Class Switch Recombination and Somatic Hypermutation of Virus-Neutralizing Antibodies Are Not Essential for Control of Friend Retrovirus Infection

Toll-like receptor 7 and Myd88 are required for antiretroviral antibody and germinal center responses, but whether somatic hypermutation and class-switch recombination are required for antiretroviral immunity has not been examined. Mice deficient in activation-induced cytidine deaminase (AID) resisted Friend virus infection, produced virus-neutralizing antibodies, and controlled viremia. Passive transfer demonstrated that immune IgM from AID-deficient mice contributes to Friend virus control in the presence of virus-specific CD4⁺ T cells.     

Subunits of IgM reconstitute defective contact sensitivity in B-1 cell-deficient xid mice: kappa light chains recruit T cells independent of complement

The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti-TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP kappa L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while kappa L chains act via another C-independent but mast cell-dependent pathway.     

De novo central nervous system processing of myelin antigen is required for the initiation of experimental autoimmune encephalomyelitis

We demonstrate the absolute requirement for a functioning class II-restricted Ag processing pathway in the CNS for the initiation of experimental autoimmune encephalomyelitis (EAE). C57BL/6 (B6) mice deficient for the class II transactivator, which have defects in MHC class II, invariant chain (Ii), and H-2M (DM) expression, are resistant to initiation of myelin oligodendrocyte protein (MOG) peptide, MOG(35-55)-specific EAE by both priming and adoptive transfer of encephalitogenic T cells. However, class II transactivator-deficient mice can prime a suboptimal myelin-specific CD4(+) Th1 response. Further, B6 mice individually deficient for Ii and DM are also resistant to initiation of both active and adoptive EAE. Although both Ii-deficient and DM-deficient APCs can present MOG peptide to CD4(+) T cells, neither is capable of processing and presenting the encephalitogenic peptide of intact MOG protein. This phenotype is not Ag-specific, as DM- and Ii-deficient mice are also resistant to initiation of EAE by proteolipid protein peptide PLP(178-191). Remarkably, DM-deficient mice can prime a potent peripheral Th1 response to MOG(35-55), comparable to the response seen in wild-type mice, yet maintain resistance to EAE initiation. Most striking is the demonstration that T cells from MOG(35-55)-primed DM knockout mice can adoptively transfer EAE to wild-type, but not DM-deficient, mice. Together, these data demonstrate that the inability to process antigenic peptide from intact myelin protein results in resistance to EAE and that de novo processing and presentation of myelin Ags in the CNS is absolutely required for the initiation of autoimmune demyelinating disease.     

CD27 is acquired by primed B cells at the centroblast stage and promotes germinal center formation

Studies on human B cells have featured CD27 as a marker and mediator of the B cell response. We have studied CD27 expression and function on B cells in the mouse. We find that B cells acquire CD27 at the centroblast stage and lose it progressively upon further differentiation. It is not a marker for somatically mutated B cells and is present at very low frequency on memory B cells. Enrichment of CD27 among centroblasts and the presence of its ligand CD70 on occasional T and B cells in or near germinal centers (GCs) suggested a role for CD27/CD70 interactions in clonal B cell expansion. Accordingly, GC formation in response to influenza virus infection was delayed in CD27 knockout mice. CD27 deficiency did not affect somatic hypermutation or serum levels of virus-specific IgM, IgG, and IgA attained in primary and recall responses. Adoptive transfer of T and B cells into CD27/CD28(-/-) mice revealed that CD27 promotes GC formation and consequent IgG production by two distinct mechanisms. Stimulation of CD27 on B cells by CD28(+) Th cells accelerates GC formation, most likely by promoting centroblast expansion. In addition, CD27 on T cells can partially substitute for CD28 in supporting GC formation.     

Phenotypic analysis of pneumococcal polysaccharide-specific B cells

The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.     

Oral administration of hapten inhibits in vivo induction of specific cytotoxic CD8+ T cells mediating tissue inflammation: a role for regulatory CD4+ T cells

We investigated whether oral tolerance could block the development of an inflammatory response mediated by CD8+ T cells, using a mouse model of oral tolerance of contact sensitivity (CS) to the hapten 2, 4-dinitrofluorobenzene (DNFB). In this system, the skin inflammatory response is initiated by hapten-specific class I-restricted cytotoxic CD8+ T (CTL) cells, independently of CD4 help. Oral delivery of DNFB before skin sensitization blocked the CS response by impairing the development of DNFB-specific CD8+ effector T cells in secondary lymphoid organs. This was shown by complete inhibition of DNFB-specific CTL and proliferative responses of CD8+ T cells, lack of specific IFN-gamma-producing CD8+ T cells, and inability of CD8+ T cells to transfer CS in RAG20/0 mice. RT-PCR and immunohistochemical analysis confirmed that recruitment of CD8+ effectors of CS in the skin at the site of hapten challenge was impaired in orally tolerized mice. Sequential anti-CD4 Ab treatment showed that only depletion of CD4+ T cells during the afferent phase of CS abrogated oral tolerance induction by restoring high numbers of specific CD8+ effectors in lymphoid organs, whereas CD4 depletion during the efferent phase of CS did not affect oral tolerance. These data demonstrate that a single intragastric administration of hapten can block in vivo induction of DNFB-specific CD8+ CTL responsible for tissue inflammation and that a subset of regulatory CD4+ T cells mediate oral tolerance by inhibiting expansion of specific CD8+ effectors in lymph nodes.     

Overexpression of activation-induced cytidine deaminase in B cells is associated with production of highly pathogenic autoantibodies

Defective receptor editing or defective B cell checkpoints have been associated with increased frequency of multireactive autoantibodies in autoimmune disease. However, Ig somatic hypermutation and/or class switch recombination may be mechanisms enabling the development of pathogenic multireactive autoantibodies. In this study, we report that, in the BXD2 mouse model of autoimmune disease, elevated expression of activation-induced cytidine deaminase (AID) in recirculating follicular CD86(+) subsets of B cells and increased germinal center B cell activity are associated with the production of pathogenic multireactive autoantibodies. CD4 T cells from BXD2 mice that expressed increased levels of CD28 and an increased proliferative response to anti-CD3 and anti-CD28 stimulation are required for this process. Inhibition of the CD28-CD86 interaction in BXD2 mice with AdCTLA4-Ig resulted in normalization of AID in the B cells and suppression of IgG autoantibodies. This treatment also prevented the development of germinal center autoantibody-producing B cells, suggesting that an optimal microenvironment enabling AID function is important for the formation of pathogenic autoantibodies. Taken together, our data indicate that AID expression in B cells is a promising therapeutic target for the treatment of autoimmune diseases and that suppression of this gene may be a molecular target of CTLA4-Ig therapy.     

Altered migration, recruitment, and somatic hypermutation in the early response of marginal zone B cells to T cell-dependent antigen

The early responses of follicular (Fo) and marginal zone (MZ) B cells to T cell-dependent Ag were compared using anti-hen egg lysozyme (HEL+) B cells capable of class switch recombination and somatic hypermutation (SHM). Purified CD21/(35int)CD23high Fo and CD21/35(high)CD23low MZ splenic B cells from SW(HEL) Ig-transgenic mice were transferred into wild-type recipients and challenged with HEL-sheep RBC. Responding HEL+ B cells from both populations switched efficiently to IgG1, generated syndecan-1+ Ab-secreting cells, and exhibited equivalent rates of proliferation. However, the expansion of HEL+ MZ B cells lagged significantly behind that of HEL+ Fo B cells due to less efficient homing to the outer periarteriolar lymphatic sheath and reduced recruitment into the proliferative response. Despite the equivalent rates of class switch recombination, the onset of SHM was delayed in the MZ subset, indicating that these two activation-induced cytidine deaminase-dependent events are uncoupled in the early response of MZ B cells. Migration of HEL+ B cells into germinal centers coincided with the onset of SHM, occurring more rapidly with Fo vs MZ responders. These results are consistent with the concept that Fo and MZ B cells have evolved to specialize in T cell-dependent and T-independent responses respectively.     

CD4 and CD8 T cells in susceptibility/protection to collagen-induced arthritis in HLA-DQ8-transgenic mice: implications for rheumatoid arthritis

To investigate the role of CD4 and CD8 T cells in arthritis, we generated transgenic mice deficient in CD4 and CD8 molecules expressing RA-susceptible gene HLA-DQ8. DQ8.CD4(-/-) mice were resistant to developing collagen-induced arthritis (CIA). However, DQ8.CD8(-/-) mice developed CIA with increased incidence and more severity than DQ8 mice. Both DQ8.CD8(-/-) and DQ8 mice produced rheumatoid factor. In addition, DQ8.CD8(-/-) mice produced antinuclear Abs. The B cell compartment and expression of DQ8 were normal in all the strains, although frequency of cells expressing DQ8 was less in CD4(-/-) mice. An increased frequency of CD3(+) double-negative (DN) T cells was found in DQ8.CD8(-/-) compared with DQ8.CD4(-/-) and DQ8 mice. These CD3(+) DN T cells produced high amounts of IL-10 in CD8-deficient mice. Analysis of cell division using a cell cycle tracking dye showed a higher rate of division of CD3(+) and CD3(+) DN T cells in DQ8.CD8(-/-) mice compared with DQ8.CD4(-/-) and DQ8 mice. Decreased apoptosis was seen in CIA-susceptible DQ8 and CD8-deficient mice, indicating a defect in activation-induced cell death. These observations suggest that CD4 cells are necessary for initiation of CIA in DQ8 mice. We hypothesize that CD8(+) T cells are not capable of initiating CIA in DQ8-transgenic mice but may have a regulatory/protective effect.     

Multichannel fluorescence spinning disk microscopy reveals early endogenous CD4 T cell recruitment in contact sensitivity via complement

Contact sensitivity (CS) is one of the primary in vivo models of T cell-mediated inflammation. The presence of CS-initiating CD4 T lymphocytes at the time of challenge is essential for transfer and full development of the late phase CS inflammatory response. From this observation investigators have speculated that early recruitment of CD4 T cells to the site of challenge must occur. Moreover, there must be rapid synthesis/release and disappearance of an important mediator during the first hours after hapten challenge. Using spinning disk confocal microscopy, we observed the very early effector events of the immune response. Simultaneous, real-time visualization of predominant neutrophil and extremely rare CD4 T cell trafficking in the challenged skin vasculature was noted (one rolling CD4 T cell for every 10-18 rolling and adherent neutrophils). We demonstrate that neutrophil adhesion during the early CS response was reduced in C5a receptor-deficient (C5aR-/-) mice or leukotriene B4 receptor antagonist-treated mice, whereas CD4 T cell recruitment was only inhibited in C5aR-/- mice. In line with these observations, leukocyte infiltration and the associated tissue damage were significantly reduced in C5aR-/- mice but not in leukotriene B4 receptor antagonist-treated wild-type mice 24 h after challenge. C5a receptor expression on T cells and not on tissue resident cells was important for the development of a CS response. Thus, by using spinning disk confocal microscopy we visualized the early events of an adaptive immune response and identified the rare but essential recruitment of CD4 T cells via the complement pathway.     

Foxp3+ follicular regulatory T cells control the germinal center response

Follicular helper (T(FH)) cells provide crucial signals to germinal center B cells undergoing somatic hypermutation and selection that results in affinity maturation. Tight control of T(FH) numbers maintains self tolerance. We describe a population of Foxp3(+)Blimp-1(+)CD4(+) T cells constituting 10-25% of the CXCR5(high)PD-1(high)CD4(+) T cells found in the germinal center after immunization with protein antigens. These follicular regulatory T (T(FR)) cells share phenotypic characteristics with T(FH) and conventional Foxp3(+) regulatory T (T(reg)) cells yet are distinct from both. Similar to T(FH) cells, T(FR) cell development depends on Bcl-6, SLAM-associated protein (SAP), CD28 and B cells; however, T(FR) cells originate from thymic-derived Foxp3(+) precursors, not naive or T(FH) cells. T(FR) cells are suppressive in vitro and limit T(FH) cell and germinal center B cell numbers in vivo. In the absence of T(FR) cells, an outgrowth of non-antigen-specific B cells in germinal centers leads to fewer antigen-specific cells. Thus, the T(FH) differentiation pathway is co-opted by T(reg) cells to control the germinal center response.     

Clearance of Pneumocystis carinii in mice is dependent on B cells but not on P carinii-specific antibody

Both CD4(+) T cells and B cells are critical for defense against Pneumocystis carinii infection; however, the mechanism by which B cells mediate protection is unknown. We show that P. carinii-specific IgM is not sufficient to mediate clearance of P. carinii from the lungs since CD40-deficient mice produced normal levels of specific IgM, but were unable to clear the organisms. Using chimeric mice in which the B cells were deficient in CD40 (CD40KO chimeras) we found that clearance of P. carinii infection is delayed compared with wild-type controls. These CD40KO chimeric mice produced normal levels of P. carinii-specific IgM, but did not produce class-switched IgG or IgA. Similarly, clearance of P. carinii was delayed in mice deficient in FcgammaRI and III (FcgammaRKO), indicating that P. carinii-specific IgG partially mediates opsonization and clearance of P. carinii. Opsonization of organisms by complement did not compensate for the lack of specific IgG or FcgammaR, since C3-deficient and C3-depleted FcgammaRKO mice were still able to clear P. carinii. Finally, micro MT and CD40KO chimeric mice had reduced numbers of activated CD4(+) T cells in the lungs and lymph nodes compared with wild-type mice, suggesting that B cells are important for activation of T cells in response to P. carinii. Together these data indicate that P. carinii-specific IgG plays an important, but not critical, role in defense against P. carinii. Moreover, these data suggest that B cells also mediate host defense against P. carinii by facilitating CD4(+) T cell activation or expansion.     

Skin inflammation during contact hypersensitivity is mediated by early recruitment of CD8+ T cytotoxic 1 cells inducing keratinocyte apoptosis

Contact hypersensitivity (CHS) is a T cell-mediated, Ag-specific skin inflammation induced by skin exposure to haptens in sensitized individuals. Th1/T cytotoxic 1 cells are effector cells of CHS, whereas Th2/T regulatory CD4(+) T cells have down-regulating properties. We have previously shown that CHS to 2,4-dinitrofluorobenzene is mediated by specific CD8(+) effector cells, whose cytolytic activity is mandatory for induction of skin inflammation. In this study, using immunohistochemistry and RT-PCR analysis, we show that CD8(+) T cells are rapidly recruited into the skin at the site of hapten challenge before the onset of clinical and histological signs of skin inflammation. This early CD8(+) T cell recruitment is concomitant with: 1) transient IFN-gamma mRNA expression suggesting local activation of effector cells; and 2) induction of keratinocyte (KC) apoptosis which gradually increased to a maximum at the peak of the CHS response. Alternatively, skin infiltration of CD4(+) T cells occurred later and coincided with the peak of the CHS reaction and the beginning of the resolution of skin inflammation. Mice deficient in CD8(+) T cells did not develop CHS, whereas mice deficient in CD4(+) T cells developed an enhanced inflammatory response with increased numbers of CD8(+) T cells recruited in the skin associated with massive KC apoptosis. These data show that CHS is due to the early and selective recruitment in the skin of CD8(+) T cytotoxic 1 effector cells responsible for KC apoptosis.     

Quantitatively reduced participation of anti-nuclear antigen B cells that down-regulate B cell receptor during primary development in the germinal center/memory B cell response to foreign antigen

The peripheral B cell compartment contains high levels of "polyreactivity" including autospecificities. We have described a pathway that certain autoreactive B cells may take in gaining stable access to the foreign Ag-responsive peripheral compartment. This pathway was revealed in mice expressing a targeted Ig H chain transgene encoding BCRs with "multireactivity" for the hapten arsonate and DNA-based autoantigens. B cells expressing such BCRs develop to mature follicular phenotype and locale, and are not short-lived. These B cells express very low levels of BCR, indicating that they are not "ignorant" of self Ag, but do not display features of anergy in in vitro assays. Nonetheless, a variety of states of lymphocyte anergy has been described, and some may only be manifested in vivo. As such, we analyzed the ability of these B cells to participate in a T cell-dependent immune response to arsonate in vivo. These B cells mount an early primary response similar to control B cells, including homing to follicles, migration to the T-B interface, and induction of costimulatory molecules, proliferation, differentiation to AFCs, class switching, and entry into GCs and somatic hypermutation. Nonetheless, these B cells display reduced participation in the latter stages of the GC response and in the anamnestic AFC response. In total, these data suggest that while the autoreactivity of this type of B cell does not result in anergy, the ability of such B cells to participate in a cross-reactive immune response to foreign Ag is compromised.     

TRAIL deficiency does not rescue impaired CD8+ T cell memory generated in the absence of CD4+ T cell help

Ag-specific CD8(+) T cells immunized in the absence of CD4(+) T cell help, so-called "unhelped" CD8(+) T cells, are defective in function and survival. We investigated the role of the proapoptotic molecule TRAIL in this defect. We first demonstrate that TRAIL does not contribute to the CD8(+) T cell response to Listeria monocytogenes strain expressing OVA (LmOVA) in the presence of CD4(+) T cells. Secondly, we generated mice doubly deficient in CD4(+) T cells and TRAIL and analyzed their CD8(+) T cell response to LmOVA. Memory CD8(+) T cells in double-deficient mice waned over time and were not protective against rechallenge, similar to their TRAIL-sufficient unhelped counterparts. To avoid the effects of CD4(+) T cell deficiency during memory maintenance, and to address whether TRAIL plays a role in the early programming of the CD8(+) T cell response, we performed experiments using heterologous prime and early boost immunizations. We did not observe activation-induced cell death of unhelped CD8(+) T cells when mice were infected with followed vaccinia virus expressing OVA 9 days later by LmOVA infection. Furthermore, primary immunization of CD4(+) T cell-deficient mice with cell-associated Ag followed by LmOVA infection did not reveal a role for TRAIL-mediated activation-induced cell death. Overall, our results suggest that CD4(+) T cell help for the CD8(+) T cell response is not contingent on the silencing of TRAIL expression and prevention of TRAIL-mediated apoptosis.     

Checkpoint kinase 1 negatively regulates somatic hypermutation

Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis.