Impact of CMV Reactivation, Treatment Approaches, and Immune Reconstitution in a Nonmyeloablative Tolerance Induction Protocol in Cynomolgus Macaques

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease which can be complicated with cytomegalovirus (CMV) infection during its course. CMV reactivation can mimic an SLE flare and lead to delay in diagnosis. Here, we reported a previously diagnosed SLE patient who presented with fever, leukopenia, and cutaneous ulcers. Initially, this was diagnosed as an SLE flare and the patient was treated with higher doses of corticosteroids but no improvement was observed. Both nuclear and cytoplasmic inclusions inside the endothelial cells in the skin biopsy as well as positive immunohistochemistry (IHC) staining for CMV antigen were clues to the correct diagnosis of CMV reactivation. Treatment with ganciclovir resulted in clinical resolution. In this report, a very rare clinical form of CMV infection manifesting as cutaneous necrotizing vasculitis on the lower extremity is described and the literature regarding this case is reviewed.

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Alemtuzumab (Campath 1-H) Exposure Correlates with Risk of Chronic Graft vs Host Disease and CMV Viremia after Allogeneic Transplantation.

Blood ◽

10.1182/blood.v106.11.1818.1818 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1818-1818

Author(s):

Justin P. Kline ◽

Rajiv Swamy ◽

Dezheng Huo ◽

Laura Michaelis ◽

Richard A. Larson ◽

...

Keyword(s):

At Risk ◽

Half Life ◽

Immune Reconstitution ◽

Chronic Gvhd ◽

Absolute Lymphocyte Count ◽

Enzyme Linked Immunosorbent Assay ◽

Unrelated Donor ◽

Increased Risk ◽

Cmv Reactivation

Abstract Conditioning regimens using in vivo alemtuzumab (Campath-1H, humanized anti-CD52) are characterized by low rates of acute and chronic GVHD, but may also result in delayed immune reconstitution. Optimization of such regimens will depend on an understanding of the relation between alemtuzumab exposure, immune reconstitution and GVHD. We have conducted a prospective study of fludarabine 30 mg/m2/d x 5 days, melphalan 140 mg/m2 x 1 day and alemtuzumab 20 mg/d x 5 days as conditioning for related and unrelated allografts. Using an enzyme-linked immunosorbent assay (ELISA), we determined serum free and total Campath levels in 46 patients with hematologic malignancies (45) or sickle cell disease (1) on day 0, day 7, day 14, day 28, day 50, day 75, day 100, day 150 and at one year after transplant (HSCT). 26 (57%) had a matched sibling donor, 16 (35%) a matched unrelated donor (MUD) and 4 (9%) a mismatched related or unrelated donor. 44 pts engrafted and are included in the analysis. Median follow up for survivors was 2.8 years. Grade II–IV aGVHD occurred in 8 pts after a median of 42 days (range 22 to 60). Eight pts developed cGVHD after a median of 107 days (range 89–140). 15/41 (37%) at risk pts developed CMV reactivation. The half-life of free alemtuzumab (fA) was 26 days after HSCT with wide interpatient variation in fA pharmacokinetics (e.g. Coefficient of variation was 138% on day 0). On day 0, 1 patient had an undetectable level of fA. By day 28, 50, and 100, there were 6 (14.6%), 12 (35.3%), and 13 (52%) pts with undetectable fA, respectively. Figure 1 shows the fitted means and 95% confidence intervals of fA over time. Using log-rank and Cox proportional hazard models, there was no association between fA on day 0, day 28, or the last available fA, and development of acute GVHD. However, pts with higher average free and total Campath levels in the first month had a lower risk of developing cGVHD (p=0.02). The median fA concentration in the first month for pts with cGVHD was 0.32 (inter-quarter range IQR: 0.22–0.41), as compared with 0.97 (IQR: 0.23–3.31) in those without cGVHD. No significant association between absolute lymphocyte count and fA concentration was found after adjusting for time (p=0.28). Finally, among pts at risk, a higher fA concentration on day 0 (p=0.002), and in the first month (p=0.003) was significantly associated with CMV viremia. In summary, the estimated half-life of serum fA is 26 days after HSCT, but with considerable interpatient variability. Higher concentrations of fA were associated with a decreased incidence of cGVHD, but an increased risk of CMV reactivation. In contrast to a previous preliminary analysis, no association existed between fA and lymphocyte reconstitution. Variation in alemtuzumab pharmacokinetics may predict important clinical outcomes, such as cGVHD and CMV reactivation. Future studies are warranted to determine an optimal alemtuzumab exposure that hastens immune reconstitution while minimizing chronic GVHD. Fig 1. Fitted Mean Free Campath and 95% CI Fig 1. Fitted Mean Free Campath and 95% CI

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Donor Lymphocytes Stimulated with CMV pp65 Gene Modified Dendritic Cells for Prophylaxis of CMV Following Allogeneic Haemopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v110.11.2989.2989 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2989-2989

Author(s):

Kenneth P. Micklethwaite ◽

Leighton E. Clancy ◽

Upinder Sandher ◽

Elizabeth Snape ◽

Kenneth Francis Bradstock ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Stem Cell ◽

Graft Versus Host Disease ◽

Immune Reconstitution ◽

Adverse Reactions ◽

Specific Lysis ◽

Graft Versus Host ◽

Haemopoietic Stem Cell ◽

Cmv Reactivation

Abstract Background: Cytomegalovirus (CMV) reactivation post-allogeneic haemopoietic stem cell transplant (HSCT) causes morbidity and mortality. Pharmacological therapy is limited by bone marrow suppression that can result in opportunistic infection. Adoptive transfer of ex-vivo generated CMV-specific T-cells has the potential to rapidly restore immunity, prevent CMV infection and circumvent the need for pharmacotherapy. Methods: We have recruited 18 patients into a trial of donor-derived CMV-specific T-cells generated using dendritic cells transduced with a fiber modified adenoviral vector encoding the immunodominant CMV matrix protein pp65. Thus far, 8 patients have received T-cells prophylactically starting at day 28 post-HSCT. Recipients were monitored for adverse reactions, CMV reactivation by polymerase chain reaction (PCR) and CMV-specific immune reconstitution. Results: 18 cultures have been completed with an 8.5–30 fold increase in total cell number (mean 2.1 × 108, range 0.8–5.1 × 108). Products consisted of T-cells (median 96.5%) displaying an effector memory phenotype (CD45RO+, CD62L−) and a predominance of CD8+ (14–90%) over CD4+ (2.5–57%) cells. All cultures exhibited negligible alloreactivity against recipient derived targets (0–3.6% specific lysis at E:T ratio of 20:1) but strong killing of CMV pp65 peptide pulsed targets (43–79% specific lysis at E:T 20:1). Killing was also observed against targets labeled with adenoviral antigens (2.7–12.5% lysis at E:T 20:1) demonstrating the bi-virus specificity of these cultures. In HLA-B7 and HLA-A2 donors, percentages of tetramer binding T-cells accounted for 3.2 to 40% of cells. Enrichment of cells recognizing known HLA-A24, B8 and B35 epitopes was not observed despite these cultures exhibiting strong cytotoxic activity against pp65 pulsed targets. Reasons for non-infusion include early death post-transplant (3 patients), failure of cultures to meet release criteria (2 patients), severe graft versus host disease (1 patient) and refusal (1 patient). 3 patients are currently awaiting infusion. Patients infused have been followed from 21 to 189 days post infusion. In 5 patients we have demonstrated rapid, polyepitope, CMV pp65-specific immune reconstitution using tetramer and ELISPOT analysis. CMV reactivation by PCR has occurred in 3 patients but none have progressed to CMV disease, suggesting the functional capacity of the cells to control viral reactivation. One patient received pharmacotherapy with valaciclovir while no other patients have required anti-viral antibiotics. There have been no infusion related adverse reactions. Graft versus host disease, non-CMV infections and other adverse events have not exceeded expected rates. Conclusions: Prophylactic adoptive transfer of CMV-specific T-cells is safe, hastens CMV-specific immune reconstitution and may reduce the need for anti-CMV pharmacotherapy in allogeneic HSCT recipients. Our data indicate the potential of specific cellular therapy to control opportunistic infections in severely immunosuppressed patients post-HSCT.

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Design of an international investigator-initiated study on MOdern Treatment of Inhibitor-positiVe pATiEnts with haemophilia A (MOTIVATE)

Therapeutic Advances in Hematology ◽

10.1177/20406207211032452 ◽

2021 ◽

Vol 12 ◽

pp. 204062072110324

Author(s):

Carmen Escuriola Ettingshausen ◽

Robert F. Sidonio

Keyword(s):

Real Life ◽

Tolerance Induction ◽

Coagulation Factor ◽

Prolonged Treatment ◽

Haemophilia A ◽

Related Complication ◽

Treatment Approaches ◽

Group 2 ◽

Recombinant Fviii ◽

The Impact

Background: Inhibitor development is the most serious treatment-related complication of replacement coagulation factor VIII (FVIII) therapy for patients with haemophilia A. Immune tolerance induction (ITI), which involves intensive and prolonged treatment with plasma-derived or recombinant FVIII, is the only clinically proven strategy for eradication of inhibitors. The bispecific antibody emicizumab is approved for use in patients with and without inhibitors to prevent bleeding but does not eliminate inhibitors. MOTIVATE ( www.motivate-study.com ) aims to capture different approaches to the treatment and management of patients with haemophilia A and inhibitors, document current ITI approaches from real-world clinical experience, and evaluate the efficacy and safety of ITI, emicizumab prophylaxis and ITI with emicizumab prophylaxis. Methods: The investigator-initiated MOTIVATE study [ClinicalTrials.gov identifier: NCT04023019; EudraCT 2019-003427-38] will investigate in real-life clinical practice the management of patients with haemophilia A of any severity who have developed inhibitors to FVIII. All treatment is at the investigator’s discretion. The following treatment approaches will be evaluated: Group 1 – ITI with Nuwiq®, octanate® or wilate® and aPCC/rFVIIa if needed to treat bleeding episodes (BEs) or during surgery or for prophylaxis; Group 2 – ITI with Nuwiq®, octanate® or wilate® and emicizumab prophylaxis and aPCC/rFVIIa if needed to treat BEs or during surgery; Group 3 – routine prophylaxis with emicizumab, aPCC or rFVIIa without ITI and aPCC/rFVIIa if needed to treat BEs or during surgery. Patients will not be randomised to a treatment group and may change groups during the study. Conclusions: It is planned to enrol 120 patients who will be followed for up to 5 years. Optional sub-studies will explore factors that may influence ITI results as well as the impact of different treatment approaches on important aspects of patient health, including joint and bone health and the risk of thrombotic events.

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NKG2C+CD57+ Natural Killer Cells May Play an Important Role in Maintaining Virus-Specific Immune Reconstitution Post-Allogeneic HSCT

Blood ◽

10.1182/blood-2018-99-110779 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3327-3327

Author(s):

Debora Queiros ◽

Susanne Luther-Wolf ◽

Eva M Weissinger ◽

Arnold Ganser

Keyword(s):

T Cells ◽

Healthy Volunteers ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Immune Reconstitution ◽

Blood Samples ◽

Allogeneic Hsct ◽

Cell Counts ◽

Cmv Reactivation

Abstract Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for malignant hematological diseases in adults. Due to the delayed immune reconstitution after HSCT, human cytomegalovirus (CMV) can reactivate, leading to prolonged hospitalization and increased morbidity and even mortality. Natural Killer (NK) cells have recently been described to undergo persistent reconfiguration in response to CMV-reactivation. Here we analyzed the presence and expansion of CMV-specific NK cells in patients after allogeneic HSCT. Methods: A multicolor flow cytometry panel for monitoring the CMV-specific NK cell (NKG2C+CD57+) reconstitution and expression of activating receptors was established. Reconstitution of CMV-specific NK cells was assessed in peripheral blood samples from 67 CMV-seropositive patients. The samples were collected and analyzed between day 0 and 100 post-HSCT at intervals of 7-10 days. Monitoring of CMV-reactivation by CMV-pp65 expression and reconstitution of CMV-specific T cells (CMV-CTLs) was done routinely in our laboratory, using 7 commercially available, certified CMV-tetramers, allowing for comparison of CMV-CTL and NKG2C+CD57+ NK cells. For further immunological tests, PBMCs from CMV-seropositive healthy volunteers were isolated by density gradient centrifugation. NK cells were negatively selected by magnetic bead separation. Additional purification of NKG2C+CD57+ NK cellswas achieved by cell sorting. Selected NK cells were expanded by co-culture with irradiated allogeneic PBMCs as feeder cells and the medium was supplemented with PHA and IL-2. Expanded CD57+NKG2C+ NK cells were KIR-typed. Results: Our patient cohort consisted of 67 patients after allogeneic HSCT with a median age of 59 years (range: 20-75). Forty-two patients (62.7%) were transplanted for acute leukemia, 54 (80.6%) received reduced intensity conditioning (RIC) and 62 (92.5%) received anti-thymocyte-antibodies globulin (ATG). GvHD-prophylaxis was cyclosporine A (CsA) in combination with mycophenolate motefil (MMF) for 82.1% of the patients and 77.6% were transplanted from matched donors. Thirty-three (49.2%) patients reactivated CMV (median age: 59.5 years, range 28-75; median day of reactivation: 38 days post-HSCT, range: 19-54). A significant increase in the absolute cell counts of NKG2C+CD57+ NK cells was observed after CMV reactivation, when compared to patients who did not reactivate CMV (p<0.0001). Interestingly, we observed a decreased expression of the CD8-molecule on NK cells during CMV-reactivation. CD8-expression on NK cells was previously described to be associated with a more cytotoxic phenotype of NK cells, this decrease may be a consequence of apoptosis following lytic activity. Monitoring for an additional activation marker, NKG2D, showed a significant increased expression after CMV reactivation (p=0.006), demonstrating not only the activating regulation of NK cells, but also, the co-stimulatory effects on T cell proliferation and cytokine production. Remarkably, when comparing NKG2C+CD57+ NK cells with CMV-specific T cells (Figure 1), both cell populations show similar kinetics of expansion, with an increase in the absolute cell counts during and after CMV-reactivation. NKG2C+CD57+ preliminary expansion-studies were performed using peripheral blood samples from CMV-seropositive healthy volunteers. After two weeks in culture, an expansion of up to 3100-fold was achieved. Further studies to assess the proliferative capacity of NKG2C+CD57+ subpopulation and its functional properties post-HSCT, are ongoing. In addition, an extensive panel of cytokines and chemokines excreted by the NKG2C+CD57+cells will be studied in order to evaluate their recruitment ability of other cell-types. Conclusion: Taken together, our results indicate that NK cells undergo a dynamic modulation and expansion of this population occurs in response to CMV-reactivation. Additionally, NKG2C+CD57+ NK cells may substitute for missing CMV-specific T cells shortly after HSCT and may play an important role in sustaining the immune reconstitution after CMV-reactivation. This study shows that NKG2C+CD57+ NK cells can be selected and expanded in vitro, holding promise for adoptive transfer in patients with recurrent CMV-reactivations. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

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Survival Benefit of Asymptomatic Cytomegalovirus Reactivation and Early Lymphocyte Recovery after HLA-Identical Allogeneic Peripheral Blood Stem Cell Transplantation.

Blood ◽

10.1182/blood.v106.11.5336.5336 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5336-5336

Author(s):

Dong Hwan Kim ◽

Sang Kyun Sohn ◽

Jin Ho Baek ◽

Jong Gwang Kim ◽

Kyu Bo Lee ◽

...

Keyword(s):

Overall Survival ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Immune Reconstitution ◽

Peripheral Blood Stem Cell ◽

Survival Curve ◽

Transplant Outcomes ◽

Cmv Reactivation ◽

Blood Stem Cell Transplantation

Abstract Background: Cytomegalovirus (CMV) reactivation (CMV-R) is associated with increased morbidity after allogeneic stem cell transplantation (SCT). However, after the introduction of ganciclovir preemptive therapy, CMV-R can be successfully controlled if the recipient’s immunity recovers. Although many investigations have already focused on CMV-R as a risk factor, data related to the impact of asymptomatic CMV-R on transplant outcomes is scarce. Accordingly, the present study analyzed the differences in the transplant outcomes the immune reconstitution according to the CMV-R after allogeneic PBSCT. Method: A total of 76 patients undergoing allogeneic peripheral blood stem cell transplantation (PBSCT) were included in the current study. The transplant outcomes and immune reconstitution after 3, 6, and 12 months were analyzed according to the occurrence of CMV-R in 33 patients. Result: The analysis revealed a favorable prognosis for the group with CMV-R compared to those without CMV-R: p=0.0037 for OS, p=0.0204 for NRM, and p=0.05 for the risk of relapse. CMV-R was also found to correlate to the lymphoid reconstitution (p=0.024). In multivariate analyses, CMV-R was found to be a favorable prognostic factor in terms of OS (p=0.010, hazard ratio [HR] 2.948) and NRM (p=0.05, HR 2.665), along with a higher transplant CD34+ cell dose (p=0.003 for OS, p=0.002 for NRM), standard risk (p=0.023 for OS), and acute GVHD grade 0~2 (p=0.007 for NRM). Conclusion: In a PBSCT setting, CMV-R did not seem to be a poor prognostic factor in terms of OS and NRM, possibly due to the accelerated lymphoid immune reconstitution associated with CMV-R. Figure. Survival curve for overall survival (A) and lymphoid immune reconstitution according to CMV reactivation (B) Figure. Survival curve for overall survival (A) and lymphoid immune reconstitution according to CMV reactivation (B)

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Adequate Immune Reconstitution in Recipients of Unrelated Donor Hematopoietic Cell Transplants Given Rabbit Anti-Thymocyte Globulin During Conditioning.

Blood ◽

10.1182/blood.v114.22.2234.2234 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2234-2234

Author(s):

Devon Fletcher ◽

John M. McCarty ◽

Harold M Chung ◽

Kathryn Candler ◽

Catherine H Roberts ◽

...

Keyword(s):

Immune Reconstitution ◽

Hematopoietic Cell ◽

First Year ◽

Unrelated Donor ◽

Post Transplant ◽

Cell Counts ◽

Number Of Patients ◽

Cellular Immune ◽

Cmv Reactivation

Abstract Abstract 2234 Poster Board II-211 Anti-thymocyte globulin (ATG) is known to reduce the risk of developing acute graft vs host disease following allogeneic hematopoietic cell transplant (HCT). Its effects on long-term immune reconstitution are less well defined, particularly in the adult population undergoing unrelated donor (URD) HCT. Since 2004, rabbit ATG (Thymoglobulin, Genzyme Inc, Cambridge, MA) has been used at our institution during the conditioning of patients undergoing URD HCT. We performed a retrospective landmark analysis to compare immune reconstitution in patients who received ATG during conditioning vs. those who did not. Patients had to have completed at least 6 months of follow-up post transplant. Eighty six patients were eligible, and underwent analysis of immune reconstitution in the first year post transplant. Fifty six patients underwent matched related donor HCT and did not receive ATG (no ATG cohort); 30 patients received an URD HCT (ATG cohort). The median age for no ATG cohort was 49 years and for the ATG cohort was 48. There were 40 females in the combined cohorts. The no ATG cohort included patients with the diagnosis of AML (34%), NHL (21%), MM (16%), ALL (9%), along with CML, CLL, MDS, MF and HD (20%). The ATG cohort was comprised of AML (43%), MDS (23%), ALL (13%), CML (13%), along with NHL & SAA (8%). Conditioning regimens used in the no ATG vs. ATG cohorts were 12-Gy TBI-Cy in 18% vs. 47%, Bu-Cy in 42% vs. 40%, and others in 40% vs. 13% (Flu-Mel, Bu-Flu, Flu-Cy, 2-Gy TBI,TBI-VP16). Stem cells were GCSF mobilized PBSC in 95% of the no ATG cohort and in 44% of the ATG cohort. The ATG dose administered was either 7.5 or 10 mg/kg in 3 divided doses, given from day -3 to day -1. With a median follow up of 727 days in the no ATG cohort and 480 days in the ATG cohort, 82% of the patients survived in the no ATG cohort compared to 73% in the ATG cohort (Fisher's exact test P=0.41). Absolute lymphocyte counts at 6, 9 and 12 months following transplantation were (mean ± SD) 1.2 ± 0.6×10 3 /μL vs. 1.0 ± 0.8 (T-Test, P=0.44), 1.5 ± 0.9 vs. 1.3 ± 1.0 (P=0.51) and 1.6 ± 0.9 vs. 1.3 ± 0.9 (P=0.23) respectively in the no ATG cohort vs. ATG cohort. Lymphocyte subset enumeration data was obtained during the first year following HCT at the time of cessation of immunosuppression and was available for 32 and 12 patients in the no ATG and ATG cohorts respectively. Absolute CD3+ cell counts measured at a median of 278 days were 1226 ± 773 vs. 981 ± 442 /μL in the no ATG vs. ATG cohorts (P=0.52). Simultaneously measured absolute CD3+/4+ cell counts were 483 ± 231 vs. 242 ± 122 (P=0.001), CD3+/8+ were 717 ± 627 vs. 701 ± 444 (P=0.94), CD19+ were 250 ± 239 vs. 351 ± 233 (P=0.25) and CD56+ were 181 ± 97 vs. 178 ± 67 (P=0.75) in the no ATG vs. ATG cohorts. Surveillance for EBV and CMV reactivation was performed using PCR. No statistically significant difference was noted in rate of CMV reactivation between the two cohorts in the 6-12 month post-transplant period indicating equivalent functional cellular immune reconstitution. EBV reactivation did not occur in either cohort. During the same time period the incidence of culture proven fungal infections and viral infections was equivalent between the two groups, however there was a significantly higher number of patients who experienced bacterial infection episodes in the ATG group. We are investigating the impact of ATG administration on the relative rate of relapse in these two cohorts. We conclude that ATG administered during conditioning did not adversely impact cellular immune reconstitution in this cohort of patients, even though these high-risk patients had undergone URD HCT with bone marrow as the stem cell source in the majority. This effect may be explained by a reduction in the incidence of acute GVHD secondary to ATG use, which in turn reduces the overall immunosuppressive exposure these patients experience following transplantation. T helper cell reconstitution appears to be delayed and may contribute to the higher number of patients experiencing bacterial infections in the ATG cohort. Disclosures: Off Label Use: Thymoglobulin for GVHD prophylaxis. McCarty:Celgene: Honoraria; Genzyme: Honoraria.

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Rapid Recovery of CD4 Counts Following Low Dose Alemtuzumab Conditioning During HLA Matched Related Bone Marrow Transplant for Children with Haemoglobinopathies

Blood ◽

10.1182/blood.v116.21.2335.2335 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2335-2335

Author(s):

Lucy Cook ◽

Farah O'Boyle ◽

Leena Karnik ◽

Helen New ◽

Irene Roberts ◽

...

Keyword(s):

Cell Count ◽

Graft Versus Host Disease ◽

Immune Reconstitution ◽

Low Dose ◽

Cell Recovery ◽

Host Disease ◽

Graft Versus Host ◽

Cd4 Cell ◽

The Impact ◽

Cmv Reactivation

Abstract Abstract 2335 CMV viraemia and disease remains a major cause of morbidity and mortality following allogeneic bone marrow transplantation (BMT). We investigated the impact of low dose alemtuzumab conditioning in paediatric matched sibling allogeneic BMT for haemoglobinopathy on immune reconstitution (early and late CD3/4+, CD3/8+, CD3-/CD56+ cell recovery) and subsequent viral infections in 18 consecutive transplants (14 β thalassaemia major and 4 sickle cell anaemia). A secondary aim was to investigate the impact of immune reconstitution on graft versus host disease but due to the extremely low incidence of acute and chronic graft versus host disease with this protocol (acute grade II to IV 11.6% and chronic 4.6%) we were unable to assess this. Transplant protocol: following hypertransfusion to suppress endogenous haemopoiesis, patients were conditioned with oral busulfan 14 mg/kg (days -9 to -6), cyclophosphamide 200 mg/kg (days -5 to -2) and alemtuzumab 0.3 mg/kg (days -8 to -6). Graft versus host disease prophylaxis was provided with ciclosporin for six months and methotrexate 10 mg/m2 on days +4 and +7. All transplants used BM as source of stem cells. CMV PCR surveillance was undertaken twice weekly and CMV reactivation was treated with intravenous foscarnet or ganciclovir, followed by secondary prophylaxis with valganciclovir. Routine peripheral blood immunophenotyping of lymphocyte subsets was performed at 60-, 90-, 120-, 150- and 180- days post-BMT. Results: the median age of transplant recipients was 7 years (range 2 – 17 years) and median follow up 254 days (range 203–378). The median CD3/CD4+ cell count rose above 200 cells/μl by day 180 and above 300 cells/μl by day 254 following the withdrawal of immune suppression at six months (fig 1A), the median CD8+ cell count increased earlier, to >200 cells/μl by day 60 and 300 cells/μl by day 180 (fig 1B) and the median CD56+ cell count reached 200 cells/μl by day 60 and plateaued thereafter (fig 1C). Overall, CMV reactivation occurred in 13 patients (72.2%) after a median interval of 35 days following BM infusion (range day-1 to day +72): 10/13 patients (76.9%) in whom both recipient and donor were CMV positive (CMV +/+); 1/3 patients in whom recipient was CMV negative and donor CMV positive (CMV -/+); and in 1/1 who was CMV positive with a CMV negative donor (CMV +/−). Significantly there was no clinical CMV disease in any patient and with the use of oral valganciclovir there were no subsequent CMV reactivations. CMV reactivation occurred in patients with lower CD4+ cell counts at day 60 (67 cells/μl) compared with the CMV-unaffected group (a median CD4+ count 298 cells/μl; p= 0.0262). Four patients experienced other viral infections: HHV6 infection requiring foscarnet on day +85 (n=1), urinary BK virus not requiring treatment (n=2) and parainfluenza pneumonitis requiring ribavarin on day +12 (n=1). Conclusion: Low dose alemtuzumab conditioning is associated with rapid CD4+ cell recovery (CD4+ >200 cells/μl by day +180) in children undergoing allogeneic matched related donor BMT for haemoglobinopathy. Although there is a high frequency of CMV reactivation (72.2%) in patients with low CD4+ counts at day 60 (median CD4+ count 67 cells/μl), no clinical CMV disease was seen and subsequent reactivations of CMV were prevented by the use of valganciclovir secondary prophylaxis whilst awaiting full immune reconstitution. Disclosures: No relevant conflicts of interest to declare.

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Monitoring Early CMV-Specific CD8+ T-Cell Responses After Allogeneic Stem Cell Transplantation By Pentamer and Streptamer Staining

Blood ◽

10.1182/blood.v122.21.4597.4597 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4597-4597

Author(s):

Lorea Beloki ◽

Miriam Ciaurriz ◽

Natalia Ramirez ◽

Amaya Zabalza ◽

Cristina Mansilla ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Antiviral Therapy ◽

Allogeneic Stem Cell Transplantation ◽

Immune Reconstitution ◽

Specific Ctls ◽

Cmv Reactivation

We present the results of a pilot study using pentamer (PM) and streptamer (ST) multimer complexes for monitoring CMV-specific CD8+ T-cells (CTLs). We analysed 15 patients that underwent allogeneic Stem Cell Transplantation (HSCT). Patient characteristics are summarized in Table 1. All patients and donors were positive for the HLA-A*02:01 allele. PM and ST were directed against the epitope NLVPMVATV (495-503) of the CMV phosphoprotein 65 (pp65). Samples were obtained at 15-day intervals until day +90 and monthly thereafter.PatientCMV status (D/R)GenderAgeDiagnosisDonorConditioningEngraftment (day)Follow up (months)GVHD (day)1+/+F43NHLSIBRIC2415-2-/+F33MDSURDRIC1912-3+/+M55AMLSIBMAC1714474-/+F41AMLURDMAC2121-5+/+F32AMLSIBMAC2013256+/+F64MDSSIBRIC2714897-/+M58CLLURDRIC2313928+/+M57ALLURDMAC155-9+/+F39AMLURDMAC2012-10-/+M42ALLURDMAC2125-11-/+M44AMLURDMAC12815312+/+F65AMLSIBRIC258-13-/+M27AMLSIBMAC3031814+/+M65AMLSIBRIC1966215+/-F30SAASIBRIC133- Three patterns were observed. In 3 patients (20%) no CMV-specific-CTLs could be detected despite several CMV reactivations, requiring prolonged cumulative antiviral therapy (median 68 days; range 67-136). In 7 patients (47%) CMV reactivation occurred at a mean of 41 days (10-94) and triggered a rapid increase of CMV-specific-CTLs with a median of 22.7 x 105/L (range 1.3-279.7). The CMV-PCR became immediately negative and antiviral therapy was stopped promptly after a median of 15.5 days (6-23). Finally, 5 patients (33%) showed an early immune reconstitution with CMV-specific-CTLs detected with a median of 0.7 x 105/L (range 0.2-2.8) in the absence of CMV-PCR reactivation at a median of 21 days (10-34) post-SCT. No CMV-PCR reactivation was observed in this group with a median follow-up of 12 months (5-14). Discussion Monitoring CMV-specific-T-cells might be able to distinguish patients at higher risk of recurrent virus reactivation and in need of prolonged antiviral therapy. Patients with increasing CMV-specific-CTLs detectable at the time of CMV-PCR reactivation may only need a short course of antiviral therapy, while those with early CMV-specific-CTLs may be protected from CMV reactivation. Conclusion Using Multimer-based (Pentamer and Streptamer) monitoring of CMV-specific T-cell immune reconstitution after allogeneic HSCT may contribute to the clinical decision regarding when and for how long to commence anti-CMV therapy. Disclosures: No relevant conflicts of interest to declare.

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Cytomegalovirus reactivation following allogeneic stem cell transplantation is associated with the presence of dysfunctional antigen-specific CD8+ T cells

Blood ◽

10.1182/blood-2002-05-1387 ◽

2002 ◽

Vol 100 (10) ◽

pp. 3690-3697 ◽

Cited By ~ 144

Author(s):

Evren Özdemir ◽

Lisa S. St. John ◽

Geraldine Gillespie ◽

Sarah Rowland-Jones ◽

Richard E. Champlin ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cd8 T Cells ◽

Allogeneic Stem Cell Transplantation ◽

Immune Reconstitution ◽

Cell Responses ◽

Tetramer Staining ◽

Cmv Reactivation ◽

Cmv Antigenemia

Cytomegalovirus (CMV) infection causes significant morbidity and mortality in the setting of immunodeficiency, including the immune reconstitution phase following allogeneic stem cell transplantation (SCT). We assessed CMV-specific CD4+ and CD8+T-cell responses in 87 HLA-A*0201–positive (A2+) and/or B*0702-positive (B7+) allogeneic stem cell transplant recipients using HLA-peptide tetramer staining and cytokine flow cytometry (CFC) to examine the association of CMV-specific immune reconstitution and CMV antigenemia following SCT. Strong CMV-specific T-cell responses recovered in most subjects (77 of 87, 88%) after SCT. Frequencies of CMV-specific CD8+ T cells were significantly higher in those subjects who experienced early antigenemia relative to those who did not (2.2% vs 0.33%, P = .0002), as were frequencies of CMV-specific CD4+ T cells (1.71% vs 0.75%,P = .002). Frequencies of CMV-specific CD8+ T cells were also higher in subjects experiencing late antigenemia (2.4% vs 0.57%). When we combined tetramer staining and an assessment of cytokine production in a single assay, we found that individuals who experienced CMV antigenemia had lower tumor necrosis factor-α (TNF-α)–producing fractions of tetramer-staining CMV-specific CD8+ T cells than subjects who did not (25% vs 65%,P = .015). Furthermore, individuals at high risk for CMV reactivation, including patients with acute graft-versus-host disease and those receiving steroids, had low fractions of cytokine-producing CMV-specific CD8+ T cells (25% and 27%, respectively). These data suggest that the inability to control CMV reactivation following allogeneic SCT is due to the impaired function of antigen-specific CD8+ T cells rather than an inability to recover sufficient numbers of CMV-specific T cells.

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