YEARS

-

AUTHORS

Frances Hakim

TITLE

Immune reconstitution following autologous and allogeneic stem cell transplant

ABSTRACT

The specific projects in which the Preclinical Service has become involved were initiated by transplantation protocols implemented by the clinical staff of Experimental Transplantation and Immunology Branch. We have cryopreserved patient lymphocytes from peripheral blood and marrow, tumor and CGVHD tissue biopsies, as well as plasma, and created a searchable database linking samples to protocol and transplant time points. We have then examined these samples for lymphocyte subsets, cytokine content and T cell receptor repertoire diversity and thymopoietic activity. All data are incorporated into protocol-specific spreadsheets, linking samples to protocol arms and transplant time points, and are accessible by branch clinicians over secure NIH networks. Effects of IL-7 administration: In association with a Phase I clinical trial of IL-7 administration (P. I. Claude Sportes 03-C-0152, in collaboration with Crystal Mackall in POB), the effects of this therapy were assessed by multi-parameter flow cytometry, quantitation of T cell receptor rearrangement excision circles (TREC), and spectratyping of T cell receptor repertoire diversity. In this trial IL-7 therapy was found to significantly increase peripheral blood CD4 and CD8 T cell populations. We further analyzed patient samples to investigate mechanism of action. We determined that IL-7 treatment produced a marked increase in the relative proportion of nave and central memory T cells, at the expense of effector T cells, especially the CD45RA+CD27- effector CD8 populations. IL-7 treatment produced an increase in the percentage of peripheral blood T cells in cycle (ki-67+) and increased cellular levels of the anti-apoptotic factor bcl-2; the differential effects of IL-7 on T subset expansion were associated with the persistence of the proliferation and apoptotic protection in nave cells, as compared to effector cells. Furthermore, we determined that these shifts were associated with a dilution of TREC in the most nave CD31+CD45RA+ population of CD4 cells, consistent with a primary mechanism for IL-7 therapy of thymic-independent expansion of peripheral T cell populations. Finally, IL-7 was shown to produce a significant increase in the diversity of the overall T cell receptor repertoire. These assays contributed to an understanding of the mechanism of IL-7 effects. Furthermore they supported the use of IL-7 therapy to enhance T cell repertoire and immune functional competence in patients with limited thymopoietic capacity, such as in aging populations and in patients with immune deficits after chemotherapy. Immune reconstitution following intensive lymphodepletion and autologous CD34+ stem cell transplantation in patients with severe systemic lupus erythematosus (SLE). In an ongoing trial (P.I. Steven Pavletic 04-C-0095), T and B cell immune reconstitution has been monitored by multi-parameter flow cytometry, TREC quantitation and plasma cytokine assays to evaluate recovery of thymopoiesis, reconstitution of naive, memory and effector T cell populations and normalization of immune function in patients with severe autoimmunity. These studies assessed the efficacy of the cytoreductive regimen in depleting T and B cells and the timecourse of immune recovery. These studies contribute to development of autologous transplant as a therapeutic modality in treatment of severe lupus and to an understanding of immune system homeostasis and regeneration in the context of autoimmunity. Immune populations and dysfunction in chronic graft vs host disease: In an ongoing trial (P.I. Steven Pavletic 04-C-0281), patients who have developed chronic GVHD following allogeneic transplantation are evaluated at NIH in a multi-institute natural history trial. The PCS core has evaluated T and B cell immune reconstitution and function in these patients by multi-parameter flow cytometry, TREC and cytokine assays, and by PCR analysis and confocal microscopy of tissue biopsies stained by fluorescent immunohistochemistry. These assays support the ongoing multidisciplinary natural history study of chronic GVHD, by contributing to the identification of biomarkers and to an understanding of chronic GVHD pathogenesis. We have determined that elevated levels of B cell activating factor of the TNF family (BAFF)in chronic GVHD are significantly correlated both with elevated plasma levels of inflammatory cytokines and with reduced levels of circulating B cells. Furthermore we have linked these elevated BAFF levels with persistence of transitional B cells, consistent with a reduction in negative selection of autoreactive cells. In addition, studies of peripheral blood populations and cytokines have been correlated with immunohistochemical and PCR analyses (performed by an ETIB clinical research fellow, M. Imanguli, working in the Core) of oral biopsy tissues from patients in the chronic GVHD protocol. These studies have demonstrated the involvement of cytotoxic T cell effectors in oral CGVHD. Finally, we support a therapeutic trial for bronchiolitis obliterans, a severe complication of chronic GVHD (P. I. Ronald Gress and Kirsten Williams 08-C-0097). This trial tests the efficacy of a drug blocking the leukotriene pathway to inhibit the progression of fibrosis in this obstructive lung disorder. The core provides multi-parameter flow cytometric assessment of leukotriene receptor levels in peripheral blood leukocytes and in bronchial lavage cells. Cytokine and monokine production following Th2 and Th2/Tc2 therapy based transplant regimens: An ongoing project has assessed lymphokine and monokine production capacity post transplant, focusing on allogeneic transplants incorporating immune therapy with Th2, Th2/Tc2 or Th2.rapa donor-derived cells (P. I. Dan Fowler, 04-C-0055, 04-C-0131). In the early post-transplant period and at regular intervals thereafter, the cytokine and monokine production capacity of peripheral blood cells has been determined by generation of anti CD3/anti CD28-stimulated and bacterial lipopolysaccharide (LPS) stimulated supernatants and by the assessment of the frequencies of cytokine producing cells by flow cytometry. These assays support branch transplant protocols by assessing the use of monokine supernatants and frequency of monokine producing cells as a potential biomarkers predicting acute GVHD. Furthermore they evaluate the efficacy and durability of the cytokine shift resulting from the infusion of Th2/Tc2 and Th2-rapa cells. Immune reconstitution following lymphodepletion: In several ongoing ETIB clinical trials of allogeneic and autologous stem cell transplantation therapies (04-C-0055 and 07-C-0195; PIs Daniel Fowler and Michael Bishop) as well as in continuing follow-up of patients treated in earlier trials (96-C-0104, 99-C-0143, 03-C-0077; P.I.s Claude Sportes, Daniel Fowler, Michael Bishop), the process of immune reconstitution has been assessed by multiparameter flow cytometry, functional assays, quantitation of TREC, spectratyping analyses of repertoire diversity and ELISA assays of plasma cytokines. These studies have assessed the contributions of thymopoiesis and homeostatic cytokines in the recovery of CD4 and CD8 T cell populations following autologous stem cell transplantation. These data now are used as a basis of comparison in patient [summary truncated at 7800 characters]

FUNDED PUBLICATIONS

  • Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets.
  • Association of serum interleukin-7 levels with the development of acute graft-versus-host disease.
  • Increased T-bet+ cytotoxic effectors and type I interferon-mediated processes in chronic graft-versus-host disease of the oral mucosa.
  • TRECing long-term success in SCID.
  • Up-regulation of NK cell activating receptors following allogeneic hematopoietic stem cell transplantation under a lymphodepleting reduced intensity regimen is associated with elevated IL-15 levels.
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    1 grants:7210012034f4fe86f77eb644cd39b474 sg:abstract The specific projects in which the Preclinical Service has become involved were initiated by transplantation protocols implemented by the clinical staff of Experimental Transplantation and Immunology Branch. We have cryopreserved patient lymphocytes from peripheral blood and marrow, tumor and CGVHD tissue biopsies, as well as plasma, and created a searchable database linking samples to protocol and transplant time points. We have then examined these samples for lymphocyte subsets, cytokine content and T cell receptor repertoire diversity and thymopoietic activity. All data are incorporated into protocol-specific spreadsheets, linking samples to protocol arms and transplant time points, and are accessible by branch clinicians over secure NIH networks. Effects of IL-7 administration: In association with a Phase I clinical trial of IL-7 administration (P. I. Claude Sportes 03-C-0152, in collaboration with Crystal Mackall in POB), the effects of this therapy were assessed by multi-parameter flow cytometry, quantitation of T cell receptor rearrangement excision circles (TREC), and spectratyping of T cell receptor repertoire diversity. In this trial IL-7 therapy was found to significantly increase peripheral blood CD4 and CD8 T cell populations. We further analyzed patient samples to investigate mechanism of action. We determined that IL-7 treatment produced a marked increase in the relative proportion of nave and central memory T cells, at the expense of effector T cells, especially the CD45RA+CD27- effector CD8 populations. IL-7 treatment produced an increase in the percentage of peripheral blood T cells in cycle (ki-67+) and increased cellular levels of the anti-apoptotic factor bcl-2; the differential effects of IL-7 on T subset expansion were associated with the persistence of the proliferation and apoptotic protection in nave cells, as compared to effector cells. Furthermore, we determined that these shifts were associated with a dilution of TREC in the most nave CD31+CD45RA+ population of CD4 cells, consistent with a primary mechanism for IL-7 therapy of thymic-independent expansion of peripheral T cell populations. Finally, IL-7 was shown to produce a significant increase in the diversity of the overall T cell receptor repertoire. These assays contributed to an understanding of the mechanism of IL-7 effects. Furthermore they supported the use of IL-7 therapy to enhance T cell repertoire and immune functional competence in patients with limited thymopoietic capacity, such as in aging populations and in patients with immune deficits after chemotherapy. Immune reconstitution following intensive lymphodepletion and autologous CD34+ stem cell transplantation in patients with severe systemic lupus erythematosus (SLE). In an ongoing trial (P.I. Steven Pavletic 04-C-0095), T and B cell immune reconstitution has been monitored by multi-parameter flow cytometry, TREC quantitation and plasma cytokine assays to evaluate recovery of thymopoiesis, reconstitution of naive, memory and effector T cell populations and normalization of immune function in patients with severe autoimmunity. These studies assessed the efficacy of the cytoreductive regimen in depleting T and B cells and the timecourse of immune recovery. These studies contribute to development of autologous transplant as a therapeutic modality in treatment of severe lupus and to an understanding of immune system homeostasis and regeneration in the context of autoimmunity. Immune populations and dysfunction in chronic graft vs host disease: In an ongoing trial (P.I. Steven Pavletic 04-C-0281), patients who have developed chronic GVHD following allogeneic transplantation are evaluated at NIH in a multi-institute natural history trial. The PCS core has evaluated T and B cell immune reconstitution and function in these patients by multi-parameter flow cytometry, TREC and cytokine assays, and by PCR analysis and confocal microscopy of tissue biopsies stained by fluorescent immunohistochemistry. These assays support the ongoing multidisciplinary natural history study of chronic GVHD, by contributing to the identification of biomarkers and to an understanding of chronic GVHD pathogenesis. We have determined that elevated levels of B cell activating factor of the TNF family (BAFF)in chronic GVHD are significantly correlated both with elevated plasma levels of inflammatory cytokines and with reduced levels of circulating B cells. Furthermore we have linked these elevated BAFF levels with persistence of transitional B cells, consistent with a reduction in negative selection of autoreactive cells. In addition, studies of peripheral blood populations and cytokines have been correlated with immunohistochemical and PCR analyses (performed by an ETIB clinical research fellow, M. Imanguli, working in the Core) of oral biopsy tissues from patients in the chronic GVHD protocol. These studies have demonstrated the involvement of cytotoxic T cell effectors in oral CGVHD. Finally, we support a therapeutic trial for bronchiolitis obliterans, a severe complication of chronic GVHD (P. I. Ronald Gress and Kirsten Williams 08-C-0097). This trial tests the efficacy of a drug blocking the leukotriene pathway to inhibit the progression of fibrosis in this obstructive lung disorder. The core provides multi-parameter flow cytometric assessment of leukotriene receptor levels in peripheral blood leukocytes and in bronchial lavage cells. Cytokine and monokine production following Th2 and Th2/Tc2 therapy based transplant regimens: An ongoing project has assessed lymphokine and monokine production capacity post transplant, focusing on allogeneic transplants incorporating immune therapy with Th2, Th2/Tc2 or Th2.rapa donor-derived cells (P. I. Dan Fowler, 04-C-0055, 04-C-0131). In the early post-transplant period and at regular intervals thereafter, the cytokine and monokine production capacity of peripheral blood cells has been determined by generation of anti CD3/anti CD28-stimulated and bacterial lipopolysaccharide (LPS) stimulated supernatants and by the assessment of the frequencies of cytokine producing cells by flow cytometry. These assays support branch transplant protocols by assessing the use of monokine supernatants and frequency of monokine producing cells as a potential biomarkers predicting acute GVHD. Furthermore they evaluate the efficacy and durability of the cytokine shift resulting from the infusion of Th2/Tc2 and Th2-rapa cells. Immune reconstitution following lymphodepletion: In several ongoing ETIB clinical trials of allogeneic and autologous stem cell transplantation therapies (04-C-0055 and 07-C-0195; PIs Daniel Fowler and Michael Bishop) as well as in continuing follow-up of patients treated in earlier trials (96-C-0104, 99-C-0143, 03-C-0077; P.I.s Claude Sportes, Daniel Fowler, Michael Bishop), the process of immune reconstitution has been assessed by multiparameter flow cytometry, functional assays, quantitation of TREC, spectratyping analyses of repertoire diversity and ELISA assays of plasma cytokines. These studies have assessed the contributions of thymopoiesis and homeostatic cytokines in the recovery of CD4 and CD8 T cell populations following autologous stem cell transplantation. These data now are used as a basis of comparison in patient [summary truncated at 7800 characters]
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