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).     

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.     

CD4+ICOS+ T lymphocytes inhibit T cell activation 'in vitro' and attenuate autoimmune encephalitis 'in vivo'

The inducible co-stimulator (ICOS, CD278) is essential to theefficient development of normal and pathological immune reactions.Since ICOS-deficient mice have enhanced susceptibility to experimentalallergic encephalomyelitis (EAE), we have functionally analyzeda CD4⁺ICOS⁺ population comprising 6–15% of all CD4⁺ Tcells in secondary lymphoid organs of unmanipulated wild-typemice and checked for their ability to suppress EAE. In C57BL/6mice, CD4⁺ICOS⁺ cells were a major source of cytokines includingIFN-, IL-2, IL-4, IL-10 or IL-17A. Upon activation, these cellsshowed preferentially enhanced production of IL-4 or IL-10 butinhibited IFN- production. In contrast, CD4⁺ICOS^– cellsmainly produced IFN-. Interestingly, CD4⁺ICOS⁺ cells partiallysuppressed the proliferation of CD4⁺ICOS^– or CD4⁺CD25^–lymphocytes ‘in vitro’ by an IL-10-dependent mechanism.Furthermore, CD4⁺ICOS⁺ activated and expanded under appropriateconditions yielded a population enriched in cells producingIL-10 and T_h2 cytokines that also suppressed the proliferationof CD4⁺CD25^– lymphocytes. CD4⁺ICOS⁺ cells, before or afterexpansion in vitro, reduced the severity of EAE when transferredto ICOS-deficient mice. In the same EAE model, lymph node cellsfrom ICOS-deficient mice receiving ICOS⁺ cells showed reducedIL-17A production and enhanced IL-10 secretion upon antigenactivation in vitro. Thus, naturally occurring CD4⁺ICOS⁺ cells,expanded or not in vitro, are functionally relevant cells ableof protecting ICOS-deficient mice from severe EAE.     

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.     

In vivo and in vitro repertoire of CD3+CD4 CD8 thymocytes

CD4-CD8- thymocytes contain a cell subset which expresses thecomplete CD3-TCR complex (/ or /) of which the ontogeny andfiliation are unknown. One of the questions is whether thispopulation can be intrathymically selected, an obligatory stepfor the mature CD4⁺ and CD8⁺ cell differentiation pathway, orif the absence of CD4 and CD8 allows them to escape thymic selection.The repertoire of CD3 ⁺ CD4 - CD8 - (CD3 ⁺ DN) thymocytes wasanalyzed in different strains of mice with different combinationsof H-2 and Mls expression. The expression of V_ß8.1in freshly isolated CD3⁺ DN cells is the highest in Mls-l^b miceand the lowest in MIS-l^a and F₁ mice, suggesting that selectionagainst this specificity can be achieved in vivo. By contrast,a low percentage of V_ß6⁺ cells is found in all thestrains, with no correlation according to MIS expression. Invitro culture of DN thymocytes with IL-1 and IL-2 leads to theproliferation of CD3⁺ DN cells. In vitro culture amplifies thein vivo pattern of V_ß8.1 expression, while V_ß6⁺cells only expand in DN cells of 66 and 61002 Mls-l^b mice withthe same genetic background. These results show: (i) CD3⁺ DNthymic cells can be intrathymically selected but the repertoireis different from that of mature T cells; (ii) V_ß6and V_ß8.1 selection do not follow the same pattern;(iii) this repertoire can be modified by In vitro culture, towarda more mature type; and (iv) comparison of DN cell repertoireof BALBlc, BALB.D2 (congenic for MLs), and other strains ofmice suggests a multigenic control for this selection, and aninvolvement of background genes.     

IFN-{gamma} expression in CD8+ T cells regulated by IL-6 signal is involved in superantigen-mediated CD4+ T cell death

Infection with pathogens containing superantigens (Sags) canresult in massive excessive CD4⁺ T cell activation and deathin such conditions as toxic shock, food poisoning and autoimmunediseases. We here showed how enhancement of IL-6 signaling suppressesSag-mediated activated CD4⁺ T cell death. Sag-induced CD4⁺ Tcell death increased in IL-6 knockout (KO) mice, whereas itdecreased in mice characterized by enhanced IL-6–gp130–STAT3signaling. The serum concentration of IFN- was inversely correlatedwith the magnitude of IL-6 signaling, and IFN- deficiency inhibitedSag-induced activated CD4⁺ T cell death, suggesting that IL-6suppresses CD4⁺ T cell death via IFN- expression. Interestingly,depletion of activated CD8⁺ T cells inhibited Sag-mediated increasesin IFN- expression in IL-6 KO mice as well as the augmentedCD4⁺ T cell death. The results demonstrate that IL-6–gp130–STAT3signaling in activated CD8⁺ T cells contributes to Sag-inducedCD4⁺ T cell death via IFN- expression, highlighting this signalingaxis in CD8⁺ T cells as a potential therapeutic target for Sag-relatedsyndromes.     

Impaired CD28-mediated co-stimulation in anergic T cells

We have investigated a CD28 co-stimulation in anergic T cellsin staphylococcal enterotoxin Binoculated mice by stimulatingthe cells with a plate-coated anti-TCR antibody in the presenceor absence of an anti-CD28 antibody. CD28 co-stimulation increasedthe levels of IL-2 and IL-4 mRNAs in nalve CD4⁺V_ß8⁺T cells. However, it did not increase the levels of IL-4 mRNAat all and only partially increased those of IL-2 mRNA in anergicT cells. It was demonstrated that CD28 co-stimulation was impairedso that it no longer stabilized cytoklne mRNAs in anergic cells.The levels of IL-4 mRNA in response to TCR stimulation werehigher in anergic T cells than those in nalve T cells in spiteof the defective CD28 co-stimulation in the former cells. Anergyinduction and generation of a T_h2-type immune response in vivoare discussed     

Induction of CD5 antigen on human CD5 B cells by stimulation with Staphylococcus aureus Cowan strain I

We have examined whether the CD5 phenotype could be inducedon human B cell surfaces by the polycional B cell stimulator,Staphyiococcus aureus Cowan strain I (SAC). Fresh tonsillarB cells were prepared by Percoll density gradient from E^–cells. The proportion of CD5⁺ B cells In the 50/60% and 60/70%interface high-density fractions varied between 1.2 and 10.2%depending on the tonsil preparations when they were placed onthe in vitro culture 12–60 h prior to flow cytometrlcanalysis. The expression of CD5 antigen obviously increasedin the presence of SAC (1:10⁵ v/v). The percentage of CD5⁺ Bcells varied from tonsil to tonsil, from 25.1 to 65.9% in aseries of experiments. The CD5⁺ B cells were found both amongCD23⁺CD25⁺CD71⁺ and CD23^–CD25^–CD71^– B cells.The level of CD5 expression was related to the cell size eniargement.The addition of anti-CD5 antibody in the culture blocked theCD5 induction by SAC without interfering with the expressionof other activation markers. A time-course study showed thatCD5 antigen appeared to be induced on the cell surface duringthe G₀ to G₁ phase transition in the cell cycle. When CD5⁺ andCD5^– B cells were separated by magnetic isolation, theCD5^– B cells showed DNA synthesis to the stimulation bySAC and expressed CD5 antigen on their cell surface. These resultssuggest that human CD5^– B cells can express the CD5 phenotypeby stimulationwith the polyclonal B cell stimulator, SAC.     

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.     

IL-7 induces proliferation of CD3 /low CD4 CD8 human thymocyte precursors by an IL-2 independent pathway

The proliferation potential of highly purified human CD3^–CD4^–CD8^–(triple negative) and CD3^low(lo)CD4^–CD8^– thymocyteprecursors in response to various cytokines was investigated.High in vitro growth ability was observed in response to recombinanthuman IL-2 (riL-2) and human riL-7, both in the absence of anyco-mitogen and in combination with phorbol 12-myristate 13-acetate(PMA). Furthermore, the proliferation of these thymocyte precursorsin the presence of rlL-7, although accompanied by a significantincrease of IL-2 receptor (IL-2R) p55 expression, appeared independentof that mediated by the autocrine IL-2 pathway, since mAbs toIL-2 and IL-2R p55 did not eliminate responsiveness to rlL-7.Synergism of rlL-7 with rlL-2 was also observed, while no cooperationwas detectable with rlL-4 or rlL-6. Analysis of surface phenotypeand cell cycle status of cells cultured in the presence of rlL-7,both plus and minus PMA, showed that CD3^– as well as CD3¹⁰cells readily proliferated to rlL-7. Upregulation of the levelsof expression of CD3 antigen was also observed in these cultures.These results, together with the previous characterization ofIL-7 as a human pre-B cell and mature T cell growth factor,Identify IL-7 as a cytokine with biologic activities on a varietyof target cells. They also suggest that IL-7, in analogy withthe mouse system, might play a role in human T cell ontogeny.     

Anergy in vivo: down-regulation of antigen-specific CD4+ Th1 but not Th2 cytokine responses

Efficient immunologic tolerance, defined as antlgen-speclflcunresponslveness, can be peripherally induced by the l.v. Injectionof syngenelc splenocytes coupled with antigen using ethylenecarbodilmlde (ECDI). We have previously reported that unresponslvenessinduced via l.v. Injection of syngenelc splenocytes coupledwith intact, UV-lnactlvated Theiler's murine encephalomyelitisvirus (TMEV-SP) resulted in ‘split tolerance’. Bothvtrus-speclflc delayed-type hypersensltlvlty and lgG2a levelswere inhibited, whereas lgG1 levels were increased when comparedwith sham tolerized controls. In the present report we demonstratethat tolerance induced by l.v. Injection of TMEV-coupled splenocytesresulted in antigen-specific inhibition of T cell proliferation,as well as IL-2 and IFN- production in response to both wholeTMEV and the immunodomlnant viral epitope. Additionally, toleranceinduction resulted in abrogation of T_h1 -derived [IL-2, IFN-and LT/tumor necrosis factor-ß (TNF-ß)]cytokine mRNA expression in response to In vitro stimulationwith UV-inactlvated TMEV as determined by reverse transcrlptasepolymerase chain reaction. In contrast, expression of T_h2-derived(IL-4, IL-6 and IL-10) cytokine mRNA was not affected in tolerizedmice. Tolerance functioned directly at the level of CD4⁺ T_h1cells at both the induction and effector limbs as depletionof CD8⁺ T cells both prior to in vivo tolerizatlon or in vitroculture had no effect on inhibition of T_h1-specific responses.The mechanism of In vivo tolerance induction appeared to beanergy of CD4⁺ T_h1 cells since IL-2, IFN- and LT/TNF-ßmRNA expression as well as virus-specific prollferatlve responsescould be restored by addition of rlL-2 to In vitro culturesof tolerant, CD4⁺ T_h1 populations. These results suggest thatin vivo ‘split tolerance’ Induced by l.v. Injectionof ECDI-flxed, antigen-coupled splenocytes involves anergy ofTMEV-speclflc, CD4⁺ T_h1 lymphocytes and concomitant primingof T_h2 cells. The induction of antlgen-speclflc, in vivo anergyhas important implications in the design of therapeutic strategiesfor immunopathologic diseases mediated by T_h1 lymphocytes, especiallyT cell-mediated autoimmune disorders.     

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.     

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     

CD4+ T cell associated cytokine gene expression during experimental infection with Listeria monocytogenes: the mRNA phenotype of granuloma formation

In murine listeriosls, elimination of bacteria and immunityto re-infection critically depend on Thy-1⁺ CM^– cells,while cell-mediated inflammatory phenomena like delayed-typehypersensltlvlty and granuloma formation are mediated by CD4⁺T cells. In an attempt to correlate T cell phenotype and functionwith a particular set of cytokines produced in vivo, we examinedthe cytokine gene expression profile associated with the presenceor absence of CD4⁺ and/or CD8⁺ cells in the livers of mice duringexperimental infection with Usterta monocytogenes. T cell subsetdepletion was achieved by l.p. administration of saturatingamounts of the appropriate mAbs, and mRNA detection was carriedout using a qualitative and semi-quantitative polymerase chainreaction-based mRNA amplification protocol. In both primaryand secondary infection, the presence of CD4⁺ cells was a prerequisitefor granuloma formation, and was found to be closely associatedwith mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increasein expression of tumor necrosis factor (TNF)- and granulocytemacrophage colony stimulating factor, and a 25-fold increasein expression of IFN- and TNF-ß mRNAs, suggestinga role for these cytokines in granuloma formation. In strikingcontrast, depletion of CD8⁺ cells did not result in reducedmRNA expression for any one of the cytokines studied, Implyingthat CD8⁺ T cell mediated cure and prevention of listerlosismay operate via qualitatively distinct mechanisms.     

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.     

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.