risk of CMV (13). Our work along with these prior studies provides a strong rationale for a large prospective longitudinal study to define the immune parameters that predict CMV immunity in lung transplantation. Once established, this approach would open the possibility of strategically measuring CMV-specific immunity after transplant to permit discontinuation of antiviral prophylaxis based on functional measures of CMV immunity rather than a fixed duration of time. Such an approach could minimize drug side effects and cost while maximizing effective CMV prevention.

In summary, this report is the first study of CMV seropositive lung transplant recipients to demonstrate CMV-specific immunity in the setting of antiviral prophylaxis by polyfunctional cytokine production. We find that after lung transplant there is a CD8 T cell response to CMV peptide stimulation that is characterized by concurrent IFNγ and TNFα production and is detectable despite ongoing antiviral prophylaxis. Levels of CMV-specific T cells and their pattern of polyfunctional cytokine production, while variable among at risk lung transplant recipients on valganciclovir, is comparable to seropositive patients off antiviral prophylaxis and to seropositive healthy nontransplant controls. Valganciclovir prophylaxis, therefore, does not appear to impair the maintenance of CMV-specific immunity in lung transplantation. The extent to which differences in these specific immune response parameters contributes to the high burden of CMV disease after lung transplantation is uncertain. Our work suggests that polyfunctional cytokine measurements represent a potentially useful strategy in the assessment of CMV-specific immunity in lung transplant recipients and should be further explored in additional prospective studies.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

National Institutes of Health KL2RR024127 and American Society of Transplantation Clinical Faculty Development Award (L.S.), National Heart Lung and Blood Institute SCCOR 1P50-HL084917-01 and K24-091140-01 (S.M.P.), Duke Translational Medicine Institute (S.S., R.M. and K.J.W.) and Duke University's CTSA grant 1 UL1 RR024128-01 from NCRR/NIH (L.S., C.C. and S.M.P.).

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References