Influence of Infused Naïve CD3+CD8+ and CD3+CD56+ NKT-Cells on Single Unit Dominance After Double Cord Blood Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 652-652
Author(s):  
Filippo Milano ◽  
Shelly Heimfeld ◽  
Ted Gooley ◽  
Ian Nicoud ◽  
Colleen Delaney
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Single Unit ◽  
Cord Blood Transplantation ◽  
Cell Subset ◽  
Cell Content ◽  
Blood Transplantation ◽  
Single Donor ◽  
The Mean

Abstract Abstract 652 Introduction: Double cord blood transplantation (dCBT) has emerged as a feasible alternative for the treatment of adults and children with hematologic malignancies. In the great majority of cases after dCBT, one unit emerges as the sole source of long-term hematopoiesis. However, the determinants and mechanism of this single unit dominance remain poorly understood. To investigate this dominance phenomenon we have analyzed the cell subset content of the cord blood (CB) units after dCBT. Methods: All CB units were thawed and washed by centrifugation prior to infusion, and a small sample removed from the final products post-wash for multicolor flow cytometric assessment of graft composition; viable cell subsets included stem/progenitors (CD34+), monocytes (CD14+), B (CD20+), NK (CD3−CD56+), NK/T (CD3+CD56+), and T-cell subsets (CD3+CD4+, CD3+CD8+, Memory, Naïve, Regulatory) expressed as infused cell subset/kg of actual recipient weight (in some cases insufficient sample was available for complete cell subset analysis on both CB units (see Table 1). Analysis of donor chimerism was performed on sorted peripheral blood CD3+, CD56+, CD14+ or CD33+ fractions on days 7, 14, 21, 28, 56, and 80 after transplantation. Single donor dominance was defined as more than 95% in all cell fractions. In the rare cases of both units engrafting, the unit contributing >60% hematopoiesis was considered the dominant one. A one-sample test of the null hypothesis that a binomial proportion is equal to 0.5 was used to test the association of concordance of higher cell dose with unit dominance. A one-sample paired t-test was also used to compare the mean differences in cell dose between winning and losing units. A total of 72 patients received dCBT between January 2008 and May 2011. Twelve patients (16%) were excluded because of death before informative chimerism data or due to graft failure. Sixty patients were included in the final analysis: 32 (54%) were conditioned with cyclophosphamide (CY) 60 mg/kg, fludarabine (FLU) 75 mg/m2 and total body irradiation (TBI) 1200–1320 cGy; 17 (28%) with Treosulfan 14 mg/m2, FLU 200 mg/m2 and TBI 200 cGy; and 11 (18%) with CY 50 mg/kg, FLU 200 mg/m2 and TBI 200 cGy. GVHD prophylaxis consisted of cyclosporine and mycophenolate mofetil. Results: Single unit dominance was observed in all recipients of myeloablative conditioning within 21 days of dCBT. Recipients of non-ablative conditioning initially had mixed donors-host chimerism but usually were 100% single donor by day 100. Similar to the findings of others, the unit containing the greater CD3 dose was statistically concordant as the dominant unit (p<0.0001). Of interest, further T-cell subset analyses showed dominance was more correlated with higher CD3+CD8+ (p<0.0001) than with CD3+CD4+ (p=0.001), and more with naive CD3+CD8+ (p=0.009) as compared with naive CD3+CD4+ (p=0.10). The CD3+CD56+ NKT cell content also showed a significant concordance (p=0.02). Furthermore, the mean difference in both naive CD8+ and NKT between what became the dominant and the non-dominant unit was 0.41 × 105/Kg and 0.11 × 104/Kg, respectively (p=0.008 and p=0.02). In contrast to other published reports, no significant associations were found with the infused dose of total cells or CD34+ cells, and no correlations with the infused dose of monocytes, B-cells, NK, memory CD3+CD4+ or CD3+CD8+, or regulatory T-cells were seen (Table1). Conclusion: These data indicate that higher T-cell content, in particular the naïve CD3+CD8+ T-cell subset, is crucial in determining which cord blood unit will ultimately predominate. Our findings provide further insight into the biology of dCBT, and support the hypothesis that dominance is an immune-mediated process in which naïve CD8+ T-cells and perhaps NKT cells within the graft play an important role. Our findings may have important implications for optimal unit selection, and for ex-vivo graft manipulations to enhance engraftment, immune reconstitution, and disease control. Disclosures: No relevant conflicts of interest to declare.

High Expression of Granzymes and Perforin along with Increased T cell Cycling In Vivo Predicts the Development of Opportunistic Infections (OI) Following Unrelated Cord Blood Transplantation (UCBT).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2238-2238
Author(s):  
Paul Szabolcs ◽  
Young-Ah Lee ◽  
Luciana Marti ◽  
Melissa Reese ◽  
Joanne Kurtzberg
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Cd8 T Cells ◽  
Viral Infections ◽  
Opportunistic Infections ◽  
Cord Blood Transplantation ◽  
Ki 67 ◽  
Granzyme A ◽  
Blood Transplantation

Abstract Introduction: Unrelated umbilical cord blood transplantation (UUCBT) is a viable option for those who lack HLA-matched sibling donors. However, opportunistic infections (OI) occurring in the first 100 days, remain the major cause of morbidity and mortality. Viral infections are the primary cause of OI death. As previously shown, cord blood T cells have significantly less preformed effector molecules available intracellularly to kill virally infected cells via the perforin-granzyme pathway than adult PB T cells. Since several virally infected patients control their infections in the absence of specific antiviral therapy ( e.g adenoviral enteris, polyoma cystitis) we postulated that the T cell compartment of those UUCBT recipients who experience early viral infections maty upregulate expression of the perforin exocytosis pathway. In parallel the impact of viral infection on T cell turnover would also be appreciable. Here we report on 19 prospectively studied pediatric patients, all full donor chimera, following myeloablative therapy. Methods: On day +50 we determined by 4-color FACS the expression of intracellular Granzyme, A, B, along with perforin. To monitor T cell turnover proliferating cells were identified by monitoring for the KI-67 nuclear antigen. The expression of the antiapoptosis gene BCL-2 was similarly monitored in both CD4+ and CD8+ T cells. We analyzed their association with the development of de novo OI up to day +100 employing Student’s t-test. Results: Mean age of patients was 6.2 years. 10 of 19 patients developed OI (adenovirus x 4, CMV x 7, EBVx1, parainfluenza x 1) with 5/10 patients experiencing more than one viral infections simultaneously) at a median of 29 days after UUCBT. Of those with OI 6/10 died due to their infections while 8/9 without OI are alive at a median of 15.8 months after UUCBT with one death due to leukemic relapse. Table I presents the correlation between the tested parameters with the development of OI. Patients experiencing viral infections had significantly higher % of their T cells in particular CD8+ T cells equipped with effectors of cytotoxicity and were proliferating in higher percentage compared to those with no active infections. However, the anti-apoptotic protein BCL-2 expression was significantly lower in patients experiencing OI that may lead to their shorter life span and overall T cell lymphopenia observed in OI patietns that we have previously detected in a larger cohort of 102 patients (ASBMT 2004 abstract#48). Conclusion: Correlating with active viral infections significant maturation of cord blood T cells is evident as early as 50 days after UUCBT towards acquiring effector molecules of the perforin pathway. Enhanced T cell proliferation is counteracted by reduced expression of BCL-2 that may lead to the lymphopenia in patients with OI. Future strategies aiming to enhance the longevity of antiviral T cells may protect from death due to viral infections. Univariate analysis VARIABLE MEDIAN VALUE FOR PATIENTS WITH OI MEDIAN VALUE FOR PATIENTS WITHOUT OI t-Test p value % Granzyme A+ T cells 52% 9% 0.006 % Granzyme A+ CD8+ T cells 91% 47% <0.001 % Granzyme B+ T cells 36% 6% 0.036 % Granzyme B+ T cells 87% 39% <0.001 % Perforin+ T cells 38% 4% 0.009 % Perforin+ CD8+ T cells 61% 21% <0.001 % Ki-67+ T cells 27% 16% 0.0041 % Ki-67+ CD8+ T cells 35% 16% 0.0037 BCL-2 expression level (MFI) 87 117 0.028

Contribution of T-Cell Reconstitution during the Early Phase after Cord Blood Transplantation to Favorable Clinical Outcomes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2227-2227
Author(s):  
Satoshi Takahashi ◽  
Nobukazu Watanabe ◽  
Jun Ooi ◽  
Akira Tomonari ◽  
Kashiya Takasugi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Cd8 T Cells ◽  
Early Phase ◽  
Cord Blood Transplantation ◽  
Interferon Γ ◽  
Cell Recovery ◽  
Memory Phenotype ◽  
Blood Transplantation

Abstract The immaturity of T cells in cord blood is well known in functional assays and phenotypic analyses. During the first several months after cord blood transplantation (CBT), the T cell compartment is recovered by peripheral expansion from those mature and naïve T cells in cord blood grafts and plays an important role in acute graft-versus-host disease (GVHD) and graft-versus-leukemia reaction. Recently, we have reported that adult patients with hematological malignancies receiving CBT from HLA-partially-mismatched unrelated donors (n=68) had a lower risk of severe acute GVHD (> grade II, 7% versus 26%) and transplant-related mortality (9% versus 29% at 1 year) and a higher probability of disease-free survival (74% versus 44% at 2 years) than HLA-matched unrelated bone marrow transplant (BMT) recipients (n=45) in our multivariate analysis (Takahashi et al., Blood, in press). We speculated that the immune reconstitution process over a period of several months after CBT might have contributed to these promising clinical results. Using four-color analysis with CD4, CD8, CD45RA, and CD62L, more than 90% of cord blood CD4+ and CD8+ T cells in the grafts belonged to the naïve fraction. Cytokine expression in cord blood T cells was also suppressed to 0.1% in CD4+ and to 0.9% in CD8+ with positive interferon-γ by intracellular staining, which were significantly lower than those in adult T cells (16.2% in CD4+ and 37.8% in CD8+). Circulating T cell counts normalized after 3 months for CD8+ and 4 months for CD4+ in our CBT recipients, both of which were significantly faster than in previously published studies, which were 9 months for CD8+ and 12 months for CD4+. After T cell recovery, peripheral blood T cells moved from the naïve to the central memory fraction immediately, and then moved to the effector memory fraction. A naïve subset of CD4+ T cells remained (median: 38 cells/μl on day 90, n=12) during the first 3 months, which was significantly higher than in the BMT control (median: 9 cells/μl on day 90, n=5, p=0.015), but showed a low level of CD8+ T cells (median: 14 cells/μl on day 90, n=12), almost the same as in BMT recipients (median: 13 cells/μl on day 90, n=5). Intracellular interferon-γ-producing T cells were detected at 3.4% (0.1–34.2%) in CD4+ and 32.3% (1.1–86.9%) in CD8+ at 1 month post-CBT (n=16), both of which were comparable to post-BMT. To investigate whether these T cells with memory phenotype are functional, we analyzed antigen-specific T-cell recovery using cytomegalovirus (CMV) as a specific antigen. CMV-responsive CD4+ T cells were detected within the first 4 months in all recipients with positive CMV antigenemia (n=13), but CD8+ T cells were detected only in 5 out of 13 cases, probably because of pre-emptive Gancyclovir administration in most antigenemia-positive patients. To conclude, naïve cord blood T cells rapidly increased in number and adopted a memory phenotype showing cytokine-production and antigen-recognition capacity in the early phase after CBT. These data suggest that mature T lymphocytes in cord blood have unique properties and contribute to the favorable clinical outcome of CBT.

Early Engraftment Kinetics of Leukocyte Subsets by Means of HLA-Specific Mabs After Double Umbilical Cord Blood Transplantation Show a Very Rapid Induction of Single Complete Donor Chimerism.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3338-3338
Author(s):  
Judith AE Somers ◽  
Yvette van Hensbergen ◽  
Anneke Brand ◽  
Ellen Meijer ◽  
Eric Braakman ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Cord Blood Transplantation ◽  
Post Transplant ◽  
Donor Chimerism ◽  
Rapid Induction ◽  
Blood Transplantation ◽  
Single Donor ◽  
Immunological Rejection ◽  
Chimerism Analysis

Abstract Abstract 3338 Poster Board III-226 Double umbilical cord blood transplantation (UCBT) results in higher engraftment rates as compared to single UCBT in adult patients. Sustained hematopoiesis is usually derived from a single cord blood unit (CBU). So far, the mechanism of predominance of a particular CBU is unresolved. Immunological rejection of one CBU has been proposed, as well as a selective growth advantage of one particular unit. Frequent serial chimerism studies in leukocyte subsets by use of HLA-specific monoclonal antibodies (mAbs) during the first month post transplant might contribute to unravel the mechanism of graft predominance. Methods. Seventeen consecutive patients (pts) with high risk hematological diseases received a double UCBT preceded by a non-myeloablative conditioning regimen (Cy 60 mg/kg/ Flu 160 mg/kg/ TBI 2×2 Gy). CBUs were selected by low resolution typing for HLA-A and –B loci and by allele typing for HLA-DRB1. The minimal required HLA-match grade was 4/6. If discriminating HLA mismatches between the 3 different parties were present, early analysis in leukocyte subpopulations was performed at day 11, 18, 25 and 32 post transplant by flowcytometry using lineage-specific (CD3, CD4, CD8, CD19, CD16/56, CD14, CD33) mAbs in combination with fluorochrome labeled HLA-antigen specific human mAbs. Results of day 32 were compared with routine chimerism analysis (VNTR) of peripheral blood TNC and T-cells. Results. Two pts were non-evaluable for engraftment due to early death and insufficient follow up, respectively. Donor engraftment in 15 pts occurred after a median of 30 days (range: 11-45). At 1 month post transplant, 9 pts showed complete single and 3 showed mixed chimerism by VNTR-analysis. In 3 patients, cells originating from both CBUs were present. Ultimately, complete single donor chimerism was established in 14 pts. Early chimerism studies with HLA-specific mAbs were performed in 8 pts. In all pts results at day 32 corresponded with chimerism analysis performed by VNTR. For 7 other pts no discriminating HLA-mAbs were available. Simultaneous 3-donor-origin detection of T-cells, monocytes and granulocytes based on HLA-disparities was possible in 4 pts. In 3 of them, all subsets showed a similar pattern: a clear predominance of a single CBU in all lineages as from day 11-18 onwards. Furthermore, the disappearing CBU was transiently detectable in these pts at day 11. In the fourth patient, T cells were of recipient origin at day 32 with predominance of a single CBU in all other subsets. The disappearing unit could be followed in another 4 pts, which revealed again a transient appearance of that unit in the various lineages at day 11. Although detectable in a total of 8 pts at day 11, all disappearing CBUs were completely lost within 18 days. The prefreeze TNC did not predict for the prevailing UCB, nor did the number of HLA-mismatches. Conclusions. These results show that double UCBT following a non-myeloablative regimen without ATG is associated with a rapid induction of complete single donor chimerism. The disappearing unit, although early detectable, was completely lost within 18 days in all patients, in whom it could be monitored by HLA-specific mAbs. We observed no disproportional increase of any leukocyte subset of the prevailing CBU nor of host leukocyte subset. This characteristic, uniform, early engraftment pattern in the presence of different HLA-disparities may rather argue for a difference in growth potential between both CBUs than an immunological rejection of the disappearing unit. Disclosures. No relevant conflicts of interest to declare.

scholarly journals Cognate CD4 T-Cell Licensing of Dendritic Cells Heralds Anti-Cytomegalovirus CD8 T-Cell Immunity after Human Allogeneic Umbilical Cord Blood Transplantation

2014 ◽  
Vol 89 (2) ◽  
pp. 1058-1069 ◽  
Author(s):  
T. W. H. Flinsenberg ◽  
L. Spel ◽  
M. Jansen ◽  
D. Koning ◽  
C. de Haar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Cord Blood Transplantation ◽  
Survival Rates ◽  
T Cell Immunity ◽  
Cell Responses ◽  
Blood Transplantation ◽  
Cell Immunity ◽  
Cognate Antigen

ABSTRACTReactivation of human cytomegalovirus (CMV) is hazardous to patients undergoing allogeneic cord blood transplantation (CBT), lowering survival rates by approximately 25%. While antiviral treatment ameliorates viremia, complete viral control requires CD8+T-cell-driven immunity. Mouse studies suggest that cognate antigen-specific CD4+T-cell licensing of dendritic cells (DCs) is required to generate effective CD8+T-cell responses. For humans, this was not fully understood. We here show that CD4+T cells are essential for licensing of human DCs to generate effector and memory CD8+T-cell immunity against CMV in CBT patients. First, we show in CBT recipients that clonal expansion of CMV-pp65-specific CD4+T cells precedes the rise in CMV-pp65-specific CD8+T cells. Second, the elicitation of CMV-pp65-specific CD8+T cells from rare naive precursors in cord blood requires DC licensing by cognate CMV-pp65-specific CD4+T cells. Finally, also CD8+T-cell memory responses require CD4+T-cell-mediated licensing of DCs in our system, by secretion of gamma interferon (IFN-γ) by pp65-specific CD4+T cells. Together, these data show that human DCs require licensing by cognate antigen-specific CD4+T cells to elicit effective CD8+T-cell-mediated immunity and fight off viral reactivation in CBT patients.IMPORTANCESurvival rates after stem cell transplantation are lowered by 25% when patients undergo reactivation of cytomegalovirus (CMV) that they harbor. Immune protection against CMV is mostly executed by white blood cells called killer T cells. We here show that for generation of optimally protective killer T-cell responses that respond to CMV, the early elicitation of help from a second branch of CMV-directed T cells, called helper T cells, is required.

In Vitro Assessment of Tolerance and Rejection Occurring After Co-Transplantation of Allogeneic Umbilical Cord Blood and Haploidentical CD34+ Cells as Treatment for Severe Aplastic Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2352-2352
Author(s):  
Nicole J. Gormley ◽  
Aleah Smith ◽  
Maria Berg ◽  
Lisa Cook ◽  
Catalina Ramos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Cord Blood Transplantation ◽  
Neutrophil Recovery ◽  
Post Transplant ◽  
Blood Transplantation ◽  
Cd34 Cells ◽  
Cord Blood Cells

Abstract Abstract 2352 Introduction/Methods: The administration of highly purified haploidentical peripheral blood CD34+ cells combined with an unrelated cord blood transplant results in earlier neutrophil engraftment than is typically seen with a cord blood transplant alone. Chimerism data from pilot trials evaluating this strategy have reported 3 phases of engraftment: 1) early myeloid engraftment from transplanted haplo-CD34+ cells followed by 2) cord blood engraftment resulting in dual chimerism and 3) the subsequent disappearance of haploidentical donor cells with resultant full donor cord chimerism. The mechanism accounting for the disappearance of haploidentical cells has not been defined. Here the clinical results and an in vitro assessment of alloreactivity in three patients that underwent combined haploidentical CD34+ cell and cord blood transplantation for severe aplastic anemia (SAA) are described. The conditioning regimen consisted of cyclophosphamide (60mg/kg/day on days -7 and -6), fludarabine (25mg/m2/day on days -5 to -1), horse ATG (40mg/kg/day on days -5 to -2), and total body irradiation (200cGy on day -1). GVHD prophylaxis consisted of tacrolimus and mycophenolate mofetil. PCR of STRs was used to assess chimerism in T-cell and myeloid lineages and mixed lymphocyte reaction assays(MLR) were performed on peripheral blood samples collected at different time-points post-transplant to assess for alloreactivity against the recipient, the haploidentical donor, or the cord unit. Stimulator cord blood cells for the MLR were obtained from residual cord blood cells remaining in the infusion bag after patient administration and expanded in vitro using anti-CD28/CD3 Dynabeads. Results: Prior to transplantation, all three pts had transfusion dependent SAA associated with severe neutropenia that was refractory to conventional immunosuppressive therapy. Pt 1 had an early transient myeloid recovery (ANC 400 on day+11) from the haploidentical donor followed by engraftment of the cord unit (Cord ANC > 500) on day 21. The patient is currently 2 years post transplant and has 100% cord blood chimerism and is transfusion independent. An MLR assay performed when donor T-cell chimerism was 100% cord, showed evidence for rejection of the haploid cells by cord blood T-cells, with the MLR response to haploidentical donor cells being seven fold higher than the response to fully HLA-mismatched 3rd party cells. In pt 2, neutrophil recovery from the transplanted haploidentical donor occurred on day +10, with chimerism studies showing no evidence for cord engraftment in either myeloid or T-cell lineages at any point post-transplant. The patient is currently 15 months post transplant and is transfusion independent with normal blood counts and sustained “split” chimerism (T-cells recipient in origin with myeloid cells being 100% haploidentical donor). MLR assays showed that the recipient was tolerant to the haploid donor, with no statistically significant difference in the alloreactive response to the haploid donor compared to self. In pt 3, neutrophil recovery from the transplanted haploidentical donor occurred on day +10, with chimerism studies showing split chimerism (T-cell chimerism >90% cord and myeloid chimerism 88–100% haploid donor in origin). MLR assays again showed evidence of rejection of the haploid cells by cord blood T-cells, with a trend towards greater alloreactivity against the haploid donor compared to an HLA mismatched 3rd party on post-transplant day +63. Conclusions: Combined haploidentical CD34+ cell and unrelated cord blood transplantation following highly immunosuppressive conditioning represents a viable treatment option for patients with SAA who lack an HLA-matched donor. Using this approach, 2 of 3 pts had cord blood engraftment associated with early neutrophil recovery from the haploidentical donor. In one pt, the cord unit failed to engraft. Remarkably, sustained engraftment from the haploidentical donor in this pt resulted in transfusion independence. MLR appears to be a useful approach to assess the in vitro alloreactivity of this unique stem cell graft source. In the two pts who had cord engraftment, in vitro MLR assessments established that the disappearance of haploid cells occurred as a consequence of rejection of the haploidentical cells by engrafting cord blood T-cells, rather than from non-immunological haploidentical cell graft failure. Disclosures: No relevant conflicts of interest to declare.

Improving Immune Reconstitution After Cord Blood Transplantation Using Ex Vivo Expanded Virus-Specific T Cells: A Phase I Clinical Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 224-224 ◽  
Author(s):  
Patrick J Hanley ◽  
Caridad Martinez ◽  
Kathryn Leung ◽  
Barbara Savoldo ◽  
Gianpietro Dotti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Dose Level ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Cord Blood Transplantation ◽  
Viral Reactivation ◽  
Blood Transplantation

Abstract Abstract 224 Adenovirus(Ad), Cytomegalovirus(CMV) and Epstein-Barr virus(EBV) frequently cause severe morbidity and mortality in patients(pts) after stem cell transplantation (SCT) and cord blood transplantation(CBT). We have shown that adoptive immunotherapy with peripheral blood(PB) donor derived multivirus-specific Cytotoxic T Lymphocytes directed against Ad, CMV and EBV can effectively prevent and treat the clinical manifestations of these viruses after SCT. CBT, while less likely to cause GvHD than conventional SCT, is unlikely to provide passive transfer of virus-specific CTL, since CBTs come from virus-naïve donors. Here we report for the first time the transfer of CB-derived multivirus-specific CTL(cbmCTL) to CBT recipients to restore cellular immunity to Ad, CMV and EBV. The development of cbmCTLs for pts undergoing CBT requires the priming and extensive expansion of naïve T cells rather than the more limited and simple direct expansion of pre-existing memory T cell populations from virus-exposed donors. We hypothesize that cbmCTL, derived from naïve T cells, will be efficacious and persist in vivo. Our protocol uses an initial round of stimulation with autologous CB-derived dendritic cells transduced with a recombinant Ad5f35 vector containing a transgene for the immunodominant CMV antigen, pp65 (Ad5f35pp65) in the presence of IL-7, IL-12 (CTEP-NCI) and IL-15. This is followed by 2 rounds of weekly stimulation with autologous Ad5f35pp65-transduced EBV-LCL in the presence of IL-15 or IL-2. Seven cbmCTL cultures generated for clinical use contained a mean of 48% CD8+, and 36% CD4+ cells with a mean of 33% CD45RA-/CD62L+ central memory T cells. In 51Cr release and/or IFNg ELISPOT assays, cbmCTL lines showed specific activity against all viruses. We have treated 7 pts who received the 80% fraction of a fractionated CB unit followed by cbmCTLs generated from the remaining 20% fraction; two pts were treated on each dose level;5×106/m2; 1×107/m2; and 1.5×107/m2 while one pt has been treated with 2.5×107/m2 – dose level 4. Pts received cbmCTLs on days 63–146 after CBT (median: day 83). No early infusion-related toxicities or subsequent GvHD was observed. All pts engrafted neutrophils by day 30 (median: day 20) despite receiving only 80% of the CB unit. Five of 7 pts had no initial infection or reactivation episodes, remaining free of CMV, EBV, and Ad from 2 months to 2 years post-CBT. Of the two remaining pts, pt 1 was transiently viremic for CMV pre-infusion and became highly viremic 4-weeks post-cbmCTL. The pt received a 2nd dose of cbmCTLs and CMV DNA/antigen became undetectable in the PB within 16 days of the 2nd dose and remains asymptomatic and virus free >2 yr post-CBT. Analysis of this pt's PB showed a rise in CMV-T cells even prior to cbmCTL #2, with a 31-fold expansion of CMV-T cells by 4 weeks after the initial CTLs. This pt also had AdV in his stool, which resolved without additional therapy. Shortly after CTL infusion, pt 4 had detectable EBV DNA in the PB that was controlled without additional antiviral therapy. The transferred cells had long-term persistence, since T cell receptor(TCR) deep-sequencing (ImmunoSEQ) allowed us to track infused T cell clones (i.e. clones present in the infused cbmCTL but absent in peripheral blood before cbmCTL infusion) up to 1 year post-CBT in 5/5 pts tested. In summary, none of the recipients of cbmCTL developed viral disease; in two pts with viral infections, the infections resolved without progression to disease, coinciding with the appearance of virus-specific T cells in peripheral blood. Hence, administration of cbmCTL to pts after CBT has so far been safe and can facilitate reconstitution of virus-specific T cells and control viral reactivation/infection in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cord blood transplantation recapitulates fetal ontogeny with a distinct molecular signature that supports CD4+ T-cell reconstitution

2017 ◽  
Vol 1 (24) ◽  
pp. 2206-2216 ◽  
Author(s):  
Prashant Hiwarkar ◽  
Mike Hubank ◽  
Waseem Qasim ◽  
Robert Chiesa ◽  
Kimberly C. Gilmour ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Cord Blood Transplantation ◽  
Cd4 T Cell ◽  
Molecular Signature ◽  
Blood Transplantation ◽  
T Cell Reconstitution ◽  
Key Points

Key Points Cord blood T cells are ontogenetically distinct from the peripheral blood T cells. Recapitulation of fetal ontogeny after cord blood transplantation results in rapid CD4+ T-cell reconstitution.

scholarly journals Functionally active virus-specific T cells that target CMV, adenovirus, and EBV can be expanded from naive T-cell populations in cord blood and will target a range of viral epitopes

Blood ◽  
2009 ◽  
Vol 114 (9) ◽  
pp. 1958-1967 ◽  
Author(s):  
Patrick J. Hanley ◽  
Conrad Russell Young Cruz ◽  
Barbara Savoldo ◽  
Ann M. Leen ◽  
Maja Stanojevic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Cord Blood Transplantation ◽  
Viral Disease ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Transplant Recipients ◽  
Blood Transplantation ◽  
Related Mortality

The naive phenotype of cord blood (CB) T cells may reduce graft-versus-host disease after umbilical cord blood transplantation, but this naivety and their low absolute numbers also delays immune reconstitution, producing higher infection-related mortality that is predominantly related to CMV, adenovirus (Adv), and EBV. Adoptive immunotherapy with peripheral blood-derived virus-specific cytotoxic T lymphocytes (CTLs) can effectively prevent viral disease after conventional stem cell transplantation, and we now describe the generation of single cultures of CTLs from CB that are specific for multiple viruses. Using EBV-infected B cells transduced with a clinical-grade Ad5f35CMVpp65 adenoviral vector as sources of EBV, Adv, and CMV antigens, we expanded virus-specific T cells even from CB T cells with a naive phenotype. After expansion, each CTL culture contained both CD8+ and CD4+ T-cell subsets, predominantly of effector memory phenotype. Each CTL culture also had HLA-restricted virus-specific cytotoxic effector function against EBV, CMV, and Adv targets. The CB CTLs recognized multiple viral epitopes, including CD4-restricted Adv-hexon epitopes and immunosubdominant CD4- and CD8-restricted CMVpp65 epitopes. Notwithstanding their naive phenotype, it is therefore possible to generate trivirus-specific CTLs in a single culture of CB, which may be of value to prevent or treat viral disease in CB transplant recipients. This study is registered at www.clinicaltrials.gov as NCT00078533.

scholarly journals Ontogeny of human mucosal-associated invariant T cells and related T cell subsets

2018 ◽  
Vol 215 (2) ◽  
pp. 459-479 ◽  
Author(s):  
Ghada Ben Youssef ◽  
Marie Tourret ◽  
Marion Salou ◽  
Liana Ghazarian ◽  
Véronique Houdouin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Cord Blood Transplantation ◽  
T Cell Subsets ◽  
Memory Phenotype ◽  
Mait Cells ◽  
Developmental Program ◽  
Blood Transplantation ◽  
Invariant T Cells

Mucosal-associated invariant T (MAIT) cells are semi-invariant Vα7.2+ CD161highCD4− T cells that recognize microbial riboflavin precursor derivatives such as 5-OP-RU presented by MR1. Human MAIT cells are abundant in adult blood, but there are very few in cord blood. We longitudinally studied Vα7.2+ CD161high T cell and related subset levels in infancy and after cord blood transplantation. We show that Vα7.2+ and Vα7.2− CD161high T cells are generated early during gestation and likely share a common prenatal developmental program. Among cord blood Vα7.2+ CD161high T cells, the minority recognizing MR1:5-OP-RU display a TRAV/TRBV repertoire very similar to adult MAIT cells. Within a few weeks of life, only the MR1:5-OP-RU reactive Vα7.2+ CD161high T cells acquire a memory phenotype. Only these cells expand to form the adult MAIT pool, diluting out other Vα7.2+ CD161high and Vα7.2− CD161high populations, in a process requiring at least 6 years to reach adult levels. Thus, the high clonal size of adult MAIT cells is antigen-driven and likely due to the fine specificity of the TCRαβ chains recognizing MR1-restricted microbial antigens.

A Single Center Experience with Allogeneic Sibling Umbilical-Cord-Blood Transplantation in Children with Malignant and Non-Malignant Disease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2042-2042
Author(s):  
Yongping Song ◽  
Yanli Zhang ◽  
Baijun Fang ◽  
Xudong Wei ◽  
Lulu Lu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Median Time ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Cd4 T Cells ◽  
Cord Blood Transplantation ◽  
Umbilical Cord Blood Transplantation ◽  
Blood Transplantation

Abstract To study the outcome of allogeneic sibling umbilical-cord-blood transplantation (CBT) in children in China, we studied the records of 38 recipients of cord blood from HLA-identical siblings from 1998 through 2004. The mean age at transplant was 10.3+/−5.5 years. Diseases of the patients were leukemia (28), neuroblastoma (3), severe aplastic anaemia (3) and inborn errors of metabolism (four). A volume of 76–208 ml of cord blood was collected from sibling infants soon after delivery. HLA antigens were identical in 32 and one to three antigens mismatched in six. The patients received busulphan and cyclophosphamide as conditioning, and antithymocyte globulin was given to prevent graft rejection. The median number of collected nucleated cells was 3.5 x 107/kg nucleated cells (range, 2.0 to 10.2). T-cell reconstitution was evaluated by using combined approaches of phenotyping analysis of lymphocytes and assessment of ex vivo thymic function by measuring T-cell receptor (TCR) rearrangement excision circles (TRECs). The overall survival is 89% at the median observation time of 72 months. The median time for neutrophil engraftment (absolute neutrophil count >500/mm3) was 16 days (range, 10 to 41days). The median time to become transfusion independent after CBT was 27 days for platelets (range, 15 to 54) and was 28 days for packed red blood cells (range, 19 to 128). Acute GVHD (aGVHD)was observed in 20/38 patients and involved only skin in 16 patients, skin and liver or gut in 2 patients and all 3 organs in another 2 patients. Seventeen of 20 patients had grade 1 to 2 aGVHD toxicity, whereas 3 patients experienced grade 3 to 4 aGVHD. Chronic GVHD (cGVHD) developed in 12 patients. Acute transplant related mortality was 5.5%. Cause of death was persisting non-engraftment till day +100 after transplant. Late mortality occurred in 2 patients: one cGVHD associated haemorrhage 22 months after CBT and one cGVHD associated septicaemia 4 years after CBT. Immunologic reconstitution demonstrated that CBT resulted in consistent and stable T-, B- and natural killer (NK) cell development. The kinetics of development was such that T-cell development occurred between 65 to 180 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, indicating de novo maturation of T cells. During the first 5 months after transplantation, TCR repertoires were highly abnormal and TREC values low. However, in a longer follow-up (one years or more after transplantation) TREC values and TCR diversity increased and became normal. This data confirms that CBT still should be the first treatment choice if an HLA-identical sibling is available.

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