Circumventing tolerance to the prion protein (PrP): vaccination with PrP-displaying retrovirus particles induces humoral immune responses against the native form of cellular PrP

Passive immunization with antibodies directed against the cellular form of the prion protein (PrPC) can protect against prion disease. However, active immunization with recombinant prion protein has so far failed to induce antibodies directed against native PrPC expressed on the cell surface. To develop an antiprion vaccine, a retroviral display system presenting either the full-length mouse PrP (PrP209) or the C-terminal 111 amino acids (PrP111) fused to the transmembrane domain of the platelet-derived growth factor receptor was established. Western blot analysis and immunogold electron microscopy of the retroviral display particles revealed successful incorporation of the fusion proteins into the particle membrane. Interestingly, retroviral particles displaying PrP111 (PrPD111 retroparticles) showed higher incorporation efficiencies than those displaying PrP209. Already 7 days after intravenous injection of PrPD111 retroparticles, PrPC-deficient mice (Prnp(o/o)) showed high immunoglobulin M (IgM) and IgG titers specifically binding the native PrPC molecule as expressed on the surface of T cells isolated from PrPC-overexpressing transgenic mice. More importantly, heterozygous Prnp(+/o) mice and also wild-type mice showed PrPC-specific IgM and IgG antibodies upon vaccination with PrPD111 retroparticles, albeit at considerably lower levels. Bacterially expressed recombinant PrP, in contrast, was unable to evoke IgG antibodies recognizing native PrPC in wild-type mice. Thus, our data show that PrP or parts thereof can be functionally displayed on retroviral particles and that immunization with PrP retroparticles may serve as a novel promising strategy for vaccination against transmissible spongiform encephalitis.     

Polylactide-coglycolide microspheres co-encapsulating recombinant tandem prion protein with CpG-oligonucleotide break self-tolerance to prion protein in wild-type mice and induce CD4 and CD8 T cell responses

Prion diseases are fatal neurodegenerative diseases that are characterized by the conformational conversion of the normal, mainly alpha-helical cellular prion protein (PrP) into the abnormal beta-sheet-rich infectious isoform (PrP(Sc)). The immune system neither shows reaction against cellular PrP nor PrP(Sc), most likely due to profound self-tolerance. In previous studies, we were able to partly overcome self-tolerance using recombinantly expressed dimeric PrP (tandem PrP (tPrP)), in association with different adjuvants. Proof of principle for antiprion efficacy was obtained in vitro and in vivo. In this study, we demonstrate the induction of a specific Th1 T cell response in wild-type mice immunized with tPrP and CpG-oligonucleotide (ODN). Biochemical influences such as refolding conditions, ionic strength, pH, and interaction with CpG-ODN affected antigenic structure and thus improved immunogenicity. Furthermore, s.c. immunization with tPrP and CpG-ODN co-encapsulated in biodegradable polylactide-coglycolide microspheres (PLGA-MS) enhanced CD4 T cell responses and, more prominent, the induction of CD8 T cells. In this vaccination protocol, PLGA-MS function as endosomal delivery device of Ag plus CpG-ODN to macrophages and dendritic cells. In contrast, PLGA-MS-based DNA vaccination approaches with a tPrP construct generated poor humoral and T cell responses. Our data show that prophylactic and therapeutic immunization approaches against prion infections might be feasible using tPrP Ag and CpG-ODN adjuvant without detectable side effects.     

Soluble dimeric prion protein binds PrP(Sc) in vivo and antagonizes prion disease

Conversion of cellular prion protein (PrP(C)) into a pathological conformer (PrP(Sc)) is thought to be promoted by PrP(Sc) in a poorly understood process. Here, we report that in wild-type mice, the expression of PrP(C) rendered soluble and dimeric by fusion to immunoglobulin Fcgamma (PrP-Fc(2)) delays PrP(Sc) accumulation, agent replication, and onset of disease following inoculation with infective prions. In infected PrP-expressing brains, PrP-Fc(2) relocates to lipid rafts and associates with PrP(Sc) without acquiring protease resistance, indicating that PrP-Fc(2) resists conversion. Accordingly, mice expressing PrP-Fc(2) but lacking endogenous PrP(C) are resistant to scrapie, do not accumulate PrP-Fc(2)(Sc), and do not transmit disease to others. These results indicate that various PrP isoforms engage in a complex in vivo, whose distortion by PrP-Fc(2) affects prion propagation and scrapie pathogenesis. The unique properties of PrP-Fc(2) suggest that soluble PrP derivatives may represent a new class of prion replication antagonists.     

Studies on the IgA-independent immunological responses in mice to influenza virus challenge after oral vaccination with irradiated whole virus and an erythrocyte complex

Previous studies have described an oral influenza vaccine comprising whole irradiated virus and an erythrocyte complex (IV-EC), which gave broad-based protection against influenza virus challenge in mice. The present study examined the immune responses generated after live virus challenge of vaccinated mice, particularly to determine whether mice vaccinated with IV-EC had enhanced CTL activity to compensate for the previously reported diminution in lung IgA response. Oral vaccine groups examined were IV-EC, live virus alone (LV) or live virus-erythrocyte complex (LV-EC), compared with irradiated virus and erythrocyte alone controls. The antibody responses of IV-EC and LV-EC vaccinated mice showed significantly elevated lung and serum IgG2a levels post live virus challenge, with no comparable increases in IgG1 levels compared to controls. Spleen cells from IV-EC mice showed an enhanced post-challenge proliferative response to antigen compared with mice that had received live oral vaccines, indicating enhanced cellular activity post IV-EC immunization. However, CTL activity was not enhanced for IV-EC mice, and live virus-vaccinated mice had reduced CTL activity compared with controls, indicating that CTL were not important for post-vaccine protection. Cytokine analysis revealed a predominant IFN-gamma response in spleen cells from orally vaccinated mice, whereas IL-4 was not detected in any lung or spleen culture analysed. The results suggest, therefore, that protection from live influenza challenge after IV-EC or LV-EC vaccination was due to an IFN-mediated IgG2a response. Definitive confirmation of the role of these factors in post-vaccine protection can now be tested in IgG2a-depleted or IFN-gamma gene knockout mouse models.     

Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons

Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme α-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.     

Cellular tolerance of prion protein PrP in yeast involves proteolysis and the unfolded protein response

Secretory proteins undergo a stringent quality control process in the endoplasmic reticulum (ER). Misfolded ER proteins are returned to the cytosol and destroyed by the proteasome. Prion protein PrP is degraded by the proteasome in mammalian cells. However, the significance of proteolysis on PrP-induced cell death is controversial. Moreover, the specific pathway involved in PrP degradation remains unknown. Here, we demonstrate that the unglycosylated form of human PrP is subjected to the ER-associated protein degradation (ERAD) process in the yeast Saccharomyces cerevisiae. We also show that unglycosylated PrP is degraded by the Hrd1-Hrd3 pathway. Accumulation of misfolded proteins triggers the unfolded protein response (UPR), which promotes substrate refolding. Interestingly, we find that the expression of PrP leads to growth impairment in cells deficient in UPR and ERAD. These findings raise the possibility that decreased UPR activity and proteolysis may contribute to the pathogenesis of some prion-related diseases.     

Breaking immune tolerance to the prion protein using prion protein peptides plus oligodeoxynucleotide-CpG in mice

The absence of a detectable immune response during transmissible spongiform encephalopathies is likely due to the fact that the essential component of infectious agents, the prion protein (PrP), is a self Ag expressed on the surface of many cells of the host. To overcome self-tolerance to PrP, we used 30-mer PrP peptides previously shown to be immunogenic in Prnp(-/-) mice, together with CFA or CpG-oligodeoxynucleotides (CpG) in IFA. Generation of anti-PrP T and B cell responses was analyzed in the spleen, lymph nodes, and serum of immunized C57BL/6 wild-type mice. Immunization with PrP peptides emulsified in CFA did not trigger an immune response to PrP. When CpG were used, vaccination with peptides P143-172 and P158-187 generated IFN-gamma-secreting splenic T cells, and only P158-187 significantly stimulated IL-4-secreting T cells. Both peptides induced few Ab-producing B cells, and low and variable serum Ab titers. In contrast, immunization with peptide P98-127 did not induce significant levels of T cell responses but elicited specific peptide Abs. T cell epitope mapping, performed using 15-mer peptides covering PrP segment 142-182, revealed that an immunogenic motif lies between positions 156 and 172. These results demonstrate that T and B cell repertoires against PrP can be stimulated in C57BL/6 when adjuvant of the innate immunity such as CpG, but not CFA, is added to PrP peptides, and that the pattern of immune responses varies according to the epitope.     

Expression of normal cellular prion protein (PrP(c)) on T lymphocytes and the effect of copper ion: Analysis by wild-type and prion protein gene-deficient mice

The purpose of this report was to determine the effect of prion protein (PrP) gene disruption on T lymphocyte function. Previous studies have suggested that normal cellular prion protein (PrP(c)) binds to copper and Cu(2+) is essential for interleukin-2 (IL-2) mRNA synthesis. In this study, IL-2 mRNA levels in a copper-deficient condition were investigated using T lymphocytes from prion protein gene-deficient (PrP(0/0)) and wild-type mice. Results showed that Cu(2+) deficiency had no effect on PrP(c) expression in Con A-activated splenocytes. However, a delay in IL-2 gene expression was observed in PrP(0/0) mouse T lymphocyte cultures using Con A and Cu(2+)-chelator. These results suggest that PrP(c) expression may play an important role in rapid Cu(2+) transfer in T lymphocytes. The rapid transfer of Cu(2+) in murine T lymphocytes could be one of the normal functions of PrP(c).     

High prion and PrPSc levels but delayed onset of disease in scrapie-inoculated mice heterozygous for a disrupted PrP gene.

BACKGROUND: It has been proposed that the prion, the infectious agent of transmissible spongiform encephalopathies, is PrPSc, a post-translationally modified form of the normal host protein PrPC. We showed previously that mice devoid of PrPC (Prn-p0/0) are completely resistant to scrapie. We now report on the unexpected response of heterozygous (Prn-p0/+) mice to scrapie infection. MATERIALS AND METHODS: Prn-p0/+, Prn-p0/0 and Prn-p+/+ mice were obtained from crosses of Prn-p0/+ mice. Mice were inoculated intracerebrally with mouse-adapted scrapie agent and the clinical progression of the disease recorded. Mice were sacrificed at intervals, PrPSc was determined as protease-resistant PrP and the prion titer by the incubation time assay. RESULTS: Prn-p0/+ mice, which have about half the normal level of PrPC in their brains, show enhanced resistance to scrapie, as manifested by a significant delay in onset and progression of clinical disease. However, while in wild type animals an increase in prion titer and PrPSc levels is followed within weeks by scrapie symptoms and death, heterozygous Prn-p0/+ mice remain free of symptoms for many months despite similar levels of scrapie infectivity and PrPSc. CONCLUSIONS: Our findings extend previous reports showing an inverse relationship between PrP expression level and incubation time for scrapie. However, contrary to expectation, overall accumulation of PrPSc and prions to a high level do not necessarily lead to clinical disease. These findings raise the question whether high titers of prion infectivity could also persist for long periods under natural circumstances in the absence of clinical symptoms.     

Genetic control of serum neutralizing-antibody response to rabies vaccination and survival after a rabies challenge infection in mice.

Quantitative differences in serum neutralizing-antibody (SNAb) responses to rabies vaccination and survival after a rabies challenge infection between two inbred mice strains, C3H/J and C57BL/6J, were shown to be under genetic control. A 99% confidence limit calculated from the SNAb response titers of 14 C57BL/6J mice resulted in an upper limit for the SNAb response titer of C57BL/6J mice at 50.63. A SNAb titer less than or equal to 50.63 in response to rabies vaccination was assigned the phenotype of hyporesponder, and a SNAb titer greater than 50.63 in response to rabies vaccination was assigned the phenotype of hyperresponder in this study. The hyper-SNAb response to rabies vaccination and the higher frequency of survival after rabies challenge infection behave as Mendelian dominant alleles in F1 hybrids (C3H/J X C57BL/6J) and backcross (BC) (F1 [C3H/J X C57BL/6J] X C57BL/6J) progeny. Both a relatively hyper-SNAb response and a higher frequency of vaccine-inducible survival phenotypes occur in C3H/J mice. On the other hand, both the relatively hypo-SNAb response and a lower frequency of vaccine-inducible survival phenotypes behave as Mendelian recessive alleles and occur in C57BL/6J mice. C3H/J mice are H-2 Kk, and C57BL/6J mice are H-2 Kb. All three phenotypic traits (H-2 type, SNAb response, and survival after rabies challenge infection) segregate as independent (unlinked) monogenic traits in BC progeny (F1 [C3H/J X C57BL/6J] X C57BL/6J). The genetically controlled survival trait is inducible by rabies vaccination, but SNAb response is not a parameter that measures successful vaccine induction of preexposure protection from a rabies challenge infection in the BC progeny. The essential role of vaccination in developing preexposure protection in genetically responsive mice is confirmed, but indicates that in vitro measurements other than SNAb titers need to be developed to identify mice that have failed to achieve preexposure protection by rabies vaccination. This study confirms Lodmell's findings (D. L. Lodmell and B. Chesebro, J. Virol. 50:359-362, 1984; D. L. Lodmell, J. Exp. Med. 157:451-460, 1983) that susceptibility to rabies infection is genetically controlled in some mice strains. Additionally, this study indicates that conventional rabies vaccination even with more potent vaccines may not induce protection from infection in some genetically susceptible individuals.     

Bovine prion (PrP) and Doppel (Dpl) proteins expression after in vitro leukocyte activation or Dpl/PrP blocking

It has been postulated that Doppel (Dpl) and Prion (PrP) proteins have yet undetermined interactions, since Dpl is overexpressed in transgenic PrP-deficient mice. In this study we investigated the expression levels of Dpl and PrP on lymphocytes, monocytes and neutrophils (PMNs) isolated from bovine blood and incubated (2 and 18 h) with TNFalpha, IL-1, IL-2, IL-8, C5a, IFNgamma, anti-PrP, and anti-Dpl antibodies by flow cytometry. The isolation procedures yielded cell populations with high purity, viability and recovery rates. After 2 h incubation, expression of PrP or Dpl was altered only in PMNs. These cells overexpressed PrP when incubated with TNFalpha and IFNgamma, and both PrP and Dpl when incubated with C5a; incubation with TNFalpha, IL-8 and IFNgamma led to down-regulation of Dpl. Lymphocytes incubated for 18 h with IL-2 and with IFNgamma overexpressed Dpl. Incubation with the anti-PrP antibody induced down-regulation of Dpl in PMNs after 2 h and overexpression of Dpl in lymphocytes after 18 h. The differences recorded after 2 h were likely due to redistribution of pre-existing PrP or Dpl molecules, while those seen at 18 h were most probably due to increased protein synthesis. The variations seen using the different activators depend on different receptors and/or signaling pathways. These results demonstrate that is possible to alter the expression of Dpl and PrP in blood cells in vitro by incubation with either cytokines or anti-PrP antibodies. This opens an interesting opportunity to study the biology of these proteins using in vitro systems.     

Metabolic cooperation between Tfm and wild-type cells in mosaic mice after induction of DNA synthesis

Mosaic mice composed of androgen-insensitive Tfm and androgen-sensitive wild-type cells are constructed by virtue of the natural X inactivation: XX mice heterozygous for X-linked testicular feminization (Tfm) are reverted to males by the sex reversal (Sxr) mutation. After stimulation with testosterone, in the epididymis of the mosaic mice, the incorporation of ³H-thymidine is compared in both cell fractions. The labeling index of Tfm and wild-type cells is in the same order of magnitude. The result indicates that the stimulus for DNA synthesis exerted by testosterone is conveyed from the androgen-sensitive wild-type to the androgen-insensitive Tfm cells by metabolic cooperation on tissue level.On the other hand, the proportion of Tfm cells in the epididymal mosaic is far less than expected from X inactivation. The reason is that in the mosaic, in spite of normal proliferation, the undifferentiated Tfm cells die off steadily. Typical dense bodies are observed as signs of physiological cell death.     

Biochemical fingerprints of prion infection: accumulations of aberrant full-length and N-terminally truncated PrP species are common features in mouse prion disease

Infection with any one of three strains of mouse scrapie prion (PrPSc), 139A, ME7, or 22L, results in the accumulation of two underglycosylated, full-length PrP species and an N-terminally truncated PrP species that are not detectable in uninfected animals. The levels of the N-terminally truncated PrP species vary depending on PrPSc strain. Furthermore, 22L-infected brains consistently have the highest levels of proteinase K (PK)-resistant PrP species, followed by ME7- and 139A-infected brains. The three strains of PrPSc are equally susceptible to PK and proteases papain and chymotrypsin. Their protease resistance patterns are also similar. In sucrose gradient velocity sedimentation, the aberrant PrP species partition with PrPSc aggregates, indicating that they are physically associated with PrPSc. In ME7-infected animals, one of the underglycosylated, full-length PrP species is detected much earlier than the other, before both the onset of clinical disease and the detection of PK-resistant PrP species. In contrast, the appearance of the N-terminally truncated PrP species coincides with the presence of PK-resistant species and the manifestation of clinical symptoms. Therefore, accumulation of the underglycosylated, full-length PrP species is an early biochemical fingerprint of PrPSc infection. Accumulation of the underglycosylated, full-length PrP species and the aberrant N-terminally truncated PrP species may be important in the pathogenesis of prion disease.     

Distinct and non-overlapping T cell receptor repertoires expanded by DNA vaccination in wild-type and HER-2 transgenic BALB/c mice

Central tolerance to tumor-associated Ags is an immune-escape mechanism that significantly limits the TCR repertoires available for tumor eradication. The repertoires expanded in wild-type BALB/c and rat-HER-2/neu (rHER-2) transgenic BALB-neuT mice following DNA immunization against rHER-2 were compared by spectratyping the variable (V)beta and the joining (J)beta CDR 3. Following immunization, BALB/c mice raised a strong response. Every mouse used one or more CD8+ T cell rearrangements of the Vbeta9-Jbeta1.2 segments characterized by distinct length of the CDR3 and specific for 63-71 or 1206-1214 rHER-2 peptides. In addition, two CD4+ T cell rearrangements recurred in >50% of mice. Instead, BALB-neuT mice displayed a limited response to rHER-2. Their repertoire is smaller and uses different rearrangements confined to CD4+ T cells. Thus, central tolerance in BALB-neuT mice acts by silencing the BALB/c mice self-reactive repertoire and reducing the size of the CD8+ T cell component. CD8+ and CD4+ T cells from both wild-type and transgenic mice home to tumors. This definition of the T cell repertoires available is critical to the designing of immunological maneuvers able to elicit an effective immune reaction against HER-2-driven carcinogenesis.     

Distribution of cellular isoform of prion protein in T lymphocytes and bone marrow, analyzed by wild-type and prion protein gene-deficient mice

In this study, the authors investigated normal cellular prion protein (PrP(C)) expression on murine immune systems using prion protein gene-deficient mouse as negative control. Immunocytes expressing PrP(C) in adult and fetal mice were detected by flow cytometry with the monoclonal antibody against PrP(C), 6H4. Cells from thymus and bone marrow reacted positively with 6H4, while spleen cells, peritoneal cells, peripheral blood leukocytes, and intestinal intraepithelial lymphocytes were nonreactive. In thymus, PrP(C) was observed in CD4(-)CD8(-) double-negative thymocytes. PrP(C+) cells of double-negative thymocytes belonged to the CD3(-) subset, but not to the CD3(+) subset. Triple-negative PrP(C+) thymocytes expressed CD44 or CD25 antigens. Furthermore, PrP(C) was observed in c-kit(+) bone marrow cells. In fetuses, PrP(C+) cells were observed in the liver and thymus at day 16.0 and 15.0 of gestation, respectively. These results demonstrated that PrP(C) is expressed on immature immunocytes.     

The cellular prion protein (PrP(C)): its physiological function and role in disease

Prion diseases are caused by conversion of a normal cell-surface glycoprotein (PrP(C)) into a conformationally altered isoform (PrP(Sc)) that is infectious in the absence of nucleic acid. Although a great deal has been learned about PrP(Sc) and its role in prion propagation, much less is known about the physiological function of PrP(C). In this review, we will summarize some of the major proposed functions for PrP(C), including protection against apoptotic and oxidative stress, cellular uptake or binding of copper ions, transmembrane signaling, formation and maintenance of synapses, and adhesion to the extracellular matrix. We will also outline how loss or subversion of the cytoprotective or neuronal survival activities of PrP(C) might contribute to the pathogenesis of prion diseases, and how similar mechanisms are probably operative in other neurodegenerative disorders.     

Accumulation of proteinase K-resistant prion protein (PrP) is restricted by the expression level of normal PrP in mice inoculated with a mouse-adapted strain of the Creutzfeldt-Jakob disease agent.

Creutzfeldt-Jakob disease (CJD) is a transmissible neurodegenerative disease of humans caused by an unidentified infectious agent, the prion. To determine whether there was an involvement of the host-encoded prion protein (PrPc) in CJD development and prion propagation, mice heterozygous (PrP+/-) or homozygous (PrP-/-) for a disrupted PrP gene were established and inoculated with the mouse-adapted CJD agent. In keeping with findings of previous studies using other lines of PrP-less mice inoculated with scrapie agents, no PrP-/- mice showed any sign of the disease for 460 days after inoculation, while all of the PrP+/- and control PrP+/+ mice developed CJD-like symptoms and died. The incubation period for PrP+/- mice, 259 +/- 27 days, was much longer than that for PrP+/+ mice, 138 +/- 12 days. Propagation of the prion was barely detectable in the brains of PrP-/- mice and was estimated to be at a level at least 4 orders of magnitude lower than that in PrP+/+ mice. These findings indicate that PrPc is necessary for both the development of the disease and propagation of the prion in the inoculated mice. The proteinase-resistant PrP (PrPres) was undetectable in the brain tissues of the inoculated PrP-/- mice, while it accumulated in the affected brains of PrP+/+ and PrP+/- mice. Interestingly, the maximum level of PrPres in the brains of PrP+/- mice was about half of the level in the similarly affected brains of PrP+/+ mice, indicating that PrPres accumulation is restricted by the level of PrPc.     

Molecular analysis of the protease-resistant prion protein in scrapie and bovine spongiform encephalopathy transmitted to ovine transgenic and wild-type mice

The existence of different strains of infectious agents involved in scrapie, a transmissible spongiform encephalopathy (TSE) of sheep and goats, remains poorly explained. These strains can, however, be differentiated by characteristics of the disease in mice and also by the molecular features of the protease-resistant prion protein (PrP(res)) that accumulates into the infected tissues. For further analysis, we first transmitted the disease from brain samples of TSE-infected sheep to ovine transgenic [Tg(OvPrP4)] and to wild-type (C57BL/6) mice. We show that, as in sheep, molecular differences of PrP(res) detected by Western blotting can differentiate, in both ovine transgenic and wild-type mice, infection by the bovine spongiform encephalopathy (BSE) agent from most scrapie sources. Similarities of an experimental scrapie isolate (CH1641) with BSE were also likewise found following transmission in ovine transgenic mice. Secondly, we transmitted the disease to ovine transgenic mice by inoculation of brain samples of wild-type mice infected with different experimental scrapie strains (C506M3, 87V, 79A, and Chandler) or with BSE. Features of these strains in ovine transgenic mice were reminiscent of those previously described for wild-type mice, by both ratios and by molecular masses of the different PrP(res) glycoforms. Moreover, these studies revealed the diversity of scrapie strains and their differences with BSE according to labeling by a monoclonal antibody (P4). These data, in an experimental model expressing the prion protein of the host of natural scrapie, further suggest a genuine diversity of TSE infectious agents and emphasize its linkage to the molecular features of the abnormal prion protein.     

Autoimmunity and immune complex disease after neonatal induction of transplantation tolerance in mice

Mice made neonatally tolerant to alloantigens were found to develop an immunologic disease resembling systemic lupus erythematosus. In BALB/c mice neonatally injected with C57BL/6 X BALB/c F1 hybrid spleen cells, features of autoimmunity were observed first. After 5-24 wk, antinuclear, anti-SS DNA, thymocytotoxic, and rheumatoid factor-like antibodies were detected in association with hypergammaglobulinemia and with the occurrence of circulating immune complexes and cryoglobulins. Some of the antinuclear antibodies were found to be produced by F1 donor B cells persisting in the host. Second, immunopathologic changes were detected in tolerant mice. In the kidneys, an immune complex glomerulonephritis of the membranous type was observed. Immunoglobulin deposits were also found in the choroid plexus and at the dermoepidermal junction. In addition, thrombocytopenia was a common finding, and a positive direct Coomb's test occasionally was detected. Features of autoimmune disease were closely associated with the effective induction of transplantation tolerance, as revealed by the inability of spleen cells to generate in vitro cytolytic responses against C57BL/6 alloantigens. It is suggested that, although transplantation tolerance is associated with a lack of cytolytic reaction of the host against F1 hybrid donor alloantigens, other types of allogeneic interactions could lead in this model to the development of autoimmunity and immunopathology.