T cell reconstitution of BB/W rats after the initiation of insulitis precipitates the onset of diabetes

One of the diabetes susceptibility genes of the BB/W (Biobreeding/Worcester) rat maps to the lyp locus on chromosome 4. The BB/W lyp allele is responsible for a severe peripheral T lymphopenia. Correction of this lymphopenia by transfer of normal, histocompatible T cells prevents diabetes, providing T cell reconstitution is initiated before insulitis. We have analyzed this time-dependent regulation of the diabetogenic process by normal T cells. We demonstrate that T cell reconstitution after the initiation of insulitis precipitates the onset of diabetes through the recruitment of donor T cells to the autoimmune process. This inability of normal T cells to regulate primed diabetogenic BB/W T cells and their own autoreactive potential were observed when normal T cells outnumbered pathogenic T cells by approximately 1000-fold. Analysis of donor-derived T cells recovered from BB/W rats that were reconstituted before insulitis, and hence protected from diabetes, demonstrates that early T cell reconstitution of BB/W rats does not result in a long term physical or functional depletion of islet cell-specific T cell precursors among donor cells or in the expansion of T cells that can regulate the activation and expansion of diabetogenic T cells.     

Absence of RT6+ T cells in diabetes-prone biobreeding/Worcester rats is due to genetic and cell developmental defects

Diabetes-prone BB/Wor (DP) rats lack the RT6+ peripheral T cell subset whereas diabetes-resistant BB/Wor rats have normal numbers of RT6+ T cells. Lymphocyte transfusion experiments and in vivo depletion studies have demonstrated that RT6+ T cells have an important regulatory role in the pathogenesis of insulin-dependent diabetes mellitus in BB/Wor rats. In the present study, the results of genetic complementation studies indicate that the DP rat contains an intact RT6 gene, but fails to express the RT6.1 alloantigen in the functional absence of an accessory factor (provided by RT6+ cells). At the cellular level, irradiation chimeras demonstrate that the absence of RT6+ T cells in DP rats is due to an intrinsic defect that results in abnormal development and/or differentiation of prothymocytes into RT6+ T cells. The inability of DP prothymocytes to generate RT6+ T cells is not due to serum autoantibodies, lack of accessory cells, or to the presence of inhibitory cells. Inasmuch as DP bone marrow can transfer the susceptibility for diabetes to irradiated recipients, our present results suggest that an important predisposing factor for insulin-dependent diabetes mellitus in DP rats is the inability of DP prothymocytes to generate RT6+ T cells.     

A regulatory CD4+ T cell subset in the BB rat model of autoimmune diabetes expresses neither CD25 nor Foxp3

Biobreeding (BB) rats model type 1 autoimmune diabetes (T1D). BB diabetes-prone (BBDP) rats develop T1D spontaneously. BB diabetes-resistant (BBDR) rats develop T1D after immunological perturbations that include regulatory T cell (Treg) depletion plus administration of low doses of a TLR ligand, polyinosinic-polycytidylic acid. Using both models, we analyzed CD4+CD25+ and CD4+CD45RC- candidate rat Treg populations. In BBDR and control Wistar Furth rats, CD25+ T cells comprised 5-8% of CD4+ T cells. In vitro, rat CD4+CD25+ T cells were hyporesponsive and suppressed T cell proliferation in the absence of TGF-beta and IL-10, suggesting that they are natural Tregs. In contrast, CD4+CD45RC(-) T cells proliferated in vitro in response to mitogen and were not suppressive. Adoptive transfer of purified CD4+CD25+ BBDR T cells to prediabetic BBDP rats prevented diabetes in 80% of recipients. Surprisingly, CD4+CD45RC-CD25- T cells were equally protective. Quantitative studies in an adoptive cotransfer model confirmed the protective capability of both cell populations, but the latter was less potent on a per cell basis. The disease-suppressing CD4+CD45RC-CD25- population expressed PD-1 but not Foxp3, which was confined to CD4+CD25+ cells. We conclude that CD4+CD25+ cells in the BBDR rat act in vitro and in vivo as natural Tregs. In addition, another population that is CD4+CD45RC-CD25- also participates in the regulation of autoimmune diabetes.     

Genetic analysis of the BB/W diabetic rat

A large colony of BB/W diabetic rats has been developed as a research model for insulin dependent, type 1 diabetes mellitus. The foundation stock had 8% diabetics which appeared in a sporadic manner. The Worcester (W) colony was inbred by brother X sister matings for 11 generations and the proportion of diabetics increased to over 50%. The age of detection varies from 46 to 250 days. For selection purposes, classification was made at 120 days, which means that 15 to 20% potential diabetics were classified as normal. Evidence from different analyses indicates that the inheritance of diabetes is by a recessive gene or gene cluster with 50% penetrance at 120 days. The selection of breeding stock from diabetic parents raised the proportion of diabetics produced by two normal parents from 12 to 43%. Diallel tests show that diabetic and normal offspring of two diabetic parents have the same diabetic genotype. Outcrosses to other strains of rat indicate that the trait is transferred as a recessive with only 3% diabetics recovered in the F2 where noninbred BB stock was used as the diabetic source, and 36% where partially inbred BB/W was used as the diabetic parent. Since the proportion of diabetics produced by all types of crosses has changed, and may continue to change with changes in the genetic background, we have used the operational term penetrance to describe the frequency of diabetes in individuals homozygous for the diabetes gene cluster. At present the penetrance at 120 days is 59% in the BB/W colony.     

Natural killer cell number and function in the spontaneously diabetic BB/W rat

The BB/W rat provides a good model of spontaneous autoimmune diabetes. Diabetes-prone (DP) rats have a virtual lack of OX 8+ OX 19+ T cytotoxic/suppressor cells in peripheral blood lymphocytes (PBL) and spleen, suggesting that the OX 8+ OX 9- natural killer (NK) cells are the predominant cytotoxic cell in this animal. In this study, we have shown that rat NK cells belong to the OX 8+ OX 19- asialo GM1 bright population, and that rat NK cell function may be depleted in vivo by administration of OX 8 antibody. Furthermore, evidence is provided to indicate that NK cell number and activity are enhanced on a per cell basis in DP rats as compared to the diabetes-resistant W line rat. DP rats had about threefold more NK cells than did W-line rats. The cytotoxic activity mediated by spleen and PBL against the YAC-1 target generally correlated with the relative number of cells having the OX 8+ OX 19- phenotype. DP lymphocytes mediated low levels of cytolytic activity against the relatively resistant NK target cell K562. To more directly compare the activity of W-line and DP NK cells, spleen NK cells were isolated by flow sorting of the OX 8+ OX 19- population. At a 5:1 E:T ratio, DP OX 8+ OX 19- cells elicited 21% +/- 3 specific lysis and W-line cells elicited 7% +/- 2 specific lysis. To determine whether the elevated levels of NK cells and NK cell activity in DP rats were a consequence of NK cell proliferation, spleen cells were size-separated by centrifugal elutriation. The NK cell activity was predominantly mediated by small to medium-size lymphocytes and not blast-size enriched populations. Moreover, when the DNA content of splenic OX 8+ cells was measured, 98% of the cells were in the G0-G1 phase of the cell cycle. These data indicate that NK cell number and activity are elevated in DP rats, and support a role for NK cells in the pathogenesis of BB/W diabetes.     

Genes of SHR rats protect spontaneously diabetic BB/OK rats from diabetes: lessons from congenic BB.SHR rat strains

Diabetes in BB rats share many common features with human type 1 diabetes. One of them is the complex and polygenic nature of disease. Analysis of cross hybrids of diabetic BB/OK rats and rats of different diabetes-resistant strains has demonstrated that beside the MHC genes, Iddm1 and the lymphopenia, Iddm2, additional non-MHC genes are involved in diabetes development. To study the importance of the non-MHC genes, Iddm4 and Iddm3, two congenic BB.SHR rat strains were generated by recombining a segment of the SHR chromosome 6 (Iddm4; termed BB.6S; 15cM) or chromosome 18 (Iddm3; termed BB.18S; 24cM) into the BB/OK background by serial backcrossing and marker-aided selection. The characterization of both congenic strains demonstrates a drastic reduction of diabetes frequency in comparison to the BB/OK strain (86% vs 14% and 34%). It is supposed that diabetes protective genes of SHR must be located on both chromosomal segments and that these suppress the action of the essential and most important genes of diabetes development in the BB/OK rat, Iddm1, and Iddm2.     

Immune attack on pancreatic islet transplants in the spontaneously diabetic BioBreeding/Worcester (BB/W) rat is not MHC restricted

Spontaneous diabetes mellitus in the BB/W rat is preceded by lymphocytic insulitis that destroys pancreatic beta cells. Cultured pancreatic islets and adrenal cortex from inbred rats of variable MHC were transplanted to RT1/u BB/W rats without allograft rejection. Islet grafts from RT1/u and non-RT1/u rats evidenced lymphocytic insulitis in BB/W recipients that became diabetic or evidenced lymphocytic insulitis within endogenous islets. These findings suggest that BB immune insulitis is not MHC restricted and may be directed against islet transplants from non-RT1/u animals.     

Deficiencies in gut NK cell number and function precede diabetes onset in BB rats

Defects in the intestinal immune system may contribute to the pathogenesis of autoimmune diseases. Intraepithelial lymphocytes represent a substantial fraction of gut-associated lymphocytes, but their function in mucosal immunity is unclear. A newly described population of NK cells that spontaneously secrete IL-4 and IFN-gamma is present in the intraepithelial lymphocyte compartment of the rat. We hypothesized that defects in the number or function of these cells would be present in rats susceptible to autoimmunity. We report that the number of NKR-P1A(+)CD3(-) intraepithelial NK (IENK) cells is deficient before onset of spontaneous autoimmune diabetes in diabetes-prone BB (BBDP) rats. The absolute number of recoverable IENK cells was only approximately 8% of that observed in WF rats. Bone marrow transplantation from histocompatible WF donors reversed the IENK cell deficiency (and prevented diabetes) in these animals, suggesting a hemopoietic origin for their IENK cell defect. Analysis of diabetes-resistant BB rats, which develop autoimmune diabetes only after perturbation of the immune system, revealed IENK cell numbers intermediate between that of BBDP and WF rats. IENK cells were selectively depleted during treatment to induce diabetes. Prediabetic BBDP and diabetes-resistant BB animals also exhibited defective IENK cell function, including decreased NK cell cytotoxicity and reduced secretion of IL-4 and IFN-gamma. IENK functional defects were also observed in LEW and BN rats, which are susceptible to induced autoimmunity, but not in WF, DA, or F344 rats, which are resistant. Defects in IENK cell number and function may contribute to the pathogenesis of autoimmune diseases including type 1 diabetes.     

Deficiency of phenotypic cytotoxic-suppressor T lymphocytes in the BB/W rat

The BB/W rat is currently the best model of type I (insulin dependent diabetes). Even though this rat develops an autoimmune disease, they are immune deficient. In this study we have demonstrated the almost complete absence of the OX 8+, OX 19+ T cytotoxic/suppressor population in diabetes prone and acute diabetic rats. This population is present in the diabetes resistant W line. The diabetes prone and acute diabetic rats have a relative increase in OX 8+, OX 19- natural killer (NK) cells. Our data suggests that virtually all OX 8+ cells in diabetes prone and acute diabetic animals are phenotypic NK cells.     

Infection of peripancreatic lymph nodes but not islets precedes Kilham rat virus-induced diabetes in BB/Wor rats.

A parvovirus serologically identified as Kilham rat virus (KRV) reproducibly induces acute type I diabetes in diabetes-resistant BB/Wor rats. The tissue tropism of KRV was investigated by in situ hybridization with a digoxigenin-labelled plasmid DNA probe containing approximately 1.6 kb of the genome of the UMass isolate of KRV. Partial sequencing of the KRV probe revealed high levels of homology to the sequence of minute virus of mice (89%) and to the sequence of H1 (99%), a parvovirus capable of infecting rats and humans. Of the 444 bases sequenced, 440 were shared by H1. KRV mRNA and DNA were readily detected in lymphoid tissues 5 days postinfection but were seldom seen in the pancreas. High levels of viral nucleic acids were observed in the thymus, spleen, and peripancreatic and cervical lymph nodes. The low levels of infection observed in the pancreas involved essentially only endothelial and interstitial cells. Beta cells of the pancreas were not infected with KRV. These findings suggest that widespread infection of peripancreatic and other lymphoid tissues but not pancreatic beta cells by KRV triggers autoimmune diabetes by perturbing the immune system of genetically predisposed BB/Wor rats.     

Cellular immunity in allograft rejection: role of lymphocyte subpopulations and T-cell subsets in rat cardiac allograft rejection

In this study, cellular requirements for rejection are examined by the use of adoptive transfer assays in the ACI to Lewis cardiac allograft model. The findings show that adoptive transfer of 1 x 10(8) spleen cells (SpL), 5 x 10(7) T-cells, and 2 x 10(7) helper T-cells (W3/25+) obtained from normal, nonsensitized donors restores acute ACI graft rejection in sublethally irradiated (750 rad) Lewis recipients. In contrast, reconstitution with 2 x 10(7) cytotoxic T-cells (0X8+) does not restore first-set graft rejection. Reconstitution of the irradiated recipients with either W3/25+ or 0X8+ T-cells obtained from specifically sensitized syngeneic donors resulted in acute rejection. The W3/25+ T-cell subset was significantly more potent (P less than 0.01) in effecting rejection on a per-cell basis. Adoptive transfer of SpL, T-cells, and 0X8+ T-cells obtained from sensitized rats led to accelerated cardiac allograft rejection in the naive secondary recipients while W3/25+ T-cells did not. This study suggests that although the W3/25+ T-cells alone have the capacity to initiate first-set graft rejection, both W3/25+ and 0X8+ subsets appear to be critical to the completion of rejection of heart allografts. We also examined the capacity of adoptively transferred B-cells from sensitized donors to influence graft rejection. Our findings suggest that while B-cells fail to restore the capacity for graft rejection in irradiated recipients, they can, however, present MHC antigens to the secondary naive host thus causing allosensitization which results in accelerated rejection of a subsequent graft.     

Impaired post-thymic development of regulatory CD4+25+ T cells contributes to diabetes pathogenesis in BB rats

One of the BB rat diabetes (diabetes mellitus (DM)) susceptibility genes is an Ian5 mutation resulting in premature apoptosis of naive T cells. Impaired differentiation of regulatory T cells has been suggested as one possible mechanism through which this mutation contributes to antipancreatic autoimmunity. Using Ian5 congenic inbred rats (wild-type (non-lyp BB) and mutated (BB)), we assessed the development of BB regulatory CD8(-)4(+)25(+)T cells and their role in the pathogenesis of DM. BB rats have normal numbers of functional CD8(-)4(+)25(+)Foxp3(+) thymocytes. The proportion of CD25(+) cells among CD8(-)4(+) recent thymic emigrants is also normal while it is increased among more mature CD8(-)4(+) T cells. However, BB CD8(-)4(+)25(+)Foxp3(+) thymocytes fail to undergo homeostatic expansion and survive upon transfer to nude BB rats while Foxp3 expression is reduced in mature CD8(-)4(+)25(+) T cells suggesting that these cells are mostly activated cells. Consistent with this interpretation, peripheral BB CD8(-)4(+)25(+) T cells do not suppress anti-TCR-mediated activation of non-lyp BB CD8(-)4(+)25(-) T cells but rather stimulate it. Furthermore, adoptive transfer of unfractionated T cells from diabetic BB donors induces DM in 71% of the recipients while no DM occurred when donor T cells are depleted of CD8(-)4(+)25(+) cells. Adoptive transfer of 10(6) regulatory non-lyp BB CD8(-)4(+)25(+) T cells to young BB rats protects the recipients from DM. Taken together, these results demonstrate that the BB rat Ian5 mutation alters the survival and function of regulatory CD8(-)4(+)25(+) T cells at the post-thymic level, resulting in clonal expansion of diabetogenic T cells among peripheral CD8(-)4(+)25(+) cells.     

Mechanisms maintaining antibody-induced enhancement of allografts. II. Mediation of specific suppression by short lived CD4+ T cells

In DA rats grafted with PVG hearts, the injection of 1 ml of Wistar-Furth x DA)F1 anti-PVG serum on the day of grafting prevents rejection and induces a state of specific unresponsiveness. An adoptive transfer assay was used to test the capacity of T cell subsets, taken from rats given enhancing serum, to either restore rejection or to transfer unresponsiveness to syngeneic hosts irradiated with 9 Gy and grafted with donor (PVG) or third party (Wistar-Furth) hearts. W3/25+ (CD4+) cells from these animals retained some capacity to restore rejection until 50 days posttransplant, after which they invariably failed to restore PVG graft rejection but retained the capacity to effect Wistar-Furth rejection. At this time CD4+ cells were also capable of inhibiting naive but not specifically sensitized CD4+ cells capacity to restore PVG graft rejection in irradiated hosts. The development of CD4+ suppressor cells was concurrent with the appearance of clinically evident unresponsiveness in the host. MRC Ox8+ (CD8+) cells from enhanced rats when mixed with naive CD4+ cells delayed rejection in adoptive recipients but did not reestablish unresponsiveness. Paradoxically, the CD4+ cells that transfer unresponsiveness to the adoptive host proliferate such as normal cells in MLC to both donor and third party alloantigen. Unfractionated cells, CD4+ or CD8+ cells did not proliferate to relevant idiotype in vitro. The CD4+ cells after 3 days in culture, with either alloantigen or idiotype-bearing stimulator cells, lost their capacity to suppress in the adoptive transfer assay. The maintenance of specific unresponsiveness was thus shown to be due to a CD4+ suppressor T cell whose function was lost in culture, and therefore could not be detected in MLC or idiotype assays.     

Recapitulation of normal and abnormal BioBreeding rat T cell development in adult thymus organ culture

Congenitally lymphopenic diabetes-prone (DP) BioBreeding (BB) rats develop spontaneous T cell-dependent autoimmunity. Coisogenic diabetes-resistant (DR) BB rats are not lymphopenic and are free of spontaneous autoimmune disease, but become diabetic in response to depletion of RT6+ T cells. The basis for the predisposition to autoimmunity in BB rats is unknown. Abnormal T cell development in DP-BB rats can be detected intrathymically, and thymocytes from DR-BB rats adoptively transfer diabetes. The mechanisms underlying these T cell developmental abnormalities are not known. To study these processes, we established adult thymus organ cultures (ATOC). We report that cultured DR- and DP-BB rat thymi generate mature CD4 and CD8 single-positive cells with up-regulated TCRs. DR-BB rat cultures also generate T cells that express RT6. In contrast, DP-BB rat cultures generate fewer CD4+, CD8+, and RT6+ T cells. Analysis of the cells obtained from ATOC suggested that the failure of cultured DP-BB rat thymi to generate T cells with a mature phenotype is due in part to an increased rate of apoptosis. Consistent with this inference, we observed that addition of the general caspase inhibitor Z-VAD-FMK substantially increases the number of both mature and immature T cells produced by DP-BB rat ATOC. We conclude that cultured DR-BB and DP-BB rat thymi, respectively, recapitulate the normal and abnormal T cell developmental kinetics and phenotypes observed in these animals in vivo. Such cultures should facilitate identification of the underlying pathological processes that lead to immune dysfunction and autoimmunity in BB rats.     

Congenic diabetes-prone BB.Sa and BB.Xs rats differ from their progenitor strain BB/OK in frequency and severity of insulin-dependent diabetes mellitus.

Two newly established congenic diabetes-prone BB rat strains designated BB.Sa and BB.Xs carrying a region of chromosome 1 (Sa-Lsn-Secr-Igf2-Tnt, 16 cM) and a region of chromosome X (DXMgh3-Mycs/Pfkb1-Ar, 36 cM) of the SHR rats, respectively, were studied to determine whether the transferred chromosomal regions influence diabetes frequency, age at onset, and clinical picture. Therefore, 4 complete litters of BB/OK (n = 43), BB.Sa (n = 45), and BB.Xs (n = 41) were observed for diabetes occurrence up to the age of 30 weeks. From these litters 6 diabetic males of each strain manifesting in an interval of 1 week were chosen to study body weight, blood glucose, insulin requirement to survive, and several diabetes-related serum constituents at onset of diabetes and after a diabetes duration of 150 days. The diabetes frequency was significantly lower in BB.Xs than in rats of the parental strain BB/OK, whereas comparable frequencies were found between BB/OK and BB.Sa rats. Obvious differences were observed 150 days after diabetes onset between BB/OK and both BB.Sa and BB.Xs rats. BB/OK rats were significantly heavier and needed significantly more insulin/100 g body weight than BB.Sa and BB.Xs rats. Comparisons of the serum constituents as lipids, proteins, and minerals revealed significant differences between diabetic BB/OK rats and their diabetic congenic derivatives in several traits studied at onset and after 150 days of insulin treatment. These results not only show the power of congenic lines in diabetes research, but indicate for the first time that there are genetic factors on chromosomes 1 and X influencing frequency and severity of diabetes in the BB/OK rat.     

Mechanism of the intensification of the immune response to Staphylococcus in mice after the transplantation of primed donor splenocytes

Experiments on CBA, C57Bl/6 mice and (CBA X X C57Bl/6)F1 hybrids were made to study the mechanism of stimulation of the immune response to staphylococci after injection of primed splenocytes. The stimulating action of immune splenocytes was reversed after their in-vitro treatment with anti-immunoglobulin serum and complement. The stimulant effect was also seen in a semi-allogeneic system (adoptive transfer of CBA mice immune cells to (CBA X C57Bl/6)F1 recipients). Preincubation of splenocytes with CBA-anti-C57Bl/6-serum and complement prior to demonstration of antibody-forming cells did not influence their number in the spleen of hybrid recipients injected with immune cells carrying parent genotype but decreased this indicator of the immune response in control mice. It is concluded that stimulation of the immune response to staphylococci after transplantation of primed splenocytes is due to the anamnestic response of donor's cells repeatedly stimulated by antigen in the recipient's host.     

Disturbance of thyroidal iodine metabolism in BB/W rat.

To investigate the thyroid function in Bio-Breeding Worcester (BB/W) rats, we have examined the iodine metabolism, serum TSH and thyroid hormone levels in 8- and 16-week-old BB/W and normal Wistar (W) rats. At 8 weeks of age, serum TSH levels were significantly higher in BB/W rats than in W rats, although there was no difference in the serum levels of free T3 and free T4. Furthermore, the thyroidal radioactive iodine incorporation at 48 h was significantly lower in BB/W rats, suggesting that they might have some defects in iodine organification. At 16 weeks of age, serum TSH levels were also significantly higher in BB/W rats than in W rats. Furthermore, serum TSH levels in 16-week-old BB/W rats were significantly higher than in 8-week-old BB/W rats. The thyroid weight was significantly greater in BB/W rats, probably due to the increased serum TSH. The thyroidal radioactive iodine uptake at 48 h and the iodine content in the thyroid homogenates were significantly lower in BB/W rats. These results suggest that BB/W rats have some defect in iodine metabolism resulting in impaired thyroid hormone synthesis.     

Functional status of the immune system after chronic administration of 2'-deoxycoformycin in the BB rat

Insulin-dependent diabetes mellitus (IDDM) is caused by autoimmune destruction of pancreatic beta cells with the primary mechanism being cell mediated. The BB rat develops insulitis and IDDM with many features analogous to the disease in man. In previous studies we reported that weekly administration of 2'-deoxycoformycin (dCF) for four months reduces significantly the incidence of IDDM in the BB rat by 70%, and that the animals remain free of diabetes for a minimum of two months after drug withdrawal. Since the diabetes-prone BB rat is lymphopenic, with a reduction of both CD4 and CD8 cells, the continuous failure of dCF treated animals to develop diabetes may have been due to generalized immunosuppression. To test this possibility, the ability of dCF treated diabetes-free BB rats to mount an immune response after challenge with Ovalbumin was examined five months after drug withdrawal. The results showed that the post-immunization levels of total IgG and specific IgG in these animals did not differ from those observed in non-dCF treated controls nor those of control diabetes-resistant non-lymphopenic BB rats. Moreover, FACS analysis indicated no change in the percentages of total numbers of CD4+ or CD8+ cells between the two groups of animals. Histological assessment of the pancreata of the post-dCF treated animals showed varying degrees of mononuclear cell infiltrates in the islets. These data demonstrate that treatment by dCF is not permanent, and may require intermittent or continuous administration to prevent development of diabetes. Further studies are needed to determine the mechanism of action of dCF in this model of IDDM.     

Effect of cyclophosphamide on suppressor cell activity in mice unresponsive to EAE.

Protection against experimental allergic encephalomyelitis (EAE) was induced in susceptible mice of (SJL/J X BALB/c)F1 hybrid, by injection of either mouse spinal cord homogenate, the small mouse basic protein, or Cop 1 in incomplete Freund's adjuvant, before EAE induction. It was demonstrated that the unresponsiveness induced by the three antigens is mediated by suppressor T cells residing in the spleen cell population and can be adoptively transferred to normal syngeneic recipients. Low dose of cyclophosphamide (20 mg/kg) administered 2 days before the encephalitogenic challenge abrogated the unresponsiveness to EAE and reverted the protected mice sensitive to disease induction. Cyclophosphamide was also active on adoptively transferred unresponsiveness, thus donors that had been treated with cyclophosphamide were unable to further transfer unresponsiveness to EAE. These results indicate the elimination by cyclophosphamide of suppressor cells that interfere with the effector mechanisms leading to EAE.