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CD22 functions primarily as a negative regulator of B-cell receptor signaling. The Cd22a allele has been proposed as a candidate allele for murine systemic lupus erythematosus. In this study, we explored the possible expression of aberrant forms of CD22, which differ in the N-terminal sequences constituting the ligand-binding site due to synthesis of abnormally processed Cd22 mRNA, in several Cd22a mouse strains, including C57BL/6 Cd22 congenic mice. The staining pattern of splenic B cells obtained with CY34 anti-CD22 mAb, which was expected to bind poorly to the aberrant CD22, was more heterogeneous in Cd22(a) mice than in Cd22b mice. Moreover, CD22 detected on B cells of Cd22a mice was expressed more weakly and as a smaller-sized protein, compared with Cd22b mice. Significantly, analysis with a synthetic CD22 ligand demonstrated that Cd22a mice carried a larger proportion of CD22 that was not bound by cis ligands on the B-cell surface than Cd22b mice. Finally, the study of C57BL/6 Cd22 congenic mice revealed that Cd22a B cells displayed a phenotype reminiscent of constitutively activated B cells (reduced surface IgM expression and augmented MHC class II expression), as reported for B cells expressing a mutant CD22 lacking the ligand-binding domain. Our demonstration that Cd22a B cells express aberrant forms of CD22, which can potentially deregulate B-cell signaling because of their decreased ligand-binding capacity, provides further support for Cd22a as a potential candidate allele for murine systemic lupus erythematosus.

Murine phagocytes express three different activating IgG FcgammaR: FcgammaRI is specific for IgG2a; FcgammaRIII for IgG1, IgG2a, and IgG2b; and FcgammaRIV for IgG2a and IgG2b. Although the role of FcgammaRIII in IgG1 and IgG2a anti-RBC-induced autoimmune hemolytic anemia (AIHA) is well documented, the contribution of FcgammaRI and FcgammaRIV to the development of IgG2a- and IgG2b-induced anemia has not yet been defined. In the present study, using mice deficient in FcgammaRI, FcgammaRIII, and C3, in combination with an FcgammaRIV-blocking mAb, we assessed the respective roles of these three FcgammaR in the development of mild and severe AIHA induced by two different doses (50 and 200 microg) of the IgG2a and IgG2b subclasses of the 34-3C anti-RBC monoclonal autoantibody. We observed that the development of mild anemia induced by a low dose of 34-3C IgG2a autoantibody was highly dependent on FcgammaRIII, while FcgammaRI and FcgammaRIV additionally contributed to the development of severe anemia induced by a high dose of this subclass. In contrast, the development of both mild and severe anemia induced by 34-3C IgG2b was dependent on FcgammaRIII and FcgammaRIV. Our results indicate differential roles of the three activating FcgammaR in IgG2a- and IgG2b-mediated AIHA.

An as-yet-unidentified mutation, Y-linked autoimmune acceleration (Yaa), is responsible for the accelerated development of lupus-like autoimmune syndrome in mice. In view of a possible role for Yaa as a positive regulator of BCR signaling, we have explored whether the expression of the Yaa mutation affects the development and activation of transgenic autoreactive B cells expressing either 4C8 IgM anti-RBC or Sp6 IgM anti-DNA. In this study, we show that the expression of the Yaa mutation induced a lethal form of autoimmune hemolytic anemia in 4C8 transgenic C57BL/6 mice, likely as a result of activation of 4C8 anti-RBC autoreactive B cells early in life. This was further supported, although indirectly, by increased T cell-independent IgM production in spleens of nontransgenic C57BL/6 mice bearing the Yaa mutation. In contrast, Yaa failed to induce activation of Sp6 anti-DNA autoreactive B cells, consistent with a lack of increased IgM anti-DNA production in nontransgenic C57BL/6 Yaa mice. Our results suggest that Yaa can activate autoreactive B cells in a BCR-dependent manner, related to differences in the form and nature of autoantigens.

A high level expression of the Ead transgene encoding the I-E α-chain is highly effective in the suppression of lupus autoantibody production in mice. To explore the possible modulation of the Ag-presenting capacity of B cells as a result of the transgene expression, we assessed the ability of the transgenic B cells to activate Ag-specific T cells in vitro. By using four different model Ag-MHC class II combinations, this analysis revealed that a high transgene expression in B cells markedly inhibits the activation of T cells in an epitope-dependent manner, without modulation of the I-E expression. The transgene-mediated suppression of T cell responses is likely to be related to the relative affinity of peptides derived from transgenic I-E α-chains (Eα peptides) vs antigenic peptides to individual class II molecules. Our results support a model of autoimmunity prevention based on competition for Ag presentation, in which the generation of large amounts of Eα peptides with high affinity to I-A molecules decreases the use of I-A for presentation of pathogenic self-peptides by B cells, thereby preventing excessive activation of autoreactive T and B cells.

Objective CD22, a B cell–restricted transmembrane glycoprotein, regulates B cell antigen receptor signaling upon interaction with α2,6-linked sialic acid–bearing glycans, which act as ligands and are expressed on B and T cells. In this study, we investigated how the expression of CD22 ligand (CD22L) is modulated following lymphocyte activation or during the course of systemic lupus erythematosus (SLE). Methods The expression levels of CD22L on B and T cells in nonautoimmune mice were assessed by flow cytometric analysis using a soluble recombinant form of CD22, following stimulation with antigen or mitogen in vitro. In addition, the expression levels of CD22L on circulating lymphocytes were correlated with the progression of SLE in lupus-prone mice. Results We observed a constitutive expression of CD22L on mature B cells, but not T cells, in nonautoimmune mice. However, CD22L levels were up-regulated selectively on T cells (but not B cells) stimulated with antigens in vitro, while their expression levels on B cells was up-modulated following polyclonal activation with lipopolysaccharide. Furthermore, expression of CD22L was increased on circulating B cells (and to a lesser extent on T cells) in parallel with progression of SLE in several different lupus-prone mice and in a cohort of (C57BL/6 × [NZB × C57BL/6.Yaa]F1) backcross mice. Conclusion The expression of CD22L is differentially regulated in B and T cells, and high expression of CD22L on circulating B cells is a marker for development of severe SLE, suggesting a role for CD22–CD22L interactions in SLE as well as in the regulation of humoral immunity.

CD22 is a B cell-restricted transmembrane protein that apparently controls signal transduction thresholds initiated through the B cell Ag receptor (BCR) in response to Ag. However, it is still poorly understood how the expression of CD22 is regulated in B cells after their activation. Here we show that the expression levels of CD22 in conventional B-2 cells are markedly down-regulated after cross-linking of BCR with anti-IgM mAb but are up-regulated after stimulation with LPS, anti-CD40 mAb, or IL-4. In contrast, treatment with anti-IgM mAb barely modulated the expression levels of CD22 in CD5+ B-1 cells, consistent with a weak Ca2+ response in anti-IgM-treated CD5+ B-1 cells. Moreover, in CD22-deficient mice, anti-IgM treatment did not trigger enhanced Ca2+ influx in CD5+ B-1 cells, unlike CD22-deficient splenic B-2 cells, suggesting a relatively limited role of CD22 in BCR signaling in B-1 cells. In contrast, CD22 levels were markedly down-regulated on wild-type B-1 cells in response to LPS or unmethylated CpG-containing oligodeoxynucleotides. These data indicate that the expression and function of CD22 are differentially regulated in B-1 and conventional B-2 cells, which are apparently implicated in innate and adaptive immunity, respectively.

▪ Abstract Precise regulation of major histocompatibility complex class II (MHC-II) gene expression plays a crucial role in the control of the immune response. A major breakthrough in the elucidation of the molecular mechanisms involved in MHC-II regulation has recently come from the study of patients that suffer from a primary immunodeficiency resulting from regulatory defects in MHC-II expression. A genetic complementation cloning approach has led to the isolation of CIITA and RFX5, two essential MHC-II gene transactivators. CIITA and RFX5 are mutated in these patients, and the wild-type genes are capable of correcting their defect in MHC-II expression. The identification of these regulatory factors has furthered our understanding of the molecular mechanisms that regulate MHC-II genes. CIITA was found to be a non-DNA binding transactivator that functions as a molecular switch controlling both constitutive and inducible MHC-II expression. The finding that RFX5 is a subunit of the nuclear RFX-complex has confirmed that a deficiency in the binding of this complex is indeed the molecular basis for MHC-II deficiency in the majority of patients. Furthermore, the study of RFX has demonstrated that MHC-II promoter activity is dependent on the binding of higher-order complexes that are formed by highly specific cooperative binding interactions between certain MHC-II promoter-binding proteins. Two of these proteins belong to families of which the other members, although capable of binding to the same DNA motifs, are probably not directly involved in the control of MHC-II expression. Finally, the facts that CIITA and RFX5 are both essential and highly specific for MHC-II genes make possible novel strategies designed to achieve immunomodulation via transcriptional intervention.

Presentation of exogenous protein antigens to T lymphocytes is based on the intersection of two complex pathways: (a) synthesis, assembly, and transport of major histocompatibility complex (MHC) class II-invariant chain complexes from the endoplasmic reticulum to a specialized endosomal compartment, and (b) endocytosis, denaturation, and proteolysis of antigens followed by loading of antigenic peptides onto newly synthesized MHC class II molecules. It is believed that expression of MHC class II heterodimers, invariant chain and human leukocyte antigen-DM is both necessary and sufficient to reconstitute a functional MHC class II loading compartment in antigen-presenting cells. Expression of each of these essential molecules is under the control of the MHC class II transactivator CIITA. Unexpectedly, however, whereas interferon gamma stimulation does confer effective antigen-processing function to nonprofessional antigen presenting cells, such as melanoma cells, expression of the CIITA transactivator alone is not sufficient. Activation of antigen-specific T cells thus requires additional CIITA-independent factor(s), and such factor(s) can be induced by interferon gamma.

The T-cell recognition of HLA-DR-peptide complexes is generally restricted by the polymorphism of the DRB molecules but pluriallelic restriction has been described. The molecular basis of restriction and promiscuity of such peptide-specific responses is poorly understood. We isolated a panel of T-cell lines specific for the tetanus toxin peptide p2 (TT830-843) exhibiting pluriallelic restriction by DR11 and DR8 alleles. Fine restriction specificity of the T-cell lines was examined in functional assays against DR oligotyped APCs expressing different variants of DR11 and DR8 alleles. Our results show that (a) polymorphisms between serologically related alleles are relevant in terms of restriction of the peptide-specific T-cell response; in some instances, a single amino acid substitution can determine the restriction of a T-cell line; (b) different patterns of restriction are not the result of specific differences in DR-p2 binding as p2 peptide binds to all DR11 and DR8 alleles tested (DRB1* 1101, -1102, -1103, -1104, 110X, -0801, -0802, -0803, and -0806); and (c) pluriallelic restriction of the peptide-specific T-cell response correlates with the presence of a DRB1 alpha-helix motif (67-71-86) shared by some DR11 and DR8 alleles. Possible implications of pluriallelic restriction of peptide-specific T-cell response in autoimmune disorders associated with DR11 and DR8 are discussed.

Replacement of aspartic acid by alanine at position 265 (D265A) in mouse IgG1 results in a complete loss of interaction between this isotype and low-affinity IgG Fc receptors (FcγRIIB and FcγRIII). However, it has not yet been defined whether the D265A substitution could exhibit similar effects on the interaction with two other FcγR (FcγRI and FcγRIV) and on the activation of complement. To address this question, 34-3C anti-RBC IgG2a and IgG2b switch variants bearing the D265A mutation were generated, and their effector functions and in vivo pathogenicity were compared with those of the respective wild-type Abs. The introduction of the D265A mutation almost completely abolished the binding of 34-3C IgG2a and IgG2b to all four classes of FcγR and the activation of complement. Consequently, these mutants were hardly pathogenic. Although oligosaccharide side chains of these mutants were found to contain higher levels of sialic acids than those of wild-type Abs, the analysis of enzymatically desialylated D265A variants ruled out the possibility that very poor Fc-associated effector functions of the D265A mutants were due to an increased level of the mutant Fc sialylation. Thus, our results demonstrate that aspartic acid at position 265 is a residue critically implicated in triggering the Fc-associated effector functions of IgG, probably by defining a crucial three-dimensional structure of the Fc region.

A high-level expression of the Ea transgene encoding the MHC class II I-E α-chain is very effective in the protection from systemic lupus erythematosus (SLE) in mice. However, it has not been elucidated whether this protection results from the induction or increased expression of I-E heterodimers or from the generation of I-E α-chain-derived peptides displaying high affinity for I-A molecules, because previous studies were conducted in lupus-prone mice expressing I-E β-chains. To address this question, we assessed the protective effect of the Ea transgene in lupus-prone BXSB mice bearing the H2q haplotype (i.e., unable to express I-E heterodimers because of a deficiency in I-E β-chains). We observed that the Ea transgene expression resulted in a marked suppression of the development of SLE in H2q BXSB mice despite the absence of I-E expression. The observed protection was not associated with any detectable levels of T cell depletion and regulatory T cell expansion. Significantly, transgenic I-E α-chains were substantially expressed on the surface of B lymphocytes and dendritic cells, but not of macrophages, without apparent formation of mixed-isotype heterodimers with I-A β-chains. Our results demonstrate for the first time that the Ea transgene is able to prevent the development of SLE without induction of I-E heterodimer expression, indicating a critical role of I-E α-chains, but not I-E heterodimers, in the Ea transgene-mediated protection from SLE. Taken together, our data favor a model of autoimmunity prevention based on competition for Ag presentation between I-E α-chain-derived peptides and autoantigens.

Interleukin (IL)-12p40, a subunit of IL-12p70 and IL-23, has previously been shown to inhibit IL-12p70 activity and interferon-gamma (IFN-gamma) production. However, recent evidence has suggested that the role of IL-12p40 is more complex. To study the contribution of IL-12p40 to immune responses against mycobacterial infections, we have used transgenic (tg) mice overexpressing IL-12p40 under the control of a major histocompatibility complex-II promoter. The IL-12p40 transgene was expressed during steady state at concentrations of 129 +/- 25 ng/ml of serum and 75 +/- 13 ng per spleen, while endogenous IL-12p40 was hardly detectable in control littermates. Bacille Calmette-Guerin (BCG) infection strongly induced the expression of IL-12p40 transgene in infected organs, and IL-12p40 monomeric and dimeric forms were identified in spleen of IL-12p40 tg mice. Excessive production of IL-12p40 resulted in a 14-fold increase in IL-12p70 serum levels in tg mice versus non-transgenic mice. IL-23 was also strongly elevated in the serum and spleens of IL-12p40 tg mice through BCG infection. While IFN-gamma and tumour necrosis factor protein levels were similar in IL-12p40 tg and non-transgenic mice, Th2 type immune responses were reduced in IL-12p40 tg mice. The number of BCG granulomas and macrophage expressing inducible nitric oxide synthase were similar in IL-12p40 tg and non-transgenic mice. IL-12p40 tg mice were as resistant as non-transgenic mice to BCG and Mycobacterium tuberculosis infections as they could efficiently control bacillary growth. These data show that high amounts of IL-12p40 promotes IL-12p70 and IL-23 formation, but that does not affect T helper 1 type immune responses and granuloma function, thus leading to normal mycobacterial clearance in infected organs.

By generating IgM and IgA switch variants of the 34-3C IgG2a anti–red blood cell (RBC) autoantibody, we evaluated the pathogenic activity of these 2 isotypes in view of the Fc-associated effector functions (ie, complement activation and polyvalency-dependent agglutination). We found that polymeric forms of 34-3C IgM and IgA anti-RBC autoantibody were as pathogenic as IgG2a, which was the most pathogenic among 4 different IgG subclasses, whereas their monomeric variants completely lacked pathogenic effects. Histological examination showed that 34-3C IgM and IgA autoantibodies caused anemia as a result of multivalency-dependent hemaggultination and subsequent sequestration of RBC in the spleen, in contrast to Fc receptor– and complement receptor–mediated erythrophagocytosis by Kupffer cells with IgG isotypes. In addition, the development of anemia induced by IgM and IgA isotypes of 34-3C antibody and by 2 additional IgM anti-RBC monoclonal autoantibodies was not inhibited at all in C3-deficient mice, indicating the lack of involvement of complement activation in the pathogenesis of IgM- and IgA-induced anemia. Our data demonstrate a remarkably high pathogenic potential of polymeric forms of IgM and IgA anti-RBC autoantibodies due to their ability to induce hemagglutination but completely independent of complement activation.

Many patients with anemia fail to respond to treatment with erythropoietin (Epo), a commonly used hormone that stimulates erythroid progenitor production and maturation by human BM or by murine spleen. The protein product of growth arrest–specific gene 6 (Gas6) is important for cell survival across several cell types, but its precise physiological role remains largely enigmatic. Here, we report that murine erythroblasts released Gas6 in response to Epo and that Gas6 enhanced Epo receptor signaling by activating the serine-threonine kinase Akt in these cells. In the absence of Gas6, erythroid progenitors and erythroblasts were hyporesponsive to the survival activity of Epo and failed to restore hematocrit levels in response to anemia. In addition, Gas6 may influence erythropoiesis via paracrine erythroblast-independent mechanisms involving macrophages. When mice with acute anemia were treated with Gas6, the protein normalized hematocrit levels without causing undesired erythrocytosis. In a transgenic mouse model of chronic anemia caused by insufficient Epo production, Gas6 synergized with Epo in restoring hematocrit levels. These findings may have implications for the treatment of patients with anemia who fail to adequately respond to Epo.

OBJECTIVE: Monocytosis is a unique cellular abnormality associated with the Yaa (Y-linked autoimmune acceleration) mutation. The present study was designed to define the cellular mechanism responsible for the development of monocytosis and to characterize the effect of the Yaa mutation on the development of monocyte subsets. METHODS: We produced bone marrow chimeras reconstituted with a mixture of Yaa and non-Yaa bone marrow cells bearing distinct Ly-17 alloantigens, and determined whether monocytes of Yaa origin became dominant. Moreover, we defined the 2 major inflammatory (Gr-1+,CD62 ligand [CD62L]+) and resident (Gr-1-,CD62L-) subsets of blood monocytes in aged BXSB Yaa male mice, as compared with BXSB male mice lacking the Yaa mutation. RESULTS: Analysis of the Ly17 allotype of blood monocytes in chimeric mice revealed that monocytes of both Yaa and non-Yaa origin were similarly involved in monocytosis. Significantly, the development of monocytosis paralleled a selective expansion of the resident monocyte subset compared with the inflammatory subset, and the former expressed CD11c, a marker of dendritic cells. Neither monocytosis nor the change in monocyte subpopulations, including CD11c expression, was observed in Yaa-bearing C57BL/6 mice, in which systemic lupus erythematosus (SLE) fails to develop. CONCLUSION: Our results suggest that Yaa-associated monocytosis is not attributable to an intrinsic abnormality in the growth potential of monocyte lineage cells bearing the Yaa mutation and that the Yaa mutation could lead to the expansion of dendritic cells, thereby contributing to the accelerated development of SLE.

To study the specific role of transmembrane tumor necrosis factor (TmTNF) in host defense mechanisms against bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis infections, we compared the immune responses of TNF/lymphotoxin (LT)-alpha(-/-) mice expressing a noncleavable transgenic TmTNF (TmTNF tg) to those of TNF/LT-alpha(-/-) and wild-type mice. Susceptibility of TNF/LT-alpha(-/-) mice to BCG infection was associated with impaired induction of systemic RANTES but not of monocyte chemoattractant protein 1 (MCP-1), the development of excessive local and systemic Th1-type immune responses, and a substantially reduced inducible nitric oxide synthase (iNOS) activity. Resistance of TmTNF tg mice to BCG infection was associated with efficient activation of iNOS in granulomas and with the regulated release of local and systemic chemokines and Th1-type cytokines. However, M. tuberculosis infection of TmTNF tg mice resulted in longer survival and enhanced resistance compared to TNF/LT-alpha(-/-) mice but higher sensitivity than wild-type mice. TmTNF tg mice exhibited reduced pulmonary iNOS expression and showed an exacerbated cellular infiltration in the lungs despite a modest bacillary content. Our data thus indicate a role for TmTNF in host defense against mycobacteria by contributing to induction and regulation of Th1-type cytokine and chemokine expression leading to development of bactericidal granulomas expressing iNOS, which critically determines susceptibility versus resistance of the host to mycobacterial infections.

BACKGROUND/AIMS: Bacillus Calmette Guerin (BCG) infection causes hepatic injury following granuloma formation and secretion of cytokines which render mice highly sensitive to endotoxin-mediated hepatotoxicity. This work investigates the role of inducible nitric oxide synthase (iNOS) in liver damage induced by BCG and endotoxins in BCG-infected mice. METHODS: Liver injury and cytokine activation induced by BCG and by LPS upon BCG infection (BCG/LPS) were compared in wild-type and iNOS-/- mice. RESULTS: iNOS-/- mice infected with living BCG are protected from hepatic injury when compared to wild-type mice which express iNOS protein in macrophages forming hepatic granulomas. In addition, iNOS-/- mice show a decrease in BCG-induced IFN-gamma serum levels. LPS challenge in BCG-infected mice strongly activates iNOS in the liver and spleen of wild-type mice which show important liver damage associated with a dramatic increase in TNF and IL-6 and also Th1 type cytokines. In contrast, iNOS-/- mice are protected from liver injury after BCG/LPS challenge and their TNF, IL-6 and Th1 type cytokine serum levels raise moderately. CONCLUSIONS: These results demonstrate that nitric oxide (NO) from iNOS is involved in hepatotoxicity induced by both mycobacterial infection and endotoxin effects upon BCG infection and that inhibition of NO from iNOS protects from liver injuries.

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of an as yet unidentified mutant gene, Yaa (Y-linked autoimmune acceleration). In view of a possible role of marginal zone (MZ) B cells in murine SLE, we have explored whether the expression of the Yaa mutation affects the differentiation of MZ and follicular B cells, thereby implicating the acceleration of the disease. In this study, we show that both BXSB and C57BL/6 Yaa mice, including two different substrains of BXSB Yaa males that are protected from SLE, displayed an impaired development of MZ B cells early in life. Studies in bone marrow chimeras revealed that the loss of MZ B cells resulted from a defect intrinsic to B cells expressing the Yaa mutation. The lack of selective expansion of MZ B cells in diseased BXSB Yaa males strongly argues against a major role of MZ B cells in the generation of pathogenic autoantibodies in the BXSB model of SLE. Furthermore, a comparative analysis with mice deficient in CD22 or expressing an IgM anti-trinitrophenyl/DNA transgene suggests that the hyperreactive phenotype of Yaa B cells, as judged by a markedly increased spontaneous IgM secretion, is likely to contribute to the enhanced maturation toward follicular B cells and the block in the MZ B cell generation.

Expression of MHC class II, DM and Ii genes is controlled by the transactivator CIITA, a mediator of the activation of these genes by IFN-gamma. Surprisingly, MHC class II molecules expressed on CIITA transfectants behave very differently from those expressed at the same level on IFN-gamma-induced cells in terms of peptide binding and peptide-specific T cell activation. MHC class II-positive CIITA transfectants exhibit an unusually high capacity for binding exogenous peptides, with a higher percentage of DR molecules occupied by a given peptide and are much more efficient at peptide-specific, HLA-DR-restricted activation of T lymphocytes. This unexpected phenotype reflects the antigen processing defect observed in CIITA transfectants. It suggests novel strategies for the use of CIITA-transformed cells in peptide-based immunization.

The DRB1 sequence of the homozygous cell line HAG (DR13-DwHAG-DQ7) represents a new DRB allele assigned DRB1*1103, whereas its DRB3 sequence corresponds to the previously described DRB3*0101 (DR52a) allele. The DRB1*1303 gene product is undetectable by current sera used in routine serology typing. We report here direct evidence that the MHC molecule encoded by the DRB1*1303 gene is -functional in antigen presentation and in T-cell restriction. We describe a T-cell clone specific for tetanus toxin whose restriction pattern strictly follows the DRB1*1303 allele, as defined by oligonucleotide typing. It also follows the serologic reactivity with the serum LYGUE and also the DwHAG MLC-defined specificity pattern, with one exception. The potential functional sites for the DRB1*1303 gene product involved in T-cell restriction were deduced from sequence comparisons between DRB1*1303 and closely related DRB1 alleles. The relevant aa substitutions were located within close proximity to each other on the HLA class II structural model. Our results demonstrate that 1) DRB1*1303 is functional in antigen presentation and T-cell restriction 2) the functional region involved in antigen presentation and T-cell restriction by DRB1*1303 can be defined structurally.

Many patients with anemia fail, for unknown reasons, to respond to erythropoietin (Epo) - the key mediator of the erythropoietic response. Here, we report that loss of the growth arrest-specific gene 6 (Gas6), a polypeptide binding the receptor tyrosine kinases Tyro 3, Axl and Mer, caused a significant depletion of the reserve of erythroid progenitors in mice. As a result, the compensatory erythropoietic response to anemia induced by hemolysis or blood loss was defective in mice lacking Gas6 (Gas6−/ −). The impaired erythropoietic response was attributable to the hyporesponsiveness of Gas6−/ − erythroblasts to the survival activity of Epo. Only when recombinant Gas6 (rGas6) was administered together with Epo, did these factors synergistically rescue the mutant mice from anemia. Administration of rGas6 alone to Gas6−/ − mice also provided protection from phenylhydrazine (PHZ)-induced anemia, presumably due to the ability of Gas6 to enhance the pro-survival effects of Epo on erythroblasts. This was supported by in vivo and in vitro findings: (i) In the absence of Gas6, more erythroblasts died in the spleen of PHZ-treated mice, despite markedly elevated levels of Epo. (ii) Compared to WT, Gas6−/ − erythroblasts responded less to the pro-survival effects of Epo in vitro, suggesting that Gas6 signaling influences Epo receptor (EpoR) signaling. Absence of Gas6 did not alter tyrosine phosphorylation of the EpoR, but reduced Akt phosphorylation in erythroblasts when treated with Epo. Akt phosphorylation in Gas6−/ − erythroblasts was, however, restored to WT levels, when co-treated with Epo and rGas6. These findings indicate that Epo/EpoR-mediated erythroblast survival and proliferation is stimulated by Gas6 via the PI3K/Akt pathway. (iii) WT erythroblasts secrete Gas6 when treated with Epo. Together, erythroblasts, via a “Gas6 feedback system”, regulate, in an autocrine manner, their own responsiveness to Epo by reinforcing signaling downstream of EpoR. This mechanism is delicately balanced, since treatment of WT anemic mice with rGas6 alone reversed the acute anemia induced by hemolysis without causing polycythemia. A similar beneficial response in hematocrit restoration was observed when a transgenic mouse model with chronic anemia was treated with rGas6. In these mice, co-administration of rGas6 and Epo resulted in sustained rise in the hematocrit, to higher levels than achieved with Epo or rGas6 alone. Gas6 is therefore effective in chronic anemia, augments the effects of Epo, and may have Epo dose-sparing effects, and thus may provides novel and safe therapeutic approach to treat patients with Epo-resistant anemia.