CD4+ T Cell Dependent B Cell Recovery and Function After Autologous Hematopoietic Stem Cell Transplantation

Introduction: High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) represents a standard treatment regime for multiple myeloma (MM) patients. Common and potentially fatal side effects after auto-HSCT are infections due to a severely compromised immune system with hampered humoral and cellular immunity. This study delineates in depth the quantitative and functional B cell defects and investigates underlying extrinsic or intrinsic drivers. Methods: Peripheral blood of MM patients undergoing high-dose chemotherapy and auto-HSCT (before high-dose chemotherapy and in early reconstitution after HSCT) was studied. Absolute numbers and distribution of B cell subsets were analyzed ex vivo using flow cytometry. Additionally, B cell function was assessed with T cell dependent (TD) and T cell independent (TI) stimulation assays, analyzing proliferation and differentiation of B cells by flow cytometry and numbers of immunoglobulin secreting cells in ELISpots. Results: Quantitative B cell defects including a shift in the B cell subset distribution occurred after auto-HSCT. Functionally, these patients showed an impaired TD as well as TI B cell immune response. Individual functional responses correlated with quantitative alterations of CD19+, CD4+, memory B cells and marginal zone-like B cells. The TD B cell function could be partially restored upon stimulation with CD40L/IL-21, successfully inducing B cell proliferation and differentiation into plasmablasts and immunoglobulin secreting cells. Conclusion: Quantitative and functional B cell defects contribute to the compromised immune defense in MM patients undergoing auto-HSCT. Functional recovery upon TD stimulation and correlation with CD4+ T cell numbers, indicate these as extrinsic drivers of the functional B cell defect. Observed correlations of CD4+, CD19+, memory B and MZ-like B cell numbers with the B cell function suggest that these markers should be tested as potential biomarkers in prospective studies

Single-cell clonal tracking of persistent T-cells in allogeneic hematopoietic stem cell transplantation

The critical balance between intended and adverse effects in allogeneic hematopoietic stem cell transplantation (alloHSCT) depends on the fate of individual donor T-cells. To this end, we tracked αβT-cell clonotypes during stem cell mobilization treatment with granulocyte-colony stimulating factor (G-CSF) in healthy donors and for six months during immune reconstitution after transfer to transplant recipients. More than 250 αβT-cell clonotypes were tracked from donor to recipient. These clonotypes consisted almost exclusively of CD8+ effector memory T cells (CD8TEM), which exhibited a different transcriptional signature with enhanced effector and cytotoxic functions compared to other CD8TEM. Importantly, these distinct and persisting clonotypes could already be delineated in the donor. We confirmed these phenotypes on the protein level and their potential for selection from the graft. Thus, we identified a transcriptional signature associated with persistence and expansion of donor T-cell clonotypes after alloHSCT that may be exploited for personalized graft manipulation strategies in future studies

Antithymocyte globulin and T-cell reconstitution after allogeneic hematopoietic stem cell transplantation in adult patients

Einleitung: Die zeitgerechte Rekonstitution eines effektiven Immunsystems gehört zu den entscheidenden Faktoren für einen positiven klinischen Verlauf nach allogener hämatopoetischer Stammzelltransplantation (alloHSZT). Die wirksame Prophylaxe der Transplantat-gegen-Empfänger-Erkrankung (englisch: Graft-versus-Host-Disease, GvHD) ist ebenfalls wichtig für das Gesamtüberleben nach alloHSZT. Aus Kaninchen gewonnenes Antithymozyten-Globulin-Genzyme® (ATG-G [Thymoglobuline®]) wird häufig zur GvHD-Prophylaxe verwendet. Durch seine Lymphozyten-depletierende Wirkung trägt ATG-G jedoch zur Lymphopenie nach Transplantation bei. Die vorliegende Arbeit soll zum besseren Verständnis der Wirkung von ATG-G auf die Rekonstitution verschiedener T-Lymphozyten- Subpopulationen und die thymische Funktion beitragen. Methodik: Vielfarben- Durchflusszytometrie wurde für phänotypische Analysen genutzt, um die Rekonstitution verschiedener T-Lymphozyten-Subpopulationen nach alloHSZT in 20 erwachsenen Patienten mit malignen hämatologischen Erkrankungen, die mit (n=12) und ohne (n=8) ATG-G behandelt wurden, zu bestimmen. Hierfür wurden Blutproben vor und bis zu 6 Monate nach Transplantation gesammelt. Apoptose und Nekrose von humanen Thymozyten und peripheren mononukleären Blutzellen wurden nach Behandlung mit verschiedenen ATG-Präparaten in vitro analysiert. Außerdem wurden Blutproben kryokonserviert für eine spätere Analyse des Gehalts an T-Zell-Rezeptor-Exzisions-Kreisen (englisch: T cell receptor excision circle, TREC) sowie kappa-deletierenden Rekombinations-Exzisions- Kreisen (englisch: kappa-deleting recombination excision circle, KREC). Ergebnisse: Patienten, die im Rahmen einer alloHSZT mit ATG-G behandelt wurden, zeigten nachhaltig erniedrigte Werte naiver konventioneller (englisch: conventional T cells, Tcon) sowie regulatorischer (englisch: regulatory T cells, Treg) CD4+ T-Lymphozyten. Die thymische Produktion dieser Zellen – gemessen anhand des Oberflächenmoleküls CD31 auf naiven (CD45RA+) Tcon und Treg – war für 6 Monate nach Transplantation signifikant niedriger in ATG-G-behandelten als in nicht mit ATG-G behandelten Patienten. Im Gedächtniskompartiment (CD45RA-) wurde sowohl für CD4+ als auch für CD8+ T-Lymphozyten eine Verschiebung hin zu einem Effektor-Gedächtnis-Phänotyp beobachtet, ohne dass dies mit erhöhten GvHD-Raten einherging. Weiterhin hatte ATG-G zytotoxische Effekte auf humane Thymozyten in vitro. Die simultane Messung des TREC- und KREC-Gehalts in den gesammelten Blutproben (durchgeführt durch andere Gruppenmitglieder) zeigte sich geeignet zur Verlaufskontrolle der T- und B-Zellrekonstitution nach alloHSZT. Schlussfolgerung: Eine ATG-G-Behandlung im Rahmen der alloHSZT beeinträchtigt die thymische Regeneration von Tcon und Treg nach alloHSZT, und ATG-G hat vergleichsweise starke zytotoxische Effekte auf Thymozyten in vitro. Dies lässt vermuten, dass insbesondere ATG-G-behandelte Patienten von Thymus-protektiven Strategien bei alloHSZT profitieren könnten. Verschiedene ATG-Präparate sollten in kontrollierten Studien verglichen werden. Eine potenziell durch ATG-G verursachte Verschiebung hin zu einem Effektor-Gedächtnis-Phänotyp in CD4+ und CD8+ T-Lymphozyten geht nicht mit stark erhöhten GvHD-Raten einher.Introduction: Timely and effective immune reconstitution is one of the essential factors for a positive outcome of allogeneic hematopoietic stem cell transplantation (alloHSCT). Equally important is the potent prophylaxis of graft-versus-host disease (GvHD). Rabbit antithymocyte globulin-Genzyme® (ATG-G [Thymoglobuline®]) is commonly used to prevent GvHD. As a lymphocyte- depleting agent it adds, however, to lymphopenia post transplantation. The aim of this study was to contribute to the understanding of the effect of ATG-G on the reconstitution of different T-lymphocyte subsets and on thymic function in particular. Methods: Multicolor flow cytometry was used for phenotypic analysis of T-lymphocyte subset reconstitution after alloHSCT in 20 adult patients with hematologic malignancies treated (n=12) or not treated (n=8) with ATG-G. Blood samples were taken before and up to 6 months after transplantation. Apoptosis and necrosis of human thymocytes and peripheral mononuclear blood cells (PBMC) after treatment with different ATG-preparations was analyzed in vitro. Additionally, blood samples were frozen for later simultaneous evaluation of T cell receptor excision circle (TREC) and kappa- deleting recombination excision circle (KREC) content. Results: Patients undergoing alloHSCT and treated with ATG-G showed sustained low levels of naive conventional (Tcon) as well as regulatory (Treg) CD4+ T-lymphocytes. Thymic output of CD4+ T-lymphocytes in this group measured as CD31+ naive (CD45RA+) Tcon and Treg remained significantly lower for 6 months after transplantation than in patients not treated with ATG-G. In the memory (CD45RA-) compartment for both, CD4+ and CD8+ T-lymphocytes, characteristic shifts towards an effector memory phenotype could be observed in ATG-G treated patients without increased levels of GvHD. Furthermore, ATG-G had cytotoxic effects on human thymocytes in vitro. Simultaneous measurement of TREC and KREC content in the collected blood samples (conducted by other group members) proved suitable for immune monitoring of T cell (TREC) and B-cell (KREC) reconstitution after alloHSCT. Conclusion: ATG-G treatment in combination with the conditioning regimen impairs thymic production of Tcon and Treg in alloHSCT patients, and ATG-G has comparatively strong cytotoxic effects on human thymocytes in vitro. These results indicate, that especially patients treated with ATG-G could benefit from thymus-protective strategies and that controlled trials comparing different ATG-G-preparations are required. A shift towards an effector memory phenotype of CD4+ and CD8+ T lymphocytes potentially induced by ATG-G does not accelerate GvHD

Hospital payment systems based on diagnosis-related groups: experiences in low- and middle-income countries

OBJECTIVE: This paper provides a comprehensive overview of hospital payment systems based on diagnosis-related groups (DRGs) in low- and middle-income countries. It also explores design and implementation issues and the related challenges countries face. METHODS: A literature research for papers on DRG-based payment systems in low- and middle-income countries was conducted in English, French and Spanish through Pubmed, the Pan American Health Organization’s Regional Library of Medicine and Google. FINDINGS: Twelve low- and middle-income countries have DRG-based payment systems and another 17 are in the piloting or exploratory stage. Countries have chosen from a wide range of imported and self-developed DRG models and most have adapted such models to their specific contexts. All countries have set expenditure ceilings. In general, systems were piloted before being implemented. The need to meet certain requirements in terms of coding standardization, data availability and information technology made implementation difficult. Private sector providers have not been fully integrated, but most countries have managed to delink hospital financing from public finance budgeting. CONCLUSION: Although more evidence on the impact of DRG-based payment systems is needed, our findings suggest that (i) the greater portion of health-care financing should be public rather than private; (ii) it is advisable to pilot systems first and to establish expenditure ceilings; (iii) countries that import an existing variant of a DRG-based system should be mindful of the need for adaptation; and (iv) countries should promote the cooperation of providers for appropriate data generation and claims management