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.     

CD69 cell surface expression identifies developing thymocytes which audition for T cell antigen receptor-mediated positive selection

CD69, an ‘activation marker’ that is rapidly inducedon mature T cells after stimulation through the T cell antigenreceptor (TCR) was found to be expressed on 10% of normal thymocytes.All of these CD69⁺ thymocytes express ß TCR, and theyinclude both TCR^lowCD4⁺CD8⁺ and TCR^highCD4⁺CD8^– or CD4^–CD8⁺thymocytes. The CD69⁺ cells can be further segregated into heat-stableantigen (HSA)⁺TCR^*HSA^–TCR^high and HSA⁺TCR^high thymocytepopulations. None of CD69⁺ cells express the mature T cell markerQa-2. Thus CD69⁺ cells present in vivo appear phenotyplcallyto represent transitional cell populations between immatureTCR^lowHSA⁺Qa-2^– double-positive cells and mature TCR^highHSA^–QA-2⁺single-positive cells. In addition, TCR engagement by MHC moleculesis required for CD69 expression in the thymus. Taken together,the CD69 ⁺ thymocytes appear to represent the cells auditioningin positive selection process or they are the cells that havebeen positively selected recently. Analysis of a TCR transgenicmouse model revealed an increased number of CD69⁺ thymocytesin a positively selecting thymus, whereas no CD69⁺ transgenicTCR⁺ thymocytes were observed in the non-selecting thymus. Basedon the results of this study, we suggest that the surface expressionof CD69 serves as a useful marker to identify and trace thosethymocytes that are engaged in the TCR-mediated positive selectionprocess in the thymus.     

Entry of CD4 CD8 immature thymocytes into the CD4/CD8 developmental pathway is controlled by tyrosine kinase signals that can be provided through TCR components

Entry of thymus-migrated precursor cells into the CD4/CD8 developmentalpathway was analyzed by using the short-term organ culturesof day 14 fetal mouse thymus lobes. Organ cultures of CD4^–CD8^–day 14 fetal thymocytes for 1-2 days resulted in the generationof CD4^–CD8⁺ cells, which were mostly immediate precursorcells for CD4⁺CD8⁺ thymocytes. This differentiation of CD4^–CD8^–thymocytes into CD4^–CD8⁺ cells was strongly enhanced byanti-CD3 antibodies. The anti-CD3-induced generation of CD4^–CD8⁺cells was even found in the immunodeficient scid fetal thymuscultures, and the cell surface CD3 expression on the scid fetalthymocytes could be directly visualized, indicating that functionalCD3 could be expressed on CD4^–CD8^– immature thymocyteswithout being associated with rearranged TCR components. Theanti-CD3-lnduced generation of CD4^–CD8⁺ cells from scidand normal fetal thymus cultures was inhibited by tyrosine kinaseinhibitors Herblmycin A and Tyrphostin. The generation of CD4^–CD8⁺cells in unstimulated normal fetal thymus cultures was alsomarkedly inhibited by the tyrosine kinase inhibitors but notby Cyclosporin A, suggesting that tyrosine klnase-dependentbut calclneurin-lndependent signals were essential for the differentiationof CD4^–CD8^– thymocytes. Interestingly, the generationof CD4^–CD8⁺ cells from the normal fetal thymus cultureswas modestly but consistently enhanced by anti-TCRßantibody, suggesting that functional TCRß in additionto CD3 was expressed on normal CD4^–CDS⁺ immature thymocytes.On the other hand, anti-TCR antibody did not affect this differentiationin the normal fetal thymus cultures and the generation of CD4^–CD8⁺cells from the normal fetal thymus cultures of TCR-deficientmice was still enhanced by anti-TCRß or anti-CD3 antibodies,indicating that either TCR chains or TCR⁺ cells were not involvedin the control of the differentiation into CD4^–CD8⁺ cells.These results indicate that the entry of CD4^–CD8^–immature thymocytes into the CD4/CD8 developmental pathway iscontrolled by tyrosine kinase signals and that these signalscan be provided through the engagement of TCR-CD3 complexeswith or without TCRß chains expressed on the CD4^–CD8^–immature thymocytes.     

Functional CD4+T cell subsets defined by expression of CD45RC and NTA260 antigens and age-associated polarization in murine lupus

Using two mAb, one specific to the alternative exon 6-dependentepitope of CD45 molecules(JH6.2) and one a natural thymocytotoxicautoantibody (NTA) with an unknown reactive epitope (NTA260),we subdivided splenic CD4⁺ T cells from 2-month-old BALB/c miceinto five phenotypically distinct subsets. CD45RC⁺NTA260^–(SI) cells were phenotypically analogous to CD4⁺ T cells predominatingin newborn mice and produced a significant amount of IL-2, butnot so IL-4, IL-10 or IFN- when stimulated with immobilizedanti-CD3 mAb in vitro. They appeared to consist mainly of naiveT_hP cells. The CD45RC⁺;NTA260⁺ (S II) subset also produced IL-2,but not other cytokines; however, the IL-2 levels produced weremuch higher than seen with the S I subset, thereby suggestingthe predominance of further maturated T_hP cells. The D45RC^–NTA260⁺(S III) subset mainly produced IL-4, IL-10, IFN- and less IL-2,and contained memory cells that helped the secondary antibodyresponse to a recall antigen, and hence contained T_h2 and probablya mixture of T_h0 and T_h1 cells. The CD45RC^–NTA260^–(S IV) subset was a poor responder to the immobilized anti-CD3mAb. The CD45RC^brightNTA260^dull(S V) subset consisted of a smallnumber of cells that were phenotypically analogous to activatedCD4⁺ T cells. While an age-associated decrease in the proportionof S I and less markedly in S II and in turn increase in S IIIsubsets of CD4⁺ T cells occurred in normal BALB/c mice, autoimmunedisease-prone (NZBxNZW)F₁ mice showed a marked age-associateddecrease in the proportion of not only S I, II but also IIIsubsets. As aged (NZBxNZW)F₁ mice carry CD4⁺ T helper cellsfor IgG anti-DNA antibody production, such age-associated polarizationto the S IV subset appears to be critical in the pathogeneslsof autoimmune disease in these mice.     

Activation and 'deletion' of self-reactive mature and immature T cells during ontogeny of Mls-1a mice: implications for neonatal tolerance induction

We assessed the kinetics of V_ß6⁺ T cell eliminationin the lymph nodes and thymus during Mls-1^a mouse ontogeny.Our results suggest that induction of tolerance to Mls-1^a antigensinvolves mechanisms other than clonal deletion of immature Tcells in the thymus. Mature CD4⁺CD8^– (CD4SP) T cells wereaffected by Mls-1^a antigens earlier than immature thymocytepopulations. Up to 2 weeks after birth, reduced frequenciesof V_ß6⁺ T cells were detected only in CD4SP cellsfrom the thymus and lymph nodes, and generation of CD4SP cellsin the thymus was blocked at least 1 week earlier than thatof their CD4⁺CD8^loTCR^hl immature precursors. The number of V_ß6⁺CD4SPT cells increased during the first 2 weeks of life and remainedconstant thereafter. We thus found no evidence of deletion ofmature V_ß6⁺CD4SP T cells, as the reduced frequenciesin adult mice can be attributed to the dilution of previouslygenerated cells in lymphoid organs of growing mice, which increasein cellulartty after birth. V_ß6⁺CD4⁺ T cells wereactivated in vivo shortly after birth, as shown by a selectiveincrease in IL-2 receptor a chain expression in the thymus andlymph nodes from day 0 to day 2 after birth. It is thereforelikely that endogenous expression of Mls-1^a antigen shortlyafter birth activates V_ß6⁺CD4SP T cells and rendersthem anergic. This process of tolerance induction may precedethe clonal deletion of immature T cells in the thymus, describedin the adult mouse.     

CD4+Thy1- thymocytes with a Th-type 2 cytokine response

We have identified a small subset of CD3⁺, CD4⁺, CD8^–thymocytes that do not express Thy1 (CD90). This Thy1^–subset represents 1–3.7% of the total number of thymocytesin a naive mouse. CD4⁺Thy1^– thymocytes express high levelsof CD3, intermediate to high levels of heat-stable antigen (HSA),and low levels of CD25, CD45RB, CD69, CD44 and CD62L. They producehigh titers of IL-4 and no IFN- upon stimulation in vitro, aresponse characteristic of T_h2 cells. In the thymi of mice infectedneonatally with a high dose of the retrovirus Cas-Br-E MuLV,the frequency of CD4⁺Thy1^– cells increased ~10-fold. High-dosevirus infection resulted in decreased HSA and increased CD44expression on CD4⁺Thy1^– cells relative to cells from naivemice. CD4⁺Thy1^– cells from high-dose infected mice alsosecreted IL-4 and not IFN- upon in vitro stimulation. We previouslyreported that infection of newborn mice with a high dose ofmurine retrovirus results in the induction of a non-protectiveanti-viral T_h2 T cell response; CD4⁺Thy1^– thymocytes witha T_h2-like cytokine profile may play a role in determining thecytokine bias of this anti-viral response.     

Defective protein tyrosine phosphorylation and altered levels of p59fyn and p56ick in CD4 T cells from HIV-1 infected patients

Early in HIV infection, CD4⁺ lymphocytes exhibit the propertiesof an anergic state characterized by unresponslveness to mltogensor to TCR stimulation and by defective IL-2 production. As tyrosinephosphorylation is the earliest of the biochemical events initiatedby stimulation of CD3-TCR, we studied protein tyrosine phosphorylationin purified CD4⁺ lymphocytes from 25 asymptomatic seropositlvepatients (CD4 T cells >350/mm³) previously stimulated invitro by immobilized antl-CD3 mAb or by co-lmmoblllzed antl-CD3and antl-CD28 mAbs. Purified CD4⁺ lymphocytes from HIV-lnfectedpatients exhibited defective early protein tyrosine phosphorylationin response to CD3 activation when compared with normal subjects.This defect was observed mainly in patients in whom prollferatlveresponses to immobilized antl-CD3 ranged from 2 to 50% of controlvalues obtained in healthy donors, and was frequently associatedwith increased cellular levels of p59^fyn and decreased cellularlevels of p56^ick. Interestingly, these defects appeared to correlatewith the degree of impairment in thymidine incorporation. SinceCD28 mAbs have been reported to enhance prollferatlve responsesto the CD3–TCR pathway in cloned murine or human anergicmodels and to induce tyrosine phosphorylation in human T cells,we studied the role of CD28 mAb as a co-signal. Although antl-CD28co-stlmulatlon augmented the prollferatlve responses in bothcontrols and HIV-lnfected patients, It failed to correct thetyrosine phosphorylation pattern in the latter. Our resultssuggest a relationship between defective early protein tyrosinephosphorylation and impairment of proliferatlve responses inCD4 T cells from HIV-lnfected patients, and evidence is providedthat associated altered cellular levels of the fyn and Ick tyrosinekinases might play an important role in the anergic responseobserved early during HIV infection.     

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.     

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.     

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.     

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.     

CD4 , CD8 {gamma}/{delta} cells from normal mice respond to a syngeneic B cell lymphoma and can induce its differentiation

Lymph nodes and spleens from normal unimmunized mice containsmall numbers of CD3⁺, CD4^–, CD8^– (double negative,DN) T cells. Of these, approximately one-third express the markerLy-5(8220) in a form previously seen only on normal B cellsand a population of DN T cells found in mice genetically proneto develop autolmmunity. DN T cells proliferate when co-culturedwith a syngeneic surface Ig⁺ lymphoma, CH12. After one cycleof stimulation with CH12 almost all of the responding CD3⁺ DNcells express Ly-5(B220), suggesting that it is an activationmarker for some DN T cells. The CH12 responding population alsocontains cells with two other phenotypes, Thy-1⁺, CD4^–,CD8^–, Ly-5(B220)⁺, sIgM^–, CD3^– and Thy-i⁺,CD4⁺, CD8^–, Ly-5(B220)^–, sIgM^–, CD3⁺. TheLy-5(B220)⁺, CD3^– population is no longer found afterrepeated stimulation. While the relationship between these threepopulations is unknown, DN I cells can proliferate in the absenceof CD4⁺ or CD8⁺ cells and therefore their proliferation is notdependent on the presence of other T cells or lymphokines producedby CD4⁺ or CD8⁺ T cells. Anti-CD3 Immunoprecipitation of CH12-respondlngcells reveals at least seven different receptor proteins ofwhich five can also be precipitated with an anti-(C1/C2) monoclonalantibody. Thus at least three different TCR– heterodimersare expressed by CH12-responding T cells. The Thy-1⁺, CD4^–,CD8^–, Ly-5(B220)⁺ cells can provide help to CH12 cellsfor Ig secretion even in the absence of the nominal antigenfor the B lymphoma cells, in summary, these results demonstratethat in normal mice there is a small population of CD4^–,CD8^–, Ly-5(B220)⁺ T cells with / receptors which can providehelp for a syngeneic B cell lymphoma. Received 9 May 1989, accepted 31 May 1989.     

Differential effect of transforming growth factor-{beta}1 on the activation of human naive and memory CD4+ T lymphocytes

Transforming growth factor-ß1 (TGF-ß1) canhave stimulatory or inhibitory effects on cell growth. For severalcell types, the effect of TGF-ß1 was found to correlatewith the differentiation stage of the cells and the presenceof other cytoklnes. We have studied here the influence of TGF-ß1on CD4⁺ T cell activation in relation to the differentiationstage of the cells by evaluating the effect of TGF-ß1on the prollferatlve responses of purified CD4⁺CD45RA⁺ (unprfmed)and CD4⁺CD45RO⁺ (primed) lymphocytes. Under certain conditions,TGF-ß1 exerted a co-stlmulatory effect on peripheralblood CD4⁺CD45RA⁺ T cells whereas the outgrowth of CD4⁺CD45RO⁺T cells was suppressed in any activation system tested. Theenhancement of prollferatlve responses by TGF-ß1 inTCR/CD3 or CD2 stimulated cultures of CD45RA⁺ cells involvedup-regulatlon of CD25 expression and was dependent on the presenceof exogenous IL-2 or CD28 mAbs; IL-7 driven proliferatlve responseswere suppressed by TGF-ß1. These observations wereconfirmed in experiments with purified cord blood (CB) CD4⁺T cells inasmuch as addition of TGF-ß1 caused a 2-to 7-fold increase in IL-2 driven proliferatlve responses ofthese cells. Finally we show that, in contrast to the effectof TGF-ß1 during primary stimulation of CB CD4⁺ Tcells, TGF-ß1 suppressed T cell proliferation for40% in secondary cultures of these cell. Our findings indicatethat TGF-ß1 Is a blfunctlonal regulator of CD4⁺ Tcell growth in vitro, with co-stimulatory capacities duringCD45RA⁺ T cell mediated primary responses and growth suppresslveeffects during secondary responses of CD45RO⁺ T cells.     

Mel14+CD4+ T cells from aged mice display functional and phenotypic characteristics of memory cells

Aging is accompanied by an increased fraction of memory CD4⁺T cells. Despite the fact that human memory cells have beenreported to produce high levels of IL-2, studies in mice andman indicate an age-related decline in IL-2 production. In thepresent study, we examined whether these conflicting resultsdepend on the activation pathway employed in a comparison ofphenotypically distinct CD4⁺ T cells from young and aged mice.Our data indicate an age-related decline in IL-2 productionby CD4⁺ T cells when the cells were stimulated with concanavalinA in the presence of accessory cells or the combination of immobilizedanti-CD3 and soluble anti-CD28. However, when CD4⁺ T cells wereonly stimulated with Immobilized anti-CD3, an age-related increasein IL-2 production was observed. This age-related increase inIL-2 could be attributed to the ability of CD4⁺ T cells fromaged mice to produce IL-4 on this stimulation, since anti-IL-4inhibited the IL-2 production In these cultures to levels foundwith cells from young mice. The addition of exogenous IL-4 greatlyenhanced the IL-2 production of CD4⁺ T cells from young miceto levels far beyond that of the aged counterparts, emphasizingthe dominant role of IL-4 In the induction of IL-2 stimulatedwith immobilized anti-CD3. No differences were observed in theactivation requirements of Mel14^– CD4⁺ T cells from youngand aged mice. However, Mel14⁺ CD4⁺T cells from aged mice werefunctionally and phenotypically more mature than their youngcounterparts, since they were capable of IL-2 and IL-4 productionin response to antl-CD3 without the need of CD28 triggeringand expressed Pgp-1 and ICAM-1 in a higher density. Our dataindicate therefore that Mel14 is not a stable marker for naiveCD4⁺ T cells and might not be appropriate to distinguish thesecells from memory cells.     

Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo

Clonal deletion and/or inactivation establishes tolerance toself antigens. Endogenous and exogenous (bacterial) superantigens,like the staphylococcal enterotoxlns, induce ligand-specificclonal anergy in vivo and thus are believed to mirror aspectsof post-thymic tolerance mechanisms in mature peripheral T cells.Here we analyzed the level of anergy of ligand-responsive V_ß8⁺T cells from staphylococcal enterotoxin B (SEB)-primed micein vivo and in vitro. Upon in vitro restimulation with SEB,CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells failed toproduce IL-2. However, functional IL-2 receptors were triggered,since supplementation with IL-2 induced clonal growth in virtuallyall CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells as determinedby limiting dilution analyses. Thus in vitro unresponslvenessof lymphocytes from SEB-primed mice reflects the inability ofSEB-reactlve V_ß8⁺ T cells to produce IL-2. Surprisingly,anergy as defined in vitro was at variance with that in vivo.Following further challenge with SEB, systemic and acute lymphokineproduction (Including IL-2 and tumor necrosis factor) occurredwith almost identical peak values and kinetics to primary invivo responses, and D-galactosamlne-sensltlzed mice succumbedto lethal shock. Polymerase chain reaction analyses revealedthat CD4⁺V_ß8⁺ expressed IL-2-specific mRNA in vivoupon restimulatlon with SEB. While lymphokine production andexpression of the IL-2 receptor was similar to the responseto in vivo primary stimulation, only CD8⁺V_ß8⁺ T cellsexpanded clonally upon reintroductlon of SEB in vivo. Henceprimed V_ß8⁺ T cells challenged with SEB display invitro anergy yet in vivo responsiveness, at least in part. Weconclude that the state of anergy is reversible, dependent uponthe quality of activation signals provided in in vivo ratherthan in in vitro culture conditions.     

Differential proliferative responses in subsets of human CD28+cells delineated by BB27 mAb

We report the Identification of a novel 140 kDa disulflde-llnkeddimer expressed by a subset of peripheral blood T lymphocytes.This molecule, which Is recognized by mAb BB27, is also detectedon cells of the myelomonocytlc lineage. In the T cell lineage,Its expression Is positively modulated after lymphocyte activation.A series of double-labeling experiments revealed that BB27 mAbIdentifies new CD4 and CDS cell subsets different from thosedefined by CD45RA, CD45RO, CD26, CD29, CD31, and CD38. Finally,BB27 mAb also subdivides the CD28 subset. Of the utmost interestIs the finding that a proliferative response to CD28 mAb andphorbol myrlstate acetate stimulation is exclusively obtainedin the CD28⁺BB27⁺ cell subset, whereas the CD28⁺BB27^–subset falls to proliferate.     

CD38 expression on mouse T cells: CD38 defines functionally distinct subsets of {alpha}{beta} TCR+CD4 CD8 thymocytes

We have examined CD38 expression on mouse lymphocytes usingthe rat mAb NIM-R5 and demonstrate that CD38 expression is restrictedto {small tilde}8% of thymocytes. Although CD38 is absent fromthe majority of CD4+ CD8^– and CD4^–CD8⁺ T cells,we detected a strong correlation between CD36 expression andß⁺CD4^–CD8^– T cells in the thymus, withnearly 80% of ß TCR⁺CD4^–CD8^– thymocytesbeing CD38⁺. Using heat stable antigen (HSA) and CD38, we dividedß⁺CD4⁺CD8^– thymocytes into four subsets: HSA⁺CD38^–,HSA^– CD38^hi, HSA^–CD3810^low and HSA^– CD38^–.Two established characteristics of ß TCR⁺CD4CD8^–cells, bias towards Vß 8.2 TCR expression and highlevels of IL-4 production, were used to establish a possiblerelationship between the above thymocyte subsets. Our presentdata show that the HSA⁺CD38^– subset is not biased towardsVß8.2 TCR expression whereas the HSA^– CD38^–subset does show this bias (–47%). Neither of these subsetsmake IL-4 upon CD3 mediated stimulation. In contrast, the CD38⁺subsets are heavily biased toward Vß8.2 expressionand produce large amounts of IL-4 upon stimulation, particularlythe CD38^low cells. Taken together, these data suggest that thesefour subsets represent various stages of a possible differentiationpathway for ß TCR⁺ CD4^–CD8^– cells, withthe HSA⁺CD38^– subset being the most Immature while theHSA^–CD38^low subset is the most functionally mature. Thesecharacteristics support the view that ap TCR⁺CD4^–CD8^–T cells represent an independent lineage with a distinct, butas yet obscure, role in immunity     

Developmentally regulated expression of the PD-1 protein on the surface of double-negative(CD4 CD8 ) thymocytes

PD-1, a member of the Ig superfamily, was previously isolatedfrom an apoptosis-induced T cell hybridoma 2B4.11 by subtractivehybridization. Expression of the PD-1 mRNA is restricted tothymus in adult mice. Using an anti-PD-1 mAb (J43), we examinedexpression of the PD-1 protein during differentiation of thymocytesin normal adult, fetal and RAG-2^-/- mice with or without anti-CD3mAb stimulation. While PD-1 was expressed only on 3–5%of total normal thymocytes, –34% of the CD4^-CD8^- double-negative(DN) fraction are PD-1⁺ cells with two distinct expression levels(low and high). PD-1^high thymocytes belonged to TCR lineagecells. In the DN compartment of the TCR ß lineage,PD-1 expression started at the low level from the CD44⁺CD25⁺stage and the majority of thymocytes expressed PD-1 at the CD44^-CD25^-stage in which thymocytes express TCR ß chains. Theanti-CD3 antibody administration augmented the PD-1 expressionas well as the differentiation of the CD44^-CD25⁺ DN cells intothe CD44^-CD25^- DN stage, not only in normal mice but also inRAG-2-deficient mice. The fraction of the PD-1^low cells in theCD4⁺CD8⁺ double-positive (DP) compartment was very small (>5%)but increased by stimulation with the anti-CD3 antibody, althoughthe total number of DP cells was drastically reduced. The resultsshow that PD-1 expression is specifically induced at the stagespreceding clonal selection.