Blinatumomab overcomes poor prognostic impact of measurable residual disease in pediatric high-risk first relapse B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Blinatumomab, a CD3/CD19 BiTE® (bispecific T cell engager) molecule, was superior to high-risk third course consolidation chemotherapy (HC3) in prolonging event-free survival (EFS) in children with high-risk first relapse B-cell precursor acute lymphoblastic leukemia (B-ALL). Here, we report results from a post hoc measurable residual disease (MRD) analysis of this phase 3 study (NCT02393859). PROCEDURE: Children >28 days and <18 years with high-risk first-relapse B-ALL in cytomorphological complete remission (M1 marrow, <5% blasts) or with M2 marrow (≥5% and <25% blasts) after induction and two cycles of high-risk consolidation chemotherapy (baseline) were enrolled in this trial. Patients received one cycle of blinatumomab (15 µg/m2 /day, 4 weeks, continuous intravenous infusion) or HC3. The primary endpoint was EFS. In this post hoc analysis, patients with MRD <10-4 by PCR were grouped as having positive but not quantifiable (pbnq) or undetectable disease. RESULTS: A higher proportion of patients with MRD <10-4 had undetectable versus pbnq disease after blinatumomab (day 29) than after HC3 (p = 0.0367). Of the 22 patients with MRD ≥10-4 at baseline who achieved MRD remission after blinatumomab, 20 (91%) achieved MRD <10-4 remission by day 15. Patients treated with blinatumomab had improved EFS and overall survival compared with those treated with HC3 independent of end-of-induction or baseline (end-of-second consolidation) MRD levels. CONCLUSIONS: Blinatumomab was more efficacious than HC3 regardless of MRD status before treatment. These data support the role of blinatumomab in inducing deep MRD remission, negating the poor prognostic value of MRD.

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Effect of Blinatumomab vs Chemotherapy on Event-Free Survival Among Children With High-risk First-Relapse B-Cell Acute Lymphoblastic Leukemia: A Randomized Clinical Trial.

Importance: Blinatumomab is a CD3/CD19-directed bispecific T-cell engager molecule with efficacy in children with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Objective: To evaluate event-free survival in children with high-risk first-relapse B-ALL after a third consolidation course with blinatumomab vs consolidation chemotherapy before allogeneic hematopoietic stem cell transplant. Design, Setting, and Participants: In this randomized phase 3 clinical trial, patients were enrolled November 2015 to July 2019 (data cutoff, July 17, 2019). Investigators at 47 centers in 13 countries enrolled children older than 28 days and younger than 18 years with high-risk first-relapse B-ALL in morphologic complete remission (M1 marrow, <5% blasts) or with M2 marrow (blasts ≥5% and <25%) at randomization. Intervention: Patients were randomized to receive 1 cycle of blinatumomab (n = 54; 15 µg/m2/d for 4 weeks, continuous intravenous infusion) or chemotherapy (n = 54) for the third consolidation. Main Outcomes and Measures: The primary end point was event-free survival (events: relapse, death, second malignancy, or failure to achieve complete remission). The key secondary efficacy end point was overall survival. Other secondary end points included minimal residual disease remission and incidence of adverse events. Results: A total of 108 patients were randomized (median age, 5.0 years [interquartile range {IQR}, 4.0-10.5]; 51.9% girls; 97.2% M1 marrow) and all patients were included in the analysis. Enrollment was terminated early for benefit of blinatumomab in accordance with a prespecified stopping rule. After a median of 22.4 months of follow-up (IQR, 8.1-34.2), the incidence of events in the blinatumomab vs consolidation chemotherapy groups was 31% vs 57% (log-rank P < .001; hazard ratio [HR], 0.33 [95% CI, 0.18-0.61]). Deaths occurred in 8 patients (14.8%) in the blinatumomab group and 16 (29.6%) in the consolidation chemotherapy group. The overall survival HR was 0.43 (95% CI, 0.18-1.01). Minimal residual disease remission was observed in more patients in the blinatumomab vs consolidation chemotherapy group (90% [44/49] vs 54% [26/48]; difference, 35.6% [95% CI, 15.6%-52.5%]). No fatal adverse events were reported. In the blinatumomab vs consolidation chemotherapy group, the incidence of serious adverse events was 24.1% vs 43.1%, respectively, and the incidence of adverse events greater than or equal to grade 3 was 57.4% vs 82.4%. Adverse events leading to treatment discontinuation were reported in 2 patients in the blinatumomab group. Conclusions and Relevance: Among children with high-risk first-relapse B-ALL, treatment with 1 cycle of blinatumomab compared with standard intensive multidrug chemotherapy before allogeneic hematopoietic stem cell transplant resulted in an improved event-free survival at a median of 22.4 months of follow-up. Trial Registration: ClinicalTrials.gov Identifier: NCT02393859.

AIEOP-BFM consensus guidelines 2016 for flow cytometric immunophenotyping of Pediatric acute lymphoblastic leukemia.

Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish "simple" from bilineal/"complex" mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter-laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society.

Flow diagnostics essential code: a simple and brief format for the summary of leukemia phenotyping.

BACKGROUND: Flow cytometry is a valuable part in the routine diagnostics of acute leukemia (AL). Although internationally recognized definitions of main AL subsets are available, there is currently no consensus format for the short summary of clinical flow cytometry reports. Since clinical reports are too long for most database purposes, there is a need for a standardized format of their short summaries. METHODS: The Associazione Italiana Ematologia Oncologia Pediatrica--Berlin Frankfurt Muenster (AIEOP-BFM) Flow Network that encompasses reference diagnostics laboratories in Australia, Austria, Czechia, Germany, Israel, Italy, and Switzerland have designed a pro-forma for the summary of flow cytometry results in the diagnosis of leukemia. The process involved several meetings and other communications, during which the group established a consensus on the essentials that lead to the diagnostic conclusions in childhood AL. RESULTS: The "Flow Diagnostics Essential (FDE) Code" is a result from an agreement within the AIEOP-BFM Flow Network. In a standardized format, it reports the extent of the infiltration by a malignant clone, followed by description antigen expression as strong, weak or negative, and a diagnostic conclusion. CONCLUSIONS: A consensus brief format (the "FDE Code") has been designed as a brief summary of the diagnostic immunophenotype of childhood AL. It is also applicable for the diagnostic investigation of other malignancies by flow cytometry. The FDE code may be included in the final clinical report and/or used in the setting of a multicenter clinical trial database.

Transforming growth factor beta (TGFß) plays a crucial role in prolonging allograft survival in an allodepletion ("pruning") skin transplant model.

Adoptive cell therapies involving cell manipulation to achieve tolerance are increasingly being studied in animal models and in human trials. We have demonstrated that the specific removal of allo-stimulated dividing cells (or "pruning") promotes long-term allograft survival across a major MHC mismatch in transplant models including skin, heart and islet transplants. In this study, we examine the role of transforming growth factor beta (TGFß), an important regulatory cytokine, on allograft survival in our allodepletion or "pruning" skin transplant model. Increased proliferation of CD4(+) T cells was observed following allo-stimulation of BALB/c spleen cells (labeled with CFSE) in the presence of the regulatory cytokines TGFß and (interleukin-2) IL-2 in a mixed lymphocyte culture (MLC). Expression of the regulatory gene forkhead box-3 (FoxP3) was increased in both the allo-stimulated non-dividing (ND) (CFSE(high)) and dividing (D) (CFSE(low)) CD4(+) T cell populations, with the highest expression found in the D CD4(+) T cell population. Mice reconstituted with allo-stimulated ND CD4(+) T cells following TGFß/IL-2 stimulation showed prolonged allograft survival, similar to previous data. Significantly, TGFß/IL-2 stimulation prevented acute rejection of allografts across a major MHC mismatch in the presence of highly activated allo-stimulated D CD4(+) T cells. Blockade of TGFß promoted rejection of allografts even following depletion of allo-stimulated D CD4(+) T cells. These studies support a crucial role for TGFß in the survival of allografts and shows that regulatory cytokines TGFß/IL2 can delay the rejection of allografts, even in the presence of highly activated alloreactive T cells.

Alterations in chemokine receptor CCR5 expression on blood dendritic cells correlate with acute graft-versus-host disease.

BACKGROUND: Dendritic cells (DC) are important in the development of acute graft-versus-host disease (GVHD) after allogeneic hemopoietic cell transplantation (alloHCT). The trafficking of immature DC from blood to GVHD target organs is likely to be regulated by chemokine receptors. METHODS: We performed flow cytometry to document the expression of chemokine receptors on circulating DC and correlated the findings after alloHCT with occurrence of acute GVHD. RESULTS: In normal individuals, plasmacytoid DC (pDC) expressed high levels of CCR5, whereas the major CD16 myeloid DC subpopulation lacked CCR5. However, its expression on CD16 cells was induced by culture in allogeneic mixed lymphocyte reaction supernatant, an effect largely mediated by interferon-γ. CCR5 was expressed on a significant proportion of CD16 DC in 42 alloHCT patients, whereas it was down-regulated on pDC. The maximum percentage of CCR5CD16 DC, at any time after transplantation, correlated with acute GVHD, whereas the minimum CCR5 on pDC showed a similar correlation. Before developing signs of GVHD, the maximum percentage CCR5CD16 DC was higher in patients with GVHD grades II to IV than in GVHD grades 0 and I, whereas the minimum percentage CCR5 on pDC was lower in GVHD grades II to IV than in GVHD grades 0 and I. CCR5 levels more than 20.5% on CD16 myeloid DC and less than 22.6% on CD123 pDC correlated with subsequent GVHD grades II to IV with high sensitivities and specificities. CONCLUSIONS: These observations may reflect DC activation and altered homing during the alloimmune response and could allow early diagnosis and therapeutic intervention before the clinical diagnosis of GVHD.

Failure to achieve a threshold dose of CD34+CD110+ progenitor cells in the graft predicts delayed platelet engraftment after autologous stem cell transplantation for multiple myeloma.

To predict platelet engraftment more accurately post autologous stem cell transplantation (SCT), we retrospectively analyzed the CD34(+)CD110(+) (CD110 or c-mpl, thrombopoietin receptor) content in the grafts of 70 patients undergoing transplantation for multiple myeloma (MM) with an in-house flow cytometric assay. We found that infusing at least 3.0 x 10(4) CD34(+)CD110(+) cells/kg clearly separated the cohort into those who achieved platelet engraftment before or after 21 days. This early megakaryocyte cell marker correlated more closely with early versus delayed platelet engraftment than CD34(+) measurements. Of the 70 patients, 4 required > or = 21 days to achieve platelet transfusion independence. Three of the 4 received a CD34(+)CD110(+) cell dose of <3.0 x 10(4) cells/kg, whereas 66 of 70 patients who received >3.0 x 10(4) CD34(+)CD110(+) cells/kg achieved platelet transfusion independence in <21 days (P < .001). Infusing >3.0 x 10(4) CD34(+)CD110(+) cells/kg was sensitive (100%) and specific (75%) for achieving platelet engraftment within 21 days. Patients with delayed platelet engraftment received a median of 2.28 x 10(4) CD34(+)CD110(+) cells/kg versus 12.1 x 10(4) cells/kg in those without this complication (P = .033). No effect was seen with neutrophil engraftment. Patients with early engraftment required a median of 1 platelet transfusion post transplant versus 2.5 in those with late engraftment (P = .009). A subthreshold absolute CD34(+)CD110(+) cell dose in the graft is a reliable predictor of delayed platelet engraftment, and could be used to guide the timing and number of peripheral blood stem cell (PBSC) collections for patients with MM undergoing an SCT.

Clinical-scale elutriation as a means of enriching antigen-presenting cells and manipulating alloreactivity.

BACKGROUND AIMS: Clinical-scale elutriation using the Elutra(c) has been shown to enrich monocytes reliably for immunotherapy protocols. Until now, a detailed assessment of the four (F1-F4) non-monocyte fractions derived from this process has not been performed. METHODS: Using fluorescence-activated cell sorting (FACS), we performed phenotypic analyses to investigate the possible enrichment of T, B, natural killer (NK) and dendritic cells (DC) or their subsets in one or more Elutra fractions. RESULTS: Blood DC were enriched up to 10-fold in some fractions (F3 and F4) compared with the pre-elutriation apheresis product. This increased the number of DC that could be isolated from a given cell number by immunomagnetic separation. It was also found that CD62L(-) effector memory CD4(+) T cells were enriched in later fractions. In four of five cases tested, cells from F3 demonstrated decreased alloreactive proliferation in a mixed lymphocyte reaction compared with cells from the apheresis product. B cells were enriched in F1 compared with the apheresis product. CONCLUSIONS: In addition to providing enrichment of monocytes for the generation of DC, the Elutra enriches cell subsets that may be incorporated into and enhance existing immunotherapy and stem cell transplantation protocols.

Prophylactic infusion of cytomegalovirus-specific cytotoxic T lymphocytes stimulated with Ad5f35pp65 gene-modified dendritic cells after allogeneic hemopoietic stem cell transplantation.

Cytomegalovirus (CMV) and its therapy continue to contribute to morbidity and mortality in hemopoietic stem cell transplantation (HSCT). Many studies have demonstrated the feasibility of in vitro generation of CMV-specific T cells for adoptive immunotherapy of CMV. Few clinical trials have been performed showing the safety and efficacy of this approach in vivo. In this study, donor-derived, CMV-specific T cells were generated for 12 adult HSCT patients by stimulation with dendritic cells transduced with an adenoviral vector encoding the CMV-pp65 protein. Patients received a prophylactic infusion of T cells after day 28 after HSCT. There were no infusion related adverse events. CMV DNAemia was detected in 4 patients after infusion but was of low level. No patient required CMV-specific pharmacotherapy. Immune reconstitution to CMV was demonstrated by enzyme linked immunospot assay in all recipients with rapid increases in predominantly CMV-pp65 directed immunity in 5. Rates of graft-versus-host disease, infection, and death were not increased compared with expected. These results add to the growing evidence of the safety and efficacy of immunotherapy of CMV in HSCT, supporting its more widespread use. This study was registered at www.anzctr.org.au as #ACTRN12605000213640.

Long-term cardiac allograft survival across an MHC mismatch after "pruning" of alloreactive CD4 T cells.

Specific tolerance to allografts has been achieved by a variety of means. We have previously shown that ex vivo removal of dividing CD4(+) T cells from an MLR or "pruning" delays skin allograft rejection. We tested pruning of alloreactive T cells as a strategy for retaining a broad T cell repertoire while removing alloreactive T cells in a model of cardiac allograft transplant. Using CFSE staining of responder BALB/c cells with stimulator C57BL/6 cells in an MLR, SCID mice were reconstituted with either dividing (D) or nondividing (ND) CD4(+) T cells derived from an MLR and then challenged with heterotopic cardiac allografts. Mice reconstituted with D CD4(+) T cells rejected cardiac allografts from the stimulator strain with a median survival time (MST) of 29 days, while mice reconstituted with ND CD4(+) T cells maintained allografts from the stimulator strain (MST of >100 days) while rejecting third-party allografts (B10.BR) (MST = 11 days). ELISPOT assays demonstrate donor-specific hyporesponsiveness of the ND CD4(+) T cells. TCR beta-chain V region (TRBV) repertoire analysis demonstrates clonal expansion within both rejecting D cardiac allografts and ND cardiac allografts surviving for the long-term. Histology showed greater allograft infiltration by the D CD4(+) T cells. The surviving ND cardiac allografts demonstrated reduced cellular infiltration and reduced incidence of allograft vasculopathy, but with the development of chronic fibrosis. Thus, pruning of alloreactive T cells allows long-term-specific cardiac allograft survival while retaining the ability to reject third-party allografts.

Ex vivo expansion and prophylactic infusion of CMV-pp65 peptide-specific cytotoxic T-lymphocytes following allogeneic hematopoietic stem cell transplantation.

Cytomegalovirus reactivation and infection post-allogeneic hematopoietic stem cell transplant continue to cause morbidity and mortality. Current pharmacologic therapies are limited by side effects. Adoptive transfer of ex vivo generated cytomegalovirus-specific T cells has the potential to restore immunity, prevent cytomegalovirus, and circumvent the need for pharmacologic therapies. We have generated donor-derived cytomegalovirus-specific cytotoxic T cells using dendritic cells pulsed with the HLA-A2 restricted nonapeptide NLVPMVATV (NLV) derived from the cytomegalovirus-pp65 protein. These cytotoxic T cells have been given prophylactically to 9 recipients aged 4 to 65 years on or after day 28 post-allogeneic hematopoietic stem cell transplant. Only 2 of 9 recipients received T cell depletion in vivo or in vitro. There were no immediate adverse reactions to the infusions. During 97-798 days of follow-up, 2 recipients developed cytomegalovirus reactivation; neither developed cytomegalovirus disease or required pharmacotherapy. Three recipients developed acute graft versus host disease after infusion. Two recipients died, 1 from thrombotic thrombocytopenia purpura secondary to cyclosporine, 1 from complications of graft versus host disease. A transient increase in numbers of cytomegalovirus-specific T cells demonstrated by NLV-tetramer binding was seen in 6 recipients. Prophylactic adoptive transfer of NLV-specific T cells is safe and may be effective in preventing cytomegalovirus reactivation.

Activated circulating dendritic cells after hematopoietic stem cell transplantation predict acute graft-versus-host disease.

BACKGROUND: Dendritic cells (DC) are central to the development of acute graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (alloHSCT). We hypothesized that DC activation status determines the severity of GVHD and that activated DC may be detected in the circulation prior to clinical presentation of GVHD. METHODS: Following transplant, blood samples were obtained twice weekly from alloHSCT patients. Myeloid (CD11c+) and plasmacytoid (CD123hi) DC were enumerated by flow cytometry, and activated myeloid DC were identified using the CMRF-44 monoclonal antibody. RESULTS: Of 40 alloHSCT patients, 26 developed acute GVHD. Severity of GVHD was associated with low total blood DC counts (P=0.007) and with low myeloid and plasmacytoid DC numbers (P=0.015 and 0.003). The CMRF-44 antigen was expressed on blood CD11c+ DC in all cases prior to GVHD onset, whereas of the 14 patients without GVHD, seven had no CMRF-44+ CD11c DC. Patients with CMRF-44+ CD11c+ DC in more than 20% of samples were more likely to subsequently develop acute GVHD (P=0.001, odds ratio=37.1), while patients who developed grade 2-4 GVHD had prior higher percentages of CMRF-44+ CD11c+ DC compared to grade 0-1 GVHD patients (P=0.001). CMRF-44 expression on >7.9% CD11c+ DC predicted for subsequent development of GVHD with a sensitivity of 87.5% and specificity of 79.2%. CONCLUSIONS: Activation status, as assessed by CMRF-44 antigen expression, of blood CD11c+ DC is highly associated with acute GVHD and these cells may be targets for therapeutic intervention.

Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray.

A novel antibody microarray has been developed that provides an extensive immunophenotype of leukaemia cells. The assay is a solid phase cell-capture technique in which 82 antigens are studied simultaneously. This paper presents the analysis of 733 patients with a variety of leukaemias and lymphomas from peripheral blood and bone marrow. Discriminant Function Analysis of the expression profiles from these 733 patients and 63 normal subjects were clustered and showed high levels of consistency with diagnoses obtained using conventional clinical and laboratory criteria. The overall levels of consensus for classification using the microarray compared with established criteria were 93.9% (495/527 patients) for peripheral blood and 97.6% (201/206 patients) for bone marrow aspirates, showing that the extensive phenotype alone was frequently able to classify the disease when the leukaemic clone was the dominant cell population present. Immunophenotypes for neoplastic cells were distinguishable from normal cells when the leukaemic cell count was at least 5 x 10(9) cells/l in peripheral blood, or 20% of cells obtained from bone marrow aspirates. This technique may be a useful adjunct to flow cytometry and other methods when an extensive phenotype of the leukaemia cell is desired for clinical trials, research and prognostic factor analysis.

"Pruning" of alloreactive CD4+ T cells using 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester prolongs skin allograft survival.

Removal of alloreactive cells by either thymic deletion or deletion/anergy in the periphery is regarded as crucial to the development of tolerance. Dyes, such as CFSE, that allow monitoring of cell division suggest that in vitro proliferation could be a used as a way of "pruning" alloreactive cells while retaining a normal immune repertoire with retention of memory to previously encountered pathogens. This would overcome the problems occurring as a result of therapies that use massive depletion of T cells to allow acceptance of organ transplants or bone marrow grafts. We therefore used a skin graft model of CD4-mediated T cell rejection across a major H-2 mismatch (C57BL/6 (H-2(b)) to BALB/c (H-2(d)) mice) to evaluate whether nondividing CD4(+) T cells derived from a mixed lymphocyte culture would exhibit tolerance to a skin graft from the initial stimulator strain. We demonstrate that selective removal of dividing alloreactive CD4(+) T cells resulted in marked specific prolongation of allogeneic skin graft survival, and that the nondividing CD4(+) T cells retained a broad TCR repertoire and the ability to maintain memory. This novel way of depleting alloreactive T cells may serve as a useful strategy in combination with other mechanisms to achieve transplant tolerance.

Matching T-cell receptors identified in renal biopsies and urine at the time of acute rejection in pediatric renal transplant patients.

Urinary monitoring of kidney allograft function has been used for many years. More recently, molecular identification of cytotoxic T-cell products has been used as a diagnostic tool in acute rejection. Monitoring of T-cell infiltrates by analysis of the T-cell receptor (TcR) gene usage has been performed on biopsies with acute and chronic rejection, but not on urine samples. The aim of this study was to identify and compare TRBV gene usage assessing the CDR3 (Complementarity Determining Region 3) length distribution and sequence in urine and biopsies of pediatric renal allograft patients at the time of acute rejection and compare them with peripheral blood. We studied four pediatric renal transplant recipients with acute cellular rejection. We identified restricted and matched TRBV CDR3 spectratypes with overexpressed TRBV families and show identical, clonally expanded TRBV CDR3 sequences in all four patients present in the urine and renal allograft. We demonstrate that urinary monitoring can detect graft-infiltrating lymphocytes in acute rejection and may have a role in the monitoring of renal transplants.

Human CD62L- memory T cells are less responsive to alloantigen stimulation than CD62L+ naive T cells: potential for adoptive immunotherapy and allodepletion.

Selective depletion of alloreactive T cells from allogeneic stem cell grafts can reduce graft-versus-host disease (GVHD) while preserving beneficial effects of T cells including facilitation of engraftment, protection against opportunistic infection, and reduced relapse risk. Memory T cells (CD62L(-)) represent a population of T cells that have previously encountered pathogens and may contain fewer T cells capable of recognizing neoantigens including recipient allogeneic antigen (aAg). We investigated whether human naive (CD62L(+)) or memory (CD62L(-)) T cells had different capacities to respond to aAg by assessing their ability to proliferate in response to and lyse HLA-mismatched Epstein-Barr virus-transformed B cells. Freshly sorted and in vitro expanded CD62L(-) memory T cells were less responsive to aAg stimulation than were CD62L(+) naive T cells but contained higher levels of cytomegalovirus (CMV)-specific T cells. Analysis of T cell receptor (TCR) repertoire showed restricted TCR diversity in the memory T-cell population possibly due to selection associated with chronic exposure to common pathogens. Memory T cells may represent a donor cell subpopulation suitable for enhancing immune reconstitution without increasing the risk of GVHD.

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.