Ex Vivo Generation of Donor Antigen-Specific Immunomodulatory Cells: A Comparison Study of Anti-CD80/86 mAbs and CTLA4-lg Costimulatory Blockade.

Adoptive transfer of alloantigen-specific immunomodulatory cells generated ex vivo with anti-CD80/CD86 mAbs (2D10.4/IT2.2) holds promise for operational tolerance after transplantation. However, good manufacturing practice is required to allow widespread clinical application. Belatacept, a clinically approved cytotoxic T-lymphocyte antigen 4-immunoglobulin that also binds CD80/CD86, could be an alternative agent for 2D10.4/IT2.2. With the goal of generating an optimal cell treatment with clinically approved reagents, we evaluated the donor-specific immunomodulatory effects of belatacept- and 2D10.4/IT2.2-generated immunomodulatory cells. Immunomodulatory cells were generated by coculturing responder human peripheral blood mononuclear cells (PBMCs) (50 × 106 cells) with irradiated donor PBMCs (20 × 106 cells) from eight human leukocyte antigen-mismatched responder-donor pairs in the presence of either 2D10.4/IT2.2 (3 µg/106 cells) or belatacept (40 µg/106 cells). After 14 days of coculture, the frequencies of CD4+ T cells, CD8+ T cells, and natural killer cells as well as interferon gamma (IFN-γ) production in the 2D10.4/IT2.2- and belatacept-treated groups were lower than those in the control group. The percentage of CD19+ B cells was higher in the 2D10.4/IT2.2- and belatacept-treated groups than in the control group. The frequency of CD4+CD25+CD127lowFOXP3+ T cells increased from 4.1±1.0% (preculture) to 7.1±2.6% and 7.3±2.6% (day 14) in the 2D10.4/IT2.2- and belatacept-treated groups, respectively (p<0.05). Concurrently, delta-2 FOXP3 mRNA expression increased significantly. Compared with cells derived from the no-antibody treated control group, cells generated from both the 2D10.4/IT2.2- and belatacept-treated groups produced lower IFN-γ and higher interleukin-10 levels in response to donor-antigens, as detected by enzyme-linked immunospot. Most importantly, 2D10.4/IT2.2- and belatacept-generated cells effectively impeded the proliferative responses of freshly isolated responder PBMCs against donor-antigens. Our results indicate that belatacept-generated donor-specific immunomodulatory cells possess comparable phenotypes and immunomodulatory efficacies to those generated with 2D10.4/IT2.2. We suggest that belatacept could be used for ex vivo generation of clinical grade alloantigen-specific immunomodulatory cells for tolerance induction after transplantation.

Optimized processing for pathogen inactivation of double-dose buffy-coat platelet concentrates: maintained in vitro quality over 7-day storage.

BACKGROUND AND OBJECTIVE: Efficient pathogen inactivation (PI) offers the possibility of increasing the number of buffy coats per pool without the concurrent increased risk of pathogen transmission. Here, we describe the findings of in vitro analyses of platelets from pools of eight buffy coats treated with amotosalen and UVA light (INTERCEPT Blood System for Platelets) using INTERCEPT disposable processing sets with plastic materials sourced from alternate suppliers and split afterwards to obtain two therapeutic transfusion doses. METHODS: Double-dose platelet concentrates were prepared from pools of eight buffy coats in additive solution (SSP+) using either previous 6-lead or new 8-lead pooling sets and PI processing sets in previous or alternate supplier sourced plastics (AS). Platelets were treated with the INTERCEPT Blood System then stored for up to 7 days and tested for in vitro quality. RESULTS: All tested units (n = 30) were in conformity with European guidelines. Using AS sets more effectively maintained glucose reserves (P < 0·01), reduced lactate production (P < 0·01), reduced CD62P expression (P < 0·01) and downregulated levels of surface CD42b (P < 0·01) overtime. AS set maintained JC-positive cells (NS) between day 2 and day 7 and sustained platelet integrin activation (PAC-1) between day 2 and day 7 (NS). Overall sCD40L and PGDF accumulated in an equivalent way (P < 0·01) within series. SUMMARY/CONCLUSIONS: In summary, our data demonstrate that PI treatment using AS sets, in combination with the new pooling set for double-dose platelet preparation, maintained the platelet in vitro quality over 7 days of storage.

Change of apheresis device decreased the incidence of severe acute graft-versus-host disease among patients after allogeneic stem cell transplantation with sibling donors.

BACKGROUND: The composition of the graft used for allogeneic hematopoietic stem cell transplantation (HSCT) is important for the treatment outcome. Different apheresis devices may yield significant differences in peripheral blood stem cell graft cellular composition. We compared stem cell grafts produced by Cobe Spectra (Cobe) and Spectra Optia (Optia) with use of the mononuclear cell (MNC) protocol, and evaluated clinical outcome parameters such as graft-versus-host disease (GvHD), transplant-related mortality (TRM), relapse, and overall survival. STUDY DESIGN AND METHODS: During 5 years, 31 Cobe Spectra and 40 Spectra Optia grafts were analyzed for CD34, CD3, CD4, CD8, CD19, and CD56 cell content. Clinical outcome parameters were correlated and compared between the two patient groups using different apheresis devices. RESULTS: Optia grafts contained fewer lymphocytes compared to Cobe (p < 0.001). Optia grafts had a significantly lower incidence of acute GvHD Grades II through IV (Cobe 45% vs. Optia 23%; p = 0.039) and TRM (16% vs. 2.5%; p < 0.05) but higher chronic GvHD (32% vs. 67%; p = 0.005) compared to Cobe grafts. Finally, the multivariate analysis showed a significant correlation among the different apheresis devices and both acute GvHD II through IV and severe chronic GvHD. The multivariate analysis also showed a significant correlation between the CD3+ cell dose and the incidence of severe acute GvHD. CONCLUSION: Optia-obtained grafts yielded a lower acute GvHD Grades II-IV and TRM risk, but had no impact on relapse or overall survival in this study. Understanding and further improvement of peripheral blood stem cell (PBSC) apheresis techniques may be used in the future to personalize HSCT by, for example, fine-tuning the GvHD incidence.

Assessment of TREC, KREC and telomere length in long-term survivors after allogeneic HSCT: the role of GvHD and graft source and evidence for telomere homeostasis in young recipients.

Reconstitution of the adaptive immune system following allogeneic hematopoietic stem cell transplantation is crucial for beneficial outcome and is affected by several factors, such as GvHD and graft source. The impact of these factors on immune reconstitution has been thoroughly investigated during the early phase after transplantation. However, little is known about their long-term effect. Similarly, leukocyte telomere length (TL) shortening has been reported shortly after transplantation. Nevertheless, whether TL shortening continues in long-term aspect is still unsettled. Here, we assessed T-cell receptor excision circle (TREC), kappa deleting recombination excision circle (KREC) and leukocyte TL in recipients and donors several years post transplantation (median 17 years). Our analysis showed that, recipients who received bone marrow (BM) as the graft source have higher levels of both TREC and KREC. Also, chronic GvHD affected TREC levels and TL but not KREC levels. Finally, we show that recipient's TL was longer than respective donors in a group of young age recipients with high KREC levels. Our results suggest that BM can be beneficial for long-term adaptive immune recovery. We also present supporting evidence for recipient telomere homeostasis, especially in young age recipients, rather than telomere shortening.

Functionality testing of stem cell grafts to predict infectious complications after allogeneic hematopoietic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Allogeneic hematopoietic stem cell transplantation (HSCT) is a routine clinical procedure performed to treat patients with haematological malignancies, primary immune deficiencies or metabolic disorders. Infections during lymphopenia after allogeneic HSCT are associated with high mortality and morbidity. Typical infectious agents are Epstein-Barr virus, cytomegalovirus, herpes simplex virus, varicella-zoster virus and fungi. The study aim was to evaluate whether measurement of the responses of antigen-specific T-cells, recognizing infectious pathogens would correlate to protective functions in the stem cell recipient post-transplant. MATERIALS AND METHODS: Twenty-one grafts were analysed by flow cytometry and cells were stimulated in vitro with relevant infectious antigens, followed by evaluation of T-cell proliferation and cytokine production. Results were compared to the recipients' clinical records 1-year post-transplantation. RESULTS: We show that an extensive repertoire of transferred antigen-specific T-cells from allogeneic donor grafts against infectious agents, involved in post-transplant infections, are linked to an absence of infectious complications for the recipient up-to 1-year post-transplant. The protective effect was associated with antigen-specific T-cell proliferation and IL-1ß secretion. CONCLUSION: Our results suggest that assaying T-cell function before HSCT could determine individual risks for infectious complications and thus aid in clinical decision-making regarding prophylactic and pre-emptive anti-infective therapy.

Alpha/beta T-cell depleted grafts as an immunological booster to treat graft failure after hematopoietic stem cell transplantation with HLA-matched related and unrelated donors.

Allogeneic hematopoietic stem cell transplantation is associated with several complications and risk factors, for example, graft versus host disease (GVHD), viral infections, relapse, and graft rejection. While high levels of CD3+ cells in grafts can contribute to GVHD, they also promote the graft versus leukemia (GVL) effect. Infusions of extra lymphocytes from the original stem cell donor can be used as a treatment after transplantation for relapse or poor immune reconstitution but also they increase the risk for GVHD. In peripheral blood, 95% of T-cells express the αß T-cell receptor and the remaining T-cells express the γδ T-cell receptor. As αß T-cells are the primary mediators of GVHD, depleting them from the graft should reduce this risk. In this pilot study, five patients transplanted with HLA-matched related and unrelated donors were treated with αß T-cell depleted stem cell boosts. The majority of γδ T-cells in the grafts expressed Vδ2 and/or Vγ9. Most patients receiving αß-depleted stem cell boosts increased their levels of white blood cells, platelets, and/or granulocytes 30 days after infusion. No signs of GVHD or other side effects were detected. A larger pool of patients with longer follow-up time is needed to confirm the data in this study.

Cord blood graft composition impacts the clinical outcome of allogeneic stem cell transplantation.

INTRODUCTION: Despite routine use of umbilical cord blood (CB) grafts as stem cell source for allogeneic stem cell transplantations, much remains unknown regarding their cell composition and correlation with clinical outcome. METHODS: We analyzed material from 30 CB units used for allogeneic hematopoietic stem cell transplantation by multicolor flow cytometry. Phenotypic data were correlated with various clinical outcomes such as survival, graft-versus-host disease (GVHD), relapse, rejection, viral reactivation, and bacteremia. RESULTS: We found that above-median frequencies of CD69+ T cells, naïve CD8+ T cells, and CD127+ B cells in the CB graft were each associated with significantly improved patient survival. Moreover, a statistically significant correlation was seen between higher levels of CD94+ T cells and herpes simplex virus and varicella zoster virus reactivation post transplantation. A similar correlation was seen for the frequency of CD95+ cells in total CD3+, as well as CD4+ and CD8+ T-cell subsets, and viral reactivation. Finally, a higher frequency of naïve CD8+ T cells was associated with the incidence of acute GVHD. CONCLUSION: Our study highlights the importance of further exploration of graft composition before CB transplantation as a tool for risk prediction.

Characterization of long-term mixed donor-donor chimerism after double cord blood transplantation.

Double cord blood transplantation (DCBT) with two matched or partially matched cord blood units has been implemented successfully to circumvent the limitations of graft cell dose associated with single CBT. After DCBT, sustained haematopoiesis is derived almost exclusively from only one of the donated units. None the less, we previously observed two of six evaluable DCBT patients still having mixed donor-donor chimerism at 28 and 45 months post-transplantation, respectively. In the present study we utilize flow cytometry techniques to perform the first thorough analysis of phenotype and functionality of cord blood units in patients with mixed donor-donor chimerism. Our results suggest that the two stable cord blood units are different phenotypically and functionally: one unit shows more naive T cells, lower T cell cytokine production and higher frequencies of natural killer cells, the other shows higher frequencies of well-differentiated and functional lymphocytes. Additionally, in comparison with control patients having a single prevailing cord blood unit, the patients with donor-donor chimerism exhibit less overall T cell cytokine production and a smaller fraction of memory T cells. Furthermore, our results indicate that human leucocyte antigen-C match of donor units may partly explain the development of a donor-donor mixed chimerism.

Cytotoxic crossmatch analysis before allo-SCT is a poor diagnostic tool for prediction of rejection.

The predictive value of cytotoxic crossmatch analysis before allo-SCT remains unclear. We retrospectively analyzed the clinical impact of cytotoxic T- and B-cell crossmatch testing before allo-SCT between January 2000 and June 2005. Cytotoxic crossmatches were performed in 157 patients receiving stem cells from matched unrelated donors or an HLA-A, -B or -DRB1 allele mismatched graft. Ninety patients are still alive. Eleven patients rejected their grafts. One of 11 patients with rejection was positive in a T-cell crossmatch before allo-SCT and 4 of 11 in B-cell crossmatches. T-cell crossmatches showed a sensitivity of 9% and a specificity of 97% compared with 36 and 86% for B-cell crossmatches. Positive T- and/or B-crossmatch before SCT had no predictive value for survival in this study as compared with patients with a negative crossmatch. In conclusion, the pretransplant cytotoxic T- and/or B-crossmatch is a poor predictor of rejection after allo-SCT.

Unrelated cord blood and mismatched unrelated volunteer donor transplants, two alternatives in patients who lack an HLA-identical donor.

The aim was to evaluate two transplant strategies for patients who lack HLA-identical donors, namely HLA-A, HLA-B or -DR beta 1 mismatched unrelated donor (MM URD) transplants (n=14) and umbilical cord blood transplants (UCB, n=27). Diagnosis, disease stage and age were similar in the two groups. Cell dose was lower in the UCB group (P<0.001). Median time to ANC of >0.5 x 10(9)/l was 30 days in the UCB group and 17 days in the MM URD group (P=0.002). Engraftment of plt was delayed in the UCB group (P=0.03). The UCB patients required fewer erythrocyte transfusions (P=0.001). At 100 days, complete donor chimerism for CD3 was 63 and 44% in the UCB and MM URD groups, respectively. Acute GVHD of grades II-IV were 30% in the UCB group and 21% in the MM URD group. The corresponding figures for chronic GVHD were 9 and 20%, respectively. TRM was 30% in the UCB patients and 50% in the MM URD patients. Three-year survival was 66% in the UCB group and 14% in the MM URD group (P=0.006). Although the material is small and heterogeneous, engraftment was delayed, leukocyte chimerism was not significantly different and survival was superior using UCB rather than MM URD transplants.

Is the activity of partially agonistic MHC:peptide ligands dependent on the quality of immunological help?

CD8(+) cytotoxic T lymphocytes (CTL) are important for the immunological control of infections and tumours. Engagement of the T-cell receptor (TCR) with major histocompatibility complex (MHC) class I/peptide complexes on antigen-presenting cells (APC) is the key interaction, which initiates the process of T-cell activation. Depending on the affinity of this interaction, different arrays of signalling pathways and functional outcomes can be activated in the specific T cells. Molecular alterations in the peptide bound to the MHC class I can lead to a lower affinity of the MHC:TCR interaction resulting in incomplete or qualitatively different T-cell responses. Altered peptide ligands (APL) exhibiting such activity are referred to as partial agonists and often occur naturally through genetic instability, which affects T-cell epitopes derived from rapidly mutating viruses or tumour-associated cellular antigens. Partial agonists are usually viewed as peptide variants, which escape efficient CTL recognition. Our recent data suggest that APL can not only trigger incomplete activation but also induce and modulate intrinsic T-cell programmes leading to the shut-off of specific CTL responses. This APL-induced suppression appears to be more prominent in the absence of immunological help, suggesting that under conditions of immune deregulation APL may actively inhibit CTL responses against infectious agents or tumours. In this review, we discuss experimental data supporting this model and possible role of APL-induced immunosuppression in different pathological conditions.

Pharmacological disintegration of lipid rafts decreases specific tetramer binding and disrupts the CD3 complex and CD8 heterodimer in human cytotoxic T lymphocytes.

Accumulating evidence strongly supports the role of lipid rafts in the regulation of T-lymphocyte activation, but the organization and molecular composition of these cholesterol- and sphingolipid-rich membrane microdomains in different subsets of T cells remain poorly investigated. Here, we show that pharmacological disruption of lipid rafts in human CD8+ cytotoxic T-lymphocyte (CTL) clones disturbs the integrity of CD3 complex and CD8 heterodimer, without affecting the reactivity with T-cell receptor (TCR)-specific antibodies. This demonstrates that interaction with completely assembled CD3 complex is not required for the stable expression of TCR at the cell surface. The effect of raft disruption on CD3 and CD8 expression correlates with failure to bind specific tetrameric complexes by a proportion of surface TCR molecules. However, the interaction of specific tetramer with the rest of surface TCR pools appears to be unaffected, demonstrating that TCR-signalling complexes may differ in their requirement for cholesterol to stably maintain their composition and to rearrange for efficient tetramer binding. Together with previously published data, our results support the existence of molecular and/or structural heterogeneity of lipid rafts that may play an important role in controlling distinct functional properties of T-cell subsets.