Immunospecific depletion of graft-versus-host-reactive lymphocytes using sensitized syngeneic initiator T lymphocytes.

We investigated a model of a lethal graft-versus-host (GVH) reaction with the aim of depleting donor spleen cells of immunospecific GVH-reactive lymphocytes. In previous studies of the recruitment of effector T lymphocytes by sensitized syngeneic initiator T lymphocytes (ITLs) we found, using a local GVH reaction, that precursors of specific GVH-reactive lymphocytes were recruited to a draining lymph node. In this study, adult F1 hybrid mice were lethally irradiated and reconstituted with 2 x 10(6) syngeneic bone marrow cells and varying numbers of spleen cells from parental strain mice. To deplete donor spleen cells of GVH-reactive lymphocytes, parental strain mice were given injections in the hind footpads 6 days earlier of syngeneic ITLs that had been sensitized in vitro against allogeneic fibroblasts. We found that injection of ITLs sensitized against the relevant allogeneic antigens led to a marked decrease in the specific GVH potential of donor spleen cells. These findings show that GVH-reactive lymphocytes can be depleted selectively by activating their recruitment to particular lymph nodes, using syngeneic ITLs.     

Comparative analysis of immunological reconstitution induced by vascularized bone marrow versus bone marrow cell transplantation

Abstract  We have reported previously that vascularized bone marrow transplantation (VBMT) in an orthotopic hind limb graft brings about complete repopulation of bone marrow cavities in lethally irradiated syngeneic recipients within 10 days. Intravenous infusion of an equivalent volume of bone marrow cell suspension was evidently less effective. The purpose of this study was to investigate the reconstitution of immunocompetent compartments of lethally irradiated syngeneic rats after VBMT. Lewis rat hind limbs were transplanted orthotopically into irradiated recipients. Ten days after irradiation and bone marrow transplantation, bone marrow, mesenteric lymph nodes, and sera from rats were harvested. Mesenteric lymph node lymphocytes were analyzed. The responsiveness fomesenteric lymph node lymphocytes (MLNL) to mitogens and cell proliferation in the presence of sera and bone marrow cell (BMC) culture supernatants were measured. Our studies have shown that vascularized bone marrow transplantation brings about rapid replenishment of lymphoid organs of lethally irradiated syngeneic recipients. The re-populating subsets are fully responsive to mitogens. Sera from reconstituting rats had no effect on the proliferation of mature lymphocytes. Intravenous infusion of a number of BMC in suspension equivalent to that grafted in hind limb transplant was less efficient in reconstitution of lymphoid tissue.     

Successful semiallogeneic and allogeneic bone marrow reconstitution of lethally irradiated adult mice mediated by neonatal spleen cells

Spleens of fetal/newborn mice less than 3-4 days of age contain a naturally occurring cell population capable of suppressing T-dependent and T-independent immune responses of third-party adult cells both in vitro and in vivo. We have utilized newborn spleen cells to prevent acute graft-versus-host (GVH) disease in lethally irradiated adult hosts reconstituted with semiallogeneic or even allogeneic bone marrow cells. Pretreatment of reconstituting cell populations with newborn spleen cells reduced the incidence of GVH disease from 100% to 20% in semiallogeneic and from 100% to 40% in allogeneic combinations. Long-term-surviving reconstituted hosts proved immunologically unresponsive to both donor and host histocompatibility antigens, yet possessed a fully chimeric lymphoid system responsive to T and B cell mitogens, as well as unrelated third-party alloantigens.     

Progress toward Production of Immunologic Tolerance with No or Minimal Toxic Immunosuppression for Prevention of Immunodeficiency and Autoimmune Diseases

With donor and recipient matched at the major histocompatibility complex (MHC) locus, peripheral lymphoid tissue transplantation can be carried out without producing a graft-versus-host reaction or graft-versus-host disease (GVHD), thus correcting profound T cell immunodeficiencies of neonatally thymectomized mice. This analysis set the stage for clinical application of bone marrow transplantation (BMT) to provide for the first time cure of a human disease. With successful BMT, we cured immunologic deficiencies of a patient with XL severe combined immunodeficiency; thereafter we were the first to employ BMT to cure aplastic anemia. BMT regularly corrects immune and hematologic deficiencies caused by fatal irradiation without producing GVHD if the bone marrow (BM) used for the transplants has been purged of postthymic T cells. Over two decades in conjunction with Ikehara et al., we have shown that lethal total body irradiation (TBI) plus allogeneic BMT prevents or cures many organ-specific and systemic experimental autoimmune diseases. Animal models successfully treated by BMT include type I diabetes in nonobese diabetes (NOD) mice, type II diabetes in insulin-insensitive, glucose intolerant, diabetes mellitus (KK/Ay) mice, and autoimmune lupus erythematosus (LE) and glomerulonephritis in New Zealand Black × New Zealand White first generation hybrid (NZB × NZW)F1 females. El-Badri extended Ildstad's original research showing a high frequency of survival with a normal functioning immune system after stable mixed chimerism is produced by mixed BMT in C57BL/6 (normal long-lived black strain) mice transplanted with T cell-depleted marrow (TCDM) from BALB/c (“normal” long-lived strain) allogeneic donors and C57BL/6 syngeneic donors. We showed that osteoblasts act as facilitator cells for allogeneic BMT and promote engraftment of allogeneic hematopoietic stem cells. Wang et al. then showed that the autoimmunities and fulminating renal disease of BXSB (C57BL × SB cross and selective lupus-like systemic autoimmunity) male mice was prevented and could be cured by transplantation using TCDM from both BALB/c (resistant) and BXSB (susceptible) strains given to BXSB recipients after lethal TBI. This treatment produced mixed BMT and a stable mixed chimerism, increased longevity, corrected all manifestations of autoimmunity, and cured fulminant glomerulonephritis in these recipients. These studies generated a new perspective on the potential usefulness of BM and stem cell transplants to cure major diseases that can possibly be treated by BMT. Mixed BMT from TCD BALB/c and BXSB mice cured autoimmunities and fulminant glomerulonephritis in BXSB mice. LE disease plus coronary vascular disease that occurs in (NZW × BXSB)F1 mice, along with idiopathic thrombocytopenic purpura, is also cured in lethally irradiated (NZW × BXSB)F1 mice by BMT from C57BL/6 donors. Furthermore, hemolytic anemia, autoimmune phenomena, and hyalinizing glomerular renal disease of New Zealand Black (NZB) mice were prevented or cured by stem cell transplants using purified stem cells from MHC-matched DBA/2 donors or NZB donors. Consequently, we reasoned that autoimmunities reside in stem cells. More recently, we found that transplants of both BM cells and bones can completely and permanently prevent otherwise highly resistant autoimmune diseases of MRL/lpr lpr mice and an autoimmune polyarthritis of NZB/Kn mice. Ildstad concluded that lethal preparative measures would not be acceptable for preparations to treat autoimmune diseases, so we now employ a gentle method for producing stable mixed chimerism described by Sharabi and Sachs to achieve mixed marrow transplantation and mixed hematopoietic chimerism. Other diseases we are approaching using this gentle manipulation include two forms of diabetes: insulin-dependent diabetes mellitus (IDDM) type I in NOD mice and non-insulin-dependent diabetes mellitus (NIDDM) type II in KK/Ay mice, atherosclerosis of apolipoprotein-E + knockout ApoE^−/− knockout (K.O.) mutant mice that can be cured by BMT, and senility-accelerated disease in systemic amyloidosis and acceleration of aging model (SAM-P) mice accompanied by and driven by autoimmunities. Today, clinical BMT sometimes cures 75 otherwise lethal diseases, including acute and chronic resistant leukemias, lymphomas, aplastic anemias, cancers, hematologic anomalies, and inborn errors of metabolism. The new findings suggest that BMT may become an effective treatment for immunodeficiencies, autoimmunities, and hematologic diseases for which current treatments remain inadequate.     

Immune dysfunction associated with graft-versus-host reaction in mice transplanted across minor histocompatibility barriers. I. Depressed antigen-specific antibody responses to bacteriophage phi chi 174

The effect of the graft-versus-host reaction in response to minor histocompatibility antigens on the antibody response to a T-dependent antigen was studied in four strains of mice. Lethally irradiated mice were transplanted with bone marrow plus graded numbers of spleen cells from H-2-compatible donors. Recipients of syngeneic bone marrow transplants and recipients of allogeneic bone marrow depleted of T cells made normal antibody responses to bacteriophage phi chi 174 when immunized on day 28 (primary) and day 56 (secondary) after marrow transplantation. Recipients of allogeneic bone marrow plus spleen cells made only small amounts of specific antibody and failed to make IgG antibody after secondary immunization. The pattern of the depressed antibody response suggests that the primary mechanism of immune dysfunction in mice with the minor antigen GVHR is a lack of T helper cell function.     

Prevention of graft-versus-host mortality in mice by preincubation of the graft with highly purified spleen-derived immunosuppressive peptides

Two non-species-specific spleen-derived immunosuppressive peptides (SDIP) have been highly purified from bovine spleen and are referred to as SDIPe and SDIPHP with reference to the last step of the purification procedure (electrophoresis or high pressure liquid chromatography, respectively). Preincubation of lymphoid cells with very dilute preparations of both SDIPs significantly reduced their capacity to induce a lethal graft-versus-host reaction (GVHR), but did not diminish their capacity to form hematopoietic colonies in the spleens of lethally irradiated syngeneic hosts (CFUs). Therefore, such pretreatment of bone marrow grafts by SDIP could be useful candidate for GVHR prevention. In contrast, preincubation with thymulin (serum thymic factor (FTS) plus ZnCl2), which shares several physicochemical properties with SDIP, significantly improved the ability of lymphoid cells to induce lethal GVHR. The opposite immunomodulatory effects of these highly similar peptides are discussed.     

Bone marrow transplantation in the rat. V. Lymphoid cell subclasses in the target organs during acute graft-versus-host disease

The distribution of white cell subclasses in different lymphoid (bone marrow, spleen, and blood) and parenchymal (liver, skin, lungs, and gut) target organs was studied after bone marrow transplantation in the rat. BN rats were irradiated and transplanted with 60-80 X 10(6) Lew (allogeneic) or BN (syngeneic) bone marrow cells. The recovery of lymphocytes was somewhat elevated in the bone marrow and spleen, slightly decreased in the blood, and markedly higher in the liver and skin in the allograft compared with the syngeneic graft recipient. A mild lymphocytic bronchitis was present in the lungs of the allografted animal, and the gut was hypocellular throughout the observation period. The total recovery of different lymphocyte subclasses; pan T, T helper, T suppressor-killer, class-II-positive cells, and surface-Ig-positive B cells in the different lymphoid organs--i.e., bone marrow, spleen, and blood--was similar in allogeneic compared with syngeneic graft recipients. In the liver and skin, which are the major target organs of acute graft-versus-host disease (aGVHD) in the rat, there was a massive infiltration of different T cell subclasses; high numbers of B cells were also seen in the liver. There was no difference in the T helper/T suppressor-killer ratio in the lymphoid organs or the liver of allograft compared with syngeneic graft recipients; in the skin and lungs the ratio was reduced more in the allograft compared with syngeneic graft recipient, whereas in the gut the situation was the opposite. These observations emphasize regional differences in the structure of inflammation in the different parenchymal target organs of aGVHD in the rat.     

Induction of stable chimerism and elimination of graft-versus-host disease by depletion of T lymphocytes from bone marrow using immunomagnetic beads

The goal of transplantation is the induction of immunologic tolerance. At present, nonspecific immunosuppression is used to prevent graft rejection and, commonly, graft-versus-host disease (GVHD). Nevertheless, nonspecific immunosuppressive therapy is frequently complicated by infection, malignant tumors, and drug toxicity. In order to examine whether hematopoietic chimerism can be used to induce specific allograft tolerance, we have reconstituted lethally irradiated Lewis rats with ACI bone marrow that has been depleted of T cells with use of immunomagnetic beads. This technique consists of binding OX-19, a mouse anti-rat pan-T lymphocyte monoclonal antibody, to magnetic polymer beads. Mixing of bone marrow or splenocytes with the bead/OX-19 complexes, followed by magnetic separation, results in significant depletion of T cells with minimal nonspecific cell loss. Immunomagnetic T-cell depletion of bone marrow, followed by reconstitution of a lethally irradiated host, allows for the development of stable, mixed hematopoietic chimerae without evidence of GVHD. These hosts are immunocompetent by clinical criteria. Recipients of untreated donor bone marrow that did or did not receive nonspecific immunosuppression demonstrated varying degrees of GVHD and reduced survival. The ability to rapidly and simply deplete T lymphocytes from bone marrow and produce stable, immunocompetent hematopoietic chimerae without GVHD may be an important method for tolerance induction to vascularized allografts.     

Viral interleukin-10 gene therapy to induce tolerance to solid organ transplants in mice

In this study, a novel gene therapy approach to prolong allograft survival was designed. Autologous (syngeneic) hematopoietic stem cell-enriched bone marrow cells (HSC; lin(-)) engineered with the vIL-10 gene (vIL-10-HSC) were injected (4 to 6 x 10(6) cells, i.v.) into lethally (9.5 Gy) or sublethally (4 Gy) irradiated CBA/J mice 6 weeks prior to allogeneic heart (C57BL/6) transplantation (Tx). Cardiac allograft survival was significantly (P <.004) prolonged in lethally (71 +/- 40 days) and sublethally (114 +/- 15 days) irradiated mice that received vIL-10-HSC compared to controls that received no HSC (11 +/- 1 days), unengineered HSC, or vector-DNA-engineered HSC (12 to 16 days). Tolerant graft histopathology demonstrated mild arteritis/venulitis (grade 0.7) and rejection (grade 1.0). Intragraft expression of costimulatory molecules (B7.1, B7.2), cytokines (IL-2, IL-4, mIL-10, IFN-gamma), and iNOS molecules were markedly lower in tolerant grafts that survived for >100 days; recipient T lymphocytes demonstrated hyporeactivity to donor and third-party antigens in mixed lymphocyte cultures. These findings have important implications and potential therapeutic applications in transplantation and autoimmune diseases.     

Bone marrow transplantation with T-cell-depleted grafts. I. Reconstitution of immunohemopoietic functions in lethally irradiated mice transplanted with unseparated or T-cell-depleted syngeneic bone marrow grafts

The potential role of donor's mature T lymphocytes on the recovery of various immunological functions and hematopoiesis was investigated in lethally irradiated BALB/c mice by studying reconstitution with normal, as compared with T-cell-depleted, syngeneic marrow grafts. Recovery of total, as well as mononuclear, peripheral white blood cell counts, platelets, hemoglobin levels, proportion of Thy 1.2+ cells, responses to concanavalin A, phytohemagglutinin and lipopolysaccharide, mixed lymphocyte response, cell-mediated lympholysis response, anti-SRBC agglutinins and natural killer activity were basically similar in recipients of unmanipulated (as compared with T cell depleted) syngeneic marrow grafts. The data suggest that in a syngeneic murine bone marrow transplantation setting, mature donor T lymphocytes do not seem to play a major role in immunohemopoiesis. Normal T cell number and T-cell-dependent immune function can be readily regenerated out of the stem cell reservoir of adult donors following transplantation into lethally ablated recipients.     

Protection against murine ascites tumours by lymphoid cell populations with T memory or cytotoxicity.

It was possible to protect irradiated C3H mice against lethal doses of allogeneic ascitic tumor cells (RBL-3 or P-815 Y) by systemic administration of low doses of syngeneic sensitized lymphoid cells. Two types of cell populations were active at similar doses: (1) spleen cells harvested 2 months after a nonlethal inoculation of tumour cells, at which time no cytotoxic lymphocytes wer in vivo sensitization or in vitro sensitized spleen cells; sucll and dose dependent and target cell specific. The findings imply that T memory cells as well as cytotoxic cells are able to protect, although it was not possible to exclude the presence of memory lymphocytes in the cytotoxic cell population. Antiserum to cytotoxic T cells (CTL) was prepared in an attempt to distinguish memory and cytotoxic effector cells. The antiserum did not react with the majority of T cells in a normal spleen or thymus, but showed specificity for the CTL cell lineage. Antiserum treatment and complement abolished protection in all types of sensitized cell populations. Since memory and cytotoxic cells appeared to share differentiation antigens, we could not establish whether memory cells are precursors or products of cytotoxic cells.     

Fast lymphoid reconstitution after vascularized bone marrow transplantation in lethally irradiated rats.

BACKGROUND: Bone marrow (BM) transplantation for treatment of hematological and solid malignancies is routinely carried out in conjunction with radio- and chemotherapy. Many patients achieve complete remission of the malignant process; however, their lymphohematopoietic recovery remains in most cases incomplete. This is probably due to the functional changes in the recipient BM stromal cells subsequent to myeloablative therapy. Transplantation of BM hematopoietic cells in a spatial relationship with stromal cells would give an insight into the kinetics of hematological repopulation of the recipient. The aim of this study was to investigate the lymphopoietic reconstitution of irradiated rats after vascularized bone marrow transplantation (VBMT) in comparison with i.v. bone marrow cell (BMC) infusion. METHODS: Lewis rats were totally irradiated with 8Gy and repopulated with syngeneic BMC introduced i.v. or in orthotopic hind limb graft. Ten days after irradiation and BMC graft BM, peripheral blood (PB) and mesenteric lymph nodes (MLN) were collected. The yield and the phenotype of cells were analyzed. RESULTS: VBMT brings much higher cell repopulation of BM cavities of lethally irradiated rats than BMC infusion. Orthotopic hind limb graft promotes also rapid lymphocyte replenishment of PB and MLN of lethally irradiated syngeneic recipients. The population rate of BMC, PB lymphocytes, and MLN lymphocytes was higher after VBMT than BMC injection in suspension. The percentage of T and B lymphocytes in PB and MLN on day 10 after VBMT was comparable with control values. Reconstituted PB lymphocytes showed two subsets of CD4+ cells: "bright" and "dull." All CD4+ cells in PB lymphocytes of i.v. BMC infused recipients expressed low level of these molecules ("dull" subset). CONCLUSIONS: The results of our studies indicate that the presence of stromal cells in their close relationship with stem cells is essential for the fast lyphohematopoietic repopulation of irradiated recipients. The population of CD4+dull cells may represent immature cells. These cells were not found in MLN of VBMT rats. All MLN CD4+ cells represented the "bright" subset, what suggests that the process of cell maturation may occur in the lymphoid organs.     

Reciprocal allogeneic bone marrow transplantation between NOD mice and diabetes-nonsusceptible mice associated with transfer and prevention of autoimmune diabetes

Reciprocal allogeneic bone marrow transplantations were carried out between diabetes-susceptible nonobese diabetic (NOD) and diabetes-nonsusceptible C57BL/6 or B10.BR/cd mice to examine the role of the immune system and host environment in the development of autoimmune diabetes. Serotyping of lethally irradiated hosts reconstituted with allogeneic bone marrow showed the hematopoietically derived cells to be of donor origin. Our results showed that lethally irradiated NOD mice reconstituted with a B10.BR/cd hematopoietic cell system remained totally free of insulitis, failed to develop diabetes, and thrived to old age. In contrast, lethally irradiated C57BL/6 or B10.BR/cd mice reconstituted with an NOD hematopoietic cell system all developed insulitis, but only approximately 10% progressed to overt diabetes. Direct adoptive transfer of insulitis and diabetes by mature T-lymphocytes apparently was not required; analogous results were obtained when diabetes-nonsusceptible hosts were reconstituted with NOD hematopoietic cells containing T-lymphocytes or devoid of Thy-1+ cells. The difference in frequency for the development of insulitis versus insulitis plus overt diabetes in C57BL/6 and B10.BR/cd mice suggests that the hematopoietically derived immune cells from NOD mice were sufficient to induce anti-islet reactivity but may require the diabetogenic host environment to develop the frequency and severity of diabetes observed in NOD mice.     

Avoidance of graft versus host reactions in cured W-anemic mice.

Graft-versus-host reactions of parental cells in F1 hybrids were studied with two unrelated inbred strains of mice that differed at the mouse histocompatibility locus. W-anemic F1 recipients were compared with lethally irradiated normal F1 recipients. Both sets of recipients were populated by marrow and spleen cell grafts from parental and F1 donors. Most W-anemic F1 recipients were cured by parental and F1 cell grafts (except B6 spleen). Even after 13 to 18 months, they showed little or no effect from GVH reactions. Lethally irradiated normal F1 recipients tolerated parental marrow grafts almost as well, but gave dramatically different results with parental spleen grafts. Seventy-nine of 80 irradiated F1 recipients of parental spleen grafts died within 1 month. Unlike lethally irradiated recipients, W-anemic recipients have substantial numbers of their own cells along with the donor cells in their lymphoid tissues. These F1 lymphocytes may interact with parental lymphocytes in vivo to restrain reactions against F1 allogeneic antigens.     

Induction of intestinal graft-versus-host reactions across mutant major histocompatibility antigens by T lymphocyte subsets in mice

We have examined the ability of highly purified subsets of C57B1/6 L3T4+ and Lyt2+ cells to cause intestinal graft-versus-host reactions in H-2 mutant mice expressing isolated class I (bm1) or II (bm12) MHC mutations. Heavily irradiated B6xbm12)F1 mice given B6 L3T4+ T cells (anti-class II GVHR) developed an acute, lethal GVHR that was associated with intense jejunal crypt hyperplasia and an early rise in the density of intraepithelial lymphocytes. Irradiated (B6xbm1)F1 mice given B6 Lyt2+ T cells (anti-class I GVHR) showed a similar phase of crypt hyperplasia within the first 2 weeks, but this was less marked than for anti-class II GVHR and was not associated with an increase in IEL count. Only very minor gut pathology was observed when B6 Lyt2+ T cells were transferred to irradiated mice carrying the bm9 class I mutation, which is much weaker than the bm1 mutation and does not stimulate Lyt2+ helper T cells. The GVHR mediated by B6 L3T4+ and Lyt2+ T cells was H-2 class-specific, as no pathology was seen in bm12 recipients of Lyt2+ cells or in bm1 recipients of L3T4+ cells. B6 L3T4+ and Lyt2+ T cells both induced splenomegaly and intestinal GVHR in nonirradiated, 4-5-day-old neonatal (B6xbm12)F1 or (B6xbm1)F1 mice, respectively, a form of intestinal GVHR that does not require specific cytotoxic T lymphocytes. Again, Lyt2+ T cells were less efficient than L3T4+ T cells in the induction of GVHR. Thus, both class I and class II MHC-restricted T cells can mediate different forms of intestinal GVHR under appropriate circumstances, but class II MHC-restricted T cells may be more efficient.     

混合脐血移植后造血及免疫重建特性的研究

目的　探讨混合脐血移植重建造血和免疫功能的特点及规律。方法　分别将两份人HLA半相合、不相合混合脐血或单份脐血输入经亚致死量照射后的严重联合免疫缺陷 (SCID)小鼠。观察三组脐血在SCID小鼠体内植入状况及造血和免疫功能重建特性。结果　单份和混合脐血均可在受鼠体内植入 ,形成供 受混合嵌合体 ,植入率差异无显著性 (P >0 .0 5 ) ,并能重建人类多系造血 ,且易于向NK和B淋巴细胞方向分化。用多聚酶链反应 序列特异性寡核苷酸 (PCR SSO)探针检测人HLA DQB1基因发现 ,混合脐血移植可有 1份或 2份脐血植入 ,其中造血祖细胞含量和体外集落形成能力高者 ,更易于植入 ,且供者间HLA差异小者 ,趋向两份脐血同时植入。结论　混合脐血移植可重建SCID小鼠造血和免疫功能 ,但造血细胞各谱系分化不均衡。     

Induction by thymic fractions of T cell subsets capable of modulating GVHR intensity.

Two thymic stromal fractions previously shown to have specific enhancing effects on anatomically distinct T cell populations were tested for their capacity to induce functionally distinct T cell subsets. Parental mice were injected with either soluble thymic fraction (STF) or insoluble thymic fraction (ITF), and their lymphoid cells were harvested 11 days later. It was shown that ITF elicited a splenic corticosensitive T cell subset endowed with enhanced graft-versus-host reaction (GVHR)-inducing capacity. On the other hand, STF increased, mainly in lymph nodes, the number of corticoresistant T cells significantly less active in GVHR. Furthermore, lymphocytes from ITF-treated parental donors could become corticoresistant with reduced GVHR activity after a 1-hr in vitro incubation with STF. We have thus shown that two different elements of the thymic microenvironment could modulate the intensity of the GVHR by modifying the equilibrium between two T cell subsets. These are believed to represent two consecutive differentiation stages.     

Despite efficient intrathymic negative selection of host-reactive T cells,autoimmune disease may develop in porcine thymus-grafted athymic mice: evidence for failure of regulatory mechanisms suppressing autoimmunity

BACKGROUND: CD4 T-cell reconstitution and xenogeneic tolerance is achieved in T cell-depleted, thymectomized C57BL/6 (B6) mice and nude mice by grafting of fetal pig thymus (FP THY). Sixty percent of grafted nude mice and 10% of grafted thymectomized B6 mice develop a clinical illness resembling chronic graft-versus-host disease. METHODS: Negative selection of mouse T cells in FP THY grafts was studied in "AND" TCR transgenic mice with a negative selecting MHC. Pathologic and immunohistochemical examinations and adoptive transfer assays were performed to determine the role of mouse CD4+ cells in the occurrence of autoimmune disease in this model. RESULTS: Marked clonal deletion of mouse thymocytes bearing a transgenic TCR ("AND"), which recognizes H2s expressed by host hematopoietic cells, was observed in FP THY grafts. Pathologic and immunohistochemical examinations of the liver, skin, lungs, and kidneys of mice with wasting syndrome showed marked mouse CD4+ T-cell infiltration without detectable pig cells. After adoptive transfer of splenocytes, but not of CD4+ cell-depleted splenocytes, from sick mice along with B6 bone marrow cells to lethally irradiated syngeneic B6 mice, the secondary recipients developed a similar autoimmune syndrome as the donors. Cotransfer of na?ve syngeneic splenocytes prevented the occurrence of autoimmune disease in secondary recipients of splenocytes from healthy FP THY-grafted BALB/c nude mice. CONCLUSION: These results demonstrate a key role for mouse CD4+ T cells in causing autoimmune disease in this model and suggest the importance of regulatory mechanisms in addition to intrathymic clonal deletion for the maintenance of tolerance to recipient antigens.     

Analyses of development of insulitogenic T lymphocytes in NOD mice by transplantation of bone marrow, thymus, and pancreas

To estimate the functional participation of the insulitogenic genes in the development of insulitogenic lymphocytes, we attempted to induce insulitis in normal allogeneic BALB/c hosts by bone marrow transplantation from NOD mice. The development of the insulitogenic lymphocytes was histologically examined using the host's pancreas and pancreatic tissue from NOD mice which had been grafted under the renal capsules 2 weeks before sacrifice. Adult-thymectomized NOD mice that had been reconstituted with the thymus and bone marrow from NOD mice showed insulitis in both the host's pancreas and grafted pancreatic tissue from newborn NOD mice. When BALB/c mice were lethally irradiated and then reconstituted with NOD bone marrow cells, no insulitis developed in the pancreatic grafts from NOD and BALB/c mice or in the host's pancreas. Since the specificities and functions of T lymphocytes are controlled by the thymic microenvironment during the differentiation within the thymus, the thymus of BALB/c genotype may be responsible for the failure to induce the insulitogenic lymphocytes. Therefore, athymic BALB/c mice were reconstructed with bone marrow cells and thymus of NOD genotype. No insulitis developed, however, in either the host's pancreas or grafted pancreatic tissue. These results suggest that the bone marrow cells and thymus of NOD genotype are not sufficient to induce insulitogenic lymphocytes in the allogeneic BALB/c environment.     

Alloimmunization: induction of antileukemic reactivity without modification of anti-host reactivity in H-2-compatible mice

Transplanted immunocompetent cells from CBA (H-2k) mice immunized against individual or pooled lymphoid cells from various allogeneic strains resulted in the elimination of widely disseminated leukemia cells in AKR (H-2k) host mice as measured in a GVL bioassay. In most cases, alloimmunization resulted in reactivity against AKR-L comparable to that observed when CBA donors were specifically immunized with irradiated AKR-L cells. Cells from unimmunized or isoimmunized CBA mice had no detectable GVL reactivity. Alloimmunization caused no increase in the mild GVH reactivity of unimmunized or isoimmunized CBA lymphoid cells transplanted into lethally irradiated nonleukemic AKR hosts; whereas, specific immunization with AKR-LX cells resulted in significantly increased GVH-related mortality.