Immunological Consequences of In Utero Exposure to Foreign Antigens

Immunologic tolerance refers to a state of immune nonreactivity specific to particular antigens as an important issue in the field of transplantation and the management of autoimmune diseases. Tolerance conceptually originated from Owen’s observation of blood cell sharing in twin calves. Owen’s conceptual framework subsequently constituted the backbone of Medawar’s “actively acquired tolerance” as the major tenet of modern immunology. Based upon this knowledge, the delivery of genetically distinct hematopoietic stem cells into pre-immune fetuses represented a novel and unique approach to their engraftment without the requirement of myeloablation or immunosuppression. It might also make fetal recipients commit donor alloantigens to memory of their patterns as “self” so as to create a state of donor-specific tolerance. Over the years, the effort made experimentally or clinically toward in utero marrow transplantation could not reliably yield sufficient hematopoietic chimerism for curing candidate diseases as anticipated, nor did allogeneic graft tolerance universally develop as envisaged by Medawar following in utero exposure to various forms of alloantigens from exosomes, lymphocytes or marrow cells. Enduring graft tolerance was only conditional on a state of significant hematopoietic chimerism conferred by marrow inocula. Notably, fetal exposure to ovalbumin, oncoprotein and microbial antigens did not elicit immune tolerance, but instead triggered an event of sensitization to the antigens inoculated. These fetal immunogenic events might be clinically relevant to prenatal imprinting of atopy, immune surveillance against developmental tumorigenesis, and prenatal immunization against infectious diseases. Briefly, the immunological consequences of fetal exposure to foreign antigens could be tolerogenic or immunogenic, relying upon the type or nature of antigens introduced. Thus, the classical school of “actively acquired tolerance” might oversimplify the interactions between developing fetal immune system and antigens. Such interactions might rely upon fetal macrophages, which showed up earlier than lymphocytes and were competent to phagocytose foreign antigens so as to bridge toward antigen-specific adaptive immunity later on in life. Thus, innate fetal macrophages may be the potential basis for exploring how the immunological outcome of fetal exposure to foreign antigens is determined to improve the likelihood and reliability of manipulating fetal immune system toward tolerization or immunization to antigens.

The GABA receptor GABRR1 is expressed on and functional in hematopoietic stem cells and megakaryocyte progenitors

GABRR1 is a rho subunit receptor of GABA, the major inhibitory neurotransmitter in the mammalian brain. While most investigations of its function focused on the nervous system, its regulatory role in hematopoiesis has not been reported. In this study, we found GABRR1 is mainly expressed on subsets of human and mouse hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs). GABRR1-negative (GR−) HSCs led to higher donor-derived hematopoietic chimerism than GABRR1-positive (GR+) HSCs. GR+ but not GR− HSCs and MkPs respond to GABA in patch clamp studies. Inhibition of GABRR1 via genetic knockout or antagonists inhibited MkP differentiation and reduced platelet numbers in blood. Overexpression of GABRR1 or treatment with agonists significantly promoted MkP generation and megakaryocyte colonies. Thus, this study identifies a link between the neural and hematopoietic systems and opens up the possibility of manipulating GABA signaling for platelet-required clinical applications.

Translational impact of NIH-funded nonhuman primate research in transplantation

The National Institutes of Health (NIH) has long supported using nonhuman primate (NHP) models for research on kidney, pancreatic islet, heart, and lung transplantation. The primary purpose of this research has been to develop new treatments for down-modulating or preventing deleterious immune responses after transplantation in human patients. Here, we discuss NIH-funded NHP studies of immune cell depletion, costimulation blockade, regulatory cell therapy, desensitization, and mixed hematopoietic chimerism that either preceded clinical trials or prevented the human application of therapies that were toxic or ineffective.

Droplet digital PCR for the simultaneous analysis of minimal residual disease and hematopoietic chimerism after allogeneic cell transplantation

Background Minimal residual disease (MRD) and hematopoietic chimerism testing influences clinical decision and therapeutic intervention in patients after allogeneic stem cell transplantation (HSCT). However, treatment approaches to induce complete donor chimerism and MRD negativity can lead to complications such as graft-versus-host disease (GvHD) and marrow aplasia. Therefore, there is a need for comprehensive characterization of the molecular remission status after transplantation. Methods We analyzed 764 samples from 70 patients after HSCT for the simultaneous measurement of chimerism and molecular targets used for MRD testing with a digital PCR (dPCR) platform. Results Mixed chimerism (MC) was detected in 219 samples from 37 patients. The mean percentage of host derived DNA in these clinical samples was 4.3%. Molecular relapse with a positive MRD marker and/or increased WT1 expression was observed in 15 patients. In addition to WT1 overexpression, other MRD positive markers were: NPM1 (Type A, B, K), DNMT3A (R882H), MLL-PTD, IDH1 (R132H) and KRAS (G12S). Increasing MC was observed in 15 patients. This group of patients showed either a positive MRD marker, increased WT1 expression or both. Next, we analyzed whether MC or the molecular target for MRD was first detected. MC and MRD marker positivity in this group was first detected in six and two patients, respectively. In the remaining seven patients MC and MRD positivity was detected simultaneously. Conclusions The combination of MRD and chimerism markers in a dPCR platform represents a practical, sensitive and accurate diagnostic tool for the comprehensive assessment of the molecular remission status of patients undergoing HSCT.

Intestinal Immune Conditioning with Helminths Employs Host T Helper 2 (Th2) Pathway to Induce Mixed Chimerism and Regulate Graft-Versus-Host Disease

Background Achieving mixed chimerism suppresses donor T cell alloreactivity and reduces graft-versus-host disease (GVHD)-related mortality after bone marrow transplantation (BMT). Mixed hematopoietic chimerism is the only condition proven to lead to immune tolerance after transplantation. It can be achieved with protocols utilizing chemotherapeutic agents or strategies that block T cell activation or deplete host T cells. Selective irradiation of the thymus or lymphoid organs can also lead to mixed hematopoietic chimerism. After total lymphocyte irradiation (TLI), mixed chimerism is mediated by host iNKT lymphocytes and the T helper 2 (Th2) cytokine IL4 generated by those cells. IL4 signals through IL4 receptor (IL4R) and STAT6. After a toxic myeloablative pretransplant regimen, total body irradiation (TBI), intestinal immune conditioning with helminthic commensals regulates the host immune system and promotes a transient mixed chimerism. Conditioning with helminths also induces IL4 production by host cells and regulates GVHD while preserving the graft-versus-tumor effect. We hypothesize that induction of host Th2 pathway is critical for achieving mixed chimerism by intestinal immune conditioning Methods We evaluated the role of recipient Th2 signaling in the establishment of mixed chimerism in an MHC I/II major mismatch (H2b→H2d) model that utilizes TBI. Three weeks after infection with the mouse nematode, Heligmosomoides polygyrus-bakeri (Hpb), GVHD was induced by the delivery of T cell-depleted bone marrow (TCD-BM) and splenic T cells from uninfected WT C57BL/6 (MHC:H2b) donors into lethally-irradiated WT BALB/c, interleukin 4 (IL4) -/-, IL4Rα-/- and STAT6-/- recipients (MHC: H2d). Cellular composition for donor vs recipient cells, GVHD-mediated inflammation in end-organs (colon and lung), and survival of mice in each setting were assessed Results In 3 models of disruption of the Th2 pathway, we demonstrated that Th2 signaling in the host is essential for the establishment of mixed chimerism. Helminth infection promotes mixed chimerism in WT BALB/c BMT recipients; recipient T cells constitute 3-5% of peripheral population at 6 days post-BMT, whereas recipient T cells constitute <1-2% of population in uninfected BMT recipients (p<0.001). By contrast, when recipient cell IL4, IL4Rα or STAT6 is silenced, the percentage of recipient T cells remains negligible (<1% of total T cell population) in uninfected or Hpb-infected BMT recipients (p: NS between uninfected and Hpb-infected) (fig 1). Hpb infection promotes chimerism in mesenteric lymph nodes (MLN) of iNKT-deficient (Jα18-/-; MHC: H2d) BMT recipients of C57BL/6 donors (p<0.05 between uninfected and Hpb-infected Jα18-/- BMT recipients). Although helminths regulate GVHD-related inflammation in lungs and the colon of WT BALB/c recipients (p<0.05 between uninfected and Hpb-infected WT BALB/c BMT recipients), they fail to do so in these organs in IL4-, STAT6- or IL4Rα-deficient BMT recipients (p: NS between uninfected and Hpb-infected BMT recipients for each Th2 gene deficient group)(fig 2). Moreover, helminth infection did not promote survival of Th2-/- BMT recipients, where all mice died of lethal GVHD, although helminth infection promotes survival in WT BALB/c BMT recipients (P<0.05 between Hpb-infected WT BMT recipients and each other group). Because tolerance of alloreactive cells in mixed chimerism occurs with the induction of CTLA4 on T cells (a check point inhibitor and critical immune regulatory protein in GVHD), we also explored CTLA4 expression on peripheral T cells and found that helminth-induced CTLA4 production on splenic T cells was dependent on expression of IL4 and STAT6. In IL4-/- or STAT6-/- mice, there was no increase in the percentage of CTLA4+ T cells after Hpb infection (p: NS between uninfected and Hpb-infected BMT recipients for each Th2 gene deficient group), whereas helminth infection increased 3-fold the percentage of CTLA4+ T cells (p<0.05 between uninfected and Hpb-infected WT BALB/c mice) Conclusions Intestinal immune conditioning with helminths promotes mixed chimerism after toxic, host cell depleting myeloablative TBI and regulates lethal GVHD following BMT. Host cell Th2 pathway is critical for achieving mixed chimerism. Helminths stimulate CTLA4 - a cellular regulatory protein known to promote mixed chimerism after BMT- in a Th2 dependent manner Disclosures Blazar: Kadmon Corporation, LLC: Consultancy, Research Funding.