IL-2 receptor {alpha} chain (CD25JAC) expression defines a crucial stage in pre-B cell development

The analysis of the expression of the a chain of the IL-2 receptor(CD25.TAC) on the surface of B lineage cells In mouse bone marrowreveals that it is a useful marker to distinguish pre-B-I frompre-B-II cells. CD25 Is not expressed on CD45R(B220)⁺ c-kit⁺CD43⁺ TdT⁺ ₅⁺ C_µ^– slg^– lgH chain locus DJ_H-rearrangedpre-B-I cells of mouse bone marrow. It is expressed on largecycling CD45R(B220)⁺ c-kit⁺ CD43⁺ TdT⁺ ₅⁺ C_µ^– sig^–and on small resting CD45R(B220)⁺ c-kit⁺ CD43^– TdT⁺ ₅⁺C_µ^– sig^– sig- IgH chain locus VHDJH-rearrangedpre-B-II cells. Therefore, the transition from pre-B-I to largepre-B-II cells is marked by the downregulation of c-kit andterminal deoxynucleotldyl transferase (TdT), and by the upregulattonof CD25. SCID, RAG-2T, _µMT and ₆T mutant mice do havenormal, If not elevated numbers of pre-B-I cells but lack allCD25⁺ pre-B-II cells in their bone marrow. The expression ofa transgenic H chain under control of the _µH chain enhancerin RAG-2T bone marrow B lineage precursors allows the developmentof large and small CD25⁺ pre-B-II cells. The results suggestthat the differentiation of pre-B-I to pre-B-II cells in mousebone marrow requires the expression of _µH chains and surrogateL chains in membranes, probably on the surface of precursorB cells.     

CD40 expression and function in murine B cell ontogeny

The CD40 antigen, a member of the nerve growth factor/tumornecrosis factor receptor family, is expressed on all matureB lymphocytes and plays a crucial role in B cell activation,T cell- dependent antigen-driven isotype switching and germinalcenter formation. We have analyzed C040 expression and functionduring mouse B cell development by examining B cell precursorsin normal mice and in transgenic animals in which B cell developmentis frozen at discrete stages. These models included RAG-2 ^-/-mice, and transgenlc littermates that express µ heavychain and/ or the bcl-2 proto-oncogene transgene. CD40 was undetectableat the pro-B cell stage, but was expressed, although at lowlevels, on pre-B cells. However, pre-B cells failed to respondto CD40 triggering either by expression of CD23 or by proliferationin the presence of lL-4. Overexpression of bcl-2 increased thedensity of CD40 expression on pre-B cells: these cells respondto CD40 ligation by expressing CD23 and by proliferating inthe presence of IL-4.     

Early expression of Ig {micro} chain from a transgene significantly reduces the duration of the pro-B stage but does not affect the small pre-B stage

During B cell development, V-J rearrangements at the Ig heavyµ chain (IgH µ chain) locus occur in early cyclingprecursors (pro-B stage). Subsequently, rearrangements at theIg light (IgL) chain locus occur in late resting precursors(small pre-B stage). To study the effects of µ chain expressionon the rate of B cell development, purified hematopoletic stemcells (HSC) bearing a µ chain transgene or wild-type HSCwere transferred Into Immunodeficlent RAG-2^-/-mice and B celldevelopment was followed over time. In addition, cycling B cellprecursors were pulse-labeled by the Injection of BrdU intotransgenlc and wild-type mice, and the production of BrdU-labeledk⁺ and ⁺ B cells was followed over time. These experiments suggestedthat early expression of the µ chain from the transgenesignificantly shortened the duration of the pro-B stage andImmediately drove the precursors to differentiate into smallpre-B cells. By contrast, the presence of the transgene didnot affect the small pre-B stage, where IgL rearrangements occur.Thus, k and rearrangements occurred only after the arrest ofcell cycling as previously shown in wild-type mice, even whenthe µ chain is artificially expressed earlier in B celldevelopment.     

Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus

B lymphocyte differentiation is characterized by an orderedseries of Ig gene assembly and expression events. In the majorityof normal B cells, assembly and expression of Ig heavy (H) chaingenes precedes that of light (L) chain genes. To determine therole of the Ig heavy chain protein in B cell development andL chain gene rearrangement, we have generated mice that cannotassemble Ig H chain genes as a result of targeted deletion ofthe J_H gene segments in embryonic stem cells. Mice homozygousfor this deletion are devoid of slg⁺ B cells in the bone marrowand periphery. B cell differentiation in these mice is blockedat the large, CD43⁺ precursor stage. However, these precursorB cells do assemble L chain genes at a low level in the absenceof µ H chain proteins. These data demonstrate that rearrangementand expression of the µ H chain gene is not absolutelyrequired for L chain gene rearrangement in vivo. Expressionof µ chains may facilitate either efficient L chain generearrangement or the survival of cells that have rearrangedlight chain genes by promoting the differentiation of large,CD43⁺ to small, CD43^– pre-B cells.     

Altered response of CD4+ T cell subsets to Plasmodium chabaudi chabaudi in B cell-deficient mice

Mice depleted of B cells from birth by treatment with anti-_µantibodies can control but not clear an infection with the malariaparasite Plasmodium chabaudi chabaudi (AS). Splenic CD4⁺ T cellsfrom these mice were unable to mount a significant T_h2 responseto the parasite in vitro as shown by much lower precursor frequenciesof T_h cells for antibody production and of IL-4-producing cellscompared with the response of control-treated mice. CD4⁺ T cellsof the anti-_µ-treated mice which respond to antigens ofP. chabaudi chabaudi maintained a T_h1 phenotype throughout primaryinfection, in contrast to control mice in which a sequentialappearance of T_h1 and T_h2 responses was observed. These datashow that T_h1 responses in anti-_µ-treated mice are sufficientto control parasitemia but not to eliminate an infection. Thedata further suggest that depletion of B cells by treatmentwith anti-_µ; antibodies reduces the generation of theT_h2 subset during a primary response to P. chabaudi chabaudi.     

Critical role of dendritic cells in determining the Th1/Th2 balance upon Leishmania major infection

The onset of T_h1 immunity is in part regulated by genetic background.To elucidate the cell type carrying critical factors determiningthe T_h1 response, we employed Rag-2^–/– mice on Leishmaniamajor-susceptible BALB/c and -resistant B10.D2 backgrounds.By using bone marrow (BM) chimeras generated by the transplantationof B10.D2 BM cells into BALB/c-Rag-2^–/– mice, andvice versa, it was shown that hematopoietic cells carry factorsdetermining the disease outcome and T_h1 response against L.major infection. B10.D2-Rag-2^–/– mice reconstitutedwith BALB/c CD4⁺ T cells exhibited a T_h1 response and controlledL. major infection. Wild-type BALB/c mice inoculated with L.major-parasitized B10.D2-Rag-2^–/– splenocytes alsoexhibited a T_h1 response and a mild disease outcome, whereassuch a T_h1 response was not induced when CD11c⁺ dendritic cells(DCs) were depleted from parasitized B10.D2-Rag-2^–/–splenocytes. T_h1 response was reconstituted by the additionof L. major-parasitized B10.D2 DCs but not L. major-parasitizedBALB/c DCs to DC-depleted parasitized B10.D2-Rag-2^–/–splenocytes. These results indicate that DCs determine the outcomeof the disease upon L. major infection.     

B-lineage cells in {micro}-transgenic scid mice proliferate in response to IL-7 but fail to show evidence of immunoglobulin light chain gene rearrangement

The severe combined immunodeficiency (scid) mouse mutation impairsthe recombination of Ig and TCR genes. Mice homozygous for thismutation (scid mice) lack pre-B, B, and T lymphocytes. Earlierwe introduced a functionally rearranged µ-heavy chaingene into the scid mouse genome and found that this resultedin the development of pre-B cells in the bone marrow of thesemice; however, slgM⁺ B cells were not detected. We have nowinvestigated the growth properties and rearrangement statusof Ig genes in early B-lineage cells arising in µ-transgenlcscid mice. We find that the presence of a functional µ-transgeneallows pro-B cells from these mice to proliferate in short-termculture with IL-7. Nevertheless, rearrangements of Ig lightchain genes are not detected in the bone marrow of such mice.Furthermore, the frequency of rearrangement detected at theendogenous Ig heavy chain locus in scid pro-B and pre-B cellsis reduced relative to that in wild-type cells.     

Co-stimulation via CD28 induces activation of a refractory subset of MRL-Ipr/Ipr T lymphocytes

Peripheral lymphoid tissues of Ipr mice contain a large proportionof TCRß/CD3⁺CD4^–CD8^– T cells that lacksurface CD2 and express the B cell isoform of CD45, B220. Thissubset of T cells does not proliferate or produce IL-2 in responseto mitogenic signals or TCR–CD3 ligation. At the sametime, these abnormal T cells display several characteristicsof an activated phenotype. Collectively, these properties ofIpr CD4^–CD8^– T cells have functional parallels withanergic T cells. A critical co-stimulatory molecule implicatedin the prevention of or recovery from anergy is CD28, whichbinds the ligand BB1/B7 on certain accessory cells. Ipr CD4^–CD8^–T cells express normal levels of CD28 which is capable of transducinga strong proliferative signal to these cells in co-stimulationwith mitogens. However, proliferation of Ipr CD4^–CD8^–T cells in response to CD28 co-stimulation does not reach thelevels observed in normal T cells stimulated under similar conditions.Stimulation with anti-CD28 mAb in conjunction with phorbol myristateacetate and lonomycin promotes cell cycling in the CD2^–subset of CD4^–CD8^– T cells, and results in a slightinduction of CD2 levels during the course of the culture period.However, the majority of cells obtained at the end of the cultureperiod remain TCRß+ CD4^–CD8^–, CD2^low/–and B220^high, similar to freshly isolated CD4^–CD8^–Ipr T cells. In contrast, if IL-2 is included in the cultures,a strong shift toward a CD2⁺ phenotype is observed by a majorityof the Ipr T cells. Upon repeat stimulation, these Ipr CD4^–CD8^–T cells can now proliferate in an IL-2-dependent manner whenstimulated with only anti-CD3 mAb or mitogens, in the absenceof exogenous IL-2 or anti-CD28 mAb. These data show that thehyporesponsiveness of Ipr CD4^–CD8^– T cells doesnot result from a lack of CD28 expression, that it is not afixed state, and that it can be reversed by the induction ofcell cycling in the presence of IL-2. These observations extendthe parallels between Ipr CD4^–CD8^– T cells and anergicT cells.     

Rearrangement and expression of {chi} light chain genes can occur without {micro} heavy chain expression during differentiation of pre-B cells

The kinetics of light (L) chain gene rearrangement and expressionon mRNA and protein level has been studied with four stromalcell/IL-7 reactive, long-term in vitro proliferating pre-B celllines and clones, two from fetal liver of normal mice and twofrom fetal liver of E_µH-bcl-2 transgenic (bcl-2-tg) mice.These pre-B cell lines and clones are DJ_H-rearranged on bothH chain alleles. Two of the clones harbor H chain rearrangementswhich do not allow the expression of V_HDJ_H rearranged H chaingenes as _µH chain proteins. Upon removal of IL-7 fromthe pre-B cell cultures all four cell lines rearrange V_H-DJ_Hand V_L-J_L gene segments, loose the surface expression of c-kit,CD43, and surrogate light chain, as well as the capacity tobe clonable on stromal cells in the presence of IL-7. Pre-Bcells from normal mice die by apoptosis during differentiation,while those from bcl-2-tg mice do not. All four lines and clonesexpress comparable levels of mRNA for _µH and _µLchains with the same time kinetics during 3 days of differentiation.However, only two of the four pre-B cell lines and clones express_µH chain protein, whereas all four pre-B cell lines andclones express _µL chain protein at comparable levels between2x10⁵ and 1.40x10⁶ _µL chain molecules per cell. Theseresults suggest that _µH chain expression is not mandatoryfor rearrangement and normal expression of _µL chain geneswhen pre-B cells differentiate to B cells.     

Stromal cell independent growth of bipotent B cell macrophage precursors from murine fetal liver

We have established stromal cell independent culture conditionsunder which single murine hematopoletlc progenitor cells giverise to B lymphocytes and macrophages. IL-11, mast cell growthfactor, and IL-7 are sufficient for the growth of B lineagecells from AA4.1⁺ B220^–Mac-1^–Ly6A⁺ fetal liver cellsIsolated at day 12 of gestation. These conditions are also sufficientfor commitment to myelold differentiation, although CSF-1 Isrequired for substantial growth of macrophages. The progenitorsresponding under these culture conditions: (I) require 8 daysin culture before they become competent to respond to B cellmltogens in the presence of stromal cells and (II) undergo aminimum of three cell divisions prior to Ig heavy chain allotyperestriction. The findings suggest that the stromal cell dependencyof Immature hematopoletlc cells may reflect a requirement forstromal cell derived growth factors. Further, this providesan in vitro system in which commitment and differentiation canbe studied in single cells.     

Role of T-cell-associated lymphocyte function-associated antigen-1 in the pathogenesis of experimental colitis

The ß₂ integrin lymphocyte function-associated antigen-1(LFA-1; CD11a/CD18) is important for lymphocyte traffickingand activation as well as recruitment to sites of tissue inflammation.The objective of this study was to assess the role of ‘T-cell-associated’LFA-1 in the pathogenesis of chronic colitis in vivo. Transferof CD4⁺CD25^– T cells isolated from wild-type (wt) miceinto immunodeficient recipients [recombinase-activating gene-1-deficient(RAG-1^–/–)] produced moderate to severe colitis,whereas RAG-1^–/– mice injected with CD11a-deficient(CD11a^–/–; LFA-1^–/–) donor T cells displayedminimal macroscopic and histological evidence of colitis. Surfaceexpression of L-selectin, ₄, ₄ß₇ and chemokine receptor-7were similar for wt and CD11a^–/– donor T cells.Attenuated disease in the CD11a^–/– RAG-1^–/–animals was associated with decreased numbers of CD4⁺ T cellsin the mesenteric lymph nodes (MLNs), spleen and intestinallamina propria (LP). In addition, significant reductions inT_h1 cytokines were observed following ex vivo stimulation ofmononuclear cells obtained from the MLNs and colonic LP. Interestingly,mononuclear cells obtained from the spleens of CD11a^–/– RAG-1^–/– exhibited enhanced pro-inflammatory cytokineproduction compared with splenocytes obtained from wt RAG-1^–/–colitic mice. Taken together, our data suggest that T-cell-associatedCD11a (LFA-1) expression plays a dual role in the initiationof chronic gut inflammation by facilitating naive T-cell priming/activationand expansion within MLNs and by augmenting pro-inflammatorycytokine production following secondary stimulation by antigen-presentingcells in the colonic interstitium.     

NK1.1+ T cells from IL-7-deficient mice have a normal distribution and selection but exhibit impaired cytokine production

Particular subsets of T cells expressing the NK1.1 antigen havebeen proposed to play an immune regulatory role by their fastand strong production of cytokines, in particular IL-4. We soughtto determine factors driving the functional differentiationof NK1.1⁺ T cells. Since NK1.1⁺ T cells are exquisitely sensitiveto IL-7 stimulation, we analyzed the development, selectionand IL-4 production of NK1.1⁺ T cells in IL-7 deficient mice(IL-7–/–mice). Besides a sharp reduction of allT cell subsets, NK1.1⁺ T cells develop at normal relative frequenciesin IL-7–/–;mice. They also undergo a normal selectionprocess, as revealed by the biased V_ß TCR repertoireidentical to the one in IL-7+/+ mice. However, NK1.1₊ T cellsfrom IL-7+/+ mice were found to be impaired in IL-4 and IFN-production in in vitro and in vivo models. In addition, IL-7was able to restore IL-4 production by NK1.1⁺ thymocytes fromIL-7–/– mice. Finally, IL-7 but not IL-4 was ableto maintain and increase IL-4 production by NK1.1⁺ thymocytesfrom normal mice. These data suggest that the functional maturationof NK1.1⁺ T cells requires a cytokine-driven differentiationprocess, in which IL-7 plays a major role.     

Proliferation and apoptosis of B220+ CD4 CD8 TCR{alpha}{beta}intermediate T cells in the liver of normal adult mice: implication for Ipr pathogenesis

Small numbers of T cells have been isolated from the normalmouse liver and many of these are of the CD4^–CD8^–TCRß⁺phenotype. Larger numbers of such cells are present in the liversof mice homozygous for the Ipr mutation and the liver has beenproposed to be the site of an extrathymlc T cell developmentpathway that is expanded in Ipr/lpr mice. Using a modified separationprocedure that increases the liver T cell yield, we have beenable to characterize a subset of CD4^–CD8^–TCRß^intermediateT cells that express the B220 epltope of the CD45 molecule,and resemble in this and many other ways the accumulating Tcells in Ipr lymph nodes. These cells are an actively dividingpopulation and even in healthy, unmanipulated mice a large proportionof them are undergoing apoptosis. We propose the model thatthe normal liver is a major site for T cell destruction andthat the Ipr defect results in failure of this process withleakage of B220⁺CD4^–CD8^–TCRß⁺ cells fromthe liver to peripheral lymphoid tissues, particularly lymphnodes.     

Pervasive and stochastic changes in the TCR repertoire of regulatory T-cell-deficient mice

We hypothesize that regulatory T-cell (Treg)-deficient strainshave an altered TCR repertoire in part due to the expansionof autoimmune repertoire by self-antigen. We compared the Vβfamily expression profile between B6 and Treg-lacking B6.Cg-Foxp3^sf/Y(B6.sf) mice using fluorescent anti-Vβ mAbs and observedno changes. However, while the spectratypes of 20 Vβ familiesamong B6 mice were highly similar, the Vβ family spectratypesof B6.sf mice were remarkably different from B6 mice and fromeach other. Significant spectratype changes in many Vβfamilies were also observed in Treg-deficient IL-2 knockout(KO) and IL-2R KO mice. Such changes were not observed withanti-CD3 mAb-treated B6 mice or B6 CD4⁺CD25^– T cells.TCR transgenic (OT-II.sf) mice displayed dramatic reductionof clonotypic TCR with concomitant increase in T cells bearingnon-transgenic Vβ and V families, including T cells withdual receptors expressing reduced levels of transgenic V andendogenous V. Collectively, the data demonstrate that Treg deficiencyallows polyclonal expansion of T cells in a stochastic manner,resulting in widespread changes in the TCR repertoire.     

B-lineage cell deficits in bone marrow of lpr/lpr mice

Analyses of bone marrow (BM) lymphocytes in C57BL/6 mice homozygousfor the lpr mutation (BS.Ipr) disclosed low numbers of pre-Band B cells, as compared with age-matched control B6 mice. BMdepletion in B6.lpr mice was selective for B-lineage cells,appeared in young adults, and developed markedly with age anddisease progression, contrasting with the peripheral lymphocytehypercellularity. Normalization of pre-B and B cellularity inBM of B6.lprmice was observed after administration of polyclonalIg, that also markedly improved the clinical condition. Isolatedpre-B (B220⁺ IgM^–) cells from B6 or B6.lpr mice, however,showed essentially the same rates of IL-7-dependent proliferationand differentiation to B (lgM⁺) cells in culture, indicatingthat the BM B-lineage deficit is not the result of an intrinsicdefect inB cell generation.     

B cells play a regulatory role in mice infected with the L3 of Brugia pahangi

Mice infected with the L3 of the filarial nematode Brugia pahangimake a strong T_h2 response characterized by elevated levelsof antigen-specific IL-4, IL-5 and IL-10. Here we show thatB cells from these animals are the major proliferating populationin vitro with depletion of B cells or infection of µMTmice, resulting in reduced levels of antigen-specific proliferation.B cells also act as antigen-presenting cells (APC) to CD4⁺ cellsas demonstrated by the switch in cytokine profiles upon B celldepletion. The efficiency of B cells in antigen presentationis attenuated by IL-10 which down-regulates the expression ofB7-1 and B7-2 on the surface of B cells both in vitro and invivo. Thus, IL-10 may modulate CD4 responses in L3-infectedmice by suppressing the expression of B7 ligands on B cells.In support of this hypothesis, blockade of the IL-10R in vivoresults in increased proliferation of CD4⁺ cells. We proposethat B cells participate in a negative feedback loop: IL-10elicited by infection with L3 and produced by B cells (and CD4⁺cells) down-regulates the expression of B7 molecules on theB cell surface, attenuating their efficiency as APC to CD4⁺T cells and restricting their expansion.     

Frequent deletion of the transgene in T cell receptor {beta} chain transgenic mice

TCR V_ß8.1 transgenic mice were generated using a genomicTCR V_ß gene construct under the control of its promoterand enhancer. Among three lines of transgenic mice, one lineexpressed the transgenic TCR on only 70% of peripheral T cells,while the other two lines expressed it on almost all matureT cells. T cells which lacked expression of the transgenic TCRß chain expressed endogenous TCR ß chains.The molecular basis underlying the lack of transgene expressionin T cells of this line of transgenic mice was investigated.The transgenic TCR^– cells were isolated by two methods.First, Thy-1⁺ V_ß8.1/8.2^– cells were purifiedfrom peripheral T cells using cell sorting. Second, transgenicTCR^– T cell clones were established. In both cases, Southernblotting indicated that V_ß8.1^– T cells had deletedthe transgenic TCR gene. Thus, deletion of the transgenic TCRcan occur in a high proportion of T cells, which allows rearrangementand expression of endogenous TCR ß chains.     

Correction of gld autoimmunity by co-infusion of normal bone marrow suggests that gld is a mutation of the Fas ligand gene

lpr and gld mice develop phenotypically indistinguishable systemicautoimmune diseases and marked lymphadenopathy dominated byCD4^–CD8^– In vivo chimera experiments have demonstratedthat both ipr T and ipr B cells are intrinsically defective.Analogous experiments were conducted using gld mice. Lethallyirradiated gld mice were given mixtures of congenic gld andnormal (+/+) bone marrow differentially marked by lg heavy chainallotype. In sharp contrast to ipr-+/+ mixed chimeras, gld-+/+chimeras had little autoantibody production at 5 months andminimal adenopathy at 6 months, indicating that the normal marrow-derivedcells corrected the gld defect. Thus, aberrant autoantibodyproduction is due to a defect extrinsic to the gld B cell andlymphoproliferation is due to a defect extrinsic to the gldT cell. These data support the hypothesis that gld mice lackan apoptosis-inducing ligand. The receptor for this ligand maybe the Fas molecule, which is defective in ipr mice T cells.     

Induction of phosphocholine-specific antibodies in X-linked immune deficient mice: in vivo protection against a Streptococcus pneumoniae challenge

X-linked Immune deficient (XID) mice are susceptible to infectionwith Streptococcus pneumoniae because they fail to mount animmune response to the Immunodomlnant phosphochollne (PC) epltopeon the bacterial cell wall. It is difficult to induce PC-speclflcantibodies in XID mice because PC-specific B cells expressingthe T15-, M167- and M603 Idiotype (Id), which provide protectionagainst S. pneumoniae, are deleted in these mice via an antigen-specific,receptor-mediated process. In addition, the standard PC hapten,p-dlazophenylphosphochollne (DPPC), induces high affinity phenylphosphochollne(PPC)-speciflc antibodies in XID mice, which are not protectiveagainst S. pneumoniae. We have used a novel PC hapten, p-nitrophenyl-6-(O-phospho-choline)hydroxyhexanoate(EPC), to induce PC-specific antibodies in XID mice. The immuneresponse to EPC-keyhole limpet hemacyanln (KLH) is dominatedby IgGi, V_H1⁺, US-Id^–, PC-inhlbitable antibodies. A smallIgM antl-PC response having a consistent T15-ld⁺ component isalso induced in XID mice, whereas normal mice produce a largeIgM response dominated by T15-ld⁺ antibodies. The immune responseto EPC-KLH remains predominantly PC-lnhlbltable even after multipleimmunizations, while the response to DPPC–KLH becomesdominated by PPC-speclflc antibodies. C.CBA/N mice immunizedtwice with EPC–KLH are protected against 10⁴ S. pneumoniaewhile as few as 10 bacteria are 100% lethal for the unlmmunlzedcontrols. The ability of EPC-protein to induce a long-lived,PC-speclflc response should make this hapten a potential TDvaccine candidate for S. pneumoniae