Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of ...structure-specific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transplant the 2F5 epitope into select acceptor scaffolds. The resultant “2F5-epitope scaffolds” possessed nanomolar affinity for antibody 2F5 and a range of epitope flexibilities and antigenic specificities. Crystallographic characterization of the epitope scaffold with highest affinity and antigenic discrimination confirmed good to near perfect attainment of the target conformation for the gp41 molecular graft in free and 2F5-bound states, respectively. Animals immunized with 2F5-epitope scaffolds showed levels of graft-specific immune responses that correlated with graft flexibility (p < 0.04), while antibody responses against the graft—as dissected residue-by-residue with alanine substitutions—resembled more closely those of 2F5 than sera elicited with flexible or cyclized peptides, a resemblance heightened by heterologous prime-boost. Lastly, crystal structures of a gp41 peptide in complex with monoclonal antibodies elicited by the 2F5-epitope scaffolds revealed that the elicited antibodies induce gp41 to assume its 2F5-recognized shape. Epitope scaffolds thus provide a means to elicit antibodies that recognize a predetermined target shape and sequence, even if that shape is transient in nature, and a means by which to dissect factors influencing such elicitation.
We report, for the first time, a replication-defective retroviral vector–associated neoplasia in a nonhuman primate. Five years after transplantation with CD34+ cells transduced with a retroviral ...vector expressing enhanced green fluorescent protein (eGFP) and a drug-resistant variant of the dihydrofolate reductase gene (L22Y), a rhesus macaque developed a fatal myeloid sarcoma, a type of acute myeloid leukemia. Tumor cells contained 2 clonal vector insertions. One insertion was found in BCL2-A1, an antiapoptotic gene. This event suggests that currently available retroviral vectors may have long-term side effects, particularly in hematopoietic stem and progenitor cells.
Opportunistic infections contribute to morbidity and mortality after peripheral blood progenitor cell (PBPC) transplantation and are related to a deficient T-cell compartment. Accelerated T-cell ...reconstitution may therefore be clinically beneficent. Keratinocyte growth factor (KGF) has been shown to protect thymic epithelial cells in mice. Here, we evaluated immune reconstitution after autologous CD34+ PBPC transplantation in rhesus macaques conditioned with myeloablative total body irradiation in the absence or presence of single pretotal body irradiation or repeated peritransplant KGF administration. All KGF-treated animals exhibited a well-preserved thymic architecture 12 months after graft. In contrast, thymic atrophy was observed in the majority of animals in the control group. The KGF-treated animals showed higher frequencies of naive T cells in lymph nodes after transplantation compared with the control animals. The animals given repeated doses of KGF showed the highest levels of T-cell receptor excision circles (TRECs) and the lowest frequencies of Ki67+ T cells, which suggest increased thymic-dependent reconstitution in these animals. Of note, the humoral response to a T-cell–dependent neo-antigen was significantly higher in the KGF-treated animals compared with the control animals. Thus, our findings suggest that KGF may be a useful adjuvant therapy to augment T-cell reconstitution after human PBPC transplantation.
Antiviral CD8(+) T cells can elaborate at least two effector functions, cytokine production and cytotoxicity. Which effector function is elaborated can determine whether the CD8(+) T cell response is ...primarily inflammatory (cytokine producing) or antiviral (cytotoxic). In this study we demonstrate that cytotoxicity can be triggered at peptide concentrations 10- to 100-fold less than those required for cytokine production in primary HIV- and CMV-specific human CD8(+) T cells. Cytolytic granule exocytosis occurs at peptide concentrations insufficient to cause substantial TCR down-regulation, providing a mechanism by which a CD8(+) T cell could engage and lyse multiple target cells. TCR sequence analysis of virus-specific cells shows that individual T cell clones can degranulate or degranulate and produce cytokine depending on the Ag concentration, indicating that response heterogeneity exists within individual CD8(+) T cell clonotypes. Thus, antiviral CD8(+) T cell effector function is determined primarily by Ag concentration and is not an inherent characteristic of a virus-specific CD8(+) T cell clonotype or the virus to which the response is generated. The inherent ability of viruses to induce high or low Ag states may be the primary determinant of the cytokine vs cytolytic nature of the virus-specific CD8(+) T cell response.
Immunodeficiency after peripheral blood progenitor cell (PBPC) transplantation may be influenced by graft composition, underlying disease, and/or pre‐treatment. These factors are difficult to study ...independently in humans. Ex vivo culture and genetic manipulation of PBPC grafts may also affect immune reconstitution, with relevance to gene therapy applications. We directly compared the effects of three clinically relevant autologous graft compositions on immune reconstitution after myeloblative total body irradiation in rhesus macaques, the first time these studies have been performed in a large animal model with direct clinical relevance. Animals received CD34+ cell dose‐matched grafts of either peripheral blood mononuclear cells, purified CD34+ PBPCs, or purified CD34+ PBPCs expanded in vitro and retrovirally transduced. We evaluated the reconstitution of T, B, natural killer, dendritic cells, and monocytes in blood and lymph nodes for up to 1 year post‐transplantation. Animals receiving selected‐transduced CD34+ cells had the fastest recovery of T‐cell numbers, along with the highest T‐cell‐receptor gene rearrangement excision circles levels, the fewest proliferating Ki‐67+ T‐cells in the blood, and the best‐preserved thymic architecture. Selected‐transduced CD34+ cells may therefore repopulate the thymus more efficiently and promote a higher output of naïve T‐cells. These results have implications for the design of gene therapy trials, as well as for the use of expanded PBPCs for improved T‐cell immune reconstitution after transplantation.
Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, fatal disorder of infancy. We report here a 17-day-old female infant who presented with high fever, hepatosplenomegaly, ...hypertriglyceridemia, hypofibrinogenemia, thrombocytopenia, and liver failure. Leukocytosis was detected with circulating “atypical” lymphoid cells. Flow cytometric studies revealed expanded subpopulations of CD8+ T cells with unusual immunophenotypic features, including a subset that lacked CD5 expression. A liver biopsy showed hemophagocytic lymphohistiocytosis with exuberant infiltrates of CD8+ T cells that lacked perforin. Mutational studies revealed a 666C→A (H222Q) missense mutation in the perforin gene. T-cell receptor studies on flow-sorted T-cell subpopulations revealed no evidence of monoclonality. Analysis of T-cell receptor excision circle levels indicated long proliferative history in the aberrant CD8+ T-cell subsets. This case provides an instructive example of uncontrolled reactive proliferation of CD8+ T cells in FHL, resulting in atypical morphology and unusual immunophenotypic features that might suggest malignancy in other clinical settings.
Abstract
Opportunistic infections have an major impact on morbidity and mortality after peripheral blood progenitor cell transplantation, and are caused by deficient T cell numbers and activities, ...which correlate with thymic dysfunction. Keratinocyte growth factor (KGF) has been shown to protect thymic epithelial cells from injury induced by chemotherapy and radiation in mice, associated with improved thymopoiesis. We for the first time evaluated the effect of KGF on immune reconstitution after myeloablative total body irradiation (TBI) and autologous transplantation in non-human primates, a clinically relevant large animal model. Four animals received KGF 250mg/kg body weight given once (day −5) before TBI (low dose KGF) and CD34+ purified PBPC transplantation, and two animals received the same dose given 3 times (day −5, −4, −3) before and 3 times (day +1, +2, +3) after TBI (day −2, −1) and transplantation (day 0) (high-dose KGF). These KGF-treated animals were compared to an age and CD34+ cell dose-matched control group of five monkeys transplanted with purified CD34+ cells but without KGF treatment. We evaluated the reconstitution of T and B cells in blood and lymph nodes (LN) for a year post transplantation. All animals engrafted (neutrophils >500/ml) day by day 11 post transplantation. The animals treated with KGF had higher frequencies of naïve T cells in blood and in peripheral LN post transplantation as compared to the control animals, for instance at one month after transplantation the percentage of LN naïve CD4+ T-cells was 35±19% in the high dose KGF group, 14±2% in the low dose KGF group and 15±4% in the control group. At 3 months after transplantation the percentage of naïve CD4+ in LN was 56±5% in the high dose and 59±9% in the low dose KGF groups versus 27±7% in the control group. We observed similar findings in the CD8+ subset. All KGF treated animals had higher peripheral blood TREC levels, broader TCR repertoires as determined by spectratyping of the CDR3 region, and lower frequencies of Ki-67+ proliferating T cells, presumably due to a lower drive for peripheral expansion in animals with better thymic output of naïve T cells. The recovery of circulating B-cells was not altered with KGF treatment compared to the controls. At 12 months after transplantation there was uniform preservation of thymic architecture with proliferating thymocytes in the animals treated with KGF, in comparison to the control group of animals, with thymic atrophy seen in the majority. Thus, peri-TBI treatment with KGF resulted in protection of thymic epithelial cells, improved thymic repopulation, and increased naïve T-cell numbers in secondary lymphatic tissues, and correlated with better immune responses to newly induced (HIV envelope) as well as recall antigens including measles and CMV. KGF-mediated protection and preservation of thymic epithelial cells from TBI-mediated damage, with improved output of naïve T cells post-transplantation may improve immune reconstitution and thus outcomes in patients undergoing autologous or allogeneic transplantation.
Opportunistic infections have an major impact on morbidity and mortality after peripheral blood progenitor cell transplantation, and are caused by deficient T cell numbers and activities, which ...correlate with thymic dysfunction. Keratinocyte growth factor (KGF) has been shown to protect thymic epithelial cells from injury induced by chemotherapy and radiation in mice, associated with improved thymopoiesis. We for the first time evaluated the effect of KGF on immune reconstitution after myeloablative total body irradiation (TBI) and autologous transplantation in non-human primates, a clinically relevant large animal model. Four animals received KGF 250mg/kg body weight given once (day −5) before TBI (low dose KGF) and CD34+ purified PBPC transplantation, and two animals received the same dose given 3 times (day −5, −4, −3) before and 3 times (day +1, +2, +3) after TBI (day −2, −1) and transplantation (day 0) (high-dose KGF). These KGF-treated animals were compared to an age and CD34+ cell dose-matched control group of five monkeys transplanted with purified CD34+ cells but without KGF treatment. We evaluated the reconstitution of T and B cells in blood and lymph nodes (LN) for a year post transplantation. All animals engrafted (neutrophils >500/ml) day by day 11 post transplantation. The animals treated with KGF had higher frequencies of naïve T cells in blood and in peripheral LN post transplantation as compared to the control animals, for instance at one month after transplantation the percentage of LN naïve CD4+ T-cells was 35±19% in the high dose KGF group, 14±2% in the low dose KGF group and 15±4% in the control group. At 3 months after transplantation the percentage of naïve CD4+ in LN was 56±5% in the high dose and 59±9% in the low dose KGF groups versus 27±7% in the control group. We observed similar findings in the CD8+ subset. All KGF treated animals had higher peripheral blood TREC levels, broader TCR repertoires as determined by spectratyping of the CDR3 region, and lower frequencies of Ki-67+ proliferating T cells, presumably due to a lower drive for peripheral expansion in animals with better thymic output of naïve T cells. The recovery of circulating B-cells was not altered with KGF treatment compared to the controls. At 12 months after transplantation there was uniform preservation of thymic architecture with proliferating thymocytes in the animals treated with KGF, in comparison to the control group of animals, with thymic atrophy seen in the majority. Thus, peri-TBI treatment with KGF resulted in protection of thymic epithelial cells, improved thymic repopulation, and increased naïve T-cell numbers in secondary lymphatic tissues, and correlated with better immune responses to newly induced (HIV envelope) as well as recall antigens including measles and CMV. KGF-mediated protection and preservation of thymic epithelial cells from TBI-mediated damage, with improved output of naïve T cells post-transplantation may improve immune reconstitution and thus outcomes in patients undergoing autologous or allogeneic transplantation.
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