Co-transfer of B cells converts resistance into susceptibility in T cell-reconstituted, Leishmania major-resistant C.B-17 scid mice by a non-congnate mechanism

Resistance to infection of mice with Leishmania major parasitesis dependent on the production of IFN- by CD4⁺ T helper cells.C.B-17 scid mice, lacking both T and B cells, succumb very quicklyto the infection, but develop resistance if reconstituted withappropriate numbers of T cells from BALB/c mice. In this model,we studied the role of B cells with regard to their abilityto influence disease outcome and to function as antigen-presentingcells for T cells. For this purpose, we reconstituted scid mice(H-2^d) with either T cells or with T and B cells obtained from(BALB/c x BALB.B)F₁ mice (H-2^d x ^b), and infected them withL. major parasites 1 day after reconstitution. Mice reconstitutedwith T cells alone cured the disease, whereas additional B cellreconstitution led to susceptibility. Healing was associatedwith a predominant T_h1-type response. In all mice, L. mayor-specificT cell proliferation was restricted to the MHC phenotype ofthe recipient (H-2^d) but not to that of the donor (H-2^d x ^b),indicating that there was no detectable contribution of donorB cells in the priming of a T cell response. Furthermore, Bcells, when purified from infected BALB/c mice, were unableto stimulate a L. mayor-specific CD4⁺ T cell clone (L1/1) withoutaddition of exogenous antigen, in contrast to macrophages fromthe same animal. These data suggest that B cells, in vivo, donot carry L. major antigen in a form capable of activating specificCD4⁺ T cells. Therefore, B cells promote disease by means otherthan cognate interaction with CD4⁺ T cells.     

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.     

Over coming the crypticity of a viral T cell Determinant by insertion into a chimeric bacterial protein

Among the potential T cell determinants contained In a proteinantigen, the T cell response only focuses on a few immunodomlnantT cell determinants, whereas cryptic epitopes remain hiddento the Immune system. In the present work, we have studied theantigen processing and presentation of the C3: 93-115 sequenceof Mahoney pollovlrus VP1 protein, which Is Immunodomlnant InH-2^d but cryptic In H-2_s and H-2_q mouse MHC haplotypes. Forthis purpose, we genetically Inserted the C3 determinant Intofive internal sttes of a bacterial protein, the maltose bindingprotein of Escherichla coll (MalE). In four out of five Insertionsites of MalE, the C3 determinant retained Its Immunodominancewhen the purified hybrid proteins were Injected to BALB/c (H-2_d)mice. Moreover, In SJL/J (H-2^d) mice, in three out of five MalE-C3constructs, the new structural environment of the cryptic C3epltope rescued Its processing and its in vivo presentationto T cells. In contrast, In DBA/¹ (H-2^d) mice, although MalE-C3chimeric proteins were correctly processed in vitro, the C3epitope remained cryptic in vivo. In this case, the impairmentto stimulate a T cell response in vivo was correlated with ashort time persistence of C3 peptides bound to Aq moleculesat the surface of live antigen-presenting cells. These resultsemphasize the role of flanking residues on the lack of processingof cryptic determinants and the Importance of the life spanof peptlde-MHC complexes to stimulate T cell responses.     

Correlation of effector function with phenotype and cell division after in vitro differentiation of naive MART-1-specific CD8+ T cells

Adoptive transfer of antigen-specific CD8⁺ T cells may representan effective strategy for immunotherapy of tumors such as melanoma,but is limited by the number and functionality of in vitro expandedT cells. Here, we document that although ELAGIGILTV-specificCD8⁺ T cells from different donors initially possessed a naivephenotype, after antigen-induced in vitro expansion two distinctphenotypes correlating with cell proliferation rate emergedin the different donors. Those cultures achieving fewer cumulativepopulation doublings (CPDs) were cytotoxic and displayed a CD45RA⁺CCR7^–phenotype. In contrast, cultures reaching higher CPDs were non-cytotoxicT cells with a CD45RA^–CCR7^– phenotype. Thus, thegeneration of larger numbers of ELAGIGILTV-specific CD8⁺ T cellscorrelates negatively with the acquisition of a CD45RA⁺CCR7^–phenotype and cytotoxic capacity. A better understanding ofthe differentiation pathways of cytotoxic T cells to obtainoptimally efficient cells for adoptive transfer will allow thedevelopment of new immunotherapy protocols.     

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.     

Human CD4/CD45RA+ and CD4/CD45RA T cell subsets express CD4-p56lck complexes, CD4-associated lipid kinases, TCR/CD3-p59fyn complexes, and share similar tyrosine kinase substrates

T cell activation appears to be regulated by an interplay betweenprotein tyrosine kinases (PTKs) and protein tyrosine phosphatases(PTPases). p56^lck and p59^fyn have been found to associate withCD4 and TCR-CD3 respectively. The CD45 family of transmembranePTPases has been shown to be able to regulate the activitiesof these receptor-associated PTKs in vitro. In man, CD45 containsfive different isoforms whose distribution defines subsets ofT cells having distinct activation requirements and in vitrofunctions.Several groups have reported a physical interaction betweendistinct isoforms of CD45 and CD2, CD4, and the TCR-CD3 complex.Given the potential regulatory interaction between CD45 andPTKs in CD4⁺ subsets expressing different CD45 isoforms, wehave examined CD4 associated and TCR-CD3^– associated PTKactivities, associated phosphatidyl inositoi (PI) kinases andsubstrates of tyrosine phosphoryiation in CD45RA⁺and CD45RA^–CD4⁺ T cell lines derived from peripheral blood. Both subsetsexpress CD4-assoclated p56^lck and TCR-CD3-associated p59^fynkinases which exhibit identical in vitro phosphoryiation atthe Y-394 and Y-420 autophosphorylation sites respectively.Further, both subsets exhibited PI kinases activity associatedwith CD4-p56^lck. Consistent with these observations, anti-CD3crosslinklng induced the phosphoryiation of a similar spectrumof intracellular substrates in these CD45RA⁺and CD45RA^–CD4⁺ T cell lines. These observations indicate that despitethe possible interaction between CD45 isoforms and CD4 or TCR-CD3,the mere expression of the CD45RA isoform does not in and ofitself alter the presence of receptorassociated kinases or theirintracellular targets.     

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.     

Differential effects of cordycepin on the induction of sister-chromatid exchange and chromatid breaks in BALB/Mo mouse lymphocytes treated with mitomycin C

BALB/Mo mice lymphocytes, carrying endogenous Moloney murineleukaemia virus (M-MuLV), show significantly higher in vitrobaseline frequencies of sister chromatid exchange (SCE) thanlymphocytes from control (M-MuLV free) BALB/c mice. In vitrotreatment of lymphocytes with cordycepin (10 µg/ml), anantiviral substance, lowers the level of SCE in BALB/Mo cellsto the same value of BALB/c cells. The drug also reduces thehigher sensitivity of BALB/Mo compared to BALB/c lymphocytesto the induction of SCE by mitomycin C (MMC) administered eitherin vitro (3 x 10^–8, 10^–7 M) or in vivo (0.3, 3 mg/kg).BALB/Mo lymphocytes treated in vivo with a high dose of MMC(10 mg/kg) show reduced susceptibility to the induction of SCEbut increased frequencies of chromatid breaks and micronuclei.In this situation, cordycepin increases the level of SCE inBALB/Mo lymphocytes to exactly the level seen in BALB/c cells,but it does not affect the frequency of chromosomal aberrations.Since cordycepin is known to inhibit poly(A) synthesis, thusblocking RNA maturation, it is suggested that M-MuLV proviralintegration is not the sole factor responsible for the alteredsusceptibility of BALB/Mo lymphocytes to SCE induction, butthat it is most likely viral gene expression that is neededfor this effect to occur. On the contrary, the high susceptibilityof BALB/Mo lymphocytes to the induction of chromatid aberrationsby a high dose of MMC administered in vivo seems to be independentof viral maturation. ²To whom correspondence should be addressed     

An MRL/MpJ-lpr/lpr substrain with a limited expansion of lpr double-negative T cells and a reduced autoimmune syndrome

The autosomal recessive mutant gene, lpr, has been shown toaccelerate the progression of lupus-like autoimmune disease,which is associated with a massive expansion of a unique CD4^–CD8^–double-negative T cell subset, in MRL/MpJ mice. Here we reporta substrain of MRL/MpJ-lpr/lpr (MRL-lpr) mice which live almosttwice as long with delayed development of glomerulonephritis,compared with conventional MRL-lpr mice. This substrain, termedMRL-lpr.II (II for long-lived), develops generalized lymphadenopathycharacteristically seen in MRL-lpr mice. However, the expansionof a double negative lpr T cell subset is markedly limited witha mean value of 15% in their lymph nodes compared to about 70%in conventional MRL-lpr mice. Overall production of autoantibodies,such as anti-DNA and rheumatoid factors, does not significantlydiffer between the two MRL-lpr mice. However, serum levels ofcryoglobulins, whose major component is lgG3, are markedly diminishedin MRL-lpr.ll mice with a parallel decrease in lgG3. Since MRL-lpr.llmice still carry the lpr mutation, as documented by the presenceof defects in the Fas antigen, a possible new mutation in thissubstrain may play a significant role in the pathogenesls oflupus-like autoimmune syndrome.     

Engagement of CD45 during in vitro priming enhances antigen-specific Th cell frequencies

CD45 is a transmembrane protein tyrosine phosphatase expressedby all lymphoid cells including T cells. Substantial experimentaldata has shown that CD45 maintains a permissive state for TCRsignaling. The highly glycosylated extracellular domain of CD45may be the site of Interaction with regulatory lectin-like counter-receptorson antigen-presenting cells. The mAb NDA5, recognizing a uniquebut broadly distributed epitope of CD45, was used to study thepossible immunoregulatory role of CD45 during antl-CD3 and antigen-specificCD4⁺ T cell activation. In vitro priming of peripheral bloodmononuclear cells with peptlde antigens in the presence of mAbNDA5 results in a higher frequency of antigen-specific T cells.The responses of both naive and memory T cells to peptide antigenswere sensitive to mAb NDA5-enhanced priming. Anti-CD3 activationof normal resting T cells, in the presence of mAb NDA5, resultedIn enhancement of tyrosine phosphorylation of specific intracellularproteins associated with TCR signal transduction. In cultureswithout antigen, mAb NDA5 down-regulated the cell surface expressionof both CD3 and CD4, yet did not stimulate proliferation ofresting T cells. Together these results suggest that engagementof CD45 during in vitro priming has a significant effect onthe development of antigen-specific T cell populations.     

Development of retrovirally marked human T progenitor cells into mature thymocytes

Retroviral vectors have been used in most human gene therapytrials that have been undertaken. Many of these therapies havefocused on the introduction of genes into hematopoietic stemcells with the goal of obtaining expression in the mature Tlymphocytic progeny. It has proven difficult to achieve expressionin the lymphoid lineage, although several groups have demonstratedlow expression of transduced genes in the myelold lineage. Inthis study we used an in vitro thymic organ culture in whichstem/progenitor cells can develop into T cells and all intermediatestages can be studied and manipulated to investigate the fateof a retrovirally introduced Escherlchla coll LacZ gene in thissystem. Here we show that certain conditions can transduce JurkatT cells, three different antigen-specific T cell clones andCD34⁺CD3^–CD4^–CD8^– thymocytes (progenitor Tcells) with high (>80%) efficiency. Moreover, retroviraltransduction with the LacZ gene does not inhibit T and NK celldifferentiation of progenitor cells in fetal thymic organ cultures(FTOC). The LacZ gene also is functionally expressed at allstages of development, although the expression decreases somewhatduring differentiation. This experimental system, combiningFTOC and retroviral transduction, provides a genetic tool forthe study of human T cell development.     

Migration pathways of CD4 T cell subsets in vivo: the CD45RC- subset enters the thymus via {alpha}4 integrin-VCAM-1 interaction

The present investigation examines the localization and migrationof purified T cell subsets in comparison with B cells, CD8 Tcells and CD4⁺CD8^– single-positive thymocytes. CD4 T cellsubsets in the rat are defined by mAb MRC OX22 ( anti-CD45RC),which distinguishes resting CD4 T cells (CD45RC⁺) from those(CD45RC^–) which have encountered antigen in the recentpast– subpopulations often referred to as ‘naive’and ‘memory’. Purified, ⁵¹Cr-labelled CD45RC⁺ CD4T cells broadly reflected the migration pattern of CD8 T cellsand B cells. Early localization to the spleen was followed bya redistribution to mesenteric lymph nodes (MLN) and cervicallymph nodes ( CLN) , B cells migrating at a slightly slowertempo. There was almost no localization of these subpopulationsto the small or large intestine [Peyer's patches (PP) excluded].In contrast, CD45RC^– CD4 T cells (indistinguishable insize from the CD45RC⁺ subset) localized in large numbers tothe intestine; they were present here at the earliest time point(0.5 h) , persisted for at least 48 h but did not accumulate,indicating a rapid exit. Numerically, localization of CD45RC^–CD4 T cells in the MLN could be accounted for entirely by afferentdrainage from the intestine. Unexpectedly, CD45RC^– CD4T cells (but not other subsets) localized and accumulated inthe thymus. In vivo treatment with mAb HP2/1 against the integrin₄ subunit inhibited almost entirely CD45RCT^– CD4 T cellmigration into the PP (98.1%), intestine (87.1%) , MLN (89.1%)and thymus (93.5%) migration into the CLN was only reduced byhalf. To distinguish between recognition of MAdCAM-1 and VCAM-1by ^4–containing integrins, recipients were treated withmAb 5F10 against rat VCAM-1. Except for the thymus and a smallreduction in CLN, localization of CD45RC^– CD4 T cellswas unaffected; entry to the thymus was almost completely blocked(92.3%) by anti-VCAM-1. The results indicated (i) that CD45RC^–CD4 T cells alone showed enhanced localization to the gut andPP, probably via ₄ß₇-MAdCAM-1 interaction; ( II) thatmany CD45RC^– cells entered nonmucosal LN independentlyof ₄ integrin or VCAM-1; and (III) that entry of mature recirculatlngCD45RC^– CD4 T cells into the thymus across thymic endothellumwas apparently regulated by ₄ integrln-VCAM-1 interaction.     

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.     

Progression of T cell lineage restriction in the earliest subpopulation of murine adult thymus visualized by the expression of lck proximal promoter activity

The proximal promoter of lck directs gene expression exclusivelyin T cells. To investigate the developmental regulation of thelck proximal promoter activity and its relationship to T celllineage commitment, a green fluorescence protein (GFP) transgenic(Tg) mouse in which the GFP expression is under the controlof the proximal promoter of lck was created. In the adult GFP-Tgmice, >90% of CD4⁺CD8⁺ and CD4⁺CD8^– thymocytes, andthe majority of CD4^–CD8⁺ and CD4^–CD8^– [double-negative(DN)] thymocytes were highly positive for GFP. Slightly lowerbut substantial levels of expression of GFP was also observedin mature splenic T cells. No GFP⁺ cells was detected in non-Tlineage subsets, including mature and immature B cells, CD5⁺B cells, and NK cells, indicating a preserved tissue specificityof the promoter. The earliest GFP⁺ cells detected were foundin the CD44⁺CD25^– DN thymocyte subpopulation. The developmentalpotential of GFP^– and GFP⁺ cells in the CD44⁺CD25^–DN fraction was examined using in vitro culture systems. Thegeneration of substantial numbers of ß and T cells aswell as NK cells was demonstrated from both GFP^– and GFP⁺cells. However, no development of B cells or dendritic cellswas detected from GFP⁺ CD44⁺CD25^– DN thymocytes. Theseresults suggest that the progenitors expressing lck proximalpromoter activity in the CD44⁺CD25^– DN thymocyte subsethave lost most of the progenitor potential for the B and dendriticcell lineage. Thus, progression of T cell lineage restrictionin the earliest thymic population can be visualized by lck proximalpromoter activity, suggesting a potential role of Lck in theT cell lineage commitment.     

ER-MP12 antigen, a new cell surface marker on mouse bone marrow cells with thymus-repopulating ability: II. Thymus-homing ability and phenotypic characterization of ER-MP12-positive bone marrow cells

In the accompanying paper we showed that six distinct subsetsof bone marrow (BM) cells can be identified using the mAb ER-MP12and ER-MP20 in two-colour immunofluorescence analysis. Uponintrathymic transfer into sublethally irradiated mice thymus-repopulatingability was restricted to ER-MP20^– BM cells expressingeither high or intermediate levels of the ER-MP12 antigen (1–2%and –30% of BM nucleated cells respectively). The highestfrequency of thymus-repopulating cells was found in the minorsubset of ER-MP12⁺⁺20^– BM cells. In the present studywe demonstrate that upon intravenous transfer, thymus-homingand-repopulating BM cells are exclusively confined to the ER-MP12⁺⁺20^–and ER-MP12⁺20^– subpopulations, the highest frequencybeing detected among ER-MP12⁺⁺20^– BM cells. Analysis ofthe peripheral blood leucocytes of reconstituted mice showedthat not only prothymocytes but also progenitorcells of theB cell lineage as well as the myelold lineage were present withinboth subsets. Three-colour flow cytometric analysis revealedthat ER-MP12⁺⁺20^– BM cells in particular were phenotyplcallyheterogeneous with respect to the expression of the cell surfacemarkers Thy-1, Sca-1, CD44, B220 and c-kit. Taken together ourdata demonstrate that ER-MP12 positively identifies BM cellswith the ability to home to and repopulate the thymus. The phenotypicheterogeneity displayed by the ER-MP12⁺⁺20^– BM subset,containing the highest frequency of thymus-homing and-repopulatingcells, provides a basis for further separation of prothymocyteactivity from other haematopoietic activities in the BM of themouse.     

The nature of the immune response (Ir) gene defect for pigeon cytochrome c in [B10.A(4R) x B10.PL]F1 mice: A Comparison Between Thymic Selection and Antigen Presentation

[B10.A(4R) x B10.PL]F₁ mice are low responders to pigeon cytochromec, while [B10.A(2R) x B10.PL]F₁ and B10.A mice are high responders.The in vivo site at which the different aliomorphs of the E_aIa molecule exert their Ir gene effect on the immune responseto pigeon cytochrome c was examined by creating two differentsets of radiation-induced bone marrow chimeras. [B10.A(4R) xB10.PL]F₁(b.m.) B10.A(lrr.) chimeras, which possess antigen-presentingcells (APC) of the low responder, but whose T cells are educatedin a high responder environment, were found to be low respondersto pigeon cytochromec. In contrast, B10.A(b.m.) [B10.A(4R)x B10.PL]F₁(lrr.) chimeras, which possess APC of the high respondertype, but whose T cells are educated in a low responder environment,responded to pigeon cytochrome c Addition of B10.A APC to thefirst type of chimera, both prior to antigen priming and atthe time of the secondary challenge in vitro, converted 50%of the animals to responders Furthermore, [B10.A(4R) x B10.PL]F₁mice responded to pigeon cytochrome c If they were primed witha 10-fold greater antigen dose and restimulated in vitro Inthe presence of B10.A APC. These results suggest that the primarysite of the Ir gene defect in this system is at the level ofantigen presentation and not in the T cell repertoire.     

Evaluation of presence and functional activity of potentially self-reactive T cells in aged mice

Autoimmunity is known to increase in aging. A possible factorcould be an alteration in the T cell repertoire wIth advancingage. Antibodies to the variable region of the ß chainof the TCR activate T cells and can serve as probes for analysisof the T cell repertoire. We have used V_ß3 and V_ß17aantibodies to determine the presence and functionality of normallydeleted T cells bearing potentially self-reactive TCR in peripherallymphoid tissue and blood from aged (SJL/J x BALB/c) F₁ LAF₁and BALB/c mice. Although an occasional 20- to 24-month-oldmouse exhibited V_ß3⁺ or V_ß17a⁺ T cells intheir lymph nodes or peripheral blood lymphocytes (PBL) slightlyabove the range for normal young mice of these I-E⁺ strains,there was no striking ‘escape’ from the normal thymicdeletion process. However, responsiveness to anti-V_ß3and anti-V_ß17a was slightly higher In aged, and particularlyIn aged thymectomlzed (TX), than in young mice. This was incontrast to proliferative responses to stimulation with antibodyto the normally expressed V_ß8 which were lower inthe lymph nodes from aged than from young mice. The PBL of some30- to 36-month-old mice were also examined. Enhanced numbersof ‘forbidden’ V_ß bearing T cells wereseen more frequently at this age. In spite of the age-relateddecrease in overall CD4/CD8 T cell ratios in all organs, themice with relatively high V_ß17a⁺ T cells exhibitedproportionally more CD4⁺ cells in that V_ß population.We conclude that the ‘forbidden’ T cells that respondto anti-V_ß stimulation in the 20- to 24-month-oldmice are most likely of extra-thymic origin, since they weremore readily detectable in aged TX mice. Potentially self-reactiveCD4 (and CD8) single-positive T cells were detectable in PBLonly in very aged (30–36 months old) euthymic mice.     

Immunosuppression induced by hepatic portal venous immunization spares reactivity in IL-4 producing T lymphocytes.

Immunization of naive or specifically primed C3H/HEJ with irradiated B10.BR spleen cells via the hepatic portal vein leads to an antigen specific decrease in the proliferative and cytotoxic response to B10.BR antigen assayed in vitro (and to increased graft survival of B10.BR grafts in vivo). This effect seems to be mediated in the main by a decrease in IL-2 production from CD4+ T lymphocytes of mice given antigen by the portal route, which is in turn caused by a decreased precursor frequency of IL-2-producing cells. No clear decrease in IL-4 production was seen. Hepatic APC isolated from mice receiving antigen via the portal vein were unable to induce IL-2 production from a C3H/HEJ anti-B10.BR cell line in vitro, in contrast to splenic APC derived from the same mice. Even when antigen was given by conventional (systemic) intravenous routes (in this case via the lateral tail vein) hepatic APC isolated from those mice were unable to stimulate IL-2 production from this cell line. Furthermore, 24 h exposure of a cell line to antigen pulsed hepatic APC left those cells refractory to a subsequent restimulation with antigen presented by splenic APC. Spleen lymphoid cells from primed mice challenged in vivo with B10.BR liver cells (i.v.) were similarly unable to produce IL-2 on rechallenge in vitro with irradiated B10.BR spleen cells, though no defect was seen if in vivo challenge was with B10.BR spleen cells. These data imply that presentation of multiple minor cell surface antigens by hepatic APC leads to specific anergization of IL-2 producing T cells, in a fashion which seems to be distinct from that previously reported as due to 'veto-like' activity.