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.     

Kinetics of B cell subpopulations in peripheral lymphoid tissues: evidence for the presence of phenotypically distinct short-lived and long-lived B cell subsets

A small proportion of the slg⁺ B lymphocytes in peripheral lymphoidorgans [22% in spleen and 6 % in lymph node (LN)] in rat carriesthe Thy-1 antigen. These Thy-1⁺ B cells represent newly formedbone marrow (BM) derived (or Immature) B cells. In this studywe investigated the kinetic behavior of Thy-1⁺ and Thy-1^–B cells in various lymphoid tissues. The renewal rates of theseB cells in young adult rats were determined by continuous administrationof 5-bromo-2-deoxyuridine (BrdU) for up to 6 weeks. At severaltime intervals, Thy-1⁺ and Thy-1^– B cell subpopulationsin BM, blood, spleen and popllteal LN were analyzed for thepresence of incorporated BrdU, using three-color immunocytologyon cytospin preparations. In all tissues studied, the Thy-1⁺B cells were rapidly renewed with a rate that varied between58 % (BM) and 22 % (LN) per day. Virtually all Thy-1+ B cellswere labeled by BrdU within a period of 8 –16 days, indicatingthat all these cells are relatively short-lived. By contrast,the replacement of Thy-1^– B cells in these tissues was30 –40 times lower, and ranged between 2.0 and 0.8 % perday. In absolute numbers we estimate that 57 million Thy-1⁺B cells are renewed per day in the BM whereas only 10 millionThy-1^– B cells are replaced in the pool of long-livedperipheral B cells. This implicates a cell loss of 80 % at thetransition of the Thy-1⁺ to the Thy-1^– B cell stage. Thefew B cells that actually become incorporated into the poolof mature B cells are most probably selected on the basis ofthe specificity of their slg.     

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.     

Somatic mutations in antibodies expressed by germinal centre B cells early after primary immunization

We have studied the maturation of the immune response by lookingat the generation of antibody diversity in germinal centre Bcells. Mice were immunized with the antigen 2-phenyloxazolone.Germinal centre B cells, defined by their strong binding topeanut agglutinin (PNAh^hl), were sorted from the spleen andfused. Ten days after imrnunization high numbers of antigenspecific hybridoma lines were obtained from the PNAM subsetof B cells, suggesting that the small fraction of B cells whichare PNA^hl harbour the antigen activated population. The majorityof the day 10 quences from PNAh^hl cells were shown to be mutated.However, in contrast to results from later stages of the lmmuneresponse, most of the mutations found were silent. The preferentialexpansion of B cell clones expressing the mutations characteristicof the mature response was not observed at this stage. Amongthese hybridoma lines was at least one which, apparentty throughsomatic mutation, had lost the ability to bind antlgen. We concludethat in the micro-environment of the germinal centre the B cellrepertoire is diversified by hypermutation.     

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.     

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.     

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.     

Characterization of a 50 kDa surface membrane protein on thymic stromal cells as an important factor for early T cell development

We previously reported that the nude mouse-derived splenic Tcell clone, N-9F, exhibits a prollferative response to the SL10.3thymic epithelial cell clone. In the present study we generatedan Armenian hamster mAb, HS9, specific for SL10.3, which Inhibitedthe N-9F's proliferative response to SL10.3. We performed thymocyterepopulation experiments using fetal liver cells and 2'-deoxyguanosine-treatedthymic rudiments. After 14 days of culture, donor fetal livercells proliferated and differentiated to CD4⁺CD8⁺ and CD4^–CD8⁺with some CD4⁺CD8^– cells in the host thymic rudiments.However, most of the thymocytes remained at a CD4^–CD8^–immature stage in the presence of HS9 and the cell recoverywas reduced to 30% of the control. Immunohistostaining and flowcytometry studies revealed that HS9 reacted with stromal cellsof fetal thymus at the earliest from day 14 gestation. Neitherthymocytes nor lymph node T cells were stained with HS9. HS9antigen was distributed not only on thymic subcapsular and corticalstromal cells, but also on peripheral B cells in adult mice.The antigen that HS9 detected was found to be a 50 kDa surfacemembrane protein on thymic stromal cells. On the other hand,the 50 kDa molecule is associated with two other molecules of80 and 100 kDa on the B cells. These data indicate that theHS9 antigen may have an important role for early T cell development,especially at a stage from CD4^–CD8^– to CD4⁺CD8⁺,and may have some unknown function on B cells.     

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.     

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

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

The status of Ig loci rearrangements in single cells from different stages of B cell development

Differential expression of c-kit, CD25 (TAC), surrogate L chainand cytoplasmic µH chain, and surface expression of IgMand IgD allows the separation of B220 (CD45⁺)B cell subpopulations.PCR analyses with DNA of single cells developed by others andby us have been used to monitor the conformation of the Ig Hand L chain gene loci in these different B lineage subpopulations.The results of these analyses indicate that B220⁺/c-kit⁺/CD25^–cells are the precursors of large B220⁺/CD25⁺/slgM^– which,in turn, are the precursors of small B220⁺/CD25⁺/slgM^–cells. The majority of B220⁺/c-kit⁺/CD25^– cells are D_HJ_H-rearranged,with L chain loci in germline configuration and are thus pre-BI cells. More than 90% of all large B220⁺/CD25⁺/slgM^–cells have at least one H chain locus V_HD_HJ_H rearranged; halfof them have also the second locus V_HD_HJ_H rearranged and arethus large pre-B II cells. Rearrangements of at least one alleleof the kL chain loci become detectable in 65% of the small B220⁺/CD25⁺/slgM^–cells, 67% of the immature B and >75% of the mature B cells.The ratio of kL to L gene rearrangements in all three subpopulationsis {small tilde}10:1, indicating that the kL/L ratio is establishedas soon as rearrangements are made.     

IL-2 expands and maintains IgM plasmablasts from a CD5+ subset contained within the germinal centre cell-enriched (surface IgD /CD39 buoyant) fraction of human tonsil

IL-2 was found to promote the rapid growth of a minority populationcontained within the germinal centre (GC) cell-enriched (CD39^–and/or lgD^– buoyant) fraction of human tonslllar B lymphocytes.The cells emerging in response to IL-2 had a high mitotic indexand morphologically resembled plasmablasta. Cultures could bemaintained in the absence of feeder cells for up to 3 weeksin IL-2 and were characterized by large amounts of IgM in theirsupernatants: 40% of the cells contained readily detectablecytoplasmic IgM by day 10 of culture. Negligible quantitiesof IgG and IgA were found. The target population for IL-2-drlvenexpansion and IgM secretion was 8mlg⁺/CD38⁺ and was subjectto suppression by anti-IgM antibody. While only 8% of cellswithin the GC cell-enriched fraction were CD5⁺ (compared with15% of high density resting B cells), their removal led to an83% reduction in the amount of IgM produced in response to IL-2.IL-2 selectively expanded this minor CD5⁺ subset such that byday 6 of culture they comprised 57% of all viable cells. Culturesestablished with IL-2 showed increasing expression of cytoplasmicBcl-2 and withdrawal of growth factor resulted in cell deathvia apoptosls. We discuss these results in relation to CD5⁺B cells and their potential role in antibody responses to TDantigens.     

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     

Cellular morphometric changes in cat hearts subjected to three hours of regional ischaemia

Summary We have studied the histogenesis of malignant lymphoma (ML), small cleaved cell of the B-cell type and intermediate lymphocytic lymphoma (mantle zone lymphoma) by comparing immunophenotypes and ALP-activity of neoplastic cells with those of germinal center cells (follicular center cells) anti mantle zone (MZ) cells of secondary follicles in non-neoplastic lymphoid tissues. The neoplastic cells in 3 cases of ML, follicular, small cleaved cell and 1 case of ML, small cleaved cell expressed the phenotypes similar to those of germinal center (GC) B lymphocytes (SIgM⁺, B1⁺, B2⁺, CALLA⁺, SigD^–, IL-2R^–, Leu-1^– and ALP^–). The neoplastic cells in 2 cases of ML, follicular, small cleaved cell and 12 cases of ML, diffuse, small cleaved cell displayed the characteristic phenotypes of MZ B lymphocytes (SIgM⁺, SIgD⁺, BA-1⁺, IL-2R⁺, Leu-1⁺ and ALP⁺). The phenotypes of 2 cases of mantle zone lymphoma were closely comparable with those of MZ B lymphocytes. These findings indicate that the histogenesis of ML, small cleaved cell of the B-cell type is heterogeneous and can be divided phenotypically into 2 types (GC B lymphocyte origin and MZ B lymphocyte origin). It is also apparent that intermediate lymphocytic lymphoma (mantle zone lymphoma) is derived from MZ B lymphocytes of secondary follicles.This work was supported by a Grant-in-Aid for Cancer Research From the Ministry of Health and Welfare (NO. 61-2)     

Herpesvirus saimiri immortalization of {alpha}{beta} and {gamma}{delta} human T-lineage cells derived from CD34+ intrathymic precursors in vitro

Herpesvirus saimiri (HVS), an agent that can infect many humancell types, has been shown to immortalize selectively TCR ß⁺CD3⁺T lymphocytes. Human T cell precursors defined as CD34⁺CD3^–CD4^–CD8^–were isolated from thymic samples and exposed to HVS in thepresence of either IL-2 or IL-7. Cultures lacking the viruswere non-viable by day 15. Test cultures, in contrast, showeda sustained proliferative activity lasting >5 months, allowingthe phenotypical and molecular analysis of the cellular progeny.In the presence of IL-7, TCR ß⁺ cells with three differentphenotypes (mainly CD4⁺CD8^–, but also CD4⁺CD8⁺ and CD4^–CD8⁺)were immortalized, whereas no TCR ⁺ cells were recovered. Kineticstudies showed that the expansion of immortalized TCR ß⁺cells was preceded by a gradual loss of CD34⁺ cells followedby a transient accumulation of two distinct cell subsets: firstCD1⁺CD4⁺CD3^– cells and then CD4⁺CD8⁺ thymocytes. Thisresembles early phenotypic changes occurring during normal intrathymicT cell development. In the presence of IL-2, in contrast, onlyTCR ⁺ cells were immortalized (mainly CD4^–CD8⁺, but alsoCD4^–CD8^–). The results show that HVS can be usedto read the CD3⁺ cellular outcome of T cell differentiationassays, including ⁺ CD4^–CD8⁺, ⁺CD4^–CD8^–, ß⁺CD4⁺CD8^–,ß⁺CD4^–CD8⁺ and ß⁺CD4⁺CD8⁺ T cells.A clear role for different cytokines (IL-2 for ⁺ cells, IL-7for ß⁺ cells) in early T cell commitment was alsoapparent.     

Activation of CD45-deficient T cell clones by lectin mitogens but not anti-Thy-1

CD45, the leukocyte-common antigen, Is a transmembrane proteintyrosine phosphatase uniquely expressed by cells of hematopoletlcorigin. We have developed CD4⁺ and CD8⁺ T cell clones that aredeficient in the expression of CD45 and have previously shownthat these cells fall to proliferate in response to antigenor cross-linked CD3. These studies have now been extended toshow that stimulation with antl-Thy-1, a mltogenlc signal forthe CD4⁺CD45⁺ and CD8⁺CD45⁺ T cells, falls to induce proliferationin the CD45^– T cells. Examination of the CD8⁺CD45^–T cells correlates antl-Thy-1 unresponslveness with a failureto increase in tyrosine phosphorylatlon. Furthermore, stimulationof CD8⁺CD45⁺ T cells with antl-Thy-1 results in an increasein p56^ick activity but not in CD8⁺CD45^– T cells. In contrastto the results with antl-Thy-1, both the CD4^+– CD45 andCD8⁺CD45^– T cells respond to treatment with lectin mitogens,concanavalln A or phytohemagglutlnln. Lectin-lnduced proliferationwas inhibited by the addition of cyclosporln A. Treatment ofCD45^– T cells with PMA and lonomycln also results in proliferationindicating that activation of protein kinase C in conjunctionwith an increase in intracellular calcium rescues the defectcafsed by CD45 deficiency. The data suggest that CD45 Is requiredfor the activation of tyrosine kinase activity Immediate orprior to transmembrane signaling.     

Antigen-specific down-regulation of myelin basic protein-reactive T cells during spontaneous recovery from experimental autoimmune encephalomyelitis: further evidence of apoptotic deletion of autoreactive T cells in the central nervous system

Experimental autoimmune encephalomyelitis (EAE) was inducedin Lewis rats by the I.v. injection of 10⁷ cloned V_ß8.2⁺T cells specific for the 72–89 peptide of guinea pig myelinbasic protein(MBP). Some animals were injected simultaneouslywith 10⁷ cloned T cells specific for ovalbumin(OVA). Lymphocyteswere isolated from the spinal cord and from the peripheral lymphoidorgans of these rats and the frequencies of MBP-peptide-specificor OVA-specific proliferating cells were estimated by limitingdilution analysis at different times after cell transfer. Thefrequencies of cells specific for MBP_72–89 or OVA in thespinal cord were highest 5 days after cell transfer (MBP_72–89,1 in 1149; OVA, 1 in 1116). On day 7, when the rats were recovering,the frequency of cells specific for MBP_72–89 in the spinalcord fell dramatically to <1 in 10⁵, while that of OVA-specificcells decreased to a much lesser extent (1 in 7001). The frequenciesof MBP_72–89-specific cells in the peripheral lymphoidorgans during and after recovery were also much lower than thoseof OVA-specific cells. A similar pattern of down-regulationof the MBP-peptide-specific, but not the OVA-specific, T cellresponse was observed in the spleen and mesenteric lymph nodes(MLN) of rats 38 days after the active induction of EAE by immunizationwith equal amounts of MBP and OVA in adjuvants. In the passivelytransferred model, cells isolated from the spinal cord and MLNon day 7 did not regain responsiveness to MBP_72–89 afterincubation with high levels of IL-2, indicating that the unresponsivenesswas not due to T cell anergy. Thus this study demonstrates thatthere is a specific down-regulation of the MBP_72–89-specificT cell response during spontaneous recovery from EAE. This conclusionis consistent with our previous observation that V_ß8.2⁺T cells are selectively eliminated from the CNS by apoptosisduring recovery from EAE induced by the passive transfer ofV_ß8.2⁺ T cells reactive to this MBP peptide. In contrastto autoreactive T cells, the non-autoreactive T cells that accumulatein the CNS during EAE appear to recirculate to the peripherallymphoid organs.     

Emergence of CD52 , glycosyiphosphatidylinositol-anchor deficient lymphocytes in rheumatoid arthritis patients following Campath-1H treatment

CD52 is a glycosylphosphatidyl-inositol (GPI)-llnked glycoproteinexpressed at high levels on normal T and B lymphocytes and atlower levels on monocytes, while being absent on granulocytesand bone marrow stem cell precursors. The emergence of CD52^– lymphocytes of both T and B cell lineages was observedIn three out of 25 rheumatoid arthritis patients treated withthe humanized antibody Campath-1H in phase II clinical trial.Whereas the majority of CD52^– B cells had disappearedfrom the peripheral blood by 3 months post-treatment, both CD52^–CD4⁺ and CD8⁺ T cells persisted in the circulation for at least20 months. In the two patients that were tested, the GPI-anchoredsurface molecules CD55 and CD59 were also absent on the CD52^–cells, although expression of other cell surface transmembraneproteins (CD3, CD4 and CD2) was unaffected. The CD52^–cells maintained a stable phenotype in vitro despite repeatedre-stimulation in culture. Both CD52^– and CD52⁺ clones,established from one of the patients, responded to a similarextent to several T cell mitogens, as assessed by proliferation,suggesting that a general defect in expression of GPI-llnkedmolecules does not impair T cell activation. These data showthat an immune attack against a GPI-anchored surface moleculecan result in the selection of a GPI-anchor-deficient cell population.Despite the persistence of CD52^– T cells in the peripheralblood, no adverse reactions associated with the presence ofthese cells were noted in any of the patients; in fact theyresponded with longer remission times after Campath-1H treatmentthan the other patients in the trial. Received 16 May 1995, accepted 27 November 1995.     

Human naive T cells activated by cytokines differentiate into a split phenotype with functional features intermediate between naive and memory T cells

We have recently shown that CD45RA⁺CD4⁺ naive T cells can beactivated to proliferate by a combination of IL-2, TNF- andIL-6, but, at variance with TCR-medlated activation, they donot acquire the CD45RO molecule. This prompted us to investigatethe phenotype of these cells and the functional features theydisplay upon TCR stimulation. Naive T cells expanded by cytokines,though remaining CD45RA⁺ express a variety of activation andadhesion molecules which are peculiar to effector or memoryT cells. Naive cells primed by cytokines, when activated withantl-CD3 mAb, produce a broad spectrum of cytokines, expressCD40 ligand, but are unable to help B cells for Ig synthesis.A subset of CD4⁺CD45RA ⁺ RO^– T cells with a phenotype(HLA-DR^–, VLA-2⁺ or IL-2R⁺) similar to that of cells activatedby cytokines In vitro can be found In vivo. These results demonstratethat activation signals delivered by cytokines, in the absenceof TCR stimulation, can activate naive T cells to proliferateand differentiate into a ‘split phenotype’ withelements common to both naive and memory T cells. This novelantigen-Independent activation may help to maintain the naiveT cell repertoire and facilitate the antigen-responsivenessof naive T cells.