Intersite helper function of T cells specific for a protein epitope that is not recognized by antibodies

Humoral responses to a protein require T-B cell communication for B cell activation by T cells. Previous studies from this laboratory have mapped the T and B cell recognition sites (epitopes) on sperm-whale myoglobin (Mb) and several other proteins. It was found that, five of six regions on Mb recognized by T cells are also recognized by B cells (i.e. antibodies). There is, however, one region (E6) residing within Mb residues 61-77, that is recognized only by T cells and to which no antibody (Ab) responses are detectable. To investigate the function of this exclusive T cell epitope, we established, from E6-primed BALB/c mice, an E6-specific T cell line (T(e6)) which comprised Th2-type cells. These T cells provided help in vitro to B cells from Mb-primed BALB/c mice and activated them to produce anti-Mb Abs of the IgM (58.2%) and IgG (41.8%) isotypes. The helper activity of T(e6) cells was dependent on the concentration of the challenging Ag (intact Mb or peptide E6) in culture. Action of soluble factors released from E6-activated T(e6) cells on B(mb) cells led to low production of anti-Mb Abs, suggesting that activation of the B cells was more dependent on their contact with T cells. Mapping of the epitope recognition of the anti-Mb Abs produced in vitro by B(mb) cells on activation by T(e6) revealed that this activation was not general to all antigenic regions recognized by anti-Mb Abs in BALB/c mice. E6-specific T cells caused in vitro activation and differentiation of B(mb) cells into plasma cells that secreted anti-Mb Abs directed, in decreasing order, against the following Mb regions: E4 (107-120) > E3 (87 - 100) > E1 (10 - 22). Little or no Ab responses could be detected against peptides E2 (50 - 62), E5 (141 - 153) and E6 (61 - 77). With B cells of peptide-primed BALB/c mice, T(e6) cells activated strongly E4-, E3- or E1, and only very slightly E2- or E6-, primed B cells to secrete Abs against the correlate peptide, but failed completely to activate E5-primed B cells. The results show that a protein T cell epitope, to which no Abs are detectable, plays an active role in B cell responses against other epitopes within the same protein.     

Induction of native protein reactive antibodies by immunization with peptides containing linear B-cell epitopes defined by anti-porcine ZP3 beta monoclonal antibodies

To identify pertinent target epitopes for contraceptive vaccine development, rabbit polyclonal antibodies were raised against four peptides synthesized from the deduced amino acid (aa) sequence of porcine zona pellucida macromolecule ZP3 beta and coupled to diphtheria toxoid (DT). Synthetic peptides consisted of: P1, 23-37 aa; P2, 164-179 aa with an additional C-terminal cysteine; P3, 246-263 aa with an extra C-terminal cysteine; and P4, 310-321 aa residues corresponding to pZP3 beta precursor protein. Selected sequences were based upon B cell epitopes identified previously by monoclonal antibodies. Immune sera reacted with their respective peptides and DT in an ELISA, and also recognized porcine SIZP and pZP3 beta both in ELISA and Western blot and zona pellucida of porcine oocytes in an indirect immunofluorescence assay. None of the four anti-peptide sera recognized pZP3 alpha in Western blot, emphasizing the specificity of these antibodies to pZP3 beta. The anti-peptide sera, individually, failed to inhibit in vitro attachment of boar sperm to antibody treated zona encased porcine oocytes. However, combinations of immune sera against peptides such as P1 + P4, P2 + P4 and P1 + P2 + P4, did significantly inhibit porcine sperm-oocyte interaction. These results identify combinations of peptides that could potentially be used in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to pZP3 beta or its homologues in other species.     

B-cell activation in vitro by helper T cells specific to a protein region that is recognized both by T cells and by antibodies

The effects of Trypanosoma evansi on efferent lymphocyte phenotypes draining from a lymph node primed with Pasteurella haemolytica vaccine were studied in sheep. The prefemoral efferent lymphatic ducts of the infected sheep along with those of two uninfected sheep were surgically cannulated. Lymph was collected and lymphocytes recovered from it analysed by two-colour indirect immunofluorescence staining and cytofluoremetry in a fluorescence activated cell analyser (FACSCAN). The study showed the appearance and persistence of T. evansi in the efferent lymph for a long period of time and the appearance of CD4+CD8+ (double positive, DP) T lymphocytes in the efferent lymph of infected animals. The infection also resulted in increases in CD5+ B cells in the prefemoral efferent lymph. In addition, there were decreases in the output of conventional B cells, CD5+ and CD4+ T cell subsets but large increases in CD8+ cells followed by terminal depletion of all cell subsets. In contrast, inoculation of sheep with pasteurella vaccine antigen alone produced little alterations in the proportions, but large increases in the numbers of all T cell subsets except that of CD8+ cells which also showed little variation; and there was a concurrent increase in the numbers and proportions of efferent B cells. In addition, the abnormal expression of DP and CD5+ B cells did not occur in the uninfected vaccinated sheep. It is concluded that these abnormal changes in the kinetics of efferent lymphocyte phenotypes are likely to play a role in the genesis of the generalized immunosuppression seen in trypanosome-infected hosts.     

Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected to elicit antibodies preferentially neutralizing mutant variants of HIV-BRU lacking the N306 glycan. Therefore, two guinea pigs were immunized with monomeric wild-type HIV-BRU gp120 possessing the N306 glycan and immune sera were tested for neutralization against target viruses HIV-BRU, -A308, and -A308T321. HIV-A308 and HIV-A308T321 lack the N306 glycan; HIV-A308T321 contains an additional mutation at the tip of V3 rendering it resistant to MAb binding at this epitope. Both immune sera preferentially neutralized the two mutant virus variants lacking the N306 glycan, with a 10- to 20-fold increase in neutralization titer compared with the wild-type HIV-BRU. Thus, immunization with monomeric HIV-BRU gp120 elicited antibodies preferentially neutralizing HIV variants lacking the N306 glycan. In addition to antibodies directed against the tip of V3, other antibodies directed against epitopes shielded by the N306 glycan on the envelope oligomer were elicited by the immunization, as demonstrated by the ability of the immune sera to neutralize HIV-A308T321. One such epitope was overlapping the NEA-9284 epitope located at the amino-terminal flank of the V3 loop. Our results demonstrate that monomeric gp120 contains immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation.     

Induction of an epitope-specific humoral immune response by lipopeptide-hapten conjugates: enhancement of the anti-melittin response by a synthetic T helper (Th)-cell epitope

Lipopeptides of bacterial origin constitute potent immunoadjuvants when combined with antigens. After the immunization with lipopeptides covalently coupled to non-immunogenic low-molecular-mass antigens or haptens, a hapten-specific humoral immune response can often be obtained. The response against synthetically prepared melittin fragments was further enhanced by the additional introduction of a T helper (Th)-cell epitope into the lipopeptide-hapten conjugate. The Th-cell epitope applied, which is presented by the MHC class II molecule of the BALB/c (H-2d) haplotype, consisted of a synthetic 16-amino-acid oligopeptide derived from sperm whale myoglobin. The immune-enhancing effect was most pronounced for the melittin-derived peptide fragments [Mel(1-16)] and [Mel(17-26)-CONH2]. Antibodies obtained after 3 immunizations with the conjugates recognized the synthetic as well as the native melittin molecule. Our results show that it is possible to markedly enhance a weak hapten-specific immune response by coupling the haptens to a lipopeptide conjugated to a haplotype-specific T helper-cell epitope. The novel conjugates are well suited for the optimization of immunization procedures, and for the development of novel synthetic vaccines.     

Expansion by self antigen is necessary for the induction of experimental autoimmune encephalomyelitis by T cells primed with a cross-reactive environmental antigen

Cross-reactivity with environmental antigens has been postulated as a mechanism responsible for the induction of autoimmune disease. Experimental autoimmune encephalomyelitis is a T cell-mediated autoimmune disease model inducible in susceptible strains of laboratory animals by immunization with protein constituents of myelin. We used myelin proteolipid protein (PLP) peptide 139-151 and its analogues to define motifs to search a protein database for structural homologues of PLP139-151 and identified five peptides derived from microbial Ags that elicit immune responses that cross-react with this self peptide. Exposure of naive SJL mice to the cross-reactive environmental peptides alone was insufficient to induce autoimmune disease even when animals were treated with Ag-nonspecific stimuli (superantigen or LPS). However, immunization of SJL mice with suboptimal doses of PLP139-151 after priming with cross-reactive environmental peptides consistently induced experimental autoimmune encephalomyelitis. Furthermore, T cell lines from mice immunized with cross-reactive environmental peptides and restimulated in vitro with PLP139-151 could induce disease upon transfer into naive recipients. These data suggest that expansion by self Ag is required to break the threshold to autoimmune disease in animals primed with cross-reactive peptides.     

Complement consuming antibodies against a modified human serum protein in pigeon breeders' disease

The sera of patients with pigeon breeders' disease contain precipitating and human complement consuming antibodies against pigeon dropping antigens. Cessation of antigen exposure results in a decrease of precipitins below the level of detection in immunodiffusion. Complement consuming antibodies remain present, however, despite antigen avoidance. A close correlation is observed between human complement consumption tests with pigeon dropping antigens PDF1-A or pigeon cropmilk IgA and a modified human serum gamma-globulin. Isolation of this protein is readily achieved by its non-specific adsorption onto activated Sepharose 4B and subsequent elution with 1 M acetic acid. This modified protein may act as an autoantigen in pigeon breeders' disease, maintaining human complement consuming antibodies for years in subjects with no further bird antigen contact.     

Induction of protective T cells against Listeria monocytogenes in mice by immunization with a listeriolysin O-negative avirulent strain of bacteria and liposome-encapsulated listeriolysin O

Only listeriolysin O (LLO)-producing strains of Listeria monocytogenes generate protective immunity in mice. Based on the findings that endogenous gamma interferon (IFN-gamma) production was induced only by such strains and that purified LLO could induce IFN-gamma from NK cells, we have postulated that LLO may play a pivotal role in the induction of Th1-type protective T cells, which are highly dependent on IFN-gamma. In this study, mice were immunized with L. monocytogenes ATCC 15313, an LLO-nonproducing avirulent strain, along with LLO encapsulated in liposome (LLO-liposome). LLO-liposome was highly potent in the induction of various cytokines, including IFN-gamma. Immunization of mice with either LLO-liposome or the viable strain ATCC 15313 alone did not induce protection against challenge infection. In contrast, the combination of LLO-nonproducing bacteria plus LLO-liposome induced a significant level of protective immunity mediated mainly by Th1-type cells capable of producing a large amount of IFN-gamma in an antigen-specific manner. The protection afforded by the combination was not dependent on LLO-specific cytotoxic T cells. These results support the idea that the inability of an LLO-nonproducing avirulent strain or killed bacteria to induce the generation of protective T cells is due not to the lack of a central T-cell epitope(s) but to the lack of ability to induce the production of endogenous cytokine during the early stage of immunization; the results also suggest that an appropriate use of LLO at least in an animal model may be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria.     

Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein

Immunization with irradiated sporozoites protects animals and humans against malaria, and the circumsporozoite protein is a target of this protective immunity. We now report that adjuvant-free intramuscular injection of mice with plasmid DNA encoding the Plasmodium yoelii circumsporozoite protein induced higher levels of antibodies and cytotoxic T lymphocytes against the P. yoelii circumsporozoite protein than did immunization with irradiated sporozoites. Mice immunized with this vaccine had an 86% reduction in liver-stage parasite burden after challenge with 5 x 10(5) sporozoites (> 10(5) median infectious doses). Eighteen (68%) of 28 mice that received two or three doses of vaccine were protected against challenge with 10(2) sporozoites, and the protection was dependent on CD8+ T cells. These studies demonstrate the utility of plasmid DNA immunization against a nonviral infection. By obviating the requirement for peptide synthesis, expression and purification of recombinant proteins, and adjuvants, this method of immunization provides an important alternative for rapid identification of protective B- and T-cell epitopes and for construction of vaccines to prevent malaria and other infectious diseases.     

Heterotypic protection against influenza by immunostimulating complexes is associated with the induction of cross-reactive cytotoxic T lymphocytes

Here we report the isolation and characterization of mouse testicular cDNAs encoding the mammalian homologue of the Xenopus germ cell-specific nucleic acid-binding protein FRGY2 (mRNP3+4), hereafter designated MSY2. MSY2 is a member of the Y box multigene family of proteins; it contains the cold shock domain that is highly conserved among all Y box proteins and four basic/aromatic islands that are closely related to the other known germline Y box proteins from Xenopus, FRGY2, and goldfish, GFYP2. Msy2 undergoes alternative splicing to yield alternate N-terminal regions upstream of the cold shock domain. Although MSY2 is a member of a large family of nucleic acid-binding proteins, Southern blotting detects only a limited number of genomic DNA fragments, suggesting that Msy2 is a single copy gene. By Northern blotting and immunoblotting, MSY2 appears to be a germ cell-specific protein in the testis. Analysis of Msy2 mRNA expression in prepubertal and adult mouse testes, and in isolated populations of germ cells, reveals maximal expression in postmeiotic round spermatids, a cell type with abundant amounts of stored messenger ribonucleoproteins. In the ovary, MSY2 is present exclusively in diplotene-stage and mature oocytes. MSY2 is maternally inherited in the one-cell-stage embryo but is not detected in the late two-cell-stage embryo. This loss of MSY2 is coincident with the bulk degradation of maternal mRNAs in the two-cell embryo.     

Specificity of the T-cell responses in covalently linked peptides each comprising of a T helper epitope

The selection of T-cell specificities in chimeric peptides comprising of two T helper epitopes (288-302 and 240-252) from the fusion protein of measles virus was investigated. The resulting chimeric peptides (288-P-240 and 240-P-288) were shown to be immunogenic by inducing proliferative responses in both B10.s and C57BL/6 strains of mice. In B10.s mice immunization with the chimeric peptides resulted in proliferative T-cell responses only to the constituent 288-302 peptide, whereas in C57BL/6 mice no proliferative responses to the constituent 288-302 or 240-252 peptides were detected. In vivo competition studies between the 288-302 and 240-252 peptides for binding to the I-As molecule have shown that the peptide 288-302 was dominant in B10.s mice and competed with the non-dominant 240-252 peptide for the induction of an in vivo response. The absence of any proliferative T-cell response to the constituent 288-302 and 240-252 peptides after immunization of C57BL/6 mice with the 288-P-240 or 240-P-288 chimeric peptides, suggests that the dominant T-cell responses might have shifted to newly formed T cell epitope(s) as a result of the covalent linkage of the two peptides. In conclusion, these results indicate that the selection of Th epitopes within chimeric peptides is dependent not only on the amino acid composition of the epitope but also on the context of the epitope within the chimera and the haplotype of the mouse strain used.     

Passive immunization with antibodies against three distinct epitopes on Plasmodium yoelii merozoite surface protein 1 suppresses parasitemia

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an approximately 42-kDa fragment (MSP-142) that is derived from the C terminus of MSP-1. MSP-142 is further cleaved to an N-terminal approximately 33-kDa polypeptide (MSP-133) and a C-terminal approximately 19-kDa polypeptide (MSP-119) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-142 but not with either of the constituents MSP-133 and MSP-119, B4 recognized an epitope within the N terminus of MSP-133, and B6, B10, F5, and G3 bound to MSP-119. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.     

Induction of long-term depression (with anxiety and fear components) by immunization of rats against pargyline

The active immunization of albino rats against pargyline (a MAO B inhibitor) induced the formation of antibody to pargyline and results in deep depressive changes and fear. These changes were observed within 6 weeks after the first immunization. Therefore, it opens the possibility to model depression long by exerting the minimum influences. There was also a long-term modulation of craving for alcohol.     

Induction of neutralizing antibody in mice by immunization with recombinant 56 kDa protein of Orientia tsutsugamushi

Anti-oriential antibody inhibits Orientia tsutsugamushi attachment to, and penetration of, host cells. However, O. tsutsugamushi antigens that induce the production of a neutralizing antibody have not been identified. The authors immunized mice and rabbits with the recombinant 56 kDa protein of O. tsutsugamushi fused to the maltose binding protein of Escherichia coli (MBP-Bor56) and analysed their effect on O. tsutsugamushi attachment to or penetration of L929 cells. O. tsutsugamushi attachment and penetration were measured by using an indirect immunofluorescent antibody assay (IFA). O. tsutsugamushi growth in L929 cells was determined by [3H]thymidine uptake assay. By IFA, we observed a 96% reduction of attachment or penetration of O. tsutsugamushi treated with rabbit anti-MBP-Bor56 sera. [3H]thymidine uptake showed that mouse anti-MBP-Bor56 sera caused a 91% reduction in O. tsutsugamushi growth, when compared to mouse anti-MBP sera. These results suggest that the 56 kDa protein of O. tsutsugamushi plays an important role in O. tsutsugamushi attachment to or penetration of cells.     

Eliciting T cell immunity against poorly immunogenic tumors by immunization with dendritic cell-tumor fusion vaccines

Dendritic cells (DCs) are the most effective APCs and are being studied as natural adjuvants or Ag delivery vehicles to elicit T cell-mediated antitumor immunity. This study examined whether inoculation of DCs fused with poorly immunogenic tumor cells elicited tumor-reactive T cells for adoptive immunotherapy. DCs derived from bone marrow of C57BL/6 (B6) mice were fused with syngeneic B16 melanoma or RMA-S lymphoma cells by polyethylene glycol. The B16/DC and RMA-S/DC fusion hybrids expressed MHC class I, class II Ags, costimulatory molecules, as well as DC-specific and tumor-derived surface markers. The tumor/DC hybrids were capable of processing and presenting tumor-derived Ags, and immunization of B6 mice with irradiated B16/DC or RMA-S/DC vaccine elicited tumor-specific CTL activities. Vaccination of B6 mice with irradiated B16/DC fusion preparations induced partial host protective immunity against B16 tumor challenge. Reduced tumor incidence and prolonged survival time were observed. Adoptive transfer of T cells derived from B16/DC vaccine-primed lymph nodes into B16 tumor-bearing mice greatly reduced the number of established pulmonary metastases with or without in vivo administration of IL-2. Moreover, adoptive transfer of RMA-S/DC vaccine-primed, cultured lymph node T cells eradicated disseminated FBL-3 tumor. The results demonstrate that tumor/DC fusion products are effective cellular vaccines for eliciting T cell-mediated antitumor immunity.     

Plasmodium vivax: epitope mapping of monoclonal antibodies against the N-terminal region of the merozoite surface protein 1

Plasmodium vivax is the most widely distributed human malaria with an estimate of 35 million cases per year. The deduced amino acid sequence comparisons of the Merozoite Surface Protein 1 (MSP1) from several plasmodial species, including that of P. vivax (PvMSP1), revealed the existence of highly conserved blocks and polymorphic blocks. We had previously shown that sequences within conserved blocks from the N-terminal region of the PvMSP1 were poorly immunogenic in natural human infections. These results suggest that these regions code for important and unknown structural and/or functional features and thus they could potentially be tested as a sub-unit PvMSP1 vaccine. In the present study, a battery of monoclonal antibodies (Mabs) was produced against the N-terminal region of the PvMSP1 in an attempt to determine whether these N-terminal ICBs contained all the epitopes exposed on the native molecule. The results suggest that the most terminal ICB2 and ICB3 blocks are not exposed on the surface of the PvMSP1 native molecule and clearly eliminate the possibility of considering the N-terminal domains as unique components of a sub-unit PvMSP1 vaccine candidate.     

Epitope-specific antibody and suppression of autoantibody responses against a hybrid self protein

This study addresses the relationship of epitope-specific Ab responses and alternative autoantibody responses in a model system in which an antigenized self protein serves as the carrier for a defined heterologous B cell epitope. Ubiquitin, a nonimmunogenic self protein, was engineered to present heterologous B and T cell epitopes in the recombinant molecule. Fusion to the C terminus introduced a universal T cell epitope from a Mycobacterium tuberculosis Ag. The B cell epitope was created by inserting a 12-residue loop sequence of HIV-1 gp120 at a surface-exposed position of ubiquitin. These modifications preserved the ubiquitin fold, allowing a new conformational epitope to be presented among native self epitopes. Mice immunized with the hybrid protein bearing only the mycobacterial T cell epitope elicited a strong autoantibody response to native ubiquitin. In contrast, antisera elicited against hybrid ubiquitin presenting the HIV B cell epitope reacted specifically with the foreign epitope but not with native ubiquitin. Absence of autoantibody in the response was attributed to poor competition of autoreactive B cells for limiting T cell help. Both types of responses were associated with Th responses to defined epitopes of the ubiquitin hybrid protein. These results may have implications for a tolerance mechanism dependent on B-T cell cooperation.     

Induction of protective immunity against Japanese encephalitis in mice by immunization with a plasmid encoding Japanese encephalitis virus premembrane and envelope genes

A DNA vaccine plasmid containing the Japanese encephalitis (JE) virus premembrane (prM) and envelope (E) genes (designated pcDNA3JEME) was evaluated for immunogenicity and protective efficacy in mice. Two immunizations of 4-week-old female ICR mice with pcDNA3JEME by intramuscular or intradermal injections at a dose of 10 or 100 microg per mouse elicited neutralizing (NEUT) antibodies at titers of 1:10 to 1:20 (90% plaque reduction), and all immunized mice survived a challenge with 10,000 50% lethal doses of the P3 strain of JE virus. A single immunization with 100 microg of pcDNA3JEME did not elicit detectable NEUT antibodies but induced protective immunity. Spleen cells obtained from BALB/c mice immunized once with 10 or 100 microg of pcDNA3JEME contained JE virus-specific memory cytotoxic T lymphocytes (CTLs). BALB/c mice maintained detectable levels of memory B cells and CTLs for at least 6 months after one immunization with pcDNA3JEME at a dose of 100 microg. The CTLs induced in BALB/c mice immunized twice with 100 microg of pcDNA3JEME were CD8 positive and recognized mainly the envelope protein. These results indicate that pcDNA3JEME has the ability to induce a protective immune response which includes JE virus-specific antibodies and CTLs.     

Thrombopoietin (TPO) knockout phenotype induced by cross-reactive antibodies against TPO following injection of mice with recombinant adenovirus encoding human TPO

Adenovirus vectors have emerged as potent agents for gene transfer. Immune response against the vector and the encoded protein is one of the major factors in the transient expression following in vivo gene transfer. A single injection of an adenovirus encoding human thrombopoietin (TPO) into mice induced transient thrombocytosis, followed by a chronic immune thrombocytopenia. Thrombocytopenic mice had anti-human TPO Abs of the IgG2a and IgG1 isotypes. Thrombocytopenic mice sera neutralized more efficiently human than murine TPO, and exhibited no detectable anti-murine TPO Abs. Despite their low affinity for murine TPO, anti-TPO Abs induced a TPO knockout-like phenotype, i.e., low number of marrow megakaryocytes and of all kinds of hemopoietic progenitors. Hybridomas derived from a thrombocytopenic mouse revealed cross-reactivity of all of the secreted anti-TPO Ab isotypes. Mice subjected to myelosuppression after virus injection showed that anti-human TPO of IgG1 and IgG2a isotypes disappeared. Thus, sustained human TPO production was responsible for platelet elevation for at least 5 mo. Compelling results showed that elevated IgG2a/IgG2b ratios are always associated with thrombocytopenia, whereas low ratios are associated with tolerance or normal platelet counts. Finally, we hypothesize that in humans some chronic thrombocytopenia associated with a low TPO plasma level are due to anti-TPO Abs.