The Cell Biology of Cytomegalovirus: Implications for Transplantation

Interpretation of clinical data regarding the impact of cytomegalovirus (CMV) infection on allograft function is complicated by the diversity of viral strains and substantial variability of cellular receptors and viral gene expression in different tissues. Variation also exists in nonspecific (monocytes and dendritic cells) and specific (NK cells, antibodies) responses that augment T cell antiviral activities. Innate immune signaling pathways and expanded pools of memory NK cells and γδ T cells also serve to amplify host responses to infection. The clinical impact of specific memory T cell anti‐CMV responses that cross‐react with graft antigens and alloantigens is uncertain but appears to contribute to graft injury and to the abrogation of allograft tolerance. These responses are modified by diverse immunosuppressive regimens and by underlying host immune deficits. The impact of CMV infection on the transplant recipient reflects cellular changes and corresponding host responses, the convergence of which has been termed the “indirect effects” of CMV infection. Future studies will clarify interactions between CMV infection and allograft injury and will guide interventions that may enhance clinical outcomes in transplantation.     

B Cell Depletion With an Anti‐CD20 Antibody Enhances Alloreactive Memory T Cell Responses After Transplantation

Alloreactive memory T cells mediate accelerated allograft rejection and transplant tolerance resistance. Recent studies have shown that B cell deficient–μMT mice fail to mount donor‐specific memory T cell responses after transplantation. At the same time, other studies showed that pretransplant B cell depletion using rituximab (IgG1 anti‐CD20 mAb) combined with cyclosporine A promoted the survival of islet allografts in monkeys. In this study, we investigated the effect of anti‐CD20 antibody‐mediated B cell depletion on the memory T cell alloresponse in mice. Wild‐type and anti‐OVA TCR transgenic mice were treated with an IgG2a anti‐CD20 monoclonal antibody, which depleted nearly all B cells in the peripheral blood and secondary lymphoid organs but spared some B cells in the bone marrow. B cell depletion did not affect the direct alloresponse but resulted in a marked increase of indirect alloresponse after skin transplantation of naïve mice. Furthermore, in allosensitized mice, anti‐CD20 mAb treatment enhanced the reactivation of allospecific memory T cells and accelerated second set rejection of skin allografts. This suggests that the effect of anti‐CD20 antibodies on alloimmunity and allograft rejection might vary upon the nature of the antibodies as well as the circumstances under which they are delivered.     

A prospective multicenter observational study of cell‐mediated immunity as a predictor for cytomegalovirus infection in kidney transplant recipients

T cell immunity is essential for the control of cytomegalovirus (CMV) infection after transplantation. We evaluated a CMV‐specific peptide‐based enzyme‐linked immunosorbent spot (ELISPOT) assay to determine whether assay results could predict subsequent CMV events. Adult kidney transplant recipients at 43 centers underwent ELISPOT testing to enumerate interferon gamma (IFN‐γ) binding spot‐forming units (sfu) after stimulation of cells with an overlapping peptide pool of CMV phosphoprotein 65 (pp65) and immediate early‐1 (IE‐1) protein at the end of antiviral prophylaxis (EOP) and various time points thereafter. The primary outcome was a CMV event in the first posttransplant year. In 583 kidney transplant recipients (260 seropositive donor [D+]/seronegative recipient [R?] and 277 R+), CMV events occurred in 44 of 368 eligible patients (11.8%) at a median of 227 days (range 92‐360) posttransplant. A cutoff value of >40 sfu/2.5 × 10⁵ cells for either IE‐1 or pp65 was derived as a threshold for positivity, with a negative predictive value of >97% for CMV events. CMV events were significantly lower in assay positive vs assay negative patients (3.0% vs 19.5%, P < .0001 for pp65). Time to CMV event post‐EOP was significantly greater in those with sfu >40 at EOP (P < .0001). In this large, multicenter trial of kidney transplant recipients, we show that an assessment of CMV‐specific immunity using a novel ELISPOT assay is able to predict protection from CMV infection.     

Cytomegalovirus‐Responsive CD8+ T Cells Expand After Solid Organ Transplantation in the Absence of CMV Disease

Cytomegalovirus (CMV) is a major cause of morbidity and mortality in solid organ transplant recipients. Approximately 60% of adults are CMV seropositive, indicating previous exposure. Following resolution of the primary infection, CMV remains in a latent state. Reactivation is controlled by memory T cells in healthy individuals; transplant recipients have reduced memory T cell function due to chronic immunosuppressive therapies. In this study, CD8⁺ T cell responses to CMV polypeptides immediate‐early‐1 and pp65 were analyzed in 16 CMV‐seropositive kidney and heart transplant recipients longitudinally pretransplantation and posttransplantation. All patients received standard of care maintenance immunosuppression, antiviral prophylaxis, and CMV viral load monitoring, with approximately half receiving T cell–depleting induction therapy. The frequency of CMV‐responsive CD8⁺ T cells, defined by the production of effector molecules in response to CMV peptides, increased during the course of 1 year posttransplantation. The increase commenced after the completion of antiviral prophylaxis, and these T cells tended to be terminally differentiated effector cells. Based on this small cohort, these data suggest that even in the absence of disease, antigenic exposure may continually shape the CMV‐responsive T cell population posttransplantation.     

Positive HCMV DNAemia in stem cell recipients undergoing letermovir prophylaxis is expression of abortive infection

Letermovir (LMV) inhibits HCMV replication by binding to components of the HCMV-terminase complex showing a potential role in prevention of HCMV-related complications in allogenic hematopoietic stem cell transplant recipients (allo-HSCTRs). However, little is known about breakthrough HCMV infection and the relevance of HCMV DNAemia during prophylaxis. We reported the results of a multicenter prospective study involving five Italian centers in the management of HCMV DNAemia in 75 adult HCMV-seropositive allo-HSCTRs undergoing LMV prophylaxis. The aim of the present study was to characterize the presence of real HCMV reactivation during LMV prophylaxis. Then, the presence of circulating infectious HCMV particles was determined by virus isolation and degradation of free-floating viral DNA. This report provides the first evidence that during LMV prophylaxis the clinical relevance of HCMV DNAemia should be critically considered.     

Pretransplant CD4 Count Influences Immune Reconstitution and Risk of Infectious Complications in Human Immunodeficiency Virus–Infected Kidney Allograft Recipients

In current practice, human immunodeficiency virus–infected (HIV⁺) candidates with CD4 >200 cells/mm³ are eligible for kidney transplantation; however, the optimal pretransplant CD4 count above this threshold remains to be defined. We evaluated clinical outcomes in patients with baseline CD4 >350 and <350 cells/mm³ among 38 anti–thymocyte globulin (ATG)–treated HIV‐negative to HIV⁺ kidney transplants performed at our center between 2006 and 2013. Median follow‐up was 2.6 years. Rates of acute rejection and patient and graft survival were not different between groups. Occurrence of severe CD4 lymphopenia (<200 cells/mm³), however, was more common among patients with a baseline CD4 count 200–349 cells/mm³ compared with those transplanted at higher counts (75% vs. 30% at 4 weeks [p = 0.04] and 71% vs. 5% at 52 weeks [p = 0.001], respectively, after transplant). After adjusting for age, baseline CD4 count of 200–349 cells/mm³ was an independent predictor of severe CD4 lymphopenia at 4 weeks (relative risk [RR] 2.6; 95% confidence interval [CI] 1.3–5.1) and 52 weeks (RR 14.3; 95% CI 2–100.4) after transplant. Patients with CD4 <200 cells/mm³ at 4 weeks had higher probability of serious infections during first 6 months after transplant (19% vs. 50%; log‐rank p = 0.05). These findings suggest that ATG must be used with caution in HIV⁺ kidney allograft recipients with a pretransplant CD4 count <350 cells/mm³.     

Successful Treatment of Kaposi Sarcoma–Associated Herpesvirus Inflammatory Cytokine Syndrome After Kidney–Liver Transplant: Correlations With the Human Herpesvirus 8 miRNome and Specific T Cell Response

After transplant, patient infection with human herpesvirus 8 (HHV‐8) and Kaposi sarcoma–associated herpesvirus (KSHV) is known to cause aggressive tumors and severe nonneoplastic complications. These latter syndromes are driven by HHV‐8/KSHV lytic reactivations and related hyperinflammatory host responses typically characterized by high viral loads, elevated levels of cytokines and other inflammation biomarkers, cytopenia, organ failure, high fever, and worsening conditions (with no evidence of B cell neoplasias). These disorders are associated with a high mortality rate, often due to lack of prompt diagnosis, effective therapeutic approaches, and adequate follow‐up. These features resemble most of those defining the so‐called KSHV‐associated inflammatory cytokine syndrome (KICS), which was recently recognized in patients positive for human immunodeficiency virus (HIV). In this report, we describe—for the first time—a case of a KICS‐like nonneoplastic recurrent complication occurring after transplant in an HIV‐negative patient that was successfully treated by a combination of anti‐CD20 monoclonal therapy, antivirals, and modification of the immunosuppressive regimen. In addition to clinical and laboratory findings collected during 3‐year follow‐up, we report novel experimental data on HHV‐8–specific T cell dynamics and circulating microRNA profile, showing correlations with clinical course and other laboratory markers (including viral load, C‐reactive protein, and cytokine levels), providing useful information about abnormal cellular and cytokine dynamics underlying HHV‐8–associated inflammatory disorders in posttransplant patients.     

Potential role of CC chemokine receptor 6 in prediction of late‐onset cytomegalovirus infection following solid organ transplant

CCR6 is a chemokine receptor involved in homing memory T cells, particularly Th17 cells, to sites of mucosal inflammation. Despite the critical role of memory T cells in long‐term protective immunity against cytomegalovirus (CMV), a virus that reactivates at multiple mucosal sites, the ability of CCR6 or other Th17 marker expression to predict CMV reactivation following transplantation is not clear. Using 11‐color flow cytometry, in this prospective single‐center pilot study, we measured the expression of CCR6 and other markers of T‐cell function in peripheral blood samples obtained from 21 SOT recipients at the time of discontinuation of anti‐CMV prophylaxis. CMV viremia was monitored on a monthly basis after discontinuation of prophylaxis. Eleven patients (52%) developed CMV viremia during the six‐month follow‐up period. Late‐onset CMV infection was preceded by an immune phenotype characterized by increased CCR6 expression on bulk CD4⁺ T cells and a reduced number of circulating CMV IE‐1‐specific Th1 (CD4⁺ IFN‐γ⁺) cells. Among the markers evaluated, CCR6 was the best single predictor of late‐onset CMV infection. Our results suggest that CCR6 expression at the time of discontinuation of antiviral prophylaxis might be a useful predictor of late‐onset CMV reactivation and provide the basis for future larger prospective studies.     

BK Polyomavirus‐Specific 9mer CD8 T Cell Responses Correlate With Clearance of BK Viremia in Kidney Transplant Recipients: First Report From the Swiss Transplant Cohort Study

BK polyomavirus (BKPyV) causes premature kidney transplant (KT) failure in 1–15% of patients. Because antivirals are lacking, most programs screen for BKPyV‐viremia and, if positive, reduce immunosuppression. To evaluate the relationship of viremia and BKPyV‐specific immunity, we examined prospectively cryopreserved plasma and peripheral blood mononuclear cells at the time of transplantation (T0) and at 6 mo (T6) and 12 mo (T12) after transplant from 28 viremic KT patients and 68 nonviremic controls matched for the transplantation period. BKPyV IgG seroprevalence was comparable between cases (89.3%) and controls (91.2%; p = 0.8635), but cases had lower antibody levels (p = 0.022) at T0. Antibody levels increased at T6 and T12 but were not correlated with viremia clearance. BKPyV‐specific T cell responses to pools of overlapping 15mers (15mer peptide pool [15mP]) or immunodominant CD8 9mers (9mer peptide pool [9mP]) from the early viral gene region were not different between cases and controls at T0; however, clearance of viremia was associated with stronger 9mP responses at T6 (p = 0.042) and T12 (p = 0.048), whereas 15mP responses were not informative (T6 p = 0.359; T12 p = 0.856). BKPyV‐specific T cells could be expanded in vitro from all patients after transplant, permitting identification of 78 immunodominant 9mer epitopes including 50 new ones across different HLA class I. Thus, 9mP‐responses may be a novel marker of reconstituting CD8 T cell function that warrants further study as a complement of plasma BKPyV loads for guiding immunosuppression reduction.     

Cellular immunity predominates over humoral immunity after homologous and heterologous mRNA and vector-based COVID-19 vaccine regimens in solid organ transplant recipients

Knowledge on the immunogenicity of vector-based and mRNA-vaccines in solid organ transplant recipients is limited. Therefore, SARS-CoV-2–specific T cells and antibodies were analyzed in 40 transplant recipients and 70 controls after homologous or heterologous vaccine-regimens. Plasmablasts and SARS-CoV-2–specific CD4 and CD8 T cells were quantified using flow cytometry. Specific antibodies were analyzed by ELISA and neutralization assay. The two vaccine types differed after the first vaccination, as IgG and neutralizing activity were more pronounced after mRNA priming (p = .0001 each), whereas CD4 and CD8 T cell levels were higher after vector priming (p = .009; p = .0001). All regimens were well tolerated, and SARS-CoV-2–specific antibodies and/or T cells after second vaccination were induced in 100% of controls and 70.6% of transplant recipients. Although antibody and T cell levels were lower in patients, heterologous vaccination led to the most pronounced induction of antibodies and CD4 T cells. Plasmablast numbers were significantly higher in controls and correlated with SARS-CoV-2–specific IgG- and T cell levels. While antibodies were only detected in 35.3% of patients, cellular immunity was more frequently found (64.7%) indicating that assessment of antibodies is insufficient to identify COVID-19-vaccine responders. In conclusion, heterologous vaccination seems promising in transplant recipients, and combined analysis of humoral and cellular immunity improves the identification of responders among immunocompromised individuals.     

Cytomegalovirus DNAemia and risk of mortality in allogeneic hematopoietic stem cell transplantation: Analysis from the Spanish Hematopoietic Transplantation and Cell Therapy Group

The net impact of cytomegalovirus (CMV) DNAemia on overall mortality (OM) and nonrelapse mortality (NRM) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a matter of debate. This was a retrospective, multicenter, noninterventional study finally including 749 patients. CMV DNA monitoring was conducted by real-time polymerase chain reaction (PCR) assays. Clinical outcomes of interest were OM and NRM through day 365 after allo-HSCT. The cumulative incidence of CMV DNAemia in this cohort was 52.6%. A total of 306 out of 382 patients with CMV DNAemia received preemptive antiviral therapy (PET). PET use for CMV DNAemia, but not the occurrence of CMV DNAemia, taken as a qualitative variable, was associated with increased OM and NRM in univariate but not in adjusted models. A subcohort analysis including patients monitored by the COBAS Ampliprep/COBAS Taqman CMV Test showed that OM and NRM were comparable in patients in whom either low or high plasma CMV DNA threshold (<500 vs ≥500 IU/mL) was used for PET initiation. In conclusion, CMV DNAemia was not associated with increased OM and NRM in allo-HSCT recipients. The potential impact of PET use on mortality was not proven but merits further research.     

A Case of Recurrent Multifocal Central Giant Cell Granulomas

One case of recurrent multifocal central giant cell granulomas (CGCG) is presented. Initially, the lesions presented concurrently in the maxilla and mandible with subsequent recurrence in the mandible. Now, two recurrences are seen in the maxillary sinus and ethmoid region. The literature regarding multifocal CGCG is reviewed.     

Improving our mechanistic understanding of the indirect effects of CMV infection in transplant recipients

Cytomegalovirus (CMV) is an immunomodulatory virus that indirectly increases the risk for bacterial, fungal, and viral infections. However, the pathogenesis of this phenomenon is poorly understood. We determined whether inflammatory responses to different Toll‐like receptor (TLR) ligands are blunted during CMV infection in solid‐organ transplant (SOT) patients. Peripheral blood mononuclear cells from 38 SOT patients with and without CMV were incubated in the presence of various viral, fungal, and bacterial TLR ligands. Cytokines were measured in the supernatant by multiplex enzyme‐linked immunosorbent assay. Patients had blunted cytokine responses to bacterial, fungal, and viral ligands during CMV infection when compared to the absence of CMV infection. This was independent of viral load, clinical presentation of CMV infection or immunosuppression, supporting the clinical observation in SOT recipients that CMV infection increases susceptibility to bacterial, fungal, and other viral infections. Moreover, in the absence of CMV infection, patients with subsequent CMV infection had lower cytokines in response to TLR ligands compared to those without subsequent CMV infection, suggesting that inherent differences in patients not directly related to CMV also contribute to this increased susceptibility. In summary, these data provide novel ex vivo evidence to support indirect effects of CMV.