Bone marrow transplantation from a pediatric donor with a high frequency of cytomegalovirus-specific T-cells

A recent study reported that quantitation of cytomegalovirus (CMV)-specific CD8+ T lymphocytes in the graft and monitoring of these T cells might identify hematopoietic stem cell transplantation-recipients at the risk for progressive CMV infection. A 6-year-old girl underwent bone marrow transplantation from an HLA-identical sibling with a very high frequency of CMV specific tetramer-positive CD8+ T-cells. CMV-specific T-cell immunity was prospectively evaluated using a peptide (HLA-A2, NLVPMVATV). Tetramer assay showed that the frequency of CMV-specific CD8+ T cells of the donor in the peripheral blood was 5.3%, higher than average amongst young children. The frequency of CMV-specific CD8+ T cells of the donor in the graft was 3.7% of CD8+ T-cells. Before transplantation, the frequency of CMV specific CD8+ T cells of the recipient was 0.1% in the peripheral blood. Surprisingly, the frequency of CMV specific CD8+ T cells increased up to 30% of CD8+ T-cells at day 27 after transplantation. IFN-gamma enzyme-linked immunospot assay showed the recipient-T cells had strong responses to the A2-specific NLVPMVATV peptide. Although the phenotypic pattern of the CMV-specific T cells of the recipient was different from those of the donor before transplantation, the phenotype of the donor-derived cells retained their original phenotype in the recipient after transplantation. These finding suggested that active transferred immunity from the graft with a high frequency of CMV-specific CTL could induce a rapid reconstitution of CMV-specific T-cell mediated immunity in pediatric HLA-identical allogenetic bone marrow transplantation. The screening of peripheral blood using HLA-peptide tetramer staining might be beneficial to select donors.     

Cytomegalovirus-specific CD4+ and CD8+ T-cells follow a similar reconstitution pattern after allogeneic stem cell transplantation.

Cytomegalovirus (CMV) is a common herpes virus that can cause significant morbidity and mortality in immunocompromised individuals, particularly those undergoing allogeneic stem cell transplantation (SCT) for hematological malignancies. Recent studies have examined the kinetics of CMV-specific CD8+ T-cell reconstitution after SCT transplantation and have found virus-specific cytotoxic T-lymphocyte regeneration to be dependent on CMV serologic status and CMV reactivation events. However, the reconstitution kinetics of CMV-specific CD4+ T-cells under these same circumstances were not addressed. In this study, we used HLA class I peptide tetramer for CMV pp65 and cytokine flow cytometry to follow the reconstitution of both CD4+ and CD8+ CMV-specific T-cells after allogeneic SCT. We found that following SCT in which both donors and recipients are CMV seropositive, virus-specific CD4+ T-helper cells show the same reconstitution kinetics as CD8+ cytotoxic T-cells. Following CMV reactivation, a synchronous but temporary increase in both CD4+ and CD8+ CMV-specific lymphocytes occurs. The pattern repeats itself after subsequent episodes of CMV reactivation. These data imply that both CD4+ and CD8+ lymphocytes are necessary for an efficient immune response to CMV and suggest that CD4+ and CD8+ CMV-specific T-cells are required for the complete restoration of CMV immunity. These findings may have important implications in the development of CMV-specific adoptive immunotherapy strategies.     

Persisting posttransplantation cytomegalovirus antigenemia correlates with poor lymphocyte proliferation to cytomegalovirus antigen and predicts for increased late relapse and treatment failure.

Numerous clinical studies link cytomegalovirus (CMV) infection with incomplete posttransplantation T-cell recovery. We hypothesized that the inability of transplant recipients to handle CMV reactivation might correlate with a defective graft-versus-leukemia response and increased posttransplantation morbidity. Between May 1995 and August 2001, 82 patients who were CMV seropositive and survived the first 100 days after transplantation were identified for a day 100 landmark analysis of the effect of CMV reactivation patterns on eventual transplantation outcome. All patients underwent a myeloablative HLA-identical sibling donor T cell-depleted stem cell transplantation with scheduled donor T-cell add-back on day 45. Median follow-up was 1032 days. Forty-two patients who had either no reactivation or only 1 positive test with quick clearance were designated as a CMV immune competent group. Forty patients designated as CMV immune deficient (ID) had at least 2 positive tests. Apart from younger age (33 versus 38 years; P =.05) in the ID group, the 2 groups were balanced for clinical characteristics. In multivariate analysis, ID patients had a significantly higher incidence of leukemia relapse (58% versus 21%; P =.03) and worse disease-free survival (31% versus 66%; P =.04). There was no significant difference in week 1 to 14 posttransplantation lymphocyte counts between the 2 groups. In 67 patients tested 3 to 6 months after transplantation, a proliferative response to CMV antigen (stimulation index > or =2) occurred in 27 of 36 immune competent patients compared with 15 of 31 ID patients (P =.006). These results show that recurrent CMV reactivation in the first 100 days after transplantation predicts for reduced disease-free survival and increased leukemic relapse beyond 100 days and correlates with inferior proliferative responses to CMV. The higher relapse rate may reflect poor immune reconstitution in ID patients or an adverse effect of prolonged antiviral treatment.     

Analyzing T-cell responses to cytomegalovirus by cytokine flow cytometry

T-cell responses to human cytomegalovirus (CMV) are readily detected in chronically infected adults, and are thought to be important for protection from CMV-related pathology. Antigen-specific cytokine flow cytometry (CFC) has been used to establish the range of CMV-specific CD4 and CD8 T-cell frequencies in healthy CMV-seropositive (and seronegative) adults, as well as the dynamics of these cells over time. There are also emerging data regarding the primary CD4 and CD8 T-cell response to CMV in children and adults. Finally, CFC has been used to analyze CMV responses in chronic human immunodeficiency virus infection, as well as during immune reconstitution after bone marrow or stem cell transplantation. These data will be reviewed in terms of what they suggest about the threshold of protective T-cell immunity to CMV, and other factors in addition to T-cell frequencies that could be important in protecting from CMV-associated disease.     

Clinical aspects of CMV infection after stem cell transplantation

Cytomegalovirus (CMV) infection is one of the most important infectious complications after stem cell transplantation (SCT). Major improvements in the management of CMV infection have been achieved during the last decade, including the introduction of safe blood product support for CMV-seronegative patients, the development of early pre-emptive antiviral therapy based on sensitive diagnostic tests, and antiviral prophylaxis. With the better control of CMV infection during the first 100 days after allogeneic SCT an increase in the incidence of CMV infection and disease after day 100 after transplantation was observed. New methods that allow for the reconstitution of CMV-specific immune responses such as adoptive T-cell therapy are promising tools that might help to improve the management of late CMV infection and disease.     

Longitudinal Analysis of T-Cell Receptor Variable β Chain Repertoire in Patients with Acute Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

T-cell receptor variable β chain (TCRBV) repertoire spectratyping involves the estimation of CDR3 length distributions for monitoring T-cell receptor diversity and has proven useful for analyses of immune reconstitution and T-cell clonal expansions in graft-versus-host disease (GVHD) and graft-versus-leukemia after allogeneic stem cell transplantation. We performed a longitudinal spectratype analysis of 23 TCRBV families in 28 patients who underwent allogeneic T cell–depleted peripheral blood stem cell transplantation. Sixteen patients subsequently developed acute GVHD. We recently developed statistical methods that bring increased power and flexibility to spectratype analysis and allow us to analyze TCRBV repertoire development under appropriately complex statistical models. Applying these methods, we found that patients with acute GVHD demonstrated TCRBV repertoire development statistically distinct from that repertoire development in patients without GVHD. Specifically, GVHD patients showed spectratypes indicative of lower diversity and greater deviation from the spectratypes expected in healthy individuals at intermediate times. Most individual TCRBV subfamilies had spectratypes statistically distinguishable between GVHD and non-GVHD patients at 6 months after transplantation. These results suggest that the T-cell receptor repertoire perturbations associated with acute GVHD are widely spread throughout the TCRBV families.     

Unrelated donor status and high donor age independently affect immunologic recovery after nonmyeloablative conditioning.

The risk of cytomegalovirus (CMV) infection is higher after HLA-matched unrelated donor (URD) than after HLA-matched related donor (MRD) nonmyeloablative hematopoietic cell transplantation (HCT). We therefore investigated factors affecting immune recovery in 94 patients given HCT from MRDs (n = 51) and URDs (n = 43) after 2-Gy total body irradiation with or without fludarabine and postgrafting immunosuppression with mycophenolate mofetil and cyclosporine. CD4 T cells counts remained below normal values during the first year after HCT in both patient groups. This included abnormally low counts each of naive CD4 T cells and memory CD4 T cells. Conversely, CD8 T cell counts reached the 10th percentile of normal 6 months after HCT in MRD and URD recipients. On day 30 after HCT, URD recipients had lower counts of B cells (P = .02), naive CD4 T cells (P = .04), memory CD4 T cells (P = .005), memory CD8 T cells (P = .005), and CMV-specific T helper cells (P = .007) than had MRD recipients. This delay in CMV-specific immune reconstitution translated into increased frequency of CMV antigenemia among URD recipients during the first 100 days after HCT. Older donor age was associated with low counts of naive CD4 T cells on days 180-365 after HCT (P = .003). Further, low numbers of T cells and CD34(+) cells in the graft and development of acute graft-versus-host disease were associated with impaired immune recovery of naive CD4 T cells and B cells. In summary, immunologic recovery was poor the first year after nonmyeloablative conditioning and was delayed among URD recipients in comparison with MRD recipients. Other factors significantly associated with delayed immune recovery were advanced donor age, low numbers of CD34 and T cells in the graft, and development of graft-versus-host disease.     

Cytomegalovirus-specific cytolytic T-cell lines and clones generated against adenovirus-pp65-infected dendritic cells.

Cytomegalovirus (CMV) infection is a serious complication of allogeneic bone marrow transplantation (BMT). CMV disease can usually be prevented by passive immunization with donor-derived CMV-pp65-specific T-cell clones if provided early post-BMT. The classic method of generating CMV-specific T-cell clones requires donor-derived fibroblast lines infected with CMV as stimulators, thus limiting the availability of CMV immunotherapy to those patients for whom a donor skin biopsy can be obtained 6 to 8 weeks pretransplantation. To overcome this limitation we have used monocyte-derived dendritic cells (DCs) to induce donor anti-CMV cytotoxic T lymphocytes (CTLs). Matured, adeno-pp65-infected DCs were added at day 0 and at day 7 of a 2-week culture of donor peripheral blood mononuclear cells. DC-primed cultures were compared with cultures stimulated in an identical fashion with CMV-infected fibroblasts or with adeno-pp65-infected freshly isolated blood monocytes. Specific killing of CMV-infected fibroblasts was detected in all except the culture stimulated with pp65-infected monocytes. DCs infected after maturation elicited greater CTL activity than did DCs matured after infection. A series of 5 CD8+ clones from a fibroblast-stimulated culture and 7 CD8+ clones from a mature-DC-stimulated culture derived from a single HLA-A*0201+ individual were characterized. All 12 clones lysed autologous CMV-infected fibroblasts. All except 1 clone from the CMV-infected fibroblast arm (fibroblast arm) lysed vaccinia-pp65-infected B-lymphoblastoid cell lines (BLCLs); none lysed vaccinia-pp150-infected or noninfected BLCLs. Ten of 10 CD8+ clones tested were restricted by HLA-A*0201. Seven of the 12 clones were Vbeta6+ (2 from the fibroblast arm and 5 from the DC arm) with an identical Vbeta6.1-J1.4 sequence. Three clones from the fibroblast arm and 5 clones from the DC arm recognized the pp65 peptide NLVPMVATV (amino acids [aa], 495-503). These data show that CMV-specific T-cell clones with similar restriction patterns, T cell-receptor usage, and specificity can be generated using monocyte-derived pp65-infected-DC or CMV-infected-fibroblast stimulators. This approach should broaden the applicability of CMV-specific T-cell immunotherapy to a wider spectrum of patients by reducing the time required to generate CMV-specific T-cell clones.     

Impact of pretransplant CMV-specific T-cell immune response in the control of CMV infection after solid organ transplantation: a prospective cohort study

IntroductionAlthough solid organ transplant (SOT) recipients with pretransplant serology for cytomegalovirus (CMV-R+) are considered at intermediate risk for CMV infection post transplantation, CMV infection remains a major cause of morbidity in this population. We prospectively characterized whether having pretransplant CMV-specific cellular immunity is independently associated with controlling infection after transplantation in R + SOT recipients.MethodsA prospective cohort of consecutive R + SOT recipients that received pre-emptive treatment for CMV infection was monitored after transplantation and variables were recorded during the follow-up. The cytomegalovirus-specific T-cell immune response was characterized by intracellular cytokine staining and viral loads determined using real-time PCR.ResultsOne hundred and thirty-five R + SOT recipients were included (67 kidney, 64 liver, four liver–kidney). Only one-third of the patients (42; 31.85%) had CMV-specific T-cell immunity (CD8⁺CD69⁺INF-γ⁺ T cells >0.25%) before transplantation. Patients with negative pretransplant immunity had more CMV infection (49, 52.7% vs. 15, 35.7%; p 0.07) and received more antiviral therapy than those with immunity (32, 34.4% vs. 6, 14.3%, p 0.016). Having CMV specific immunity was an independent factor for protection from developing viraemia ≥2000 IU/mL (OR 0.276, 95% CI 0.105–0.725, p < 0.01) and lower administration of treatment (OR 0.398, 95% CI 0.175–0.905, p 0.028). Only patients with no pretransplant CMV-specific T-cell response were diagnosed with CMV-disease (8, 8.6% vs. 0, 0%, p 0.05).Discussion.Our results show that having a pretransplant CMV specific T-cell response may be associated with a lower rate of CMV viraemia and less antiviral treatment after transplantation; however, more prospective studies are needed to confirm these findings.     

Nonmyeloablative conditioning allows for more rapid T-cell repertoire reconstitution following allogeneic matched unrelated bone marrow transplantation compared to myeloablative approaches.

Nonmyeloablative pretransplantation conditioning regimens have resulted in durable engraftment of allogeneic hematopoietic stem cells. In contrast to conventional fully myeloablative approaches, nonmyeloablative regimens are associated with a marked reduction of morbidity and mortality in the early posttransplantation period. Consequently, such reduced-intensity transplantation approaches can be used in older and frailer patients who would not tolerate fully ablative regimens. However, it is currently unclear how this radically different transplantation strategy affects immunological reconstitution. To address this important issue, we used T-cell receptor Vbeta spectratype analysis to examine the distribution of complementarity-determining region 3 (CDR3)-size bands as a measure of the complexity of the redeveloping T-cell repertoire. For this study, we evaluated the T-cell repertoire of 9 patients receiving T-cell replete, matched unrelated donor transplants following fully ablative or nonmyeloablative conditioning regimens. All 4 of the myeloablative and 2 of the nonmyeloablative patients received bone marrow, whereas 3 other nonmyeloablative patients received peripheral blood stem cells. The results of the spectratype analysis demonstrated that the patients who received nonmyeloablative conditioning together with either bone marrow or peripheral blood stem cells exhibited more rapid reconstitution of T-cell repertoire complexity.     

Cytomegalovirus-specific CD8+ T cells targeting different peptide/HLA combinations demonstrate varying T-cell receptor diversity

Cytomegalovirus (CMV) infection and reactivation pose a serious threat for patients after haematopoietic stem cell transplantation. We have previously shown that CD8(+) T cells targeting different CMV epitopes correlate with protection at different threshold frequencies in those patients. To investigate if this may relate to a different quality of these cells here we analyse the T-cell receptor diversity of pp50 (245-253)/HLA-A*0101 specific CD8(+) T cells with that of CD8(+) T cells targeting various pp65 peptides. The results from this pilot study show differences in the breadth of the T-cell receptor usage of the different cell populations. We observe for the first time that the T-cell receptor Vβ CDR3 spectratypes used by CMV pp50 (245-253)/HLA-A*0101-specific CD8(+) T cells can reach higher numbers than those used by CD8(+) T cells targeting various pp65 peptides in our patient cohort. This merits further investigation into the effectiveness of the different CMV-specific T cells and their impact on immunosenescence, which is important to eventually define the most useful source of adoptive therapy and monitoring protocols for cytomegalovirus-specific immune responses.     

Deleterious Effect of Steroids on Cytomegalovirus Infection Rate after Allogeneic Stem Cell Transplantation Depends on Pretransplant Cytomegalovirus Serostatus of Donors and Recipients

This study examined the impact of prednisone (PDN) on cytomegalovirus (CMV) infection after allogeneic stem cell transplantation (allo-SCT) according to donor and recipient CMV serostatus. Seventy-five patients underwent allo-SCT from June 2010 to July 2012. The risk of CMV infection according to donor and recipient serostatus was defined as follows: high risk (HR; D–/R+), intermediate risk (IR; D+/R+?and D+/R–), and low risk (D–/R–). Forty-five patients (60%) developed CMV infection, and 46 patients (61%) received steroids (PDN?≥?1?mg/kg/day) to treat acute graft-versus-host disease. CMV infection was more common in those treated with steroids than in those not treated with steroids (70% versus 44%, respectively, P?<?.05). Overall, 40% of patients had recurrent CMV infection (50% PDN versus 24% no PDN, P?<?.05). Steroids had no impact on the incidence of CMV infection or its recurrence in HR patients; however, steroids did prolong the need for antiviral treatment. The incidence of CMV infection in IR patients was higher in those receiving PDN (80% PDN versus 41% no PDN, P?=?.01); recurrence rates were also higher (55% PDN versus 18% no PDN, P?=?.02). We analyzed CMV-specific immune reconstitution in the first 22 patients of the series and observed that patients on steroids had lower levels of CMV-specific lymphocytes TCD8 (P?<?.05 on days +60, +100, and +180) and that CMV-specific immune reconstitution (defined as lymphocytes CD8/IFN ≥ 1 cell/µL) was achieved later (after day +100 post-SCT) in the steroid group.     

Optimization of an elispot assay to detect cytomegalovirus-specific CD8+ T lymphocytes

Various arguments suggest that CD8+ T lymphocytes play a major role in the control of cytomegalovirus (CMV) infection. The detection of CMV-specific CD8+ T cells may therefore provide additional information about CMV virus detection to predict the risk of development of CMV disease, especially in immunodepressed transplant recipients. We compared and tested various experimental conditions to optimize an enzyme-linked immunospot assay (Elispot) assay for the detection of CMV-specific CD8+ T lymphocytes. The indirect Elispot assay with one six-day in vitro sensitization step was found to be the most sensitive method to detect CMV-specific CD8+ T cells compared to direct Elispot with unfractionated peripheral blood mononuclear cells or purified CD8+ T cells. We showed that low doses of interleukin-2 during the in vitro culture enhanced the sensitivity of this test, and tetramer staining was performed to verify the high efficiency of this in vitro stimulation step. We directly loaded the specific CMV peptide during the Elispot assay and demonstrated that the use of T2 cells did not improve its sensitivity. Elispot for the detection of interferon-gamma appears to be more sensitive and reliable than measurement of tumor necrosis factor alpha or granzyme B. This technique was successfully applied to detect CMV-specific CD8+ T cells in human leukocyte antigen A2 (HLA-A2) and HLA-B7 healthy patients and in one lymphopenic post-transplant patient with positive CMV serology. This highly sensitive test may be a useful tool to assess T-cell immunity directed against CMV in immunodepressed patients.     

Identification of new Th peptides from the cytomegalovirus protein pp65 to design a peptide library for generation of CD4 T cell lines for cellular immunoreconstitution

CD8 and CD4 lymphocytes control cytomegalovirus (CMV) infection in immunocompetent individuals, while patients with defective cellular immunity are prone to endogenous reactivation of latent CMV or, like seronegative subjects, prone to primary infection. Administration of CMV-specific CD8 lymphocytes was beneficial for immunocompromised hemopoietic stem cell (HSC) graft recipients. Since CD4 cells contribute to expansion of cytotoxic T lymphocytes (CTL), we defined new T(h) peptides on the immunodominant protein pp65 recognized by CD4 cells from HLA-typed subjects, in the perspective of complementing CTL administration with CMV-specific T(h) cells. Screening by ELISPOT on CD4 and CD8 subsets using overlapping peptides identified 10 novel CD4 peptides. To simplify procedures to generate T cell lines, we used a CD4 peptide library for T cell stimulation instead of ill-defined viral lysates, without the requirement of dendritic cells. This library stimulated CMV-specific CD4 cells. In fact, peptide-induced CD4 cells responded to pp65 and to the viral lysate. These cells were also devoid of alloreactivity after one stimulation cycle. Since Good Manufacturing Procedure-grade peptides can be synthesized, culture conditions are simplified and alloreactivity is rapidly lost, these procedures based on peptide stimulation can facilitate implementation of adoptive reconstitution of CD4 responses in immunocompromised patients also in the case when the HSC allodonor is available for generation of the T cell line.     

Recognition of CMV pp65 protein antigen by human CD4 T-cell lines induced with an immunodominant peptide pool

Cellular immunity against cytomegalovirus (CMV) is essential for recovery from infection and control of viral latency. In immunocompromised hosts, this balance between CMV and cellular immunity is lost. Accordingly, restoration of the CD8 compartment specific for CMV is beneficial for immunocompromised patients. It is clear that CMV-specific CD4 cells provide helper functions facilitating long-term persistence of CD8 cells. Considering the dearth of data on CMV-specific T-helper cells, we investigated the CD4 responses to the immunodominant protein pp65 to define antigenic peptides. Such peptides were pooled and used to generate long-term T-cell lines. The lines were responsive to CMV and pp65. T cells were selected with individual peptides to produce monospecific lines for accurate definition of fine epitope specificity and to confirm human leukocyte antigen HLA-DR restriction. Furthermore, these lines lost alloreactivity, suggesting that they can be generated from the allodonor for adoptive immunoreconstitution of stem cell graft recipients.     

Umbilical Cord Blood Transplantation in Severe T-Cell Immunodeficiency Disorders: Two-Year Experience

Hematopoietic stem cell transplantation is the treatment of choice for severe primary T-cell immunodeficiencies. When an HLA-identical sibling as the donor is not available, an alternative donor stem cell source is needed. In primary T-cell immunodeficiencies, T-cell-depleted HLA-haploidentical bone marrow transplantation has been particularly successful in reconstituting the immune system in many but not all of the severe T-cell immune deficiency disorders. This study reports the use of umbilical cord blood (UCB) stem cell transplantation in severe T-cell immune deficiency.Umbilical cord blood was evaluated as a stem cell source for immune reconstitution in children with severe primary T-cell immunodeficiency disorders, such as severe combined immunodeficiency syndrome (SCID), reticular dysgenesis, thymic dysplasia, combined immunodeficiency disease (CID), and Wiskott–Aldrich syndrome (WAS) when a matched sibling donor was unavailable. From 1/96 through 5/98, eight children received unrelated cord blood stem cell transplantation following a preparative regimen for the treatment of combined immunodeficiency diseases. The patients ranged in age from 2 weeks to 8 years. The cord blood units were 3/6 HLA antigen matches in two children, 4/6 in four children, and 5/6 in two child, with molecular HLA-DR mismatch in three of the children. The average time for neutrophil engraftment (absolute neutrophil count >500/mm³) was 12 days (range 10–15 days) and the average time for platelet engraftment (platelet count >20,000/mm³) was 36 days (range 24–50 days). A patient with reticular dysgenesis failed to engraft following her first transplant, but fully engrafted after a second unrelated donor cord blood transplantation. Five of six patients exhibited grade I graft-versus-host disease (GvHD), while one child had grade IV skin and gut GvHD. Immunologic reconstitution demonstrated that cord blood stem cell transplantation resulted in consistent and stable T-, B- and natural killer (NK) cell development. The kinetics of development were such that T-cell development occurred between 60 to 100 days. Initial T-cell engraftment consisted predominantly of CD45RO+, CD3+, and CD4+ T cells, and at 12 to 24 months changed to CD45RA+, CD3+, and CD4+ T cells, indicatingde novomaturation of T cells. NK cell development occurred at approximately 180 days. B cells engrafted early, and study of functional B-cell antibody responses revealed that five of six patients in whom intravenous immune globulin has been discontinued have low detectable antibody responses to tetanus and diphtheria toxoid immunizations at 18 to 24 months posttransplantation.Unrelated umbilical donor cord blood is an alternative source of stem cells for transplantation in children with severe T-cell immune deficiency disorders when a suitable HLA-matched donor is not available and when a T-depleted haploidentical preparation is not beneficial. Benefits of UCB include rapid and reliable recovery of immune function, low risk of GvHD, and low viral transmission rate.     

Simultaneous Monitoring of Cytomegalovirus-Specific Antibody and T-cell levels in Seropositive Heart Transplant Recipients

Purpose

Human cytomegalovirus (CMV) active infection (CMV infection) poses serious risks to CMV-seropositive heart transplant recipients. We evaluated the usefulness of simultaneous assessment of CMV-specific values for parameters of the humoral (antibodies) and cellular (CD4+ and CD8+ T-cells) immune responses in the identification of heart recipients at risk of developing CMV infection after transplantation.

Methods

We prospectively studied 38 CMV-seropositive heart recipients. Anti-CMV antibody titers were assessed using enzyme-linked immunosorbent assays. CD4+ and CD8+ T-cell responses to overlapping peptide pools of the CMV proteins pp65 and immediate early protein-1 (IE1) were evaluated by flow cytometry. Immunological studies were performed before transplantation and at 30?days after transplantation. Patients with CMV infection were compared with heart recipients without CMV infection.

Results

During the 6-month follow-up period, 13 (34.2%) patients developed CMV infection. At baseline, the mean anti-CMV-IgG antibody titer was lower in patients who developed CMV infection. This difference remained at 30?days after transplantation. One month after transplantation, the mean percentage of IE1-specific CD8+ T cells that are IFNg-positive (CD8/IFNg?+?IE1) was lower in CMV-infected patients. The predictive value of these variables at 30?days was increased when they were combined. Cox regression analysis revealed an association between the risk of developing CMV infection and the combination marker (low anti-CMV titer [<16,100] and low CD8/IFNg?+?IE1 percentages [<0.40%], relative hazard, 6.07; p?=?0.019). The combination marker remained significant after adjustment for clinical variables.

Conclusions

This novel approach of a simultaneous assessment of specific anti-CMV antibody titers and CD8/IFNg?+?IE1 percentages might help identify heart transplant recipients with an increased risk of developing CMV infection.     

Flow cytometric analysis of cytomegalovirus-specific cell-mediated immunity in the congenital infection

Flow cytometric analysis of CD4(+) and CD8(+) T cells specific to human cytomegalovirus (CMV) was undertaken in seven patients with congenital CMV infection, six healthy infants who had acquired infection, and six CMV-seropositive adults. Intracellular cytokine assays showed that 0.03-2.23% of CD4(+) T cells in the healthy infants and adults produced interferon-gamma (IFN-gamma) in response to CMV antigens. In contrast, such CD4(+) T cells were almost undetectable in patients with congenital CMV infection who were younger than 2 years of age. Tetrameric major histocompatibility complex/peptide complex analysis demonstrated HLA*A2402-restricted phosphoprotein 65-specific CD8(+) T cells to be present in most healthy infants and adults tested, but almost absent in the patients. Interestingly, CMV-specific CD4(+) T-cell responses were observed in two patients with congenital infection beyond the age of 5 years. The present study points to impairment of CMV-specific cellular immunity in patients in single-cell levels with congenital CMV infection during the infant period and possible restoration in later childhood.     

Strategies to accelerate immune recovery after allogeneic hematopoietic stem cell transplantation

The interplay existing between immune reconstitution and patient outcome has been extensively demonstrated in allogeneic hematopoietic stem cell transplantation. One of the leading causes of infection-related mortality is the slow recovery of T-cell immunity due to the conditioning regimen and/or age-related thymus damage, poor naïve T-cell output, and restricted T-cell receptor (TCR) repertoires. With the aim of improving posttransplantation immune reconstitution, several immunotherapy approaches have been explored. Donor leukocyte infusions are widely used to accelerate immune recovery, but they carry the risk of provoking graft-versus-host disease. This review will focus on sophisticated strategies of thymus function-recovery, adoptive infusion of donor-derived, allodepleted T cells, T-cell lines/clones specific for life-threatening pathogens, regulatory T cells, and of T cells transduced with suicide genes.     

Mobilization of Hematopoietic Progenitors from Normal Donors Using the Combination of Granulocyte-Macrophage Colony-Stimulating Factor and Granulocyte Colony-Stimulating Factor Results in Fewer Plasmacytoid Dendritic Cells in the Graft and Enhanced Donor T Cell Engraftment with Th1 Polarization: Results from a Randomized Clinical Trial

Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) both mobilize CD34⁺ stem cells into the blood when administered before apheresis but have distinct effects on dendritic cell (DC) differentiation. We previously demonstrated that the combination of GM+G-CSF results in fewer plasmacytoid DCs (pDCs) when used to mobilize peripheral blood stem cells for autologous transplantation. To test the hypothesis that the content of pDCs in an allograft can be modulated with the cytokines used for mobilization, we randomized the human leukocyte antigen–matched sibling donors of 50 patients with hematological malignancies to a mobilization regimen of either GM+G-CSF (n = 25) or G-CSF alone (n = 25). Primary and secondary endpoints included the cellular constituents of the mobilized grafts, the kinetics of posttransplantation immune reconstitution, and clinical outcomes of the transplantation recipients. Grafts from donors receiving GM+G-CSF contained equivalent numbers of CD34⁺ cells with fewer pDCs and T cells, with a higher fraction of Th1-polarized donor T cells than G-CSF mobilized grafts. Immune recovery was enhanced among recipients of GM+G-CSF. Survival was not significantly different between transplantation recipients in the two arms. The use of GM+G-CSF modulates immune function and recovery after allogeneic transplantation and should be explored in larger studies powered to evaluate clinical outcomes.