Induction of allograft tolerance after total lymphoid irradiation (TLI): development of suppressor cells of the mixed leukocyte reaction (MLR).

We searched for the presence of suppressor cells of the MLR in C57BL/Ka leads to BALB/c chimeras. The chimeras were made with total lymphoid irradiation (TLI) and marrow transplantation. Spleen cells from the old chimeras inhibited the MLR of BALB/c responder cells against C57BL/Ka stimulator cells. Inhibition was specific for the stimulator cells, since no effect on the MLR was observed with C3H or BALB.C3H stimulator cells. Maximal inhibition was achieved when the responder cells in the MLR shared the H-2 haplotype of the chimeric recipient. Spleen cells obtained from chimeras young 30 to 40 days after BM transplantation inhibited the MLR nonspecifically, since similar marked inhibition was observed regardless of the H-2 haplotype of the responder or stimulator cells. The finding of antigen-specific and nonspecific suppressor cells is similar to that observed in mice rendered tolerant to bovine serum albumin after treatment with TLI.     

Natural cell-mediated cytotoxicity in mice treated with total lymphoid irradiation (TLI)

Fractionated total lymphoid irradiation (TLI) of adult (BALB/c × C57BL/6)F₁ mice resulted in transiently augmented natural killer (NK) and natural cytotoxic (NC) cell activities. Thus, 1 day after completion of TLI, NK and NC activities in the spleens of treated mice were lower than controls but values increased and reached a maximum level of 23- to 190-fold above control at 6 days after irradiation, returning to normal levels 9 days later. Cytotoxicity was enhanced after removal of the plastic adherent population. No cytotoxicity was observed against P 815 target cells, which are sensitive to activated macrophages but not to NK. The significance of this modulation of natural cell-mediated cytotoxicity following TLI is discussed.     

Natural suppressor (NS) cells found in the spleen of neonatal mice and adult mice given total lymphoid irradiation (TLI) express the null surface phenotype

We studied the surface markers of suppressor cells of the mixed leukocyte reaction (MLR) that are transiently present in the spleens of neonatal mice after birth and of adult mice after total lymphoid irradiation (TLI). Approximately 80% of the mononuclear cells in the spleen, within the first few days after birth or after TLI, express neither the Thy-1 antigen nor surface immunoglobulin (Ig). After 30 days, less than 20% of mononuclear cells bear this null phenotype. With the use of the panning technique, we showed that the suppressors of the MLR are confined to the null cell population. The null suppressor cells are not macrophages because they did not carry macrophage markers identified by the monoclonal antibodies anti-MAC-1 and F4/80. In addition, the suppressor cells did not stain for nonspecific esterase and did not adhere firmly to plastic or glass. Spleen cells from TLI-treated mice maintained their suppressive capacity after culture in vitro for 6 to 8 wk. The cultured suppressor cells did not develop mature T cell, B cell, or macrophage markers during this time interval. Thus, the suppressor cells did not appear to be precursors of the latter cells. There was no clear relationship between the suppressor activity of the spleens and natural killer (NK) activity; the kinetics of these activities in newborn spleen appear to be inversely related. The suppressor cells, however, are similar to NK cells in that both are found in the absence of antigenic challenge, lack antigen specificity, and bear the null surface phenotype. Thus, we have termed the former cells natural suppressor (NS) cells.     

A partial characterization of suppressor cells in the spleens of mice conditioned with fractionated total lymphoid irradiation (TLI)

Total lymphoid irradiation (TLI) is a highly effective modality for inducing immunosuppression and transplantation tolerance. The cellular basis for this immunosuppression is not clear, although T cells have been implicated. To study further the effect of TLI on the immune system, we have examined the B cells and suppressor cells in the spleens from TLI-conditioned mice. Our results indicate that after TLI, the spleen is rapidly repopulated with many large, immature cells. The probable source of these cells is the shielded bone marrow (BM). The B cells from TLI-conditioned mice are transiently immature and hyporesponsive in vitro to a T-independent antigen. Spleen cells from TLI-conditioned mice nonspecifically suppress the in vitro T-independent anti-TNP response of normal B cells. The suppressor cells lack both B and T cell markers and adhere to Sephadex G-10. The suppressor cells in spleens from TLI-treated mice bear a number of similarities to those present in normal BM. When normal BM cells were analyzed by indirect immunofluorescence for the presence of the Mac-1 antigen, two populations of suppressor cells could be identified: one was Mac-1+ and the other was Mac-1-. These data are consistent with the possibility that a subpopulation of the suppressor cells found in normal BM and in the spleens from TLI-conditioned mice are immature cells of the monocytic/granulocytic lineage.     

Induction of transplantation tolerance in mice across major histocompatibility barrier by using allogeneic thymus transplantation and total lymphoid irradiation

The use of allogeneic thymus transplantation as a means of inducing tolerance across MHC barriers was investigated in thymectomized, total lymphoid irradiated BALB/c mice. In 90% of the animals long term outgrowth of histologically normal C57BL thymus grafts was observed. None of the latter animals was chimeric. All thymus graft-bearing mice showed specific nonresponsiveness for C57BL MHC Ag in mixed lymphocyte reaction and cell-mediated lympholysis. Spleen cells of the C57BL thymus-bearing mice were unable to induce lethal graft-vs-host disease in neonatal (BALB/c X C57BL) F1 mice but provoked a vigorous graft-vs-host disease reaction in (BALB/c x C3H) F1 neonates. Tolerant mice permanently accepted C57BL heart and pancreas grafts, but all rejected C3H grafts. Induction of tolerance of BALB/c pre-T cells through allogeneic thymus graft and/or specific suppressor cells seems to be involved. The present model offers new opportunities to study thymocyte maturation in a fully allogeneic environment and may yield applications for clinical organ transplantation.     

Suppression of cell-mediated immune responses after total lymphoid irradiation (TLI). I. Characterization of suppressor cells of the mixed lymphocyte reaction

Total lymphoid irradiation (TLI) was administered to (BALB/c X C57BL/6)F1 mice in eight daily doses of 200 rad (total 1600 rad). Spleen cells isolated from mice after treatment with TLI do not respond to alloantigens in vitro in a one-way mixed lymphocyte reaction (MLR), but normal reactivity recovers after approximately 2 mo. Radioresistant, antigen-nonspecific suppressor cells are documented in the spleens of TLI-treated mice immediately after radiotherapy, but suppressive capacity gradually disappears within 30 days. After TLI, the spleen is repopulated with large cells, the proportion of which is greatest at a time when theta-bearing cells are still depleted. Radioresistant suppression is mediated predominantly by the large cell subset and is thymus independent. Suppressor function can be abolished by lethal physicochemical procedures including formaldehyde fixation, multiple freeze-thawing, and heating to 56 degrees C, and it cannot be conferred by supernatants of TLI-suppressed MLR suspensions. Suppression cannot be overcome by adding various cell factors including T cell growth factor (TCGF) and lymphocyte-activating factor (LAF), nor is it affected by a prostaglandin inhibitor. Equally potent radioresistant suppressive activity is documented by co-culturing cells derived from other sources enriched in large, immature hematopoietic cells, including fetal liver cells and bone marrow cells obtained from normal and congenitally athymic mice. The presence of a large cell population and MLR suppressor function is also documented in the spleens of mice treated with single dose or fractionated doses of lethal whole body irradiation, followed by reconstitution with bone marrow cells obtained from normal mice. The data suggest that MLR suppressor cells, which are large, immature and predominantly radioresistant, can be induced after a short and well-tolerated TLI regimen.     

Effect of total lymphoid irradiation (TLI) on the primary and secondary antibody response to sheep red blood cells.

The effect of total lymphoid irradiation (TLI) on the primary and secondary hemagglutinin response to sheep red blood cells (SRBC) was studied in BALB/c mice. The primary response was eliminated for 1 month and began to return by Day 44. The latter response was IgM, and the IgG response did not return until approximately 200 days after TLI. The prolonged immunosuppression required inclusion of the thymus in the radiation field. Mice treated with single-dose whole-body irradiation (WBI) regained a normal IgG response within 28 days after irradiation. Immunization of mice prior to treatment with TLI or WBI resulted in a vigorous IgG response when mice were boosted 1 month after irradiation.     

Spleen cells from adult mice given total lymphoid irradiation (TLI) or from newborn mice have similar regulatory effects in the mixed leukocyte reaction (MLR). II. Generation of antigen-specific suppressor cells in the MLR after the addition of spleen cells from newborn mice

Spleen cells from newborn BALB/c mice were added to the mixed leukocyte reaction (MLR) between a variety of responder and stimulator cells. The newborn cells nonspecifically suppressed the uptake of (3H)-thymidine and the generation of cytolytic cells regardless of the responder-stimulator combination used. Suppressor cell activity fell rapidly during the first 4 days after birth, and could not be detected by day 20. Newborn spleen cells inhibited the generation of nonspecific suppressor cells during the MLR but did not inhibit the generation of antigen-specific suppressor cells. Thus, newborn spleen cells exhibit a pattern of regulation of the MLR similar to that reported previously for spleen cells from adult mice given total lymphoid irradiation (TLI). These regulatory interactions provide a model that explains the ease of induction of transplantation tolerance in vivo in newborn mice and in TLI-treated adult mice.     

Use of total lymphoid irradiation (TLI) in studies of the T cell-dependence of autoantibody production in rheumatoid arthritis

The effect of total lymphoid irradiation (TLI) on T cell-dependent and -independent humoral immune responses was studied in patients with intractable rheumatoid arthritis (RA). The serum levels of several autoantibodies and of antibodies to diphtheria (DT) and tetanus (TT) toxoids and to pneumococcal polysaccharide (PPS; 12 antigenic types) were studied before and after TLI. In addition, the patients were given a booster injection of DT and TT and a single injection of pneumococcal vaccine after radiotherapy. Antibody levels to DT and TT decreased about twofold after TLI and did not rise significantly (p greater than 0.05) after a booster injection. However, there was no reduction in antibody levels to PPS after TLI, and a significant rise in titers was observed after a single vaccination (p less than 0.01). The serum levels of rheumatoid factor (RF), anti-nuclear antibody (ANA), and granulocyte associated IgG rose slightly after TLI. Thus, the autoantibodies and antibodies to polysaccharides appear to be relatively independent of helper T cell function, which is markedly reduced after TLI. On the other hand, antibodies to protein antigens such as DT and TT appear to be more closely dependent upon T helper function in man, as has been reported in rodents. The findings suggest that T cell-independent autoantibody responses alone do not maintain the joint disease activity in RA, because improvement in joint disease after TLI has been reported.     

Suppression of the mixed leukocyte response and of graft-vs-host disease by spleen cells following total lymphoid irradiation (TLI)

Total lymphoid irradiation (TLI) was administered to mice as 17 fractions of 200 rads delivered to the major lymphoid organs. Spleen cells capable of suppressing the in vitro mixed leukocyte response (MLR) and in vivo graft-vs-host disease (GVHD) were found in mice after treatment with TLI. Suppression was not antigen specific and was markedly reduced by treatment of the spleen cells with anti-Thy-1.2 antiserum and complement. Suppressor activity declined with time after irradiation and disappeared within 30 to 40 days. The evidence suggests that the suppressor cells may prevent initial BM rejection and acute GVHD in allogeneic BM transplant recipients prepared with TLI.     

Late somatic effects in mice after total lymphoid irradiation

Late somatic effects of total lymphoid irradiation have been investigated in BC3F1 mice. A total X-ray dose of 34 Gy was distributed in 17 daily fractions. The cumulative mortality curve is shifted in time because all the irradiated mice died earlier than the unirradiated controls. There was a 24% shortening of life span. A marked increase of solid tumor incidence, mostly due to skin cancers, was observed (66% vs 30%). In contrast, the incidence of malignant lymphomas was greatly reduced in irradiated mice (6% vs 49%). Furthermore, nephrosclerosis was a common finding in the irradiated group (38% vs 8%). Death-rate analysis revealed an association between life shortening and the presence of solid tumors and nephrosclerosis at death.     

Comparison of the immunosuppressive effect of fractionated total lymphoid irradiation (TLI) vs conventional immunosuppression (CI) in renal cadaveric allotransplantation

Beginning in November 1981, eight patients with end stage diabetic nephropathy underwent renal cadaveric transplantation after TLI. Transplantation was done between 2 to 11 days after the end of a fractionated TLI to a total dose of 20 to 30 Gy. During the same observation period, 60 nondiabetic patients with end stage renal disease of different origin also received a cadaveric kidney graft, with a conventional regimen of immunosuppression that consists of anti-lymphocyte-globulin, tapering high doses of prednisone, and azathioprine. In the TLI-treated group only a low maintenance dose of prednisone (15 mg) was given. Immunologic monitoring was performed after transplantation at regular intervals and was compared in both groups. Phytohemagglutinin (PHA)-, concanavalin A (con A)-, and pokeweed mitogen (PWM)-induced blastogenesis, as well as the mixed lymphocyte reaction (MLR) and the cell-mediated lympholysis (CML) decreased progressively during the first months after conventional immunosuppression to 50% of the pretransplantation level, and remained there for the first year after transplantation. These tests were much more impaired after TLI (less than 15% of pre TLI value) and again no recovery occurred during the first year. Natural killer (NK) cell activity progressively decreased from a mean value of 53% lysis before transplantation to 15% lysis at the end of the first year after transplantation in the conventionally treated patients. In TLI-treated patients, however, the NK activity, which declined during irradiation from 46% specific lysis to 12%, recovered rapidly after TLI to reach levels of 35 to 40% of specific lysis from the second month on after TLI. In both groups of patients the ratio of helper-inducer (TH) to suppressor-cytotoxic (TS) lymphocytes, as determined with monoclonal antibodies, progressively declined during the first 2 mo after transplantation to a low value of about 1.2. In TLI-treated patients however, this fall progressed further, so that very low levels (less than 0.6) were noticed from the third month on after TLI. The decline of the TH:TS ratio after TLI is due to an absolute increase in the number of suppressor cells. This is in contrast with the conventionally treated patients, where the low ratio is mainly provoked by a more important decrease of the helper cell population. These changes in the balance between TH and TS subpopulations are more frequently associated with positive functional suppressor cell assays in the TLI-treated patients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Allogeneic marrow transplantation after total lymphoid irradiation (TLI): effect of dose/fraction, thymic irradiation, delayed marrow infusion, and presensitization.

BALB/c mice infused with 30 x 10(6) C57BL/Ka bone marrow (BM) cells 1 day after treatment with fractionated total lymphoid irradiation (TLI) (17 fractions of 200 rads each) became stable mixed chimeras without clinical graft-vs-host disease (GVHD). Mice given 18 fractions of 100, 50, or 25 rads each followed 1 day later by C57BL/Ka BM did not become chimeric, indicating that a critical cumulative radiation dose is required for this effect. Animals given TLI with lead shielding placed over the thymus also developed stable chimerism without GVHD. Thus susceptibility to tolerance induction and protection from GVHD after TLI and allogeneic BM transplantation is not due to alteration of the thymic microenvironment by fractionated irradiation. A delay of 7 or 21 days between completion of TLI and BM administration resulted in a high incidence of graft rejection. Sensitization to minor histocompatibility antigens of the BM donor strain by blood transfusion either before or during TLI resulted in marrow graft rejection in a high percentage of animals.     

Unsuccessful induction of low-zone tolerance to bovine serum albumin injected into the subarachnoid space

Introduction of T-dependent antigens into the subarachnoid space (isas) resulted in higher systemic antibody responses in mice than injections into the peritoneal cavity (ip) or other sites commonly used for immunization. Antibody production in isas immunized mice was not increased by treatment with cyclophosphamide (Cy) at doses known to abolish T-suppressor-cell activity, but such treatment increased antibody production in ip immunized mice toward the higher level which was observed in the isas immunized animals. Suppressor cell-dependent low zone tolerance (LZT) to TNP-BSA could not be induced by isas injections of deaggregated BSA (d-BSA). Conversely, mice which were unresponsive to ip injected d-BSA showed consistent systemic antibody responses when the antigen was injected isas. These observations indicate that immune responses initiated within the CNS are associated with relatively ineffective induction of systemic suppressor cell activity.