Mechanism of unresponsiveness to the alpha 1-6 epitope of dextran B512 in a C57BL substrain

C57BL/10ScCr mice are low responders to the alpha 1-6 epitope of dextran B512, although other C57BL mice are high responders. Both thymus-independent and thymus-dependent forms of dextran failed to induce an immune response in C57BL/10ScCr mice, but dextran functioned as a good carrier for antihapten responses in this strain. Dextran is a potent polyclonal B cell activator for cells from C57BL/10ScCr mice, although such cells are not activated by LPS. The C57BL/10ScCr mice possess the Igh-V gene coding for antibodies against dextran and the antidextran antibodies induced in (A X C57BL/10ScCr)F1 hybrids share an idiotype with antidextran antibodies produced in C57BL/10 mice. Bone marrow cells from C57BL/10ScCr mice do not respond to dextran when transferred into lethally irradiated C57BL/10 mice and C57BL/10 cells transferred into C57BL/10ScCr mice give a strong antidextran response. Thus, B cells having both the Igh-V gene coding for antibodies against dextran and activation receptors for dextran cannot be activated into antibody synthesis against any form of this immunogen. This determinant specific immunodeficiency suggests the existence of as yet unknown regulatory influences on Igh-V gene expression or B cell activation.     

Immunological unresponsiveness to thymus-independent antigens: two fundamentally different genetic mechanisms of B-cell unresponsiveness to dextran

The immune response of mice to the α-l-6 epitope of dextran (Dx) B512 was found to be under genetic control. The congenic mouse strains A, A.CA, A.SW, A.TH, and A.TL exhibited a specific defect in their response to α-l-6. Also strain CBA/N was unresponsive to α-1-6, but the mechanism of unresponsiveness was found to be different. Unresponsiveness to α-l-6 in congenic A strains was not due to suppressor cells. Although these strains failed to respond to the α-l-6 epitope, they responded strongly to the hapten Fluorescein isothiocyanate (FITC) conjugated to Dx, indicating that the Dx can function as an efficient carrier in these strains. Dx was a potent polyclonal B-cell activator in congenic A strains as well as in high responder strains. Polyclonally-activating concentrations of lipopolysaccharide (LPS) failed to induce the synthesis of anti-α- l-6 antibodies in congenic A strains, although antibodies of all other specificities studied were produced. However, in high responder strains, LPS induced the synthesis of anti-α-l-6 antibodies. It was concluded that congenic A strains do not express V genes coding for antibodies against α-l-6. In contrast, strain CBA/N failed to respond to both the α-l-6 and FITC epitope on Dx, whereas they could respond to FITC conjugated to horse erythrocytes. Dx induced a very small, if any, polyclonal antibody response in B cells from CBA/N mice or male CBA/N x DBA hybrids, whereas Dx was a very potent polyclonal B-cell activator in female hybrids. It is concluded that CBA/N mice are nonresponders to Dx or haptenated Dx, because the cell population that can respond to the polyclonal B-cell activating properties of Dx is severely depleted.     

Immune response to a thymus-dependent form of B512 dextran requires the presence of Lyb-5+ lymphocytes

Studies of the ontogeny of the immune response to B512 dextran (Dex) show that antibody responses equal to those of adult mice are not attained until 12 wk of age. We have examined the anti-Dex response after immunization with a thymus-dependent antigen isomaltohexaosyl-keyhole limpet hemocyanin (IM6-KLH) and have shown that the development of the cross-reacting anti-Dex response parallels the development of Lyb-5+ B cells. Adult levels of anti-Dex antibody after immunization with IM6-KLH are achieved in mice between 3 and 12 wk of age, a time when Lyb-5+ cells have reached adult levels. Neonatal mice, immunized at 1 d or 1 wk after birth, failed to produce a significant amount of anti-Dex antibodies, although they did produce IM6-specific antibodies after immunization with IM6-KLH. Data, which support the conclusion from these experiments that Lyb-5+ cells are required for an anti-polysaccharide response even when the immunizing antigen is thymus-dependent, include the failure of IM6-KLH to stimulate a normal anti-Dex response in mice with the xid defect and the direct demonstration in normal adult mice that elimination of Lyb-5+ cells from spleens of mice primed with IM6-KLH abolishes the ability of these cells to transfer an anti-Dex response. The data imply that the expressed B cell repertoire in adult animals is skewed such that the vast majority of B cells capable of responding to polysaccharide determinants are in the Lyb-5+ subset.     

Shared antigenic determinants by mitogen receptors and antibody molecules to the same thymus-independent antigen.

The antibody response to dextran B1355 is thymus independent, and in high responder mice, over 90% of the antibodies carry the idiotype of an alpha-1,3 binding myeloma protein (J558). The present experiments demonstrate: (a) dextran B1355 is a B-cell mitogen both in a strain which carries the J558 idiotype on antibodies and in a low-responder strain which does not express that idiotype on antibodies to dextran; (b) anti-idiotypic antibodies to J558 recognize a dextran-specific surface receptor on 10--15% of all splenic B cells in those two strains as well as in all strains so far tested; (c) as shown by inhibition experiments such surface receptors cross-react with J558, and (d) anti-idiotypic antibodies are mitogenic for spleen cells of both strains resulting in B-cell proliferation and maturation to polyclonal antibody secretion.     

Induction of immunological tolerance requires that the B cells can respond to the polyclonal B-cell-activating properties of the thymus- independent antigens

Mice were rendered specifically tolerant to the fluorescein isothiocyanatedextran (FITC) epitope by injection of FITC-dextran B512. Their spleen cells were removed at various times and cultivated in vitro with different polyclonal B-cell activators, such as lipopolysaccharide (LPS), purified protein derivative of tuberculin, and native dextran. LPS caused the appearance of high affinity anti-FITC plaque-forming cells to an equal extent with cells from untreated and tolerant animals, whereas native dextran failed to activate cells from tolerant mice, although it was a potent activator of normal cells. It was concluded that tolerance induction only affects those B cells that could respond to the polyclonal B-cell-activating properties of the tolerogen, but not other B cells having an identical set of Ig receptors directed against the tolerogen.     

Comparison of in vitro and in vivo mitogenic and polyclonal antibody and autoantibody responses to peptidoglycan, LPS, protein A, PWM, PHA and Con A in normal and autoimmune mice

We have compared the in vitro and in vivo mitogenic and polyclonal antibody (IgM-, IgG-, IgA- and IgM anti-SRBC-secreting PFC) and autoantibody (IgM anti-ssDNA-, anti-bromelin-treated [HB]- and anti-intact mouse RBC-secreting PFC) responses to peptidoglycan (PG), LPS, protein A, PWM, PHA and Con A in young (4-7 weeks) and old (7-8 months) normal (BALB/c, CBA/H, C57BL/6) and autoimmune (NZB, NZB X NZW F1, BXSB, MRL/1; old BXSB and MRL/1 were 4-5 months) mice. Our results demonstrated that: lymphocytes from young and old autoimmune mice (except old BXSB) could be further polyclonally activated in vitro by PG or LPS as well as or better than lymphocytes from young and old normal mice; lymphocytes from young or old autoimmune mice were less polyclonally activated in vitro by protein A or PWM, respectively, than lymphocytes from young or old normal mice; PG and LPS were equally effective polyclonal activators in vitro; in vivo, LPS was a stronger stimulant than PG; in vivo, LPS could induce polyclonal activation in both young and old normal and autoimmune mice, whereas, PG could only induce polyclonal activation in vivo in young and old normal mice, but did not induce further activation in young and old autoimmune mice; only some tests (anti-ssDNA and IgG PFC in vivo, and IgA and anti-HB MRBC PFC in vitro) revealed higher responses in autoimmune than in normal mice, and these higher responses were seen more often in vivo than in vitro; both autoimmune and normal mice had a high frequency of autoantibody (especially anti-ssDNA) secreting cells in polyclonal activation in vitro, whereas a high frequency of these cells in vivo was only found in autoimmune mice; in most cases in vitro, polyclonal activators did not change the frequency of autoantibody and heteroantibody secreting cells, but in vivo, both PG and LPS increased the frequency of anti-ssDNA antibody secreting cells in normal, but not in autoimmune, mice; LPS increased the in vivo, but not in vitro, frequency of cells secreting anti-HB MRBC antibodies in some strains of mice; old mice had lower mitogenic responsiveness than young mice in both autoimmune and normal strains; autoimmune mice had similar, higher or lower mitogenic responses than normal mice, depending on the strain and the age, but in most cases consistent for both B and T cell mitogens; and there was no correlation between the patterns of increased or decreased mitogenic and polyclonal antibody responses in normal and autoimmune mice.4+.     

Different mechanisms of protection by monoclonal and polyclonal antibodies during the course of herpes simplex virus infection.

Young adult C57BL mice intravaginally inoculated with herpes simplex virus type 1 and passively immunized with a monoclonal antibody directed against herpes simplex virus type 1 glycoprotein gB were shown to be more effectively protected against infection as compared with mice treated with polyclonal immune serum. In contrast to polyclonal antiserum, the monoclonal antibody markedly restricted viral multiplication in the infected mucous membranes. Consequently, skin lesions were completely prevented, and the extent of ganglionic infection was significantly reduced. The mechanism by which a monoclonal antibody, specific to glycoprotein gC, effected protection also differed from that of the hyperimmune serum, since premature latency was not induced. The data provide strong evidence that the mechanisms of protection mediated by antibodies depend on their epitope specificity. The inhibition of active antibody response after passive immunization was inducible by polyclonal antibody only, not by monoclonal antibodies.     

Relative contribution of T and B cells to hypermutation and selection of the antibody repertoire in germinal centers of aged mice

The immune system of aged individuals often produces antibodies that have lower affinity and are less protective than antibodies from young individuals. Recent studies in mice suggested that antibodies produced by old individuals may be encoded by distinct immunoglobulin (Ig) genes and that the somatic hypermutation process in these individuals is compromised. The present study employed Ighb scid mice reconstituted with normal lymphocytes from young (2-3-mo-old) and aged (20-25-mo-old) donors and immunized with a protein conjugate of the hapten (4-hydroxy- 3-nitrophenyl)acetyl (NP) to determine whether the molecular changes in antibody repertoire reflect senescence in the B cells or whether they are mediated by the aging helper T lymphocytes. The NP-reactive B cells from splenic germinal centers (GC) were recovered by microdissection of frozen tissue sections and their rearranged Ig heavy chain variable region (VH) genes of the V186.2/V3 families were sequenced. It was found that the VH gene repertoire of the GC B cells was strongly influenced by the source of the CD4+ T cells. When T cells were donated by young mice, the anti-NP response in GC was dominated by the canonical V186.2 gene, even if the responder B cells came from aged donors. However, when the mice were reconstituted with T cells from aged donors, the expression of the V186.2 gene by young B cells was diminished and the response was dominated by the C1H4 gene, another member of the V186.2/V3 family. In contrast, the somatic hypermutation process in the GC B cells followed a different pattern. The mutation frequencies in the animals that were reconstituted with both B and T cells from young donors (1/50 to 1/150 bp) were comparable to the frequencies previously reported for NP-immunized intact young/adult mice. However, when either lymphocyte subset was donated by the aged mice, the mutation frequencies declined. Thus, mice reconstituted with T cells from the aged and B cells from the young had severely compromised mutational mechanism. Likewise, the recipients of aged B and young T cells had diminished mutations even though the repertoire of their anti-NP response was dominated by the canonical V186.2 gene. It appears that the change in germine-encoded repertoire and the decrease of somatic hypermutation represent distinct mechanisms of immunosenescence and that the aging of helper T cells plays a pivotal role in both of these processes.     

Radiation leukemia in C57BL/6 mice. I. Lack of serological evidence for the role of endogenous ecotropic viruses in pathogenesis

The humoral immune response against endogenous ecotropic murine leukemia viruses (MuLV) was examined in irradiated and control C57BL/6 mice. Control mice developed antibodies against MuLV slowly throughout life. In contrast, within 2-3 mo after irradiation 90% of irradiated C57BL/6 mice had developed detectable antibodies against MuLV. The characteristics of this immune response, however, were identical in control and irradiated mice in terms of peak titers, specificity for endogenous ecotropic MuLV, and reactivity against the ecotropic viruses' glycoprotein (gp71). Moreover, the rate of appearance of antibodies against MuLV in irradiated mice and the peak titers were generally not affected by age at irradiation, dose of irradiation (two, three, or four treatments of 175 R), or bone marrow reconstitution. Although the ability of irradiation to accelerate the appearance of antibody in a population of C57BL/6 mice suggested activation of endogenous ecotropic MuLV, there was no apparent correlation between the appearance of this immune response or its persistence and the development of lymphoma. Thus, the incidence of lymphoma was comparable in mice that: (a) developed no immune response; (b) developed an immune response only transiently after irradiation; or (c) developed an immune response which persisted until death from lymphoma. Moreover, experimental conditions that alter the ability of irradiation to induce leukemia, such as age, dose, or bone marrow reconstitution did so without significantly altering either the rate of appearance of a humoral immune response to MuLV or its peak titers. The results, therefore, fail to demonstrate any seroepidemological relationship between endogenous ecotropic MuLV and radiation-induced leukemia.     

Characterization of self-reactive B cells by polyclonal B-cell activators

The existence of autoreactive B cells was predicted by theoretical considerations and, recently, confirmed by direct experiments. The aim of the present work was to investigate if the capacity of self-reactive B cells to be activated with different polyclonal B-cell activators (PBA) reflects the heterogeneity of the response as seen in all the Ig-positive cells. We injected mice with dextran sulfate, lipopolysaccharide from Escherichia coli 055:B5, and purified protein derivate of turbercle bacteria RT32 and studied the complement-dependent cytotoxicity against syngeneic spleen cells caused by the sera from injected mice with regard to the different parameters used for characterization of B-cell subpopulations. It was found that the capacity of self-reactive B cells to secrete antibodies reflects the polyclonal-activating capacity of the PBA used. The implications of these findings for the understanding of the triggering mechanism of B lymphocytes and for self-nonself discrimination are discussed.     

Identification of T-cell epitopes in clotting factor IX and lack of tolerance in inbred mice

Summary.  Immune responses to the factor IX protein pose problems for hemophilia B patients who develop antibodies against factor IX and for potential future treatment with gene therapy. To better define the response to human factor IX, we analyzed T-cell responses to human factor IX in factor IX knockout mice on BALB/c and C57BL/6 (B6) backgrounds, both strains having CD4+ T cells that proliferate in response to human factor IX. Surprisingly, wild-type mice have similar factor IX-recognizing CD4+ T cells. We defined a dominant CD4+ epitope for each strain (CVETGVKITVVAGEH for BALB/c and LLELDEPLVLNSYVTPIC for B6) that was recognized by both factor IX knockout and wild-type mice. While human factor IX did not cross-react with the mouse homologs of these epitopes, immunization with peptides from murine factor IX stimulated proliferation in factor IX knockout mice and wild-type mice, demonstrating a failure to delete murine factor IX-specific T cells in normal mice.     

Progenitors for Ly-1 B cells are distinct from progenitors for other B cells

Data from previous multiparameter fluorescence-activated cell sorter (FACS) analysis and sorting studies define a subset of murine B cells that expresses the Ly-1 surface determinant in conjunction with IgM, IgD, Ia, and other typical B cell markers. These Ly-1 B cells are physically and functionally distinct. They express more IgM and less IgD than most other B cells; they are not normally found in lymph node or bone marrow; they are always present at low frequencies (1-5%) in normal spleens, and, as we show here, they comprise about half of the B cells (10-20% of total cells) recovered from the peritoneal cavity in normal mice. Furthermore, most of the commonly studied IgM autoantibodies in normal and autoimmune mice are produced by these Ly-1 B cells, even though they seldom produce antibodies to exogenous antigens such as trinitrophenyl-Ficoll or trinitrophenyl-keyhole limpet hemocyanin. Cell transfer studies presented here demonstrate that the progenitors of Ly-1 B cells are different from the progenitors of the predominant B cell populations in spleen and lymph node. In these studies, we used FACS analysis and functional assays to characterize donor-derived (allotype-marked) B cells present in lethally irradiated recipients 1-2 mo after transfer. Surprisingly, adult bone marrow cells typically used to reconstitute B cells in irradiated recipients selectively failed to reconstitute the Ly-1 B subset. Liver, spleen, and bone marrow cells from young mice, in contrast, reconstituted all B cells (including Ly-1 B), and peritoneal "washout" cells (PerC) from adult mice uniquely reconstituted Ly-1 B. Bone marrow did not block Ly-1 B development, since PerC and newborn liver still gave rise to Ly-1 B when jointly transferred with marrow. These findings tentatively assign Ly-1 B to a distinct developmental lineage originating from progenitors that inhabit the same locations as other B cell progenitors in young animals, but move to unique location(s) in adults.     

Promotion of mouse fibroblast collagen gene expression by mast cells stimulated via the Fc epsilon RI. Role for mast cell-derived transforming growth factor beta and tumor necrosis factor alpha

Chronic allergic diseases and other disorders associated with mast cell activation can also be associated with tissue fibrosis, but a direct link between mast cell mediator release and fibroblast collagen gene expression has not been established. Using in situ hybridization, we show that the elicitation of an IgE-dependent passive cutaneous anaphylaxis (PCA) reaction in mice results in a transient, but marked augmentation of steady state levels of type alpha-1 (I) collagen mRNA in the dermis. While peak levels of collagen mRNA expression in the skin are observed 16-24 h after mast cell activation, substantial numbers of dermal cells are strongly positive for collagen mRNA at 1 and 2 h after antigen challenge, before circulating inflammatory cells are recruited into the tissues. Furthermore, experiments in mast cell- reconstituted or genetically mast cell-deficient WBB6F1-W/Wv mice demonstrate that the increased expression of collagen mRNA at sites of PCA reactions is entirely mast cell dependent. In vitro studies show that the supernatants of mouse serosal mast cells activated via the Fc epsilon RI markedly increase type alpha-1 (I) collagen mRNA levels in mouse embryonic skin fibroblasts, and also upregulate collagen secretion by these cells. The ability of mast cell supernatants to induce increased steady state levels of collagen mRNA in mouse skin fibroblasts is markedly diminished by absorption with antibodies specific for either of two mast cell-derived cytokines, transforming growth factor beta (TGF-beta 1) or tumor necrosis factor alpha (TNF- alpha), and is eliminated entirely by absorption with antibodies against both cytokines. Taken together, these findings demonstrate that IgE-dependent mouse mast cell activation can induce a transient and marked increase in steady state levels of type alpha-1 (I) collagen mRNA in dermal fibroblasts and that mast cell-derived TGF-beta 1 and TNF-alpha importantly contribute to this effect.     

Immunogenicity of a chimeric peptide corresponding to T helper and B cell epitopes of the Chlamydia trachomatis major outer membrane protein

The immunogenicity of a chimeric T/B cell peptide corresponding to antigenically characterized epitopes of the Chlamydia trachomatis major outer membrane protein (MOMP) was studied in mice to further define its potential use in the development of a subunit vaccine in preventing blinding trachoma in humans. The chimeric peptide, designated A8-VDI, corresponds to a conserved MOMP T helper (Th) cell epitope(s) (A8, residues 106-130) and serovar A VDI (residues 66-80), which contains the serovar-specific neutralizing epitope 71VAGLEK76. Mice immunized with peptide A8-VDI produced high-titered polyclonal IgG antibodies which recognized the VAGLEK-neutralizing epitope. Peptide A8-VDI primed A/J mice to produce high-titered serum-neutralizing antibodies in response to a secondary immunization with intact chlamydial elementary bodies (EBs). Peptide A8-VDI, but not peptide VDI alone, was immunogenic in six different inbred strains of mice disparate at H-2, indicating that the Th cell epitope(s) contained in the A8 portion of the chimera was recognized in the context of multiple major histocompatibility complex (MHC) haplotypes. An unexpected finding of this work was that different inbred strains of mice immunized with the chimeric peptide produced antibodies of differing fine specificities to the VDI portion of the chimera. Some mouse strains produced anti-VDI antibodies that did not recognize the VAGLEK-neutralizing epitope. The ability of mice to respond to the VAGLEK-neutralizing site was not dependent on MHC haplotype since mouse strains of the same H-2 haplotype produced anti-VDI antibodies of differing fine specificity.     

Age-related CD8 T cell clonal expansions constrict CD8 T cell repertoire and have the potential to impair immune defense

Peripheral T cell diversity is virtually constant in the young, but is invariably reduced in aged mice and humans. CD8+ T cell clonal expansions (TCE) are the most drastic manifestation of, and possible contributors to, this reduced diversity. We show that the presence of TCE results in reduced CD8+, but not CD4+, T cell diversity, and in functional inability to mobilize parts of the CD8+ T cell repertoire affected by TCE. In the model of herpes simplex virus (HSV)-1 infection of B6 mice, >90% of the responding CD8+ T cells use Vbeta10 or Vbeta8 and are directed against a single glycoprotein B (gB498-505) epitope, gB-8p. We found that old animals bearing CD8+ TCE within Vbeta10 or Vbeta8 families failed to mount an effective immune response against HSV-1, as judged by reduced numbers of peptide-major histocompatibility complex tetramer+ CD8 T cells and an absence of antiviral lytic function. Furthermore, Vbeta8 TCE experimentally introduced into young mice resulted in lower resistance to viral challenge, whereas Vbeta5+ TCE induced in a similar fashion did not impact viral resistance. These results demonstrate that age-related TCE functionally impair the efficacy of antiviral CD8+ T cell immunity in an antigen-specific manner, strongly suggesting that TCE are not the mere manifestation of, but are also a contributing factor to, the immunodeficiency of senescence.     

Spleen cells from animals tolerant to a thymus-dependent antigen can be activated by lipopolysaccharide to synthesize antibodies against the tolerogen

Immunological tolerance was induced in adult mice by the injection of 5 mg of deaggregated hapten-protein conjugate. The tolerant state was confirmed 4-19 days later by the failure of such animals to mount an immune response against an aggregated form of the same thymus-dependent hapten-protein conjugate as well as by the inability of spleen cells from tolerant animals to respond to a thymus-independent hapten-carrier conjugate. Even though the animals were fully tolerant, their spleen cells were activated by lipopolysaccharide (LPS) in vitro to produce normal numbers of plaque-forming cells against the hapten. The finding that spleen cells from tolerant animals could be activated by LPS into synthesis of antibodies against the tolerogen indicates that tolerance to thymus-dependent antigens does not affect B cells, but presumably only T cells. It is suggested that the only stringent test for the existence of B-cell tolerance is the inability of polyclonal B-cell activators to activate antibody synthesis against the tolerogen. The findings make it unlikely that B-cell tolerance to autologous thymus- dependent antigens exists and further indicate that such antigens cannot deliver activating or tolerogenic signals to B cells, although they are competent to combine with and block the Ig receptors.     

Bone marrow function. I. Peripheral T cells are responsible for the increased auto-antiidiotype response of older mice

After immunization with trinitrophenyl (TNP)-Ficoll, mice produced both anti-TNP antibodies and auto-anti-idiotype (auto-anti-Id) antibodies specific for the anti-TNP antibody. Older animals produced more auto-anti-Id than did young animals. When mice were exposed to a normally lethal dose of irradiation while their bone marrow (BM) was partially shielded, they survived and slowly (6 wk) regained immune function, as indicated by the number of nucleated cells in their spleen and the in vitro primary plaque-forming cell (PFC) response of their spleen cells to TNP-treated aminoethylated polyacrylamide beads. Recovery is presumably the result of repopulation of the peripheral lymphoid system by cells originating in the BM. By enzyme-linked immunosorbent assay (ELISA), and by hapten-augmentable PFC assay, we show that, after recovery from irradiation with their BM shielded, old animals produce low auto-anti-Id responses, like those of young animals. The transfer of splenic T cells into mice irradiated with their BM shielded provided evidence that the magnitude of the auto-anti-Id response is controlled by the peripheral T cells. Thus, mice that received splenic T cells from aged donors produced high levels of auto-anti-Id while those that received splenic T cells from young donors produce low levels of auto-anti-Id.     

The death domain kinase RIP protects thymocytes from tumor necrosis factor receptor type 2-induced cell death

Fas and the tumor necrosis factor receptor (TNFR)1 regulate the programmed cell death of lymphocytes. The death domain kinase, receptor interacting protein (rip), is recruited to the TNFR1 upon receptor activation. In vitro, rip-/- fibroblasts are sensitive to TNF-induced cell death due to an impaired nuclear factor kappaB response. Because rip-/- mice die at birth, we were unable to examine the effects of a targeted rip mutation on lymphocyte survival. To address the contribution of RIP to immune homeostasis, we examined lethally irradiated mice reconstituted with rip-/- hematopoietic precursors. We observed a decrease in rip-/- thymocytes and T cells in both wild-type C57BL/6 and recombination activating gene 1-/- irradiated hosts. In contrast, the B cell and myeloid lineages are unaffected by the absence of rip. Thus, the death domain kinase rip is required for T cell development. Unlike Fas-associated death domain, rip does not regulate T cell proliferation, as rip-/- T cells respond to polyclonal activators. However, rip-deficient mice contain few viable CD4+ and CD8+ thymocytes, and rip-/- thymocytes are sensitive to TNF-induced cell death. Surprisingly, the rip-associated thymocyte apoptosis was not rescued by the absence of TNFR1, but appears to be rescued by an absence of TNFR2. Taken together, this study implicates RIP and TNFR2 in thymocyte survival.