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.     

Epitope specificity of bee venom phospholipase A2-specific suppressor T cells which produce antigen-binding glycosylation inhibiting factor

From the spleen cells of BALB/c mice primed with bee venom phospholipaseA₂(PLA₂), we established seven T cell hybrldomas which constitutivelysecreted glycosylation inhibiting factor (GIF), expressed bothCD3 and TCRß, and responded to antigen-pulsed antigenpresenting cells (APC) for the formation of IgE-binding factor.Upon stimulation with antigen-pulsed APC, four of the sevenhybridomas produced GIF having affinity for native PLA₂. Theantigen-binding GIF could suppress the anti-hapten antibodyresponse of BALB/c mice to dinitrophenyl (DNP)-PLA₂ conjugatesin a carrier-specific manner and bound to Immunosorbents coupledwith either the mAb 14–12 or antl-TCR chain, H28-710.Analysis of the epitope specificity of the TCR on the GIF-producingT hybridomas indicated that all of the hybridomas which couldproduce antigenbinding GIF upon antigenic stimulation recognizedthe synthetic peptide representing amino acid residues 19–34in PLA₂molecules in the context of the product of the I-A^d subregionand the antigen-binding GIF formed by the cells had affinityfor the peptide. The 3-D structure of bee venom PLA₂ indicatesthat the sequence of amino acid 14–24 forms a loop inthe PLA₂ moiecule and represents an external structure of theantigen, while peptide 25–37 forms an helix. Evidencewas obtained which suggests that the sequence of 25–34contains amino acid residues interacting with la molecules,while peptide 19–24 contains residues involved in theinteraction of p19–34-la complexes with TCR on the hybridomas.It was also found that not only the synthetic peptide 19–34,but also the peptides 13–28 and 19–30 inhibitedthe binding of antigen-binding GIF to PLA₂-coupled Sepharose,while peptide 25–40 failed to do so. The results collectivelyindicate that the antigen-binding GIF and TCR on the cell sourceof the factor interact with a common epitope which is exposedon the surface of a nominal antigen.     

Major histocompatibility complex and T cell receptor interaction of the P91A tum− peptide

The P91A antigen was identified following mutation of P1 mastocytoma cells. The peptide epitope is encoded by a mutant form of the S3 subunit of the PA 700 proteasome regulatory complex. P91A stimulates a strong CD8⁺ T cell response when expressed on tumor cells or normal tissue and P91A-specific T cells express a restricted range of T cell receptors. Although it is a strong L^d-binding peptide, P91A does not conform to the established motif for this major histocompatibility complex (MHC) molecule and this has hampered elucidation of the precise epitope. L^d predominantly associates with nonamer peptides; however, using a variety of complementary approaches, the P91A epitope is identified as the octamer QNHRALDL. In the absence of the L^d motif residue proline at position 2, residues 5–7 are primarily involved in MHC interaction. P91A is thus atypical in its interaction with L^d. Residues 1, 3, and 4 are found to influence T cell recognition of P91A. Definition of the P91A peptide will allow studies on P91A processing and interactions of the P91A peptide/MHC complex with T cell receptors of differing avidity to establish the basis for restricted T cell receptor usage. The basis for the failure of the P91A tum⁺ peptide (QNRRALDL) to bind to L^d is addressed by molecular modeling.     

Analysis of T cell antigen receptors of myelin basic protein specific T cells in SJL/J mice demonstrates an {alpha} chain CDR3 motif associated with encephalitogenic T cells

Experimental autoimmune encephalomyelitis (EAE) is an animalautoimmune disease mediated by CD4⁺ T cells. Analysis of TCRexpression revealed that limited TCR elements (V^ß8.2,V2 or 4) were utilized by myelin basic protein (MBP) specificT cells In mice with H-2^U haplotype and Lewis rats. The usageof a particular ß chain complementarity determiningregion 3 (CDR3) motif has also been shown. However, It remainsunclear to what extent these observations can be extrapolated.Here we studied the TCR sequences of MBP 89–101/1-A^s specificT cell clones derived from SJL/J mice, using the polymerasechain reaction on reverse transcribed mRNA. Although the V_ßusage was less restricted than In H-2^U mice, they predominantlyutilized v_ß17a and expressed LGG or related motifsin the V_ß - D_ß - J_ß junctions.Furthermore, a single chain rearrangement between V1.1 andJBBM142 with no N region diversity was preferentially used.Concordantty, Immunization with a peptlde corresponding to the chain CDR3 was found to significantly alter the clinical courseof EAE. Comparison of the published TCR junctional regions demonstratesthat the CDR3 motifs (LGG in ß chain, CA*R*NY motifIn chains) are expressed by other encephalitogenic clones.Notably, the CA*R*NY was conserved in PL/J mice clones thatrecognize a distinct MBP–MHC determinant. It suggeststhat an antigen-independent mechanism may contribute to conservingthe a chain motif. The Implications of these observations arediscussed.     

TCR repertoire to proteolipid protein (PLP) in multiple sclerosis (MS): homologies between PLP-specific T cells and MS-associated T cells in TCR junctional sequences

In the pathogenesis of multiple sclerosis (MS), autoimmune Tcells reactive with proteolipid protein (PLP) may play a crucialrole. We determined 23 TCR (ß-chain sequences of limitingdilution T cell lines (TCL) selected against a synthetic peptide,PLP 95–116, 105–124 or 139–155, from the peripheralblood of three Japanese MS patients with the DR2, w15 haplotype(Tl, SK and OK). Fourteen sequences were originated from Tl,seven from SK and two from OK. The PLP-reactive TCL utilizedvarious V_ß and J^ß; gene segments, but therewas significant bias in the V_ß and J_ß usage.Overutilization of the V_ß2 family and dominant usageof the J_ß2.5 subfamily was seen in PLP 105–124-reactiveand 95–116-reactive TCL respectively. More remarkably,a majority of the TCL were found to express ß-chainCDR3 motifs that appear to be unique to MS brain infiltrates.In contrast, these motifs were only rarely seen in control TCRsequences from peripheral blood or from a TCL selected againsttetanus toxoid. In several cases, the _ßCDR3 homologiesbetween the PLP-reactive T cells and MS brain T cells were extensive,owing to the shared motifs in combination with the surroundingamino acid identities. These results indicate that PLP-specificT cells may be involved in the immunopathology of MS.     

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity

In this study we have analyzed the TCR V and V_ß regionsat the DNA level in the CD4⁺CD45RO⁺ memory T cell populationof synovial tissue infiltrating T lymphocytes of three rheumatoidarthritis (RA) patients and one patient with chronic arthritis.Cell lines of CD4⁺CD45RO⁺, CD4⁺CD45RO^–, CD8⁺CD45RO⁺ andCD8⁺CD45RO^– T lymphocyte populations were generated followingFACS cell sorting of freshly isolated synovial tissue mononuclearcell infiltrates (STMC) and of freshly isolated peripheral bloodmononuclear cells (PBMC) of these patients. The phenotyplc andmolecular analyses have revealed the following. (I) The TCRrepertoires of tissue infiltrating T lymphocytes in the varioussubsets were extensive on the basis of TCR V gene family usage.(II) Furthermore, each patient displayed individual specificTCR V gene expression patterns in the various STMC and PBMCderived T cell subsets. However, the majority of these arthritispatients manifested increased expression of multiple TCR V genefamilies in the synovial tissue derived CD4⁺CD45RO^– Tcell population when compared with the peripheral blood derivedCD4⁺CD45RO⁺ subset. Of these gene families, we found enhancedexpression of the TCR V7 and V_ß11 gene segments insynovial tissue to be shared by all four patients analyzed.OH) Nucleotlde sequence analysis of the CDR3 regions of a numberof TCR V regions in the CD4⁺CD45RO⁺ T cell subsets has revealedthat the CDR3 regions comprised within synovial tissue derivedTCR V regions differed from those found in peripheral bloodderived TCR V regions. These differences in CDR3 diversity mightbe the consequence of a specific interaction with particularMHC-peptlde complexes expressed at the site of inflammation.(Iv) The CDR3 region analysis also showed individual specificamlno acid motifs within the N-D-N regions of all analyzed TCRV_ß genes derived from PBMC as well as STMC.     

Identification of two binding sites in staphylococcal enterotoxin B that confer specificity for TCR V{beta} gene products

The enterotoxlns produced by Staphytococcua af1reus are potentmitogens. They stimulate T cells in an oligocional fashion thatIs dependent on the expression of particular variable regiongene elements in the ß-chaln of the TCR (V_ß).The fourth hypervarlable loop of the TCR ß-chaln Isgenerally regarded as the site of contact for both viral andmlcroblal superantigens. Recently, residues 60 and 61 of staphylococcalenterotoxin B (SEB) have been highlighted as central to theinteraction of this toxin with the TCR. We have, therefore,analysed a series of toxins with mutations at these positionsto investigate how amlno add substitutions affect the abilityof mutant toxins to stimulate both human and mouse T cells.Each of the variant toxins induced proliferation in a murineV_ß8.3 T cell clone, whereas a V_ß8.1 T cellclone only responded to native toxin. A panel of nine humanT cell clones expressing six different V_ß elements,all of which responded to native SEB, was tested for reactivityto the variant toxins. Only one V_ß19.1⁺ T cell clonewas found to be sensitive to substitution at positions 60 and61 In a manner analogous to the murine V_ß8.1 T celldone. Seml-quantitatlve analysis of the TCR V_ß expressionof human T cell lines expanded with native and mutant SEB revealedthat none of the variant toxins could stimulate T cells thatexpressed V_ß19.1. Taken together, these results suggestthat the interaction of mouse V_ß8.1 and human V_ß19.1TCRs with SEB differs from other TCRs. Sequence comparisonsof the different TCR V_ß chains indicated that residuesin the second complementarity determining region (CDR2) interactwith the 60–61 loop of SEB. Therefore, a minimum of twodistinct binding modules confer specificity to the interactionof the TCR with SEB.     

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.     

Negative selection by endogenous antigen and superantigen occurs at multiple thymic sites

The site of negative selection in the thymus has been inferredfrom a range of different experiments. Analysis of thymic deletionof Vß5⁺, V_ß11⁺ or Vß17a⁺ cellsH-2E transgenic mice led to the theory that negative selectionoccurs predominantly in the medulla (specifically, through presentationby medullary dendritic cells). Other experiments investigatedwhether transgenic TCR are deleted at the double-positive (DP)or single-positive stage following encounter with peptide ligand:by flow cytometric analysis deletion is generally found to occurat the DP thymocyte stage and as these cells are found predominantlyin the cortex, it has been inferred that this is the key siteof negative selection. The visualization of apoptotic thymocytesin situ has recently been reported for specific examples ofnegative selection. Using a panel of TCR transgenic lines inwhich negative selection occurs at different stages of thymocytedevelopment, we have used TUNEL staining to analyse the anatomicalsites of thymocyte apoptosis. For the first time we have beenable to compare directly the sites of deletion induced by theendogenous cognate peptides or by endogenous superantigen. Weshow that generalization from the medullary deletion of V_ß5⁺,V_ß11⁺ or V_ß17a⁺ cells by the endogenoussuperantigens Mtv 8 and 9 and from limited examples of corticaldeletion by exogenous peptide administered to TCR transgenicmice is over-simplified. Apoptotic thymocytes in mice lackingMtv superantigens are indeed localized in the cortex. However,when deletion is induced by cognate self peptide, apoptosiscan occur in the cortex, the medulla or at the junction betweenthe two.     

Activation of a myelin basic protein-specific human T cell clone by antigen-presenting cells from rhesus monkeys

This study addresses the capacity of peripheral blood mononuclearcells (PBMC) from rhesus monkeys (Macaca mulatta) to presentmyelin basic protein (MBP), a candidate auto-antigen for multiplesclerosis, to MBP-speclfic human CD4⁺ T cell clones. MHC-restrictlonof the human T cell clones was determined with HLA-DR-transfectedL cells, and epitope specificity was established with a panelof overlapping 20-mer peptides. The MHC-DR region of the rhesusmonkeys (Mamu) was characterized serologlcally and by sequenceanalysis. We identified one CD4⁺ HLADRB1*0301-restrlcted T_h1-llkehuman T cell clone (ES-BP8) that was activated to proliferationwith human or rhesus monkey MBP, or peptide MBP 29–48presented by PBMC from six different rhesus monkeys expressingthe Mamu-DRB1*0305 or -DRB1*0306 alleles. After transformationto continuous growth with Herpesvirus salmiri, the T cell clonecould still be stimulated by antigen (Ag) and Ag-presentlngcells (APC) from monkeys. Two other T cell clones with the sameHLA-restriction and the same peptide-specificity did not respondto MBP presented by these rhesus monkeys. The exon 2 sequencesHLA-DRB1*0301, Mamu-DRBV^*0305 and -DRB 1* 0306 differ at positions32, 47, 67, 73 and 86. These amino acid differences are notcritical for the binding of MBP 29–48 and do not abrogate-recognitionby the clone ES-BP8, but interfere with the recognition of thetwo other HLA-DRB1^*0301-restricted T cell clones. In conclusion,studying Ag-presentation from rhesus monkey may provide furtherinsight Into the Interaction of antigenic peptide, TCR and MHC.Furthermore, these results could form a useful basis for theIn vivo transfer of human auto-Ag-specific T cells into rhesusmonkey recipients.     

Skewed T cell receptor V{alpha} repertoire among superantigen reactive murine T cells

Reactivity of murine T cells with viral or bacterial superantigensis clearly correlated with the expression of TCR V_ßdomains. Thus, T cells responding to the minor lymphocyte stimulatorylocus (Mls-1^a) or staphylococcal enterotoxin B (SEB) expresspredominantly TCR V_ß6 or V_ß8.2 respectively.We have investigated the involvement of the other major variableelement of the TCR, the V domain, in these superantigen responses.Using a panel of anti-TCR V mAbs, It is demonstrated that theTCR V repertoire among superantigen stimulated V_ß6⁺or V_ß8.2⁺ blasts (responding to Mls-1^a or SEB respectivelyin vitro) is altered in comparison with anti-CD3 stimulatedcells expressing the same V domains. Furthermore, the TCR Vrepertoire is strongly skewed in TCR V_ß8.2 transgenicmice that have undergone extensive peripheral clonal deletionafter SEB injection. These data imply that the V domain influencessuperantigen recognition by sthe TCR.     

Selection of intestinal intraepithelial lymphocyte T cell receptors: evidence for a dynamic tissue-specific process

An extensive comparison of TCRß V-reglon usage byCD8ß^-CD4⁺CD8⁺ intraepithelial lymphocytes (IEL), CD4^-CD8⁺IEL, and lymph node (LN) T cell subsets in three minor lymphocytestimulating (MIs)-disparate, MHC-ldentical mouse strains revealednovel TCR selection patterns. In cases where forbidden V regionswere expressed by CD8ß^- CD4^-CD8⁺ IEL, the same TCRswere deleted from CD8ß^– CD4⁺CD8⁺ IEL, Indicatingthat lack of CD8ß expression was not solely responsiblefor forbidden V-region expression. These results also suggestedthat CD4 may be involved in negative selection of CD4⁺CD8⁺ IELTCRs. In C57BR/cdJ (Mls-1^b2^b) mice, a major increase in V_ß3⁺CD4⁺CD8⁺IEL but not in other IEL or LN subsets was noted suggestinga subset-specific expansion of V_ß3⁺ cells. Negativeselection of V_ß14⁺ cells in only the CD4⁺CD8⁺ IELsubset further supported the existence of intestine-specificTCR selection processes. Analysis of V-reglon expression ofCD8ß⁺ and CD8ß^–CD4^–CD8⁺ IELsubsets revealed that forbidden V-region expression was notstrictly confined to the CD8ß^– subset in allcases. Overall, the data point to a dynamic, gut-specific TCRselection process that may be antigen driven.     

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.     

T cell subpopulation phenotypes in filarial infections: CD27 negativity defines a population greatly enriched for Th2 cells

Three-color flow cytometric analysis was used to define surfacemarkers which identify the T_h2-type CD4⁺ cells responsible forthe eosinophilia and elevated serum IgE typical of tissue invasivehelminth infections. A group of six mAba to well known cellsurface markers were screened for differential expression onCD4⁺ CD45RO⁺ lymphocytes from normal individuals (NL; n = 6)and filaria-infected patients (PT; n = 10). The majority ofmarkers were expressed equally by both groups, but the CD4⁺CD45RO⁺ cells in the PTs showed significantly higher levelsof expression of HLA-DR than those of NLs (P = 0.014). ThisCD4⁺ HLA-DR⁺ subpopulation was then studied further for itsexpression of an additional 10 activation and adhesion molecules.CD27 showed a trend for lower intensities of expression on PTCD4⁺ HLA-DR⁺ cells than on those of NLs. Analysis of the serumfrom both NLs and PTs revealed that PTs had significantly higherlevels of soluble CD27 and CD25 (IL-2R) In the serum than NLs(P < 0.01 and P = 0.022 respectively) indicating a generalstate of Immune activation and differentiation. Functional analysisof the CD4⁺ HLA-DR⁺ and the CD4⁺ CD27^– subpopulatlonsrevealed that the CD4⁺ HLA-DR⁺ cells produced significantlyhigher levels of IL-5 than the CD4⁺ HLA-DR^– cells (P <0.04), and the CD4⁺ CD27^– cells produced significantlyhigher levels of both IL-4 and IL-5 than the CD4⁺ CD27^–cells (P <0.05 and P <0.001 respectively). Thus, whilethe CD4⁺ CD27^– and CD27⁺ subpopulatlons contain T_h1 andT_h0 cells, only the CD4⁺ CD27^– population contains theT_h2 cells (producing both IL-4 and IL-5).     

Tolerance induction by elimination of subsets of self-reactive thymocytes

Immature thymocytes expressing TCRs which confer reactivityto self-MHC molecules are subject to efficient elimination asa result of negative selection. Previously, we have identifieda lineage of H-2K^b Tg mice, CD2K^b-3, which fails to reject skingrafts from mice expressing H-2K^b even though H-2K^b-specrficcytotoxlc T cells can be generated in vitro. We now show thatbone marrow derived cells are responsible for tolerance inductionand that tolerance is acquired, at toast in part, by negativeselection in CD2K^b-3 mice. Thymocytes expressing two differenttransgenic TCR (TCR-T_g) clonotypes conferring reactivity toH-2K^b are eliminated prior to the CD8⁺CD4⁺ stage of differentiationin double Tg (CD2K^b-3xTCR-T_g)F₁ mice. As in other cases wherethymocytes from TCR-T_g mice develop in the presence of deletingligands, large numbers of TCR⁺ CD8^–CD4^– T cellsaccumulate in double Tg mice. However, these T cells fail torespond to H-2K^b in vitro but can be activated with immobilizedanti-clonotyplc antibody. Consequently, thymocytes expressingthese types of TCR molecules represent a fraction of H-2K^b-reactivethymocytes which are unable to mature into T cells capable ofmounting H-2K^b-specific cytotoxic responses. Presumably, precursorsof H-2K^b-spicific cytotoxlc T cells found in the periphery ofCD2K^b-3 mice express a distinct repertoire of TCR moleculesconferring reactivity to H-2K^b. We consider potential explanationsto account for this discrepancy and their wider implications,including the possibility that the repertoire of thymocyteaable to recognize setf-H-2Kb molecules In CD2K ^b-3 mice is dividedinto distinct subsets; those which are, and those which arenot, subject to negative selection.     

Use of the orthogonal design method to study the synergistic effects of asbestos fibres and 12-O-tetradecanoylphorbol-13-acetate (TPA) in the BALB/3T3 cell transformation system

An orthogonal design method was used to study the transformingeffects of chrysotile and crocidolite asbestos fibres in BALB/3T3cells. Three experiments, designed by tables L₉ (3⁴), L₈ (2⁷)and L₆ (3¹ x 2²) of the orthogonal method respectively, wereperformed separately. The results indicate exponential reductionsin survival of treated cells concomitant with a linear increasein exposure concentrations from 0.1 to 10.0 µg/cm², andthat chrysotile was more toxic than crocidolite; the total transformationfrequency was significantly increased with both chrysotile andTPA concentrations. There was synergism between chrysotile andTPA in sequential treatment, which suggests that chrysotileis an initiator and has a complete transforming effect at 10.0µg/cm². Crocidolite only has an initiating-like effectwithin the dose range of 0.1 – 10.0 µg/cm², andno synergistic effect when associated with TPA. ¹Present address: Department of Occupational Health, Shanghai Medical University, Shanghai 200032, People's Republic of China ²To whom correspondence should be addressed.     

Ly-49 G2+ NK cells are responsible for mediating the rejection of H-2b bone marrow allog rafts in mice

NK cells can mediate the specific rejection of bone marrow butnot solid tissue allografts in lethally irradiated mice. NKcells are also responsible for the phenomenon of ‘hybridresistance’ in which F₁ hybrid H-2 heterozygous mice canreject parental H-2 homozygous bone marrow grafts. Ly-49C andLy-49 G2 are markers identified on subsets of NK cells. WhileLy-49C⁺ NK cells have been demonstrated to mediate the specificrejection of H-2^d bone marrow allografts, the role of the Ly-49G2⁺ NK subset is unclear because depletion of this subset invivo did not affect splenic NK activity against tumor targets.Through bone marrow transplantation typing studies, we demonstratethat Ly-49 G2⁺ NK cells complement Ly-49C⁺ NK cells in thatthey specifically mediate the rejection of H-2^b bone marrowallografts in lethally irradiated mice. In support of this,depletion of the Ly-49C⁺ NK subset in vivo also enhanced theability of the mice to reject H-2^b bone marrow cells suggestingthat the depletion was augmenting the ability of the Ly-49 G2⁺NK cells to reject the marrow allografts. Depletion of Ly-49G2⁺ NK cells in F₁ hybrid mice abrogated their ability to rejectparental H-2^b but not H-2^d bone marrow grafts. Therefore, Ly-49G2 denotes, a subset of NK cells that appears to play a criticalrole in the recognition of H-2^b bone marrow cells in allogeneicand F₁ hybrid mice.     

Expression of an ovalbumin-specific V{beta}8.2 TCR transgene inhibits collagen arthritis in B10.Q mice

Previous studies have illustrated the importance of T cellsbearing ß TCRs in the induction and development ofcollagen induced arthritis (CIA) in mice. However, the scopeof TCR usage in CIA has yet to be clearly defined. Given theinherent diversity of the TCR repertoire, the relative flexibilityof the arthritogenic TCR repertoire specific for type II collagen(CII) is not clear. Therefore, we chose to examine the influenceof a highly skewed TCR repertoire on CIA. Arthritis susceptibleB10.Q (H-2^q) mice were mated with C57L (H-2^b) animals expressingan ovalbuminspecific V_ß8.2 TCR transgene (Tg) andTg⁺ offspring were further backcrossed to B10.Q. HomozygousH-2^a/q, V_ß8.2 Tg⁺ mice displayed a high level of V_ß8.2⁺T cells in peripheral blood. However, expression of some endogenousV_ß TCR, such as V_ß14, was still detected.Upon immunization with bovine CII in adjuvant, V_ß8.2Tg⁺ mice were highly resistant to CIA when compared with Tg^–littermates. Analysis of sera demonstrated a marked reductionin antibody specific for homologous mouse CII as well as heterologousbovine CII in Tg⁺ animals. Interestingly, V_ß8.2 Tg⁺mice still mounted good antibody responses following immunizationwith human thyroglobulin, indicating that the skewed TCR repertoireaffected anti-CII but not antithyroglobulin responses. Thus,our findings show that constraints placed on the TCR repertoireInhibit pathogenic responses against CII and suggest that inH-2^q mice the arthritogenlc TCR repertoire bears only limitedflexibility.     

Natural killer cell activity in lymphocytic choriomeningitis virus-infected {beta}2-microglobulin-deficient mice

We have investigated the induction and role of natural killer(NK) activity in lymphocytic choriomeningitis virus (LCMV)-infectedß₂-microglobulin-deficient (ß₂m^–)mice. We demonstrate that LCMV infection is more effective thanpolyinosinic:poiycytidylic acid (poly I:C) at stimulating NKactivity in ß₂m^– .In addition, ß₂m^–NK cells respond poorly to in vitro treatment with IL-12. Thetarget specificity of the virally induced NK cells is similarto that previously reported for chemically induced ß₂m^–NK cells. In both cases they can lyse YAC-1 tumor cells butare unable to kill ß₂m^– orß₂m⁺ T cellblasts. We have also found that the time course of inductionof NK and cytotoxic T lymphocyte (CTL) activity by LCMV in ß₂m^–mice is delayed compared with normal mice. Maximal NK and CTLactivity is attained at day 8 and 10 post-infection respectivelyin ß₂m^– compared with day 4 and 6—8 inB6 mice. Whereas normal mice die {small tilde}7 days followingintracranial infection with LCMV, the course of disease in ß₂^–mmice is protracted and characterized by a marked loss of bodyweight. We show that although the CD4⁺ CTL response in thesemice is intimately involved in mediating weight loss, the virus-inducedNK cells do not appear to play a role in the disease.