Lymphoid Chimeras Research Articles

The transcription factor XBP-1 is essential for the development and survival of dendritic cells

Dendritic cells (DCs) play a critical role in the initiation, maintenance, and resolution of an immune response. DC survival is tightly controlled by extracellular stimuli such as cytokines and Toll-like receptor (TLR) signaling, but the intracellular events that translate such extracellular stimuli into life or death for the DC remain poorly understood. The endoplasmic reticulum (ER) stress, or unfolded protein response (UPR), is a signaling pathway that is activated when unfolded proteins accumulate in the ER. The most conserved arm of the UPR involves IRE1α, an ER transmembrane kinase and endoribonuclease that activates the transcription factor XBP-1 to maintain ER homeostasis and prevent activation of cell death pathways caused by sustained ER stress. We report that XBP-1 is essential for DC development and survival. Lymphoid chimeras lacking XBP-1 possessed decreased numbers of both conventional and plasmacytoid DCs with reduced survival both at baseline and in response to TLR signaling. Overexpression of XBP-1 in hematopoietic progenitors rescued and enhanced DC development. Remarkably, in contrast to other cell types we have examined, the XBP-1 pathway was constitutively activated in immature DCs.

Engraftment of quiescent progenitors and conversion to full chimerism after nonmyelosuppressive conditioning and hematopoietic cell transplantation in miniature swine

Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoietic cell transplantation that leads to mixed chimerism and allograft tolerance in miniature swine across minor and major histocompatibility disparities. Stable chimerism persisted in most of these animals but was restricted to T cells and confined to peripheral blood. Because of the importance of myeloid and erythroid progenitors for the treatment of hematologic disorders, the objective of this study was to assess whether such cells existed in the bone marrow of these lymphoid chimeras as an indication of functional engraftment. Colony-formation assays were performed on donor inocula before infusion and on bone marrow cells harvested from the transplant recipients. Donor-origin myeloid/erythroid progenitor colonies were detected in bone marrow from 6 of 7 lymphoid chimeric recipients. A delayed donor leukocyte infusion successfully converted a stable lymphoid chimera to full multilineage chimerism within 2 weeks. Donor-origin myeloid/erythroid progenitors could be detected in the bone marrow of a host-matched recipient after myeloablation and adoptive transfer of mobilized cells from one of the engrafted lymphoid chimeras. These data suggest that even when only lymphoid chimerism is readily detected by flow cytometry, dormant myeloid/erythroid progenitors can exist and subsequent conversion to full donor chimerism can be achieved. The ability to establish multilineage engraftment and chimerism without significant toxicity may have important clinical implications for the management of nonmalignant hematopoietic disorders and hematologic malignancies.

The X-box binding protein-1 transcription factor is required for plasma cell differentiation and the unfolded protein response.

X-box binding protein-1 (XBP-1) is a transcription factor essential for plasma cell differentiation. XBP-1 transcripts are found at high levels in plasma cells from rheumatoid synovium and myeloma cell lines. Lymphoid chimeras deficient in XBP-1 have a profound defect in plasma cell differentiation, with few plasma cells in their periphery and severely reduced serum immunoglobulin levels. When introduced into B-lineage cells, XBP-1 initiates plasma cell differentiation. XBP-1 is also the mammalian homologue of the yeast transcription factor Hac1p, an important component of the unfolded protein response (UPR). The UPR allows cells to tolerate conditions of endoplasmic reticulum (ER) stress caused by misfolded proteins. Studies examining the relationship between plasma cell differentiation, XBP-1, and the UPR demonstrate that this novel signaling system is vital for plasma cell differentiation. Signals that induce plasma cell differentiation and the UPR cooperate via XBP-1 to induce terminal B-cell differentiation. Additionally, XBP-1 plays an important role in the regulation of interleukin-6 production, a cytokine essential for plasma cell survival.

Engraftment and activity of anti-CD3-activated human peripheral blood lymphocytes transferred into mice with severe combined immune deficiency.

Human PBL (huPBL) were activated with anti-CD3 mAb in vitro and then were transferred into mice with severe combined immune deficiency (SCID) to determine the effect of activation on engraftment and to determine if the engrafted human cells could provide antitumor effects in mice. Some mice were also treated with human rIL-2 after huPBL transfer. Mice were analyzed 6 to 8 wk after cell transfer and the number of human cells in the peripheral lymphoid organs was determined. Mice receiving anti-CD3-activated huPBL demonstrated a significant increase in the incidence of human T cell engraftment in the periphery as assessed by flow cytometry. Human cells were also detected in the murine thymus after anti-CD3 stimulation indicating that human T cells can migrate to the murine thymus provided that they are activated before the transfer. The transfer of anti-CD3-activated huPBL also resulted in an xenogeneic graft-vs-host reaction manifested primarily by proliferation of murine splenic hemopoietic cells. When SCID mice received the human colon carcinoma HT29, the concurrent transfer of anti-CD3-activated human cells resulted in a significant increase in survival. However, the human cells displayed low cytolytic activity toward human tumor targets when they were recovered from the lymphoid organs of the SCID recipients. Supernatants from the anti-CD3-activated cells were able to inhibit the growth of HT29 in vitro, partly because of the presence of IFN-gamma, suggesting that the human T cells are producing cytokines in vivo that have antitumor effects. Thus, the use of anti-CD3-activated huPBL in SCID mice may be of value for optimizing human cell engraftment in the human/mouse lymphoid chimeras and may be used to evaluate potential anti-neoplastic therapies that employ human effector cells.

Do donor cells function as veto cells in the induction and maintenance of tolerance across an MHC disparity in mixed lymphoid radiation chimeras?

Mixed lymphoid chimeras can be established across H-2b-->H-2d by injection of C57BL/6J (B-6) donor bone marrow cells into BALB/c hosts conditioned by sublethal irradiation, 235 cGy x3. These chimeras are specifically tolerant to both donor and host alloantigens. Tolerance cannot be broken even by injection of 4 x 10(8) normal BALB/c spleen cells (SC), suggesting a suppressor mechanism. In contrast to conventional suppression, however, in which suppressors are syngeneic to the cells they suppress, tolerance can be transferred only by cells of the allogeneic donor allotype (1,2). Thy 1+ cell depletion eliminates the capacity of the donor population to transfer tolerance and markedly reduces the capacity of B-6 BMC to induce tolerance (3). In the present studies spleen cells from tolerant chimeras (CSC), when coinjected in a 1:1 ratio, reduced the high GVHD mortality induced in irradiated H-2d/b F1 hybrids by injection of 2 x 10(7) normal BALB/c SC alone (P < .01). When coinjected at a 5:1 ratio CSC eliminated the GVHD mortality and weight loss induced by 5 x 10(6) BALB/c SC (P < .01). Depletion of B-6 cells from CSC removed their capacity to inhibit the GVHD induced by normal BALB/c SC. In contrast to conventional suppression, which requires the continued presence of suppressor cells, the BALB/c cells isolated from CSC, although unable to inhibit the GVHD of normal BALB/c SC, remained nonreactive against the H-2b allotype for a prolonged period following depletion of the B-6 cells. This prolonged response reduction dependent upon some interaction with allogeneic donor cells--which, in turn, are specifically nonreactive to host antigens--is compatible with a veto mechanism.

Human-mouse lymphoid chimeras: host-vs.-graft and graft-vs.-host reactions.

Human peripheral blood lymphocytes (huPBL) were injected into mice with severe combined immune deficiency (SCID). It was ascertained that murine natural killer (NK) cells were capable of affecting engraftment of human lymphocytes in SCID mice. The presence of host NK cells resulted in the clearance of the human lymphocytes. Human T lymphocytes were the primary cell to engraft in the SCID recipients, with human T cells being detected in the spleen, lymph nodes, bone marrow and peritoneal cavity of the mice up to several months after transfer. No human T cells were detected in the murine thymus and the level of engraftment in the periphery could be highly variable. Additionally, there appeared to be significant reactions between the human lymphocytes and the murine host resulting in a xenogeneic graft-vs.-host reaction (XGVHR). The predominant manifestation involved splenomegaly resulting from an expansion of murine hematopoietic cells in the spleens of these xenogeneic chimeras. The severity of the XGVHR could be correlated with the extent of human T cell engraftment and the recovered human T cells were found to be in a proliferative state. Thus, there appear to be significant host-vs.-graft and graft-vs.-host interactions occurring in human/mouse lymphocyte chimeras.

Cytotoxic and proliferative T-cell clones with antidonor reactivity from a patient transplanted for severe combined immunodeficiency disease

Patients who have become split lymphoid chimeras (T cells of donor origin, B cells and monocytes of host origin) following transplantation of HLA-haploidentical marrow for the treatment of severe combined immunodeficiency disease provide a unique model for the study of tolerance. One such patient, UPN 345, was transplanted with maternal marrow and was found to have antidonor proliferative reactivity without detectable donor-directed cytotoxicity when tested at 18, 23, and 66 mos following bone marrow transplantation. In bulk culture, the proliferation to donor cells could be blocked by monoclonal antibodies to HLA-DR and -DQ. Nine clones with antidonor reactivity were established by limiting dilution techniques from a mixed lymphocyte culture between engrafted T cells and irradiated donor E rosette—negative cells. All of the clones were of maternal donor origin, and all were CD3 +CD4 +CD8 −. The clones were tested for proliferative and cytotoxic activity toward donor, host, and paternal B-lymphoblastoid cell lines (B-LCL). Six clones proliferated strongly to maternal B-LCL but not to host B-LCL. Six clones were found to exclusively lyse maternal B-LCL. Four of the clones had both antidonor cytotoxic and antidonor proliferative reactivity. Monoclonal antibody blocking studies were performed on five of the six clones with cytotoxic activity. The antidonor cytotoxicity was not inhibited by monoclonal antibodies to class I determinants; however, three clones were inhibited in the presence of monoclonal antibody to DR, one clone was inhibited by anti-DQ monoclonal antibody, and one clone was inhibited by anti-DP monoclonal antibody. The cytotoxicity of all five clones was inhibited by monoclonal antibody to CD4. These data indicate that antidonor reactivity may also include a cytotoxic component which is not apparent in bulk cultures and which, based on our limiting dilution studies, is probably controlled by regulatory cells. Both the antidonor cytotoxicity and the antidonor proliferation appear to be directed primarily toward donor HLA class II antigens that are not shared with the patient.

The cellular basis for resistance to induction of tolerance in BXSB SLE male mice.

The autoimmune BXSB male mouse early in life displays a resistance to tolerance induction that is common with the other autoimmune strains (MRL/1, NZB x W, and NZB) whereas its female counterpart, which in early life is immunologically normal, is easily tolerized. The cellular basis for the BXSB male's resistance to tolerance induction with DHGG was investigated by using adoptive male-female cell transfer systems. We found that irradiated female recipients of male T-depleted bone marrow cells, plus either male or female thymus cells, resisted tolerance induction, had hyperactive splenic Ig-secreting cells, and developed early glomerulonephritis. Recipients of male thymic cells plus T-depleted female bone marrow cells could be tolerized, did not have enhanced Ig secretion, and did not develop glomerulonephritis. Furthermore, donor male T-depleted bone marrow cells--but not male thymic, female thymic, or female bone marrow cells--resisted induction of tolerance before their transfer to produce lymphoid chimeras.

Induction of partial chimerism in nonirradiated B-lymphocyte-deficient CBA/N mice.

Nonirradiated B-lymphocyte-deficient CBA/N mice given T6T6 chromosome-marked normal CBA/CaHN spleen cells became lymphoid chimeras exhibiting donor-type mitoses. Normal CBA/CaHN recipients did not exhibit significant numbers of donor-type mitoses. The lymphoid cell chimerism in the CBA/N host appeared in spleen, lymph nodes, and Peyer's patches, but not in marrow or thymus. Stimulation of CBA/N-recipient spleen cells in vitro suggested that the chimerism involved donor T6T6 cells which were responsive to the B-lymphocyte mitogen, lipopolysaccharide, but not to the T-lymphocyte mitogen, phytohemagglutinin. These data indicate that stable, long-term chimerism of a specific class of lymphocytes is possible in nonirradiated, B-lymphocyte-deficient CBA/N mice.

Further study on chimerism in tolerant mice.

SummaryBy inoculation of 40 × 106 (A × T6)F1 spleen cells intraperitoneaUy into newborn A strain mice incomplete tolerance of varying duration was established to skin grafts of the spleen donor strain. Three long-term incompletely tolerant A mice were cellular lymphoid chimeras with 2.3% of T6 positive donor cells in their spleens when sacrificed at 116 days after skin grafting. Lymphoid cells (30 × 1016) from 3 comparable animals sacrificed at 130 days produced fatal homologous disease when injected into sublethally irradiated (A × T6)F1 hybrids. The F1 hybrids succumbed after a longer latent period (19-78 days) than after injection of lymphoid cells (30 × 106) from control strain A mice (10-26 days).Seven strain A mice with short-term tolerance (15 to 55 days), did not immediately regain their immunological reactivity. A second test graft of the same origin placed after rejection of the first graft survived for periods remarkably similar to those for the first graft. When 2 comparable short-term toleran...