Desensitization with intravenous immunoglobulin and rituximab (IVIG + rituximab) followed by a kidney transplant with a single 30-mg dose of alemtuzumab as induction has been a successful strategy to improve transplant rates and outcomes for highly HLA-sensitized (HS) patients.1-3 However, profound and prolonged B and T lymphocyte depletion may result in an increased risk for viral infections.4-7
Cytomegalovirus (CMV) is the most common viral infection after solid organ transplantation, and it represents an increased risk for morbidity and mortality in transplant recipients. Viral infections are controlled primarily by antiviral CD8+ T cells.8 We have previously shown that CMV-specific CD8+ T cells (CMV-Tc), as analyzed by intracellular cytokine flow cytometry, are detected in most CMV sero (+) healthy individuals and kidney transplant recipients. Clearance of CMV DNA is associated with detection of CMV-Tc in those patients.9 T cell response to CMV pp65 as analyzed by ELISPOT was noted to be higher in non-CMV viremic kidney transplant recipients compared to viremic patients.10 The association of CMV-Tc positivity with reduced risk for CMV disease has also been reported in allogeneic stem cell transplantation.11,12 These results indicate that monitoring CMV-T cell activity posttransplant, in addition to the application of anti-CMV prophylaxis and CMV DNA monitoring, offers a more comprehensive management strategy for prevention of CMV infection.
Alemtuzumab is a monoclonal antibody, targeting CD52 positive cells (mature lymphocytes including T cell, B cell, natural killer (NK) cells and monocytes) and depleting them.13 Alemtuzumab has been used as the induction agent of choice for our HS kidney transplant recipients.1,14,15 Because alemtuzumab is a potent depleter of T cells and B cells, we were concerned about the risk for viral infections. We previously reported that CMV-Tc activity was detected by 4 months posttransplant in HS patients desensitized with IVIG + rituximab followed by a kidney transplant and alemtuzumab or daclizumab induction. In addition, anti-CMV IgG levels remained constant before and after transplantation.16 Here, we monitored both CMV-Tc and CMV-Th activities, anti-CMV IgG, and immune cell subsets in a larger cohort of HS patients undergoing desensitization and transplantation to further assess CMV immunity and risk for CMV infection.
MATERIALS AND METHODS
Patient Population and Sample Collection
This study was approved by the institutional review board at Cedars-Sinai Medical Center (protocol numbers: 10969, 12562 and 17197). The study was conducted in accordance with the ethical guideline based on federal regulations and the common rule. In addition, Cedars-Sinai Medical Center has a Federalwide Assurance.
Thirty-three patients (14 men) who were desensitized followed by kidney transplantation (17 living-donor transplants) were included in this study. Of 33 patients, 29 received an ABO-compatible transplant after desensitization consisting of 2 doses of IVIG (2 g/kg) 1 month apart with 1 dose of rituximab (1 g) in between.15 The remaining 4 patients received an ABO-incompatible transplant after desensitization consisting of 1 dose of rituximab (1 g) 2 weeks before initiation of 5 sessions of plasma exchange followed by 1 dose of IVIG (2 g/kg).17 All patients received 1 dose of alemtuzumab induction (Campath-1H, 30 mg subcutaneous injection) and maintenance immunosuppression consisted of tacrolimus, mycophenolate mofetil, and prednisone as previously described.1
All patients received antiviral prophylaxis with ganciclovir (1.25 mg/kg daily) while inpatient, and they received valganciclovir for 6 months posttransplant depending on a risk for viral infection (900 mg daily for CMV R−/D+, 450 mg daily for CMV R+/D+, R+/D−, or R−/D−) with dose adjustments for renal function and white blood cell count. Viral-PCR monitoring, including CMV-PCR, was performed at 1, 2, 3, 6, 9, 12, 18, and 24 months posttransplant or as needed as previously reported.18 CMV infection was treated with reduction of immunosuppression in conjunction with valganciclovir (900 mg twice daily for 14 to 21 days regardless of infection during or after antiviral prophylaxis, with dose adjustments for renal function and white blood cell count).
Blood samples from 20 normal adult volunteers (7 males) were included as controls in this study.
Heparinized peripheral blood samples were collected predesensitization and posttransplant, and they were submitted for flow cytometry analysis for CMV-Tc, Th, and immune cell subsets. Plasma samples were tested for anti-CMV IgG by ELISA. Ethylenediaminetetraacetic acid-anticoagulated blood samples collected posttransplant were submitted for CMV-PCR.
CMV-Specific T Cell Assay
CMV-specific T cell (CMV-T) levels were measured by cytokine flow cytometry developed in our laboratory as described elsewhere.9 Briefly, whole blood was incubated with an overlapping peptide mixture of 138 peptides spanning the sequence of CMV protein pp65 (CMV peptides) (final working concentration, 1.75 μg/mL) or sucrose density purified CMV viral lysate (CMV lysate) (final working concentration, 1.0 μg/mL) (Advanced Biotechnologies, 10-144-001, MD) together with brefeldin A and anti-CD28/CD49d overnight. The IFNγ+ cell% in CD8+ cells stimulated with CMV peptides and in CD4+ cells stimulated with CMV lysate were enumerated and defined as CMV-Tc and CMV-Th, respectively. In some of CMV-T cell results, CMV-Tc% and CMV-Th% were used to distinguish from the absolute cell number of these cell populations. CMV-Tc and CMV-Th ≥0.20% were considered positive. This was established based on the levels detected in CMV sero(+) and sero(−) normal individuals and transplant recipients.9,16
CMV DNA levels were quantified by the Transplantation and Immunology Laboratory, Cedars-Sinai Medical Center as previously described using an endpoint CMV-PCR.18,19 Briefly, 500 ng total DNA extracted from blood leukocytes was used per PCR and more than 5 copies/PCR (500 ng total DNA) was considered viremia.18
Immune Cell Subset Analysis
The number of total lymphocyte, T cells, and NK cells were enumerated by flow cytometry as previously described with modification.16 Briefly, 100 μL of heparinized peripheral blood was stained for specific surface markers by a direct single color staining method using 5 μL of Fluorescein isothiocyanate-conjugated mouse monoclonal antibodies to CD4, CD8 or CD56 (Invitrogen, Camarillo, CA). Lymphocytes, first gated by forward/side scatter, were plotted against CD4, CD8, or CD56. A percentage of each cell population in total lymphocytes was enumerated. Using the cell% of each cell subset as analyzed by flow cytometry and the total lymphocyte count (cell number/μL blood) as counted by hematologic analysis, the cell number of each cell subset was calculated, and then the results were expressed as the ratio against predesensitization level.
Anti-CMV IgG levels were quantified by ELISA (BioCheck, Inc, Foster City, CA) as per manufacturer's instruction and the optical density was used for the results.
CMV-Tc and CMV-Th levels were compared between normal individuals and patients by a Student t test (Table 1). Posttransplant CMV-Tc and CMV-Th cell% and number (Figure 2) and posttransplant immune cell numbers (Figure 4) were compared with pretransplant levels by paired t test. P value less than 0.05 was considered significant.
CMV-T Cell Levels in Predesensitized Patients and Normal Individuals
Predesensitization CMV-T cell levels in 33 patients are summarized in Table 1. Among the 30 CMV sero (+) patients, 25 showed positivity for both CMV-Tc and CMV-Th (83.3%). The remaining 5 (16.7%) did so for either CMV-Tc or Th, but not both. CMV sero (+) normal individuals showed a similar trend. Twelve individuals (75%) showed positivity for both Tc and Th, and 3 (25%) individuals were positive for either Tc or Th. The remaining 1 normal individual did not show CMV-T cell activity. No CMV-T cell activity was demonstrated in CMV sero (−) individuals. We next compared the CMV-Tc and CMV-Th levels in CMV sero (+) predesensitized patients and normal individuals who showed positivity for CMV-Tc or Th. The CMV-Th levels were significantly higher in patients compared to normal individual (2.5 ± 2.5% vs 1.1 ± 1.1%, P = 0.03), whereas CMV-Tc levels were similar (Table 1).
CMV Viremia Posttransplant
Four (11.8%) of 33 patients developed CMV viremia within 3 months posttransplant while on antiviral prophylaxis. Three of the 4 patients were CMV sero (+) at transplant and 1 was sero (−) (Table 2). The peak CMV DNA level in the CMV sero (−) patient was 1400 copies/PCR and 1 of the 3 sero(+) patients showed 544 copies/PCR. After antiviral treatment, the CMV DNA levels were quickly reduced to the negative range in both patients. The remaining 2 sero (+) patients were less than 50 copies/PCR that did not require treatment. All 3 sero (+) patients with viremia were positive for CMV-Th at transplant, but 2 of 3 were negative for CMV-Tc.
CMV-T Cell Activity Posttransplant in CMV Sero (+) Patients
The CMV-T cell activity was monitored posttransplant. The IFNγ+ cell% in CD8+ cells stimulated with CMV peptides and in CD4+ cells stimulated with CMV lysate were defined as CMV-Tc and CMV-Th, respectively. CMV-Tc and CMV-Th of 0.20% or greater were considered positive values. We first analyzed CMV-Tc or Th positivity posttransplant in patients who were positive for CMV-Tc or Th pretransplant. CMV-Tc (+) was detected in 46% of patients at 1 month, 77% at 2 months, and 96% by 3 months posttransplant (Figure 1). CMV-Th (+) was detected in 31% of patients at 1 month, 72% at 2 months, and 93% of patients by 3 months posttransplant.
We next analyzed the change in CMV-Tc and Th levels posttransplant in patients who were (+) for CMV-Tc or CMV-Th pretransplant. After alemtuzmab induction, the absolute number of total CD8+ T cells, CD4+ T cells, CMV-Tc, and CMV-Th significantly decreased (Figures 2A, D). These reductions remained up to 6 months posttransplant, although cell counts gradually improved. When the cell numbers were compared pretransplant (ratio 1.0 at the baseline) versus posttransplant, a great number of total CD8+ T cells than total CD4+ T cells were detected (0.3 ± 0.6 vs 0.1 ± 0.2 at 3 months and 0.3 ± 0.5 vs 0.2 ± 0.2 at 6 months posttransplant). In addition, greater numbers of CMV-Tc than CMV-Th were detected (0.3 ± 0.5 vs 0.1 ± 0.3 at 3 months and 0.5 ± 0.6 vs 0.2 ± 0.3 at 6 months posttransplant). In contrast to CMV-Tc and Th cell numbers, CMV-Tc% and CMV-Th% (% in the total CD8+ or CD4+ T cells, respectively) did not significantly change at 3 month posttransplant compared with pretransplant levels, and the CMV-Tc%, but not CMV-Th%, significantly increased at 6 months (Figures 2B and E). The mitogen (PHA)-stimulated IFNγ+ cell% in total CD8+ or CD4+ T cells, tested as non–antigen-specific activation control, significantly decreased at 3 months after alemtuzumab induction (Figures 2C, F).
CMV-T Cell Levels During CMV Viremia
The CMV-T cell activity and CMV DNA levels were closely monitored in 3 CMV sero (+) patients and 1 sero (−) patient that had CMV viremia. In the 3 sero (+) patients (Figures 3A-C), the CMV-Tc (+) and/or Th (+) detected at transplant became (−) for both or either Tc and Th posttransplant when CMV viremia was detected. CMV viremia was quickly cleared when the CMV-Tc and/or Th became (+). In a single CMV sero (−) patient, who showed no CMV-T cell activity at transplant, we noted that both CMV-Tc and Th became (+) when the patient developed CMV viremia (1400 copies/PCR). The viremia was cleared 1 month later (Figure 3D).
Lymphocyte Numbers and Anti-CMV IgG Levels Pretransplant and Posttransplant
The number of total lymphocytes, CD8+ and CD4+ T cells, CD56+ NK cells, and anti-CMV IgG levels were monitored pretransplant and posttransplant. The total lymphocyte numbers significantly decreased to 10% of predesensitization levels at 1 month posttransplant (post-alemtuzumab) and the levels gradually increased afterwards. However, they remained at 35% and 50% of base line at 6 and 12 months posttransplant, respectively (Figure 4A). The CD8+ and CD4+ T cell numbers were extremely low at 1 month posttransplant and remained low for 12 months, especially in CD4+ T cells (Figure 4B). In contrast, the NK cell numbers were higher than 50% of baseline from 2 months posttransplant and reached at 80% of the baseline at 12 months posttransplant (Figure 4C).
Anti-CMV IgG levels in CMV sero (+) patients at transplant did not change posttransplant from pretransplant levels (Figure 4D).
Alemtuzumab use as an induction agent in transplant recipients is well established,1,13,20 despite a risk for viral infection due to profound and prolonged lymphocyte depletion. In this study, a total of 33 HS patients who were desensitized with IVIG + rituximab followed by a kidney transplant with alemtuzumab induction were included. The rate of CMV viremia with more than 5 copies/PCR was 12%, and viremia with more than 50 copies/PCR requiring antiviral therapy was 6%. This is similar to rates observed in non-HS kidney transplant recipients.18
We monitored both CMV-Tc and CMV-Th pretransplant and posttransplant to investigate the effects of lymphocyte depletion with rituximab and alemtuzumab on these cells and assess a role of CMV-T cell activity in prevention and clearance of CMV infection. Most CMV sero (+) patients (83%) included in this study showed positivity for both CMV-Tc and Th predesensitization, and the remaining 17% showed positivity for either CMV-Tc or Th. A similar trend was observed in CMV sero (+) normal individuals. However, when the CMV-T cell levels were compared, the predesensitization CMV-Th levels in HS patients were significantly higher than normal individuals (2.5 ± 2.5% vs 1.1 ± 1.1%, P = 0.03), which is consistent with other reports. Chung et al21 have shown that effector memory CD4+ T cell percentage was significantly higher in patients with end-stage renal disease compared with healthy normal controls. Sester et al22 have also reported that hemodialysis patients, despite lower percentage of memory CD8+ and CD4+ T cells, showed robust CD4+ T cell response to influenza vaccine at the secondary vaccination compared to that in healthy young controls.
In the 30 CMV sero (+) patients who were (+) for CMV-Tc and/or Th pretransplant, approximately 50% to 70% became CMV-Tc and/or Th (−) at 1 month posttransplant (post-alemtuzumab) due to T cell depletion. However, 75% showed CMV-T cell (+) by 2 months posttransplant and 95% by 3 months posttransplant (Figure 1). To understand the effect of alemtuzumab on CMV-T cells, the absolute cell number of total CD8+ T cells, CD4+ T cells, CMV-Tc, and CMV-Th were analyzed (Figures 2A and D). The number of cells significantly decreased after alemtuzumab induction. A greater number of total CD8+ T and CMV-Tc cells were detected post-alemtuzumab compared with the total CD4+ T and CMV-Th cells, respectively (Figures 2A and D), which is consistent with other reports.23,24 Lowenstein et al23 showed that CD4+ T cells were more sensitive than CD8+ T cells to alemtuzumab-mediated complement-dependent cytotoxicity (CDC), although another study reported equal sensitivity to CDC by alembtuzumab.25 It has also been reported that the restoration of CD8+ T cells occurs more rapidly than in CD4+ T cells.24 In contrast to significant reduction of T cell numbers post-alemtuzumab, a similar percentage of CMV-Tc (% in total CD8+ T cells) or CMV-Th (% in total CD4+ T cells) was observed at 3 months post-alemtuzumab (Figures 2B and E). CMV-Tc%, but not CMV-Th%, significantly increased at 6 months post-alemtuzumab. This result suggests that CMV-Tc, memory CD8+ T cells, may be less sensitive to alemtuzumab cell depletion and/or repopulate faster than naive CD8+ T cells, whereas CMV-Th, memory CD4+ T cell, and naive CD4+ T cells are similar. Pearl et al26 and Marco et al27 showed that naive and memory CD4+ T cells were equally susceptible to alemtuzumab-mediated cytotoxicity. However, the relative proportion of memory-phenotype T cells was increased during lymphocyte repopulation after alemtuzumab cell depletion.28 This effect was most marked at 1 month post-alemtuzumab, and the memory and naive T cell proportion gradually returned to baseline by 12 months.29,30 Taken together, a few CMV-specific memory CD8+ and CD4+ cells remain after alemtuzumab cell depletion at similar and/or higher proportion to naive CD8+ and CD4+ cells, and they are capable of responding to CMV peptides and lysate resulting in IFNγ production and cytotoxic effector functions against infected cells. Abade et al31 has also reported that another lymphocyte-depleting agent, antithymocyte globulin, had no significant effect on CMV-specific T cell activity in kidney transplant recipient.
PHA-stimulated IFNγ+ cell% in total CD8+ or CD4+ T cells were also measured pretransplant and posttransplant as non–antigen-specific activation control. Interestingly, PHA-stimulated IFNγ+CD8+ cell% and IFNγ+CD4+ cell% significantly decreased at 3 months after alemtuzumab induction. These reductions persisted for 6-months in IFNγ+CD8+ cell%, but not IFNγ+CD4+ cell% cells. PHA is a monocyte-dependent T cell mitogen.32 Because monocytes are also depleted by alemtuzumab, although they are least susceptible to lysis by alemtuzumab,25 less help from monocytes might be responsible at least in part for less PHA-induced T cell activation post-alemtuzumab.
In this study, we also monitored CD56+ NK cell numbers. In contrast to T cells, the NK cell numbers returned to 30% of the pretransplant levels by 1 month posttransplant, reached 50% to 70% at 2 months, and greater than 80% by 12 months (Figure 4C). This suggests that the NK cells are less sensitive to alemtuzumab depletion and/or restoration of NK cells after cell depletion is much faster than T cells. It has been reported that NK cells were least susceptible to alemtuzumab-mediated CDC using human PBMC25 and NK cell repopulation was faster than T cells in alemtuzumab-treated cynomolgus monkeys.33 We have previously reported the lack of variation in levels of anti-CMV IgG pretransplant and posttransplant in CMV sero (+) patients who were desensitized with IVIG + rituximab followed by a transplant with alemtuzumab induction.16 This result was also seen here (Figure 4D), suggesting again that anti-CMV IgG is primarily produced by long-lived plasma cells. Taken together, consistently available anti-CMV IgG and relatively high levels of NK cells available early after alemtuzumab induction may play an important role in anti-CMV immunity via antibody-dependent cell cytotoxicity in CMV sero (+) patients posttransplant. In fact, CMV DNA was quickly cleared in 3 CMV sero (+) patients who developed CMV viremia about 1 month posttransplant when CMV-Tc and CMV-Th were not detected (Figures 3A-C). Rapid reemergence of CMV-Tc and CMV-Th was seen in patients who became viremic as well. Recent studies showing the important role of a subset of NK cells, NKG2ChiCD57hi NK cells, in controlling CMV infection are of interest. This NK cell population has been identified to be expanded exclusively at CMV infection,34 and its effector function was enhanced only in the presence of anti-CMV antibodies.35 It is possible that CMV infection in our patients might be controlled by this NK cell population.
One CMV sero (−) patient developed CMV viremia with the peak level of 1400 copies/PCR at 2 months posttransplant. In this patient, both CMV-Tc and Th rapidly developed, and the viremia was cleared within a month (Figure 3D). This result demonstrates that even CMV sero (−) patients can develop de novo proliferating CMV-T cells after lymphocyte depletion with aleumtuzumab.
In conclusion, memory CMV-T cells remaining after lymphocyte depletion with alemtuzumab are capable of efficiently proliferating to clear CMV in sero (+) desensitized patients. A sero(−) HS patient efficiently developed CMV-T cells after T cell and B cell depletion, and cleared CMV viremia. High levels of NK cells remaining posttransplant and anti-CMV IgG are consistently available posttransplant and may also contribute to clearance of CMV infection through antibody-dependent cell cytotoxicity in CMV sero (+) patients. These results suggest that IVIG + rituximab desensitization combined with alemtuzumab induction with triple immunosuppression maintenance does not increase risk for CMV infection in HS kidney transplant patients.
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