Mixed xenogeneic porcine chimerism tolerizes human anti-pig natural antibody-producing cells in a humanized mouse model.

BACKGROUND: A major obstacle to the success of organ transplantation from pigs to humans, necessitated by the shortage of human organs, is robust humoral immune rejection by pig-reactive human antibodies. Mixed xenogeneic hematopoietic chimerism induces xenoreactive B cell tolerance in rodents, but whether mixed pig/human chimerism could induce tolerance of human B cells to pig xenoantigens is unknown. METHODS: We investigated this question using a humanized mouse model in which durable mixed (pig-human) xenogeneic chimerism can be established. RESULTS: Human natural anti-pig cytotoxic antibodies, predominantly IgM, are detectable in non-chimeric humanized mouse serum, and pig-reactive antibodies were reduced in mixed chimeric versus non-chimeric humanized mice. This difference required persistent mixed chimerism and was not due to the adsorption of antibodies on pig cells in vivo. Furthermore, human B cells from spleens of mixed chimeric mice produced lower levels of anti-pig antibodies when stimulated in vitro compared with those from non-chimeric mice. CONCLUSIONS: Our findings demonstrate that mixed chimerism reduces human natural antibodies to pig xenoantigens, providing the first in vivo evidence of human B cell tolerance induction by mixed xenogeneic chimerism and supporting further evaluation of this approach for inducing human B cell tolerance to xenografts.

Lymphohematopoietic graft-versus-host responses promote mixed chimerism in patients receiving intestinal transplantation.

In humans receiving intestinal transplantation (ITx), long-term multilineage blood chimerism often develops. Donor T cell macrochimerism (≥4%) frequently occurs without graft-versus-host disease (GVHD) and is associated with reduced rejection. Here we demonstrate that patients with macrochimerism had high graft-versus-host (GvH) to host-versus-graft (HvG) T cell clonal ratios in their allografts. These GvH clones entered the circulation, where their peak levels were associated with declines in HvG clones early after transplant, suggesting that GvH reactions may contribute to chimerism and control HvG responses without causing GVHD. Consistently, donor-derived T cells, including GvH clones, and CD34+ hematopoietic stem and progenitor cells (HSPCs) were simultaneously detected in the recipients' BM more than 100 days after transplant. Individual GvH clones appeared in ileal mucosa or PBMCs before detection in recipient BM, consistent with an intestinal mucosal origin, where donor GvH-reactive T cells expanded early upon entry of recipient APCs into the graft. These results, combined with cytotoxic single-cell transcriptional profiles of donor T cells in recipient BM, suggest that tissue-resident GvH-reactive donor T cells migrated into the recipient circulation and BM, where they destroyed recipient hematopoietic cells through cytolytic effector functions and promoted engraftment of graft-derived HSPCs that maintain chimerism. These mechanisms suggest an approach to achieving intestinal allograft tolerance.

Rapid thymectomy of NSG mice to analyze the role of native and grafted thymi in humanized mice.

We developed a rapid method to remove the native mouse thymus from NSG mice, which allowed us to compare the behavior of human immune cells in the presence or absence of human T cells in human immune system mice. Removing the native mouse thymus is critical for studies of human thymopiesis in grafted thymic tissue in humanized mice.

Human Intestinal Allografts Contain Functional Hematopoietic Stem and Progenitor Cells that Are Maintained by a Circulating Pool.

Human intestinal transplantation often results in long-term mixed chimerism of donor and recipient blood in transplant patients. We followed the phenotypes of chimeric peripheral blood cells in 21 patients receiving intestinal allografts over 5 years. Donor lymphocyte phenotypes suggested a contribution of hematopoietic stem and progenitor cells (HSPCs) from the graft. Surprisingly, we detected donor-derived HSPCs in intestinal mucosa, Peyer's patches, mesenteric lymph nodes, and liver. Human gut HSPCs are phenotypically similar to bone marrow HSPCs and have multilineage differentiation potential in vitro and in vivo. Analysis of circulating post-transplant donor T cells suggests that they undergo selection in recipient lymphoid organs to acquire immune tolerance. Our longitudinal study of human HSPCs carried in intestinal allografts demonstrates their turnover kinetics and gradual replacement of donor-derived HSPCs from a circulating pool. Thus, we have demonstrated the existence of functioning HSPCs in human intestines with implications for promoting tolerance in transplant recipients.

Infection-induced type I interferons activate CD11b on B-1 cells for subsequent lymph node accumulation.

Innate-like B-1a lymphocytes rapidly redistribute to regional mediastinal lymph nodes (MedLNs) during influenza infection to generate protective IgM. Here we demonstrate that influenza infection-induced type I interferons directly stimulate body cavity B-1 cells and are a necessary signal required for B-1 cell accumulation in MedLNs. Vascular mimetic flow chamber studies show that type I interferons increase ligand-mediated B-1 cell adhesion under shear stress by inducing high-affinity conformation shifts of surface-expressed integrins. In vivo trafficking experiments identify CD11b as the non-redundant, interferon-activated integrin required for B-1 cell accumulation in MedLNs. Thus, CD11b on B-1 cells senses infection-induced innate signals and facilitates their rapid sequester into secondary lymphoid tissues, thereby regulating the accumulation of polyreactive IgM producers at sites of infection.

Natural and induced B-1 cell immunity to infections raises questions of nature versus nurture.

Mouse B-1 cells are not only major producers of steady-state natural antibodies but also rapid responders to infections and inflammation. These discrete functions may be the outcomes of distinct environmental or developmental triggers that drive B-1 cells toward IgM production or an effector cell fate. Alternatively, distinct B-1 cell subsets may exist, which differ in their functional plasticity. In this paper, we summarize existing data suggesting that B-1 cells form a heterogeneous group of cells with distinct developmental requirements and nonoverlapping functions. Most spleen B-1 cells differ in development from that of bone marrow and peritoneal cavity B-1 cells, in that they develop in the absence of natural IgM. Functional heterogeneity is revealed by findings that B-1 cells in the bone marrow and spleen, but not the peritoneal cavity, generate natural serum IgM, while the latter are rapid responders to inflammatory and infectious insults, resulting in their relocation to secondary lymphoid tissues. A clearer understanding of the developmental and functional differences within the B-1 cell pool may reveal how they might be harnessed for prophylaxis or therapy.

Protective B cell responses to flu--no fluke!

The mechanisms regulating the induction and maintenance of B lymphocytes have been delineated extensively in immunization studies using proteins and hapten-carrier systems. Increasing evidence suggests, however, that the regulation of B cell responses induced by infections is far more complex. In this study, we review the current understanding of B cell responses induced following infection with influenza virus, a small RNA virus that causes the flu. Notably, the rapidly induced, highly protective, and long-lived humoral response to this virus is contributed by multiple B cell subsets, each generating qualitatively distinct respiratory tract and systemic responses. Some B cell subsets provide extensive cross-protection against variants of the ever-mutating virus, and each is regulated by the quality and magnitude of infection-induced innate immune signals. Knowledge gained from the analysis of such highly protective humoral response might provide a blueprint for successful vaccines and vaccination approaches.