Influence of inorganic nitrogen on the inhibition of symbiotic nitrogen fixation

The absorption of nitrogen from the air and from added fertilizer was measured in legumes when varying quantities of fertilizer were added. The plants were grown in gravel cultures and supplied with mineral elements in nutrient solution through an automatic irrigating device. The ammonium nitrogen applied to the gravel culture was enriched with N15 to provide a measure of the absorption from the fertilizer and from the air;To relate the nitrogen supply obtained in the nutrient gravel culture to soil conditions, grasses were grown in both a number of typical soils and in the gravel cultures;In the legumes studied, with the exception of birdsfoot trefoil, the lowest rate of nitrogen applied to solution culture resulted in an increase in yield and in the amount fixed. The higher rates of nitrogen addition, with the exception of the case with soybeans, resulted in progressive increases in yield and nitrogen absorption, but decreases in the amount of nitrogen fixed;Under the conditions of the experiment, the fixation mechanisms did not furnish sufficient nitrogen for maximum growth of the legumes. Added nitrogen tended to increase yields and to replace the fixation processes;The influences of available nitrogen on the fixation processes were similar in all of the legumes studied when considered from the stand point of nitrogen applied in relation to the total nitrogen needs of the legumes;Recoveries of added ammonium nitrogen ranged from 76 to 94% in the legume crops, to from 79 to 97% in the grasses;Nitrate and ammonium ions from ammonium nitrate fertilizer were absorbed in about equal proportions by alfalfa, soybeans, and Sudan grass, indicating that similar results in nitrogen uptake by legumes, and inhibition of fixation should be expected had a nitrate fertilizer been employed in place of ammonium;The conclusion is reached that in soils containing from 0.2 to 0.3% of nitrogen, alfalfa and sweet clover normally fix from 60 to 75% of the nitrogen absorbed. For Ladino clover and birdsfoot trefoil, under the above soil conditions, fixation would range between 25 and 60% of the nitrogen absorbed; the lower fixations occurring in the first growing season, and particularly with birdsfoot trefoil

Regulatory CD8 T cells that recognize Qa-1 expressed by CD4 T-helper cells inhibit rejection of heart allografts

Induction of longstanding immunologic tolerance is essential for survival of transplanted organs and tissues. Despite recent advances in immunosuppression protocols, allograft damage inflicted by antibody specific for donor organs continues to represent a major obstacle to graft survival. Here we report that activation of regulatory CD8 T cells (CD8 Treg) that recognize the Qa-1 class Ib major histocompatibility complex (MHC), a mouse homolog of human leukocyte antigen-E (HLA-E), inhibits antibody-mediated immune rejection of heart allografts. We analyzed this response using a mouse model that harbors a point mutation in the class Ib MHC molecule Qa-1, which disrupts Qa-1 binding to the T cell receptor (TCR)-CD8 complex and impairs the CD8 Treg response. Despite administration of cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig), Qa-1 mutant mice developed robust donor-specific antibody responses and accelerated heart graft rejection. We show that these allo-antibody responses reflect diminished Qa-1-restricted CD8 Treg-mediated suppression of host follicular helper T cell-dependent antibody production. These findings underscore the critical contribution of this Qa-1/HLA-E-dependent regulatory pathway to maintenance of transplanted organs and suggest therapeutic approaches to ameliorate allograft rejection

mTORC1 Inhibition Protects Human Regulatory T Cells From Granzyme-B-Induced Apoptosis

Regulatory T cells (T-regs) have shown great promise as a means of cellular therapy in a multitude of allo- and auto-immune diseases-due in part to their immunosuppressive potency. Nevertheless, the clinical efficacy of human T-regs in patients has been limited by their poor in vivo homeostasis. To avert apoptosis, T-regs require stable antigenic (CD3 zeta/T-cell-receptor-mediated), co-stimulatory (CD28-driven), and cytokine (IL-2-dependent) signaling. Notably, this sequence of signals supports an activated T-reg phenotype that includes a high expression of granzymes, particularly granzyme B (GrB). Previously, we have shown that aside from the functional effects of GrB in lysing target cells to modulate allo-immunity, GrB can leak out of the intracellular lysosomal granules of host T-regs, initiating pro-apoptotic pathways. Here, we assessed the role of inhibiting mechanistic target of rapamycin complex 1 (mTORC1), a recently favored drug target in the transplant field, in regulating human T-reg apoptosis via GrB. Using ex vivo models of human T-reg culture and a humanized mouse model of human skin allotransplantation, we found that by inhibiting mTORC1 using rapamycin, intracytoplasmic expression and functionality of GrB diminished in host T-regs; lowering human T-reg apoptosis by in part decreasing the phosphorylation of S6K and c-Jun. These findings support the already clinically validated effects of mTORC1 inhibition in patients, most notably their stabilization of T-reg bioactivity and in vivo homeostasis

Type I interferons augment regulatory T cell polarization in concert with ancillary cytokine signals

In the transplant community, research efforts exploring endogenous alternatives to inducing tolerogenic allo-specific immune responses are much needed. In this regard, CD4 + FoxP3+ regulatory T cells (Tregs) are appealing candidates due to their intrinsic natural immunosuppressive qualities. To date, various homeostatic factors that dictate Treg survival and fitness have been elucidated, particularly the non-redundant roles of antigenic CD3ζ/T-cell-receptor, co-stimulatory CD28, and cytokine interleukin (IL-)2 dependent signaling. Many of the additional biological signals that affect Tregs remain to be elucidated, however, especially in the transplant context. Previously, we demonstrated an unexpected link between type I interferons (IFNs) and Tregs in models of multiple myeloma (MM)—where MM plasmacytes escaped immunological surveillance by enhancing type I IFN signaling and precipitating upregulated Treg responses that could be overturned with specific knockdown of type I IFN signaling. Here, we elaborated on these findings by assessing the role of type I IFN signaling (IFN-α and -β) on Treg homeostasis within an alloimmune context. Specifically, we studied the induction of Tregs from naïve CD4 T cells. Using in vitro and in vivo models of murine skin allotransplantation, we found that type I IFN indeed spatiotemporally enhanced the polarization of naïve CD4 T cells into FoxP3+ Tregs. Notably, however, this effect was not independent of, and rather co-dependent on, ancillary cytokine signals including IL-2. These findings provide evidence for the relevance of type I IFN pathway in modulating FoxP3+ Treg responses and, by extension, stipulate an additional means of facilitating Treg fitness via type I IFNs

Influence of inorganic nitrogen on the inhibition of symbiotic nitrogen fixation

The absorption of nitrogen from the air and from added fertilizer was measured in legumes when varying quantities of fertilizer were added. The plants were grown in gravel cultures and supplied with mineral elements in nutrient solution through an automatic irrigating device. The ammonium nitrogen applied to the gravel culture was enriched with N15 to provide a measure of the absorption from the fertilizer and from the air;To relate the nitrogen supply obtained in the nutrient gravel culture to soil conditions, grasses were grown in both a number of typical soils and in the gravel cultures;In the legumes studied, with the exception of birdsfoot trefoil, the lowest rate of nitrogen applied to solution culture resulted in an increase in yield and in the amount fixed. The higher rates of nitrogen addition, with the exception of the case with soybeans, resulted in progressive increases in yield and nitrogen absorption, but decreases in the amount of nitrogen fixed;Under the conditions of the experiment, the fixation mechanisms did not furnish sufficient nitrogen for maximum growth of the legumes. Added nitrogen tended to increase yields and to replace the fixation processes;The influences of available nitrogen on the fixation processes were similar in all of the legumes studied when considered from the stand point of nitrogen applied in relation to the total nitrogen needs of the legumes;Recoveries of added ammonium nitrogen ranged from 76 to 94% in the legume crops, to from 79 to 97% in the grasses;Nitrate and ammonium ions from ammonium nitrate fertilizer were absorbed in about equal proportions by alfalfa, soybeans, and Sudan grass, indicating that similar results in nitrogen uptake by legumes, and inhibition of fixation should be expected had a nitrate fertilizer been employed in place of ammonium;The conclusion is reached that in soils containing from 0.2 to 0.3% of nitrogen, alfalfa and sweet clover normally fix from 60 to 75% of the nitrogen absorbed. For Ladino clover and birdsfoot trefoil, under the above soil conditions, fixation would range between 25 and 60% of the nitrogen absorbed; the lower fixations occurring in the first growing season, and particularly with birdsfoot trefoil.</p

Presentation1_Type I interferons augment regulatory T cell polarization in concert with ancillary cytokine signals.pdf

In the transplant community, research efforts exploring endogenous alternatives to inducing tolerogenic allo-specific immune responses are much needed. In this regard, CD4 + FoxP3+ regulatory T cells (Tregs) are appealing candidates due to their intrinsic natural immunosuppressive qualities. To date, various homeostatic factors that dictate Treg survival and fitness have been elucidated, particularly the non-redundant roles of antigenic CD3ζ/T-cell-receptor, co-stimulatory CD28, and cytokine interleukin (IL-)2 dependent signaling. Many of the additional biological signals that affect Tregs remain to be elucidated, however, especially in the transplant context. Previously, we demonstrated an unexpected link between type I interferons (IFNs) and Tregs in models of multiple myeloma (MM)—where MM plasmacytes escaped immunological surveillance by enhancing type I IFN signaling and precipitating upregulated Treg responses that could be overturned with specific knockdown of type I IFN signaling. Here, we elaborated on these findings by assessing the role of type I IFN signaling (IFN-α and -β) on Treg homeostasis within an alloimmune context. Specifically, we studied the induction of Tregs from naïve CD4 T cells. Using in vitro and in vivo models of murine skin allotransplantation, we found that type I IFN indeed spatiotemporally enhanced the polarization of naïve CD4 T cells into FoxP3+ Tregs. Notably, however, this effect was not independent of, and rather co-dependent on, ancillary cytokine signals including IL-2. These findings provide evidence for the relevance of type I IFN pathway in modulating FoxP3+ Treg responses and, by extension, stipulate an additional means of facilitating Treg fitness via type I IFNs.</p

Table1_Type I interferons augment regulatory T cell polarization in concert with ancillary cytokine signals.xlsx

In the transplant community, research efforts exploring endogenous alternatives to inducing tolerogenic allo-specific immune responses are much needed. In this regard, CD4 + FoxP3+ regulatory T cells (Tregs) are appealing candidates due to their intrinsic natural immunosuppressive qualities. To date, various homeostatic factors that dictate Treg survival and fitness have been elucidated, particularly the non-redundant roles of antigenic CD3ζ/T-cell-receptor, co-stimulatory CD28, and cytokine interleukin (IL-)2 dependent signaling. Many of the additional biological signals that affect Tregs remain to be elucidated, however, especially in the transplant context. Previously, we demonstrated an unexpected link between type I interferons (IFNs) and Tregs in models of multiple myeloma (MM)—where MM plasmacytes escaped immunological surveillance by enhancing type I IFN signaling and precipitating upregulated Treg responses that could be overturned with specific knockdown of type I IFN signaling. Here, we elaborated on these findings by assessing the role of type I IFN signaling (IFN-α and -β) on Treg homeostasis within an alloimmune context. Specifically, we studied the induction of Tregs from naïve CD4 T cells. Using in vitro and in vivo models of murine skin allotransplantation, we found that type I IFN indeed spatiotemporally enhanced the polarization of naïve CD4 T cells into FoxP3+ Tregs. Notably, however, this effect was not independent of, and rather co-dependent on, ancillary cytokine signals including IL-2. These findings provide evidence for the relevance of type I IFN pathway in modulating FoxP3+ Treg responses and, by extension, stipulate an additional means of facilitating Treg fitness via type I IFNs.</p

Donor myeloid derived suppressor cells (MDSCs) prolong allogeneic cardiac graft survival through programming of recipient myeloid cells in vivo

Abstract Solid organ transplantation is a lifesaving therapy for patients with end-organ disease. Current immunosuppression protocols are not designed to target antigen-specific alloimmunity and are uncapable of preventing chronic allograft injury. As myeloid-derived suppressor cells (MDSCs) are potent immunoregulatory cells, we tested whether donor-derived MDSCs can protect heart transplant allografts in an antigen-specific manner. C57BL/6 (H2Kb, I-Ab) recipients pre-treated with BALB/c MDSCs were transplanted with either donor-type (BALB/c, H2Kd, I-Ad) or third-party (C3H, H2Kk, I-Ak) cardiac grafts. Spleens and allografts from C57BL/6 recipients were harvested for immune phenotyping, transcriptomic profiling and functional assays. Single injection of donor-derived MDSCs significantly prolonged the fully MHC mismatched allogeneic cardiac graft survival in a donor-specific fashion. Transcriptomic analysis of allografts harvested from donor-derived MDSCs treated recipients showed down-regulated proinflammatory cytokines. Immune phenotyping showed that the donor MDSCs administration suppressed effector T cells in recipients. Interestingly, significant increase in recipient endogenous CD11b+Gr1+ MDSC population was observed in the group treated with donor-derived MDSCs compared to the control groups. Depletion of this endogenous MDSCs with anti-Gr1 antibody reversed donor MDSCs-mediated allograft protection. Furthermore, we observed that the allogeneic mixed lymphocytes reaction was suppressed in the presence of CD11b+Gr1+ MDSCs in a donor-specific manner. Donor-derived MDSCs prolong cardiac allograft survival in a donor-specific manner via induction of recipient’s endogenous MDSCs

Regulatory T cells engineered with TCR signaling-responsive IL-2 nanogels suppress alloimmunity in sites of antigen encounter

Adoptive cell transfer of ex vivo expanded regulatory T cells (T-r(egs)) has shown immense potential in animal models of auto- and alloimmunity. However, the effective translation of such T-reg therapies to the clinic has been slow. Because T-reg homeostasis is known to require continuous T cell receptor (TCR) ligation and exogenous interleukin-2 (IL-2), some investigators have explored the use of low-dose IL-2 injections to increase endogenous T-reg responses. Systemic IL-2 immunotherapy, however, can also lead to the activation of cytotoxic T lymphocytes and natural killer cells, causing adverse therapeutic outcomes. Here, we describe a drug delivery platform, which can be engineered to autostimulate T-regs with IL-2 in response to TCR-dependent activation, and thus activate these cells in sites of antigen encounter. To this end, protein nanogels (NGs) were synthesized with cleavable bis(N-hydroxysuccinimide) cross-linkers and IL-2/Fc fusion (IL-2) proteins to form particles that release IL-2 under reducing conditions, as found at the surface of T cells receiving stimulation through the TCR. T-regs surface-conjugated with IL-2 NGs were found to have preferential, allograft-protective effects relative to unmodified T-regs or T-regs stimulated with systemic IL-2. We demonstrate that murine and human NG-modified T-regs carrying an IL-2 cargo perform better than conventional T-regs in suppressing alloimmunity in murine and humanized mouse allotransplantation models. In all, the technology presented in this study has the potential to improve T-reg transfer therapy by enabling the regulated spatiotemporal provision of IL-2 to antigen-primed T-regs