Graft-versus-host Response Research Articles

Organ weights, T-cell proliferation, and graft vs host capabilities of hypothyroidic chickens

Thiouracil-induced hypothyroidism reduced the thymus weights of young chickens. Thyroid weights were greater in thiouracil-fed (TF) chicks, and the circulating levels of triiodothyronine and thyroxine were lower. Thiouracil treatment resulted in higher relative adrenal weights and serum corticosterone concentrations. Absolute weights of adrenals were equivalent between control and TF groups. The TF chicks showed depressions of total white blood cell counts and numbers of lymphocytes. Monocyte and granulocyte numbers were unchanged from those of controls. Thiouracil treatment lowered the phytohemagglutinin M responsiveness of peripheral blood leukocytes (PBL). On the other hand, the PBL from TF chicks produced the higher graft vs host (GvH) response. Therefore, while the hypothyroidic state impaired cycling activity in T cells, this condition did not significantly influence a functional parameter of T cells, the GvH response.

Effect of Selection for Delayed Amelanosis on Immune Response in Chickens: 2. Cell-Mediated Immunity

Studies were conducted to compare the cell-mediated immune response of the Delayed Amelanotic (DAM) line of chickens with that of the line from which it originated, the Brown (BR) line, and a distantly related environmental control, the Light Brown Leghorn (LBL) line. Assays used to evaluate cell-mediated immunity were graft versus host (GvH) splenomegaly and the phytohemagglutinin (PHA) skin response. The three genetic stocks showed different responses to both tests with a variation in ranking between the tests. The GvH response of BR birds was significantly greater (P<.01) than that of DAM birds, which was in turn significantly greater (P<.001) than that of LBL birds. In the PHA skin test, the responses of BR and LBL birds did not differ but were significantly greater (P<.001) than those of DAM line birds.

Acquisition and extinction of conditioned suppression of a graft-vs-host response in the rat.

Injection of rats with cyclophosphamide (CY) after their consumption of a novel saccharin-flavored drinking solution results in a conditioned aversion to saccharin and a conditioned suppression of immune responses. In this study, female Lewis X Brown Norwegian F1 rats were conditioned by pairing saccharin with 50 mg/kg CY. Seven weeks later (day 0), a graft-vs-host response (GvHR) was induced in these animals by injecting splenic leukocytes from Lewis donors into a rear footpad. At this time, some conditioned animals were reexposed to saccharin, the conditioned stimulus. During the 7-wk interval between conditioning and immunization, subgroups of conditioned rats were given 0, 4, 9, or 18 extinction trials (saccharin followed by saline injections). Animals receiving 4, 9, or 18 extinction trials showed a greater preference for saccharin on day 0 than did animals receiving no extinction trials, but these groups did not differ among themselves; all conditioned groups showed a lower preference for saccharin than placebo-treated animals. There was a clear effect of number of extinction trials on the GvHR. Animals receiving 9 or 18 extinction trials did not differ from controls, whereas animals receiving 0 or 4 trials had a milder GvHR than did conditioned rats that were not reexposed to saccharin at the time of immunization. These results confirm a previous report of conditioned suppression of a GvHR, demonstrate that conditioned immunopharmacologic responses are subject to experimental extinction, and indicate that conditioned immunosuppression can be dissociated from conditioned taste aversion.

Genetic control and effector cells in host-versus-graft responses to H-Y antigen in mice.

T cell responses to the male-specific H-Y antigen in mice include skin graft rejection, delayed-type hypersensitivity and cytotoxic T cell responses; these are under complex H-2 and non-H-2 Ir gene control. The effector cells for these two in vivo responses are Ly 1+2-, and the cytotoxic T cell effectors generated in secondary mixed lymphocyte reactions in vitro are Ly1+2+, although their development requires the presence of Ly 1+2- T helper cells. We investigated the Ir gene control of another in vivo response to H-Y, the host-versus-graft response (HVGR), measured by popliteal lymph node enlargement. The strain distribution pattern (SDP) of primary and secondary HVG responses to H-Y indicates that there are both H-2 and non-H-2 Ir genes involved in controlling responsiveness. Cell transfer of the secondary response identifies an Ly 1+2- effector T cell for this response: this information together with the SDP of cytotoxic T cell responses suggests that the HVGR may represent activation of the T helper population involved in the generation of cytotoxic T cells.

Specific unresponsiveness to fully allogeneic kidney allografts in rats induced by procarbazine hydrochloride and antilymphocyte serum.

A short course of procarbazine hydrochloride (PCH; 50 mg/kg) and antilymphocyte serum (ALS; 5 ml/kg), administered to Lewis (LEW;RT1(1] rats in the first week following transplantation of Brown Norway (BN;RT1n) kidneys, substantially prolonged allograft survival and induced long-term survival in 62% of the grafts. The two agents acted synergistically, in that neither of them administered alone had much effect. Graft recipients did not produce detectable cytotoxic antibodies and antigen-reactive cells injected i.v. were not diverted to the liver, thus showing that neither antibodies nor immune complexes are likely to mediate the unresponsiveness. Spleen cells from graft-bearing recipients failed to cause graft-versus-host responses (GVHR) in both (LEW X BN)F1 and (LEW X DA)F1 hybrids, but they specifically suppressed the GVHR given by normal syngeneic cells to donor strain (BN) antigens. This suppression was specific because the response against third-party antigens (DA; RT1a) was unaffected. Adoptive transfer of spleen and thymus cells from PCH-ALS-treated LEW rats bearing healthy BN kidneys caused a profound prolongation of BN graft survival in sublethally irradiated LEW recipients. This transfer was specific and mediated by W3/13+ (T) lymphocytes. It is concluded that a limited regimen of PCH and ALS given in the first postoperative week incites the generation of specific suppressor T lymphocytes and that this form of immunosuppression, even without preoperative donor antigen, is an effective way of prolonging kidney allograft survival.

Calorie-Protein Deficiencies and the Immune Response of the Chicken: II. Cell-Mediated Immunity

Calorie-protein deficiency affects humoral immunity in the chicken. Secondary responses to sheep-red blood cells, a T-dependent antigen, were lower in birds fed diets two-thirds deficient in calories (C), amino acids (AA), or both (C/AA). These results led us to question what effects might be found in cell-mediated immunity after nutritional deficiency. Birds fed the same diets as in our previous study showed no reduction in total white blood cells, absolute lymphocytes, or absolute heterophils. Splenic lymphocytes from 3- or 7-week-old nutritionally deficient chickens with the exception of the AA group at 7 weeks, produced a nonsignificant increase in graft-versus-host (GvH) response of 12-day-old embryos. Thymic cells from 3-week-old nutritionally deficient birds were not deficient in the GvH potential.Thymic histology revealed reduced cellularity in AA-deficient birds. The reduced cellularity may not have been in response to stress since the mean corticosterone levels were not significantly different. Birds fed the deficient diets for 10 weeks had a significantly reduced delayed hypersensitivity response to human gamma globulin. The results indicate that the chickens’ ability to produce a GvH response was not compromised and that T-cell subpopulations may have differential sensitivities to nutritional deficiencies.

Graft Versus Host Response as Influenced by the Origin of the Cell, Age of Chicken, and Cellular Interactions

Mononuclear cells collected by Ficoll-paque or T cells collected by nylon wool elicited the same magnitude of graft versus host (GvH) response as mononuclear cells collected from the buffy coat (BC).Bone marrow, spleen, or thymic BC cells from day-old chickens did not produce a GvH response. The GvH ability of spleen and bone marrow BC cells increased rapidly during the first 4 weeks and then plateaued. Although at 4 and 12 weeks of age the GvH response produced by thymic BC cells was significantly less than spleen or bone marrow BC cells, thymic BC cells did produce a significant GvH response at these ages. Thymic BC cells from 11-month-old chickens showed significantly higher GvH response than those of previous ages. Corticosterone treatment of chickens eliminated the thymic cortex but did not enhance the GvH reactivity of thymic BC cells. Suppressor effect of thymic BC cells from day-old chickens was not detected.A synergistic effect was noted in the GvH reaction when thymic cells were combined with bone marrow or splenic BC cells of 4-week-old chickens.

Effect of cyclosporin A on the anti-leukaemia action associated with graft-versus-host disease.

Graft-versus-host disease (GVHD) was induced in Hooded (Rt1c) strain rats by means of high dose total body irradiation (TBI) and subsequent reconstitution with allogeneic bone marrow and spleen cells from WAG (Rt1u) strain donors. Untreated recipients of allogeneic cells died within 20 days of engraftment, whereas those treated daily with Cyclosporin A (CyA), given either from the day receipt of the graft (Day 0) or from Day 4, survived until the end of the experiment (Day 50). If delayed until Day 7, CyA prophylaxis was totally ineffective. Hooded rats bearing a syngeneic leukaemia were irradiated and reconstituted with allogeneic bone marrow. During the course of the ensuing graft-versus-host response (GVHR) leukaemia cells were eradicated from the spleens of the host animals. However, as a consequence of CyA prophylaxis, whether started on Day 0 or delayed until Day 4, the anti-leukaemia potential of the bone marrow allograft was completely abrogated. Anti-tumour activity after engraftment was detectable first at Day 7, i.e. the time at which the GVHR became intractable to the effects of CyA. The results indicate (1) that CyA suppresses the initial events but not the effector phase of the GVHR, and (2) that the anti-host and anti-tumour action of the GVHR may be temporally inseparable.

Synergistic interaction of H-Y and H-2K in elicitation of graft-versus-host response.

Lymph node cells from female bm-1 donors induced greater mortality among male than female (bm-1 X b)F1 hosts which had previously been sublethally irradiated. The same effect was not seen when wild type (b) parental donor cells were injected, nor in other combinations involving H-2K mutant C57BL/6 mice. The selective mortality encountered among male hosts in receipt of bm-1 female cells was shown to depend upon recognition of H-Y in the recipient, because this sex effect disappeared if the donors were first rendered tolerant of H-Y. However, absence of graft-versus-host (GVH) mortality with other combinations indicated that recognition of H-Y alone was not sufficient. When cells from donors tolerant of H-Y were mixed with cells from donors tolerant of H-2Kb, enhanced mortality among male F1 hosts was again observed. We interpret the results to indicate that there is a synergistic interaction of donor lymphocytes which recognize H-Y and H-2Kb during the GVH response.

Behaviorally conditioned suppression of a graft-versus-host response.

Cyclophosphamide (CY), previously used to condition suppression of humoral immune responses, was used to condition suppression of a graft-versus-host response (GvHR). Female (Lewis x Brown Norway) F1 rats were conditioned by pairing consumption of a saccharin solution with an intraperitoneal injection of CY at 50 mg/kg of body weight 48 days before immunization. On day 0, all animals were injected with a suspension of splenic leukocytes (2 x 10(7) cells per footpad) obtained from female Lewis donors. The regional GvHR was assessed on day 5 by weighing popliteal nodes. Conditioned animals given a single low-dose injection of CY and reexposed to conditioned stimuli had lymph node weights significantly lower than control groups and did not differ from animals given three injections of CY during the ongoing GvHR. The results suggest that conditioned immunosuppression, previously demonstrated in thymus-dependent and thymus-independent humoral immune responses, also affects the popliteal GvHR, a cellular immune response.

Role of Ia antigens in graft vs. host reactions. II. Molecular and functional analysis of T cell alloreactivity by the characterization of host Ia antigens on alloactivated donor T cells.

Graft vs. host response (GVHR)-activated donor T cells bind to stimulatory host cell-derived Ia antigens. Radioimmune cell-binding assays demonstrate that activated donor T cells acquire both host I-A and I-E alloantigens on their surface. Approximately threefold to fivefold less I-E products than I-A products are transferred. Immunoprecipitation and one-dimensional and two-dimensional gel electrophoresis analyses show that radioiodinated alpha and beta polypeptide chains of both I-A and I-E-encoded host Ia molecules may be transferred in an apparently structurally unaltered form from host cells to donor cells. Biosynthetic studies indicate that [35S]methionine-labeled activated donor T cells do not synthesize Ia antigens of the donor haplotype. Functional analyses with fluorescence-activated cell sorter sorted donor T cell subpopulations show that donor T cells that bind host I-A antigens preferentially cooperate with nonimmune host B cells. Donor T cells that do not bind detectable amounts of host I-A antigens preferentially help nonimmune donor B cells. By contrast, donor T cells that either bind or do not bind host I-A antigens display no H-2-restricted interaction and help both donor and host immune B cells. These data reveal that the Ia antigen-binding specificity of distinct functional subpopulations of alloactivated donor T cells regulates their I-region-restricted (self or allo) helper activity for nonimmune B cells but not immune B cells. Furthermore, they suggest that T cell-macrophage and T cell-B cell collaboration is mediated by a complementary anti-Ia:Ia receptor:ligand type of interaction in which the receptor of a T cell binds to the ligand of an antigen-presenting macrophage and/or B cell.

Fatal response suggestive of graft-versus-host reaction following transplantation of spleen cells from allogeneic athymic (nude) donors.

Normal female SJL/J mice were exposed to 950 R of total body irradiation (TBI) and transplanted with allogeneic spleen or marrow cells from normal or nude (athymic) C57BL/10 donors. With nude mouse donor marrow, no evidence of graft-versus-host (GVH) response was seen and all SJL/J recipients survived for more than 75 days. In contrast, when spleen cells taken from the same nude C57BL/10 donors were engrafted into SJL/J mice the incidence of fatalities among the recipients was 70% by 60 days. Furthermore, all of the recipients of nude mouse spleen cells showed signs strongly suggestive of GVH response. Comparative fatalities among the recipients of cells from normal donors were 27% for marrow at 60 days and 100% for spleen at 11 days, and these were accompanied by the characteristic signs of GVH response usually seen after transplantation of cells from normal allogeneic donors. Transplantation of normal C57BL/10 marrow mixed with small numbers at normal spleen cells resulted in an increase in the number of fatalities among the SJL/J recipients, and an increase in the severity of the signs of GVH response as compared to that seen following engraftment of normal C57BL/10 marrow alone. However, no such increases in fatalities or severity were observed when similar amounts of nude C57BL/10 spleen cells were engrafted along with normal marrow cells into SJL/J recipients. The results suggest that a factor may exist in nude mouse spleen which in allogeneic transplantation can lead to a fatal response suggestive of GVH reaction, but that nude mouse spleen lacks the T cell-related ability to enhance GVH response that has been previously demonstrated following allogeneic transplantation using normal spleen and marrow donors.

Immune Deficiency Predisposing to Epstein-Barr Virus-Induced Lymphoproliferative Diseases: The X-Linked Lymphoproliferative Syndrome as a Model

The chapter describes diseases induced by EBV in immune compromised patients. The discussion begins by considering normal immune responses that combat Epstein-Barr virus. Features of infectious mononucleosis (IM) mimicking graft-versus-host response (GVHR) have been illustrated and speculated on. A spectrum of complications of IM, including death from infiltration of vital organs by infected B cells and malignant lymphoma, has been discussed in detail. Phenotypic expressions of the X-linked lymphoproliferative syndrome (XLP) and their immunopathogenesis have been presented to exemplify EBV-induced oncogenesis in immune deficiency. The immunopathogenesis of EBV-induced lymphoproliferative diseases and methods for detecting EBV in immunodeficiency patients have been outlined. These diagnostic monitoring procedures and therapies are being attempted by the lymphoproliferative syndrome and infectious mononucleosis research groups. The results of studies of malignant lymphoproliferative diseases in patients with immune deficiencies such as those accompanying renal transplantation are elaborated. Suggested techniques for documenting EBV infections in immunedeficient patients have been provided to guide diagnosticians and researchers.

A biphasic graft vs host response in aging chickens

The graft vs host (GvH) and phytohemagglutinin (PHA) wattle responses were the parameters chosen to evaluate cell-mediated immunity in aging chickens. At 6 and 24 months of age female spleen cells were significantly more effective in eliciting a GvH response than spleen cells from 1-, 2-, 12-, and 18-month-old females. The biphasic GvH response produced by female spleen cells was not observed for male spleens. On the other hand, peripheral blood lymphocytes from both males and females exhibited a single peak, 3–6 months, in their ability to produce a GvH response. The thymic-dependent PHA wattle response of males was significantly greatest at 1 month of age and declined thereafter. Once again the females exhibited a different pattern than the males. At 1 month of age the female's wattle response was significantly greater than at 3 months, but rather than declining with age the wattles of aging females responded like 1-month-old females. Several mechanisms are offered to explain the apparent age-related regression-regeneration of cell-mediated immunity in the female.

Suppression of graft-versus-host reactions in rats bearing implants in the anterior chamber of the eye.

The graft-versus-host (GVH) response of spleen cells from rats bearing either orthotopic skin grafts or allogeneic implants in the anterior chamber of the eye was evaluated using popliteal lymph node (PLN) assay. When a viable implant remained in the anterior chamber, the spleen cells of these rats produced a popliteal lymph node enlargement in F1 hybrids which was approximately 50% of that produced by a similar number of cells from a normal animal. Conversely, the GVH response of spleen cells from orthotopically skin-grafted rats was noted to be significantly increased over the response of spleen cells from normal animals. The decrease in the GVH response of implanted rat spleen cells was a specific reaction and not because of trauma or implantation, since spleen cells from rats bearing syngeneic implants had shown no reduction in their GVH-inducing ability. The PLN weights of rats receiving mixed population of normal and implanted rat spleen cells were always less than the weights observed with an equal number of normal spleen cells. These findings permit the assumption that implant-bearing rats may be lacking or low in cells that induce GVH reactions or that there is a delayed conversion of effector cells after early immune recognition.

Toxicity of Na 251CrO 4 when used to label rat lymphocytes

Lymphocytes preincubated with various activities of Na 2 51CrO 4 were tested for their ability to induce a local, popliteal lymph node graft-versus-host (GVH) reaction. Incubation with 100 μCi of the isotope per ml of cell suspension impaired the GVH activity, 25 μCi gave a reduced response in 1 out of 3 experiments, whereas 10 μCi had no effect. The reduced GVH response was due to radiation rather than chemical damage of the cells. Preincubation of the cells with either cold Na 2CrO 4 or outdated Na 2 51CrO 4 with concentrations of Cr identical to the ones used to label cells with 100 μCi/ml cells did not impair the GVH reactivity. The cellular uptake of Na 2 51CrO 4 showed first-order kinetic over wide concentrations ranges. Mitomycin C-treated lymphocytes showed a more heavily depressed GVH reactivity than would be expected from their moderately reduced ability to accumulate in the lymph nodes. It is recommended that doses of Na 2 51CrO 4 in excess of 10 μCi/ml cells should not be used for functional studies of lymphocytes.

The host component of the popliteal lymph node graft-versus-host (GVH) reaction. Identification of cells in the node and kinetics of cell proliferation.

The cellular composition of rat popliteal lymph nodes undergoing a GVH reaction was examined. The great majority of cells isolated from the nodes were lymphocytes, while the contribution of phagocytic cells was negligible (less than 5%). Both T and B cells were present in GVH nodes. However, the proportion of B cells was higher than in normal lymph nodes (40-50% vs 30%). Cells synthesizing DNA were enumerated autoradiographically in sections of GVH nodes up to day 7 of the response. A significant increase in the proportion of 3H thymidine labelled cells had already been found on the third day of the GVH response, before lymph node enlargement was apparent. Of the differnt days tested a maximum of 6% labelled cells was found on day 7. Liquid scintillation counting of cell suspension from such nodes showed an even higher increase in 3H thymidine incorporation in GVH vs normal nodes. These findings indicate that cell proliferation is a central mechanism behind the lymph node enlargement.

Regulation of Lymphocyte Trapping: The Negative Trap

Suppression of 51Cr-labeled lymphocyte homing to the spleen occurs after administration of insoluble protein antigen and as a late sequela to the graft-vs-host response (GVHr). This decrease in splenic localization of labeled cells, termed "negative trapping," has elements of immunologic specificity in that tolerance induction or presensitization abrogates the negative trap. Increasing the dose of antigen accelerates the appearance of the negative trap, which is, however, evanescent, lasting from 24 to 48 hr. Synergistic and antergistic regulation of the GVHr-induced lymphocyte trap is produced by populations of 850 R-irradiated thymocytes and by F1 cortisone-resistant thymocytes. The time at which these subpopulations cause suppression or amplification of the lymphocyte trap correlates with the activity of the GVHr-producing inoculum. These findings suggest that regulation of lymphocyte traffic may provide a mechanism for control of immune responses in vivo.

BONE MARROW TRANSPLANTATION IN INBRED STRAINS OF MICE

Lethally irradiated mice reconstituted with allogeneic bone marrow cells usually survive the irradiation damage, but their immune‐status is not completely restored. Thus, such mice show depressed humoral antibody responses to thymus dependent antigens, prolonged allogeneic skin graft survival, and their spleen cells display reduced graft‐versus‐host (GVH) activity when inoculated into newborn F1 hybrid mice. The present study was carried out in order to explore the reason(s) for the depressed immunological activity of allogeneic irradiation chimaeric mice. It was found that these mice had an elevated level of θ positive cells compared to mice transplanted with syngeneic bone marrow cells, but their spleen cells did not show any detectable specific or third‐party GVH response. Spleen cells from allogeneic irradiation chimaeras were not able to participate in a collaborative GVH response with lymph node cells either. Possible reasons for the immunological inertness of θ positive cells in allogeneic irradiation chimaeras are discussed.

Immune responses of rats deficient in thymus-derived lymphocytes to strong transplantation antigens (Ag-B). Graft-versus-host activity, allograft rejection, and the factor of immunization.

SUMMARY A selective deficiency of thymus-derived lymphocytes was produced in adult rats by thymectomy followed by 1,000 rads of whole body y-irradiation and bone marrow restoration. By the popliteal lymph node assay, the graft-versus-host (GVH) activity of lymphocytes from such “B rats” was depressed by a factor of at least 20 compared to lymphocytes from normal rats. GVH activity was restored to normal by replenishment with 10° thymocytes, and no synergy was found between lymphocytes from B rats and lymphocytes from normal rats when these populations were injected together in varying proportions. Thus B lymphocytes are inert in this GVH response in agreement with evidence obtained by others in mice. The rejection of Ag-B-incompatible skin allografts on B rats was substantially delayed, and replenishment with thymocytes restored rejection times to normal. The time of second-set skin graft rejection in B rats was usually the same as in normal recipients but, in contrast, the GVH activity of lymphocytes from specifically immunized B rats remained very low. This suggests that the low factor of immunization found in normal animals is not simply an arithmetic consequence of the high proportion of cells which respond to strong antigens before immunization.