CD4+ T lymphocytes expressing CD40 ligand help the IgM antibody response to soluble pneumococcal polysaccharides via an intermediate cell type

Streptococcus pneumoniae causes serious infections in children, the elderly, and immunocompromised patients. Protection against infections with S. pneumoniae is mediated through Abs against the capsular polysaccharides (caps-PS). We previously showed that the murine Ab response to caps-PS is dependent on CD40-CD40L interaction. In the present paper, we addressed the question of whether the CD40-CD40L-mediated modulation of the anti-caps-PS immune reaction is the result of a direct interaction between B lymphocytes and T lymphocytes or of an indirect interaction. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice did not mount anti-caps-PS Abs. SCID/SCID mice reconstituted with B lymphocytes from wild-type mice and CD4+ T lymphocytes from wild-type mice but not CD4+ T lymphocytes from CD40L knockout mice stimulated the anti-caps-PS Ab response. This indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in a CD40L-dependent manner. SCID/SCID mice reconstituted with B lymphocytes from CD40 knockout mice and CD4+ T lymphocytes from wild-type mice generated an anti-caps-PS Ab response that could be inhibited by MR1, a blocking anti-CD40L Ab. These data indicated that CD4+ T lymphocytes stimulated the anti-caps-PS Ab response in an indirect way. Finally, lethally irradiated CD40 knockout mice reconstituted with bone marrow from wild-type mice mounted an anti-caps-PS Ab response that was comparable to the Ab response in wild-type mice, revealing that the required CD40 was on hemopoietic cells. In conclusion, we provide evidence that CD4+ T lymphocytes expressing CD40L stimulate the Ab response to soluble caps-PS by interacting with CD40-expressing non-B cells.     

Mechanism of action of combined short-term CTLA4Ig and anti-CD40 ligand in murine systemic lupus erythematosus

Short-term combination therapy with the costimulatory antagonists CTLA4Ig and anti-CD40 ligand induces prolonged suppression of disease onset in New Zealand Black/New Zealand White F(1) systemic lupus erythematosus-prone mice. To determine the mechanism for this effect, 20- to 22-wk-old New Zealand Black/New Zealand White F(1) mice were treated with six doses each of CTLA4Ig and anti-CD40 ligand Ab over 2 wk. Combination-treated mice, but not mice treated with either agent alone, had prolonged survival and the production of pathogenic IgG anti-dsDNA Ab was suppressed. Twenty weeks after completion of treatment the frequency of activated B cells producing anti-dsDNA Ab was decreased, and the abnormal transition of T cells from the naive to the memory compartment was blocked. Combination treatment partially suppressed class switching and decreased the frequency of somatic mutations in the V(H)BW-16 gene, which is expressed by pathogenic anti-DNA Abs. Treated mice were still able to respond to the hapten oxazolone when it was given 8 wk after treatment initiation, and they mounted a somatically mutated IgG anti-oxazolone response that was noncross-reactive with dsDNA. Fifty to 60% of previously treated mice, but only 14% of previously untreated mice, responded within 2-3 wk to a second course of therapy given at the onset of fixed proteinuria and remained well for a further 3-4 mo. Although this treatment had no immediate effect on serum anti-dsDNA Abs or on the abnormal T cell activation observed in sick mice, 25% of treated mice lived for >18 mo compared with 5% of untreated controls. These results suggest that the effect of costimulatory blockade in remission induction must be mediated by a different mechanism than is demonstrated in the disease prevention studies.     

Intravenous injection of a D1 protein of the Smith proteins postpones murine lupus and induces type 1 regulatory T cells

T cells that recognize nucleoproteins are required for the production of anti-dsDNA Abs involved in lupus development. SmD1 83-119 (a D1 protein of the Smith (Sm) proteins, part of small nuclear ribonucleoprotein) was recently shown to provide T cell help to anti-dsDNA Abs in the NZB/NZW model of lupus. Using this model in the present study, we showed that high dose tolerance to SmD1 (600-1000 microg i.v. of SmD1(83-119) peptide/mo) delays the production of autoantibodies, postpones the onset of lupus nephritis as confirmed by histology, and prolongs survival. Tolerance to SmD1 83-119 was adoptively transferred by CD90+ T cells, which also reduce T cell help for autoreactive B cells in vitro. One week after SmD1 83-119 tolerance induction in prenephritic mice, we detected cytokine changes in cultures of CD90+ T and B220+ B cells with decreased IFN-gamma and IL-4 expression and an increase in TGFbeta. Increased frequencies of regulatory IFN-gamma+ and IL10+ CD4+ T cells were later detected. Such regulatory IL-10+/IFN-gamma+ type 1 regulatory T cells prevented autoantibody generation and anti-CD3-induced proliferation of naive T cells. In conclusion, these results indicate that SmD1 83-119 peptide may play a dominant role in the activation of helper and regulatory T cells that influence autoantibody generation and murine lupus.     

Essential role for CD40 ligand interactions in T lymphocyte-mediated modulation of the murine immune response to pneumococcal capsular polysaccharides

Protection against infection with pneumococci is provided by anti-capsular polysaccharide (caps-PS) Abs. We investigated whether CD40 ligand (CD40L) plays a role in T lymphocyte-mediated regulation of the immune response to caps-PS, which are considered thymus-independent Ags. Administration of MR1, an antagonist mAb against murine CD40L, in BALB/c mice immunized with Pneumovax resulted in an inhibition of the IgM and IgG Ab response for various caps-PS serotypes. Evidence for the involvement of CD4(+) T lymphocytes in the Ab response to caps-PS was obtained in SCID/SCID mice that, when reconstituted with B lymphocytes and CD4(+) T lymphocytes, mounted a higher specific IgM response compared with SCID/SCID mice reconstituted with only B lymphocytes. This helper effect of CD4(+) T lymphocytes was abrogated by MR1. Blocking CD40L in vitro decreased the IgM response to caps-PS and abolished the helper effect of CD4(+) T lymphocytes. CD8(+) T lymphocyte-depleted murine spleen cells mounted a higher in vivo immune response than total murine spleen cells, which provided evidence for a suppressive role of CD8(+) T lymphocytes on the anti-caps-PS immune response. CD4(+) T lymphocyte-depleted murine spleen cells, leaving a B and CD8(+) T lymphocyte fraction, elicited only a weak in vivo and in vitro Ab response, which was enhanced after MR1 administration. In summary, our data provide evidence that T lymphocytes contribute to the regulation of the anti-caps-PS immune response in a CD40L-dependent manner.     

Cutting edge: a role for CD1 in the pathogenesis of lupus in NZB/NZW mice

Since anti-CD1 TCR transgenic T cells can activate syngeneic B cells via CD1 to secrete IgM and IgG and induce lupus in BALB/c mice, we studied the role of CD1 in the pathogenesis of lupus in NZB/NZW mice. Approximately 20% of B cells from the spleens of NZB/NZW mice expressed high levels of CD1 (CD1high B cells). The latter subset spontaneously produced large amounts of IgM anti-dsDNA Abs in vitro that was up to 25-fold higher than that of residual CD1int/low B cells. T cells in the NZB/NZW spleen proliferated vigorously to the CD1-transfected A20 B cell line, but not to the parent line. Treatment of NZB/NZW mice with anti-CD1 mAbs ameliorated the development of lupus. These results suggest that the CD1high B cells and their progeny are a major source of autoantibody production, and activation of B cells via CD1 may play an important role in the pathogenesis of lupus.     

Autoimmunity to DNA and nucleosomes in binary tetracycline-regulated polyomavirus T-Ag transgenic mice

The mechanism(s) responsible for autoimmunity to DNA and nucleosomes in SLE is largely unknown. We have demonstrated that nucleosome-polyomavirus T-Ag complexes, formed in context of productive polyomavirus infection, activate dsDNA-specific B cells and nucleosome-specific CD4(+) T cells. To investigate whether de novo expressed T-Ag is able to terminate nucleosome-specific T cell tolerance and to maintain anti-dsDNA Ab production in nonautoimmune mice, we developed two binary transgenic mouse variants in which expression of SV40 large T-Ag is controlled by tetracycline, MUP tTA/T-Ag (tet-off), and CMV rtTA/T-Ag (tet-on) mice. Data demonstrate that MUP tTA/T-Ag mice, but not CMV rtTA/T-Ag mice, are tightly controlling T-Ag expression. In MUP tTA/T-Ag transgenic mice, postnatal T-Ag expression activated CD8(+) T cells but not DNA-specific B cells, while immunization with T-Ag and nucleosome-T-Ag-complexes before T-Ag expression resulted in elevated and remarkably stable titers of anti-T-Ag and anti-dsDNA Abs and activation of T-Ag-specific CD4(+) T cells. Immunization of nonexpressing MUP tTA/T-Ag mice resulted in transient anti-T-Ag and anti-dsDNA Abs. This system reveals that a de novo expressed DNA-binding quasi-autoantigen maintain anti-dsDNA Abs and CD4(+) T cell activation once initiated by immunization, demonstrating direct impact of a single in vivo expressed molecule on sustained autoimmunity to DNA and nucleosomes.     

Antibodies and CD8+ T cells are complementary and essential for natural resistance to a highly lethal cytopathic virus

It is believed that CD8+ T lymphocytes or Abs can independently clear many primary viral infections, including those caused by Orthopoxviruses (OPV), a genus that includes the human pathogens variola and monkeypox and the vaccine species vaccinia virus. However, most experiments addressing the role of Abs and CD8+ T cells in protection have used viruses that are not specific for the host. In the present study, we used the mouse-specific OPV ectromelia virus and mice deficient in CD40, B cells, or CD8+ T cells and adoptive transfers of CD8+ T or B lymphocytes to show that the protection afforded by CD8+ T cells is incomplete. Despite sustained CD8+ T cell responses, in the absence of Ab responses ectromelia virus persists. This results in delayed disease and inexorably leads to death. Therefore, CD8+ T lymphocytes and Abs are not redundant but complementary and essential to survive infections with a highly pathogenic viruses in the natural host.     

Cutting Edge: Ectopic expression of CD40 ligand on B cells induces lupus-like autoimmune disease.

CD40 ligand (CD40L) is ectopically expressed on B cells in patients with systemic lupus erythematosus (SLE) and lupus-prone BXSB mice. To assess the role of the ectopic CD40L expression in development of SLE, we have established transgenic mice expressing CD40L on B cells. Some of the 12- to 14-mo-old CD40L-transgenic mice spontaneously produced autoantibodies such as antinuclear Abs, anti-DNA Abs, and antihistone Abs. Moreover, approximately half of the transgenic mice developed glomerulonephritis with immune-complex deposition, whereas the kidneys of the normal littermates showed either no pathological findings or only mild histological changes. These results indicate that CD40L on B cells causes lupus-like disease in the presence of yet unknown environmental factors that by themselves do not induce the disease. Thus, ectopic CD40L expression on B cells may play a crucial role in development of SLE.     

Blockade of programmed death-1 in young (New Zealand black x New Zealand white)F1 mice promotes the activity of suppressive CD8+ T cells that protect from lupus-like disease

The programmed death-1 (PD-1)/programmed death-1 ligand 1 (PD-L1) pathway regulates both stimulatory and inhibitory signals. In some conditions, PD-1/PD-L1 inhibits T and B cell activation, induces anergy, and reduces cytotoxicity in CD8(+) T cells. In other conditions, PD-l/PD-L1 has costimulatory effects on T cells. We recently showed that induction of suppressive CD8(+)Foxp3(+) T cells by immune tolerance of lupus-prone (New Zealand black × New Zealand white)F(1) (BWF(1)) mice with the anti-DNA Ig-based peptide pConsensus (pCons) is associated with significantly reduced PD-1 expression on those cells. In this study, we tested directly the role of PD-1 by administering in vivo neutralizing Ab to PD-1 to premorbid BWF(1) and healthy control mice. Anti-PD-1-treated mice were protected from the onset of lupus nephritis for 10 wk, with significantly improved survival. Although the numbers of T cells declined in aging control mice, they were maintained in anti-PD-1-treated mice, including CD8(+)Foxp3(+) T cells that suppressed syngeneic CD4(+)CD25(-) T cell proliferation and IFN-γ production, reduced production of IgG and anti-dsDNA IgG, induced apoptosis in syngeneic B cells, and increased IL-2 and TGF-β production. The administration of anti-PD-1 Ab to BWF(1) mice after induction of tolerance with pCons abrogated tolerance; mice developed autoantibodies and nephritis at the same time as control mice, being unable to induce CD8(+)Foxp3(+) T suppressor cells. These data suggest that tightly regulated PD-1 expression is essential for the maintenance of immune tolerance mediated by those CD8(+)Foxp3(+) T cells that suppress both T(h) cells and pathogenic B cells. PD-1 regulation could represent a target to preserve tolerance and prevent autoimmunity.     

Transgenic expression of the viral FLIP MC159 causes lpr/gld-like lymphoproliferation and autoimmunity

Death receptor-induced programmed cell death (PCD) is crucial for the maintenance of immune homeostasis. However, interference of downstream death receptor signaling by genetic ablation or transgenic (Tg) expression of different apoptosis inhibitors often impairs lymphocyte activation. The viral FLICE (caspase-8)-like inhibitor proteins (v-FLIPs) are potent inhibitors of death receptor-induced apoptosis and programmed necrosis. We generated Tg mice expressing the v-FLIP MC159 from Molluscum contagiosum virus under the control of the H2Kb class I MHC promoter to examine the role of death receptor-induced PCD in the control of immune functions and homeostasis. We found that expression of MC159 led to lymphoproliferation and autoimmunity as exemplified by T and B lymphocyte expansion, accumulation of TCRalphabeta+ CD3+ B220+ CD4- CD8- lymphocytes in secondary lymphoid organs, elevated serum Ig levels, and increased anti-dsDNA Ab titers. These phenotypes were caused by defective death receptor-induced apoptosis, but not by defective passive cell death in the absence of mitogenic stimulation. Lymphocyte activation was normal, as demonstrated by normal thymidine incorporation and CSFE dilution of T cells stimulated with anti-CD3 and anti-CD28 Abs. In addition, effector CD8+ T cell responses to acute and memory lymphocytic choriomeningitis virus infections were unaffected in the Tg mice. These phenotypes are reminiscent of the lpr and gld mice, and show that the v-FLIP MC159 is a bona fide PCD inhibitor that does not interfere with other essential lymphocyte functions. Thus, the MC159-Tg mice provide a model to study the effects of PCD in immune responses without hampering other important lymphocyte functions.     

CD8+ T cell-mediated suppression of autoimmunity in a murine lupus model of peptide-induced immune tolerance depends on Foxp3 expression

Systemic lupus erythematosus is an autoimmune disease caused by autoantibodies, including IgG anti-DNA. New Zealand Black/New Zealand White F(1) female mice, a model of spontaneous polygenic systemic lupus erythematosus, tolerized with an artificial peptide (pConsensus) based on anti-DNA IgG sequences containing MHC class I and class II T cell determinants, develop regulatory CD4+CD25+ T cells and CD8+ inhibitory T cells (CD8+ Ti), both of which suppress autoantibody production. CD8+ Ti inhibit primarily via secretion of TGF-beta. In the present study, we show that the inhibitory function of CD8+ T cells from tolerized mice is sustained for up to 8 wk and at all times depends on expression of Foxp3. Both CD28-positive and CD28-negative CD8+ T cells contain inhibitory cells, but the expression of mRNA for Foxp3 and for TGF-beta is higher and lasts longer in the CD28- subset. In vitro addition of TGF-beta (in the presence of IL-2) induces Foxp3 expression in a dose-response manner. Gene inhibition or blockade with small interfering RNA of Foxp3 abrogates the ability of the CD8+ Ti to inhibit anti-DNA production and the proliferation of CD4+ Th cells. Moreover, a significant correlation between expression of Foxp3 and ability of CD8+ Ti to secrete TGF-beta is observed. Therefore, CD8+ Ti in this system of tolerance are similar to CD4+CD25+ regulatory T cells in their dependence on expression of Foxp3, and there may be a bidirectional Foxp3/TGF-beta autocrine loop that determines the ability of the CD8+ T cells to control autoimmunity.     

CTL-promoting effects of CD40 stimulation outweigh B cell-stimulatory effects resulting in B cell elimination and disease improvement in a murine model of lupus

CD40/CD40L signaling promotes both B cell and CTL responses in vivo, the latter being beneficial in tumor models. Because CTL may also limit autoreactive B cell expansion in lupus, we asked whether an agonist CD40 mAb would exacerbate lupus due to B cell stimulation or would improve lupus due to CTL promotion. These studies used an induced model of lupus, the parent-into-F1 model in which transfer of DBA/2 splenocytes into B6D2F1 mice induces chronic lupus-like graft-vs-host disease (GVHD). Although agonist CD40 mAb treatment of DBA-->F1 mice initially exacerbated B cell expansion, it also strongly promoted donor CD8 T cell engraftment and cytolytic activity such that by 10 days host B cells were eliminated consistent with an accelerated acute GVHD. CD40 stimulation bypassed the requirement for CD4 T cell help for CD8 CTL possibly by licensing dendritic cells (DC) as shown by the following: 1) greater initial activation of donor CD8 T cells, but not CD4 T cells; 2) earlier activation of host DC; 3) host DC expansion that was CD8 dependent and CD4 independent; and 4) induction of acute GVHD using CD4-depleted purified DBA CD8+ T cells. A single dose of CD40 mAb improved lupus-like renal disease at 12 wk, but may not suffice for longer periods consistent with a need for continuing CD8 CTL surveillance. These results demonstrate that in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, CTL promotion is both feasible and beneficial and the CTL-promoting properties of CD40 stimulation outweigh the B cell-stimulatory properties.     

B cell depletion with anti-CD79 mAbs ameliorates autoimmune disease in MRL/lpr mice

MRL/lpr mice develop a spontaneous systemic lupus erythematosus-like autoimmune syndrome due to a dysfunctional Fas receptor, with contributions from other less well-defined genetic loci. The removal of B cells by genetic manipulation not only prevents autoantibody formation, but it also results in substantially reduced T cell activation and kidney inflammation. To determine whether B cell depletion by administration of Abs is effective in lupus mice with an intact immune system and established disease, we screened several B cell-specific mAbs and found that a combination of anti-CD79alpha and anti-CD79beta Abs was most effective at depleting B cells in vivo. Anti-CD79 therapy started at 4-5 mo of age in MRL/lpr mice significantly decreased B cells (B220(+)CD19(+)) in peripheral blood, bone marrow, and spleens. Treated mice also had a significant increase in the number of both double-negative T cells and naive CD4(+) T cells, and a decreased relative abundance of CD4(+) memory cells. Serum anti-chromatin IgG levels were significantly decreased compared with controls, whereas serum anti-dsDNA IgG, total IgG, or total IgM were unaffected. Overall, survival was improved with lower mean skin scores and significantly fewer focal inflammatory infiltrates in submandibular salivary glands and kidneys. Anti-CD79 mAbs show promise as a potential treatment for systemic lupus erythematosus and as a model for B cell depletion in vivo.     

CD40 ligand-deficient T cells from X-linked hyper-IgM syndrome carriers have intrinsic priming capability.

Deficiency in CD40 ligand (CD40L) expression is associated with impaired T cell immunity in mouse models and in humans. Previous studies have indicated that this is due to the failure of induction of extrinsic costimulatory molecules. However, other studies have suggested that CD40L is an intrinsic costimulatory molecule. The X-linked hyper-IgM syndrome (XHIM) is a primary immunodeficiency caused by mutations in CD40L, resulting in impaired Ab production and T cell immunity. CD4+ T cells from female carriers of XHIM express a variable degree of normal CD40L based on random X chromosome inactivation. We have examined T cells from XHIM carriers to investigate whether CD40L supports T cell function by acting as an intrinsic costimulator or by induction of other costimulatory molecules by examining coexpression of CD40L and markers of T lymphocyte priming. These carriers provide a unique model for comparison of CD40L-expressing and -nonexpressing lymphocytes in that all factors, including immunological experience, are equivalent between the two populations. Our results show that compared with CD40L-deficient T cells, T cells that express CD40L normally have a minimal advantage in becoming primed, as defined by CD45 RO isoform expression and production of IFN-gamma and TNF-alpha. Conversely, CD40L-deficient T lymphocytes clearly were capable of becoming primed as defined by the same parameters. These findings imply that the intrinsic costimulatory activity of CD40L is not required for attaining primed status, and that CD40L primarily supports T cell function by inducing extrinsic factors that can be shared by CD40L-deficient cells.     

A novel locus regulates both retroviral glycoprotein 70 and anti-glycoprotein 70 antibody production in New Zealand mice when crossed with BALB/c

Lupus-prone New Zealand Black and New Zealand White mice produce high serum levels of the endogenous retroviral envelope protein gp70 and develop an Ab response to this autoantigen as part of their autoimmune disease. Linkage analysis of two crosses involving New Zealand and BALB/c mice mapped these traits to a group of overlapping loci, including a novel locus on proximal chromosome 12. This locus was linked with serum gp70 and the autoimmune response against it. The linkage of serum gp70 levels to a previously described locus on distal chromosome 4 was also confirmed. Sequence analysis of a candidate gene on distal chromosome 4, Fv1, provided support that this gene may be associated with the control of serum gp70 levels in both New Zealand Black and New Zealand White mice. Linkage data and statistical analysis confirmed a close correlation between gp70 Ag and anti-gp70 Ab levels, and together gave support to the concept that a threshold level of gp70 is required for the production of anti-gp70 Abs. Serum levels of anti-gp70 Abs were closely correlated with the presence of renal disease, more so than anti-dsDNA Abs. Understanding the genetic basis of this complex autoantigen-autoantibody system will provide insight into the pathogenesis of lupus in mice, which may have implications for human disease.     

CD40 ligand blockade induces CD4+ T cell tolerance and linked suppression.

The CD40-CD40 ligand (CD40L) interaction is a key event in the initiation of an adaptive immune response, and as such the therapeutic value of CD40L blockade has been studied in many experimental models of tissue transplantation and autoimmune disease. In rodents, transplantation of allogeneic tissues under the cover of anti-CD40L Abs has resulted in prolonged graft survival but not tolerance. In this report, we show that failure to induce tolerance probably results from the inability of anti-CD40L Abs to prevent graft rejection elicited by the CD8+ T cell subset. When the CD8+ T cell population is controlled independently, using anti-CD8 Abs, then tolerance is possible. Transplantation tolerance induced by anti-CD4 mAbs can often be associated with dominant regulation, manifested as infectious tolerance and linked suppression, both of which are mediated by CD4+ T cells. We show here that CD4+ T cells rendered tolerant using anti-CD40L therapy exhibit the same regulatory property of linked suppression, as demonstrated by their ability to accept grafts expressing third party Ags only if they are expressed in conjunction with the tolerated Ags. This observation of linked suppression reveals a hitherto undocumented consequence of CD40L blockade that suggests the tolerant state is maintained by a dominant regulatory mechanism. Our results suggest that, although anti-CD40L Abs are attractive clinical immunotherapeutic agents, additional therapies to control aggressive CD8+ T cell responses may be required.     

Human rheumatoid factor production is dependent on CD40 signaling and autoantigen.

High-affinity pathologic rheumatoid factor (RF) B cells occur in autoimmune diseases such as rheumatoid arthritis, but are deleted in healthy individuals. The reasons for the survival and differentiation of these autoreactive B cells in rheumatoid arthritis are not known. Previous studies in mice transgenic for a human IgM RF have shown that peripheral encounter with soluble human IgG leads to deletion of high-affinity RF B cells; however, deletion can be prevented when concomitant T cell help is provided. This study aimed to further discern the minimal factors necessary not only for the in vivo survival of RF B cells, but also for their differentiation into Ab-secreting cells. The combination of MHC class II-reactive T cells and Ag induced the production of RF in human IgM RF transgenic mice, while either stimulus alone was ineffective. Neutralizing Abs against CD40 ligand (CD40L), but not against IL-4 or IL-15, abrogated IgM-RF production. Moreover, blockade of CD40L-CD40 allowed IgG to delete the RF precursor cells. Most importantly, activating Abs to CD40 could substitute entirely for T cell help in promoting the survival of RF precursors and in stimulating RF synthesis in T cell deficient animals. The data indicate that CD40 signaling alone can prevent deletion of RF B cells by Ag and in the presence of IgG is sufficient to trigger RF synthesis. The results suggest that selective induction of apoptosis in high-affinity RF B cells may be achieved by blockade of CD40L-CD40 interaction.     

Agonistic monoclonal antibody against CD40 receptor decreases lymphocyte apoptosis and improves survival in sepsis

Sepsis causes a marked apoptosis-induced depletion of lymphocytes. The degree of lymphocyte apoptosis during sepsis strongly correlates with survival. CD40, a member of the TNFR family, is expressed on APCs and has potent antiapoptotic activity. In this study we determined whether an agonistic Ab against CD40 could protect lymphocytes from sepsis-induced apoptosis. Secondly, we examined potential antiapoptotic mechanisms of the putative protection. Lastly, we aimed to determine whether anti-CD40 treatment could improve survival in sepsis. CD1 mice were made septic by the cecal ligation and puncture method and treated postoperatively with anti-CD40 Ab. Treatment with anti-CD40 completely abrogated sepsis-induced splenic B cell death and, surprisingly, decreased splenic and thymic T cell death as well (p < 0.001). To investigate the mechanism of protection of anti-CD40 therapy on T cells, CD40 receptor expression was examined. As anticipated, the CD40 receptor was constitutively expressed on B cells, but, unexpectedly, splenic and thymic T cells were found to express CD40 receptor during sepsis. Furthermore, CD4+CD8- T cells were the predominant subtype of T cells expressing CD40 receptor during sepsis. Additionally, the antiapoptotic protein Bcl-x(L) was found to be markedly increased in splenic B and T cells as well as in thymic T cells after treatment with anti-CD40 Ab (p < 0.0025). Lastly, mice that were made septic in a double injury model of sepsis had improved survival after treatment with anti-CD40 as compared with controls (p = 0.05). In conclusion, anti-CD40 treatment increases Bcl-x(L), provides nearly complete protection against sepsis-induced lymphocyte apoptosis, and improves survival in sepsis.     

Ig-reactive CD4+CD25+ T cells from tolerized (New Zealand Black x New Zealand White)F1 mice suppress in vitro production of antibodies to DNA

We have recently shown that tolerogenic administration of an artificial peptide (pConsensus) that is based on sequences within the V(H) regions of several murine anti-dsDNA Ig delays appearance of autoantibodies in female (New Zealand Black (NZB) x New Zealand White (NZW))F(1) (NZB/W F(1)) mice and significantly prolongs their survival. The aim of this study was to characterize the T cell population(s) involved in pConsensus-induced down-regulation of autoimmune responses in tolerized NZB/W F(1) mice. Using MHC class II dimers loaded with tolerogenic peptide, we found that pCons favored expansion of peptide-reactive CD4(+)CD25(+) regulatory T cells (T(R)) that inhibited in vitro production of anti-dsDNA Ab-forming cells. Suppression by T(R) was abrogated by the presence in culture of Ab to glucocorticoid-induced TNFR family member 18 or to TGFbeta latency-associated protein. These findings suggest possible relevance of Ag specificity in the mechanism of T(R)-mediated immune tolerance to Ig-derived peptides in NZB/W F(1) mice.     

Phenotypic and functional characterization of kidney-infiltrating lymphocytes in renal ischemia reperfusion injury

T and B lymphocytes have been implicated in the pathogenesis of renal ischemia reperfusion injury (IRI). The trafficking of lymphocytes into kidneys during IRI has been postulated to underlie this effect, but has not been rigorously studied. We therefore characterized the lymphocyte populations infiltrating into mouse kidneys 3 and 24 h after renal IRI. Immunohistochemistry and flow cytometry staining of kidney lymphocytes showed increased trafficking of CD3+ T cells and CD19+ B cells in both sham-operated and IRI mice 3 h after renal IRI. In the IRI mice, increased infiltration of NK1.1+ and CD4+ NK1.1+ cells compared with normal and sham-operated mice was observed 3 and 24 h after renal IRI, respectively. After 24 h of renal IRI, the decreased percentages of CD3+, CD19+, and NK1.1+ populations in the IRI mice compared with control groups were observed. Increased TNF-alpha and IFN-gamma production of kidney infiltration CD3+ T cells in IRI mice but not sham-operated mice was found. Unexpectedly, isolation and transfer of kidney-infiltrating lymphocytes 24 h after renal IRI into T cell-deficient mice reduced their functional and histological injury after renal IRI, suggesting that kidney-infiltrating lymphocytes could have a protective function. These quantitative, qualitative, and functional changes in kidney lymphocytes provide mechanistic insight into how lymphocytes modulate IRI, as well as demonstrating that abdominal surgery alone leads to lymphocyte changes in kidney.