Successful in vitro immortalization of human intestinal mucosal lymphocytes with Herpesvirus saimiri

Abstract: Mucosal intestinal lymphocytes form the first immune-cell line of defense in the intestine. Several methodologies, most of them cumbersome and time consuming, have been used to obtain T-cell clones to unveil their physiological role. In the present work we take advantage of the recently described technique of transformation of T lymphocytes using Herpesvirus saimiri to show that it is possible to immortalize intestinal T-cell lines derived from healthy and diseased colonic samples and thence easily obtain in vitro intestinal T-cell lines as a model for physiopathological studies. Intestinal samples were obtained by colonoscopy and digested with dispase and collagenase. Mucosal lymphocytes (assessed by the expression of the CD3 and CD103 markers) were isolated using a Percoll gradient centrifugation and transformed with Herpesvirus saimiri . Sustained growth was observed 3 months later, showing that the cells were successfully transformed, a finding further confirmed by PCR. All cell lines were CD8⁺TcRαβ⁺ and HLA-DR⁺ CD25 was expressed on 1% of Crohn's disease-derived cells and on 25% of cells derived from patients with ulcerative colitis. CD80 expression was found on 80–90% of the cells. These immortal cell lines of intestinal origin may be useful in future experiments aimed at elucidating the role of mucosal lymphocytes in health and disease.     

{alpha}{beta} and {gamma}{delta} T cells in the immune response to the erythrocytic stages of malaria in mice

Mice lacking T cells with ß TCR (TCR ß–/–)or TCR (TCR –/–) were infected with the erythrocyticstages of the malaria parasite, Plasmodium chabaudi chabaudi(AS). Mice without T cells could control and reduce a primaryinfection of P. chabaudi with a slight delay in the time ofclearance of the acute phase of infection and significantlyhigher recrudescent parasitaemias compared with control intactmice. TCR –/– mice had higher levels of both serumIg and malaria-specific antibodies of the isotypes IgG3 andIgG1 compared with control mice. TCRß–/–mice, despite a striking increase in NK1.1⁺ cells and the presenceof T cells, were unable to clear their infection. Althoughthe plasma of TCR ß–/– mice containedall Ig isotypes before and during a primary infection, theywere unable to produce significant levels of malaria-specificIgG antibodies, suggesting that in the absence of ßT cells T cells are not able to provide efficient help forantibody production.     

{gamma}{delta} T cells promote CD4 and CD8 expression by SCID thymocytes

Molecular studies of the TCR, which is expressed by a minorsubpopulatlon of T lymphocytes in all vertebrate species, havedefined a subset which expresses a receptor with extreme junctionaldiversity and a second subset, most commonly found in eplthella,which expresses a receptor of very limited diversity. In thedeveloping murine thymus, T cells appear in an ordered sequenceof specific v rearrangements, V3V, 1 on day 14, V2V1 on day17, and subsequently V4V5, V6, or V7. We demonstrate that thetransfer of expanded populations of cells from newborn thymusand cell lines expressing the invariant V3V1 receptor into SCIDmice, which lack T and B cells, results in the appearance ofCD3^–CD4⁺CD8⁺ thymocytes. Thus, one role of the early appearingV3V1 T cells in thymlc development in vivo is to promote CD4and CO8 surface expression on precursor cells.     

Human {alpha}{beta} and {gamma}{delta} T cells from unexposed individuals respond to protein antigens of the yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis, a dimorphic fungus, causes chronicgranuiomatous mycosis in susceptible individuals. Differentreports have shown that cell-mediated immunity is essentialfor protection against systemic mycosis, including paracoccldloidomycosis.We analyzed the reactivity of ß and T cells fromunexposed Caucasian donors to P. brasiliensis yeast form components.Our results indicate: (I) ß and T cells proliferateafter in vitro stimulation with lysates of P. brasiliensis;(II) similar numbers of ß T cells (f = 1/21,000) andof T cells (f = 1/8000) respond to P. braslllensls; (III) P.braslllensls-reactive T cells express the V9V2 TCR; (Iv) thestimulatory activity of P. brasilensis for both ßand T cells primarily resides in a high molecular weight (100kDa) and in a low molecular weight (<<1 kDa) fraction;(v) the ligands responsible for stimulation of both ßand T cells are sensitive to proteinase treatment We concludethat both ß and T cells from healthy individualsrespond to ubiquitous protein antigens of P. brasiliensis.     

Phenotypic and functional properties of murine {gamma}{delta} T cell clones derived from malaria immunized, {alpha}{beta} T cell-deficient mice

Six murine T cell clones expressing TCR were generated frommalaria immunized, ß T celldeficient mice. Phenotypiccharacterization of these clones has revealed that, in contrastto conventional ß T cells, there is a considerabledegree of heterogeneity among these clones with regard to theirsurface markers and their lymphokine profile. One clone wasfound to display significant anti-parasite activity in vivoupon adoptive transfer. We attempted to determine whether theprotective clone differs in one or more key characteristicsfrom the non-protective clones. Although no obvious patternpeculiar to the protective clone was observed, it appears thatmore than one parameter may, in combination, define a distinctprotective phenotype, and thus explain the functional differencebetween the protective and non-protective clones.     

Virgin {alpha}{beta} and {gamma}{delta} T cells recirculate extensively through peripheral tissues and skin during normal development of the fetal immune system

Current models of T cell migration place severe restrictionson the recirculation of virgin T cells, condemning them to migrateexclusively via high endothelial venules in lymph nodes untilthey either die or acquire the capacity to migrate to skin andperipheral tissues as memory cells following stimulation withantigen. We have demonstrated in the sheep fetus (which is Immunologicallyvirgin until after birth) that virgin T cells and dendriticcells circulate through skin and peripheral tissues during fetallife in the same non-random manner as adult T cells but in muchlarger numbers than they do in adult animals. Our data alsoshowed that T cells do not discriminate between peripheral tissuesand skin or lymph nodes on the basis of virgin or memory CD45Rphenotype, or CD2, CD58 or CD44 phenotype, and with the possibleexception of CD11a/CD18, that it is not mandatory for lymphocytesto be activated to adhesion molecule ^hl status in order to hometo fetal skin. Our results indicate that unique tissue-homingspecificities for extralymphoid tissues can be imprinted onvirgin T cells independent of foreign antigen. Virgin T cellshave previously been thought to be denied access to peripheraltissues; however, the large-scale traffic of virgin T cellsthrough extra-lymphold tissues in the fetus reported here providesa mechanism whereby direct virgin T cell interactions with self-antigensexpressed only on tissues outside the thymus can occur repeatedlyduring development of the fetal immune system.     

Differences in the tissue-specific homing of {alpha}{beta} and {gamma}{delta} T cells to gut and peripheral lymph nodes

Tissue-selective streaming of T cells is considered to be acritical element in the integration of normal immune responsesin intact animals. The results presented in this paper showthat while there were major subsets of gut-homing T cells presentin intestinal lymph, there were considerable differences inthe tissue troplsm of T cell populations circulating in lymphdraining gut and peripheral lymph nodes. Thus, while CD4⁺ cellsreturned preferentially to their tissue of origin, y8 T cellsshowed a strong migratory preference for peripheral lymph nodesregardless of their tissue of origin. In contrast, althougha population of gut-homing CD8⁺ cells was present in Heal lymph,CD8⁺ T cells from peripheral lymph nodes homed equally wellto gut and lymph nodes. There were also considerable differencesin the expression of L-selectln on T cells circulating in thetwo compartments. L-selectin was down-regulated on ßT cells present in Meal lymph but not on T cells which expressedthe highest levels of L-selectin of all T cell subsets. It issuggested that gut-homing ß T cells which have down-regulatedL-selectin are formed in the gut-associated lympnoid tissuesin response to gut antigens while the migratory properties of T cells are ontogenetically determined, independent of antigen.     

Dendritic cells and macrophages are required for Th1 development of CD4+ T cells from {alpha}{beta} TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN-{gamma} production is IFN-{gamma}-dependent

We have examined the antigen presenting cell (APC) requirementsfor primary T cell activation and T helper (Th) cell phenotypedifferentiation using naive CD4⁺ T cells from ß TCRtransgenic mice. Purified dendritic cells were the principalcell required for induction of primary ovalbumln peptide specificT cell activation and clonal expansion. However, dendritic cellsdid not induce differentiation of T cells toward Th1 or Th2phenotype. Addition of IL-4 during primary dendritic cell stimulationsof T cells resulted in the development of a Th2 phenotype whichproduced high levels of IL-4 during secondary and tertiary stimulation.In contrast, development of Th1 cells producing high levelsof IFN- could not be induced with dendritic cells alone butrequired the addition of appropriately activated macrophages.Addition of splenic or peritoneal B cells did not induce Th1development. Activated splenic macrophages induced Th1 developmentvia a non-MHC restricted mechanism. Thus, requirements for inductionof proliferation of naive CD4⁺ T cells are distinct from thosedirecting Th1 phenotype development. IL-12 could replace therequirement for macrophages to induce Th1 development when Tcells were activated with dendritic cells. Furthermore, thisIL-12 mediated development of Th1 cells producing high levelsof IFN- was dependent on IFN-.     

Phenotypic and functional assessment of intraepithelial lymphocytes bearing a 'forbidden' {alpha}{beta} TCR

Differences in the surface antigen phenotype, such as the expressionCD8 as an homodimer or the lack of Thy-1, on Intestinal Intraepitheliallymphocytes (IEL) are related, In part, to alternative differentiationpathways. The relationship of IEL lacking the pan-T cell markerCD5 to these IEL, their TCR repertoire and function has notbeen examined directly. We explored the TCR repertoire and functionof the CD5^– IEL subset In relation to the expression ofthe ‘autospecific’ V_ß6 TCR in Mls-1^a miceand to TCR. The results indicate that CD5 expression was absenton the majority of TCR IEL (96.9%) and on a significant proportionof TCR ß IEL (25.0%). Virtually all IEL In DBA/2 (Mls-1^a)mice that expressed the ‘autospecific’ V_ß6TCR were CD5^–, and this correlated with the expressionof CD8 . To assess the functional capacity of this subset ofIEL, we examined proliferation and IL-2 production in responseto TCR activation. Although CD5^– IEL proliferated in responseto anti-CD3, IEL bearing TCR V_ß6, In Mls-1^a mice,were not responsive to TCR-mediated activation. Similarly, TCR IEL were not responsive to stimulation by anti-TCR antibodies.The addition of exogenous IL-2, however, reconstituted the prollferativeresponse of both TCR IEL and the TCR V_ß6 expressingIEL. We conclude that the lack of CD5 defines a unique subsetof intraepithelial T cells expressing either TCR or ßthat Include potentially autoreactive cells that remain anergicin the absence of IL-2.     

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     

CD3{varepsilon}-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2 / mice in the absence of TCR {beta} chain expression

Recent studies have shown that TCR ß chain expressioncan effect the differentiation of CD4^–CD8^– double-negative(DN) thymocytes to CD4⁺CD8⁺ double-positive (DP) thymocytes.The TCR ß chain is expressed on the surface of DPthymocytes in association with CD3, and chains, suggestinga potential role for CD3 components in this signaling process.We now report detection of a very tow level of surface expressionof CD3 on adult DN RAG-2^–/–; thymocytes. This surfaceCD3 was associated with CD3 and chains, as detected by anti-CD3immunoprecipitation analyses. Significantly, injection of anti-CD3mAb into RAG-2^–/– mice led to the accumulation ofan IL-2R^– CD2⁺ DP cell population and a nearly 100-foldincrease in thymic cellularity to essentially normal levels.Together, these data strongly indicate that TCR ßchain-mediated developmental signals are transduced by CD3 componentsand provide potential insights into mechanisms by which TCRß chain expression may effect this process.     

Small CD4+8+TCRlow thymocytes contain precursors of mature T cells

To evaluate directly the developmental potential of cortical CD4⁺8⁺ thymocytes, highly purified populations of small, nondividing CD4⁺8⁺TCR^low and large, dividing CD4⁺8⁺TCR^high thymocytes from H-2^d mice expressing a transgenic T cell receptor restricted by H-2D^b (major histocompatibility complex class I) molecules were transferred into the thymus of normal, nonirradiated H-2^b recipient mice. The results show that both populations generate CD4^?8⁺ thymocytes under these conditions, thus providing conclusive evidence that small cortical thymocytes do not represent a “dead end” but an important intermediate stage in T cell development.     

Pathogen-induced Th1 phenotype development in CD4+ {alpha}{beta}-TCR transgenic T cells is macrophage dependent

We used an ovalbumin (OVA)-specific      

Resident CD4+ {alpha}{beta} T cells of the murine female genital tract: a phenotypically distinct T cell lineage that rapidly proliferates in response to systemic T cell activation stimuli

A population of CD4⁺ cells has been identified in the murinefemale genital tract (FGT). Phenotypic studies of FGT CD4⁺ cellsdemonstrate that they express CD3 and that the majority of thesecells are ßTCR⁺Thy-1⁺. Most of the Thy-1⁺CD4⁺ßTCR⁺ cells resemble memory T cells based on their expressionof CD44, L-selectin and CD45RB antigens. The vast majority ofThy-1⁺CD4⁺ßTCR⁺ FGT cells are CD5⁺ and all of themare B220^–. Systemic stimuli including infection with Trypanosomabrucel brucel, injection with anti-CD3, or bacterial superantigensstaphylococcal enterotoxin A or B cause a rapid accumulationof CD4+cells in the FGT exceeding that observed for CD4⁺ cellsin spleen and lymph nodes (LN). Expansion of the FGT CD4⁺ cells,which are phenotypically distinct from the splenic and LN CD4⁺T cells, is due to local proliferation rather than an influxof cells from the circulation. The CD4⁺ population in the FGTof adult nu/nu mice is dramatically reduced, indicating itsthymic dependency. In lpr/lpr mice, FGT CD4 cells do not displaychanges characteristic of splenic or LN CD4 cells in the sameanimals. These findings demonstrate that the CD4⁺ cells of themurine FGT are thymic dependent, but that they constitute aT cell lineage that phenotypically and, probably functionally,is distinct from other peripheral CD4⁺ T cell populations.     

The development of dermatitis infiltrated by {gamma}{delta} T cells in IL-7 transgenic mice

Transgenic mice constitutively expressing IL-7 developed severedermatitis with erythroderma and alopecia. The skin lesionswere characterized by massive infiltration of mononuclear cells.Immunofluorescence staining showed that most of the infiltratingcells were T cells with the majority bearing the TCR otherthan the V5 moiety. Furthermore, the number of T cells hadincreased in the lymphold organs of the dermatitis animals.These findings idicate the strong relationship between the expressionof IL-7 and the development of T cells in vivo and the pathologicalinvolvement of proliferated and/or activated T cells in skindisease.     

Emigration of selected subsets of {gamma}{delta}+ T cells from the adult murine thymus

Cells bearing the form of the TCR make up only 1–3% ofT cells in the adult murine thymus and peripheral lymphold organs.Evidence from studies of nude mice suggests that the developmentof at least some T cells is thymus dependent; however, untilnow it has not been directly demonstrated that cells are exportedfrom the thymus. In this paper we have used the technique oflabelling thymocytes in vivo with FITC, followed by flow cytometrlcanalysis to trace cells emigrating from the thymus to the spleen.Using this approach we have been able to demonstrate for thefirst time that T cells are exported from the adult murinethymus to the spleen. We also demonstrate that the cells emigratingto the spleen are a selected subset of thymocytes being heatstable antigen positive, Thy-1⁺, and expressing low levels ofCD44 (Pgp-1). In addition, investigation of TCR V; gene usageamong adult ⁺ thymocytes, recent emigrants, and spleen cells,indicated a selective emigration of cells expressing certainVgenes.     

Transforming growth factor-{beta} inhibits IL-4 and IFN-{gamma} production by stimulated human T cells

The present study investigates the effect of transforming growthfactor (TGF-ß on the production of IL-4 and IFN- bythe leukemia T_h0 type cell line HUT78, by freshly Isolated humanT cells, and by antigen specific human T cell clones. We foundthat IL-4 and IFN-ß, but not IL-2, production by stimulatedHUT78 cells was inhibited by TGF-ß1. TGF-ß1also reduced the accumulation of IL-4 and IFN- specific mRNAin stimulated HUT78 cells. However, IL-2 and IL-7 co-stimulatedIL-4 and IFN- production, whereas IL-1, IL-3, IL-5, IL-6, IL-8,tumor necrosis factor- or granulocyte macrophage colony stimulatingfactor had no effect. Because IL-2 is an Important helper cytokinefor the production of IL-4 and IFN-, we investigated whethersignal transductlon through the IL-2 receptor is Impaired byTGF-ß1. We found that tyrosine phosphorylatlon inresponse to IL-2 In HUT78 cells was strongly inhibited by ashort prelncubatlon with TGF-ß1. Evidence for an antagonisticrole for TGF-ß1 and IL-2 comes from the finding thathigh doses of IL-2 could partially overcome TGF-ß1mediated inhibition of IL-4 and IFN- production. Similar toIts effect on HUT78 cells, TGF-ß1 also inhibited IL-4and IFN- production by freshly Isolated T cells as well as byhuman T cell clones. Taken together, our experiments show thatthe IL-2 dependent cytokines IL-4 and IFN- are both negativelycontrolled by TGF-ß under conditions where IL-2 productionIs unaffected by a mechanism which partially involves an inhibitionof IL-2/1L-2R signal transductlon. These data Identify TGF-ßand IL-2 as mutual antagonists in the regulation of IL-4 andIFN- production.     

Lymph node homing cells biologically enriched for {gamma}{delta} T cells express multiple genes from the T19 repertoire

Sheep T cells have been shown serologically to express T19,a membrane protein of 180–200 kDa which is a member ofthe scavenger receptor superfamily. Previous work from thislaboratory resulted in the detection of a multigene family ofT19-like genes in the sheep genome. In this study nucleotidesequences from several T19 genes were determined and are reportedalong with the corresponding segments of a number of expressedmRNA molecules. A segment of a single sheep T19-like gene wassequenced and these data, along with the corresponding sequencesfrom cloned T19-like cDNA molecules from sheep and cow, wereused to design an ollgonucleotide primer system suitable foramplification of corresponding segments of many T19 genes andtheir cDNAs. Between 30 and 40% of cloned T19 genes were amenableto amplification using the selected primers, and sequence analysisof cloned PCR products confirmed that different T19 genes encodeunique amino acid sequences. The expression of multiple T19genes was established using cDNA molecules obtained from a singlesample of sheep lymphocyte mRNA. The possible role of the T19family of genes is discussed.     

Membrane and transmembrane signaling in Herpesvirus saimiri-transformed human CD4(+) and CD8(+) T lymphocytes is ATM-independent

In the genetic disorder ataxia telangiectasia (AT), humoral (B) and cellular (T) immunological abnormalities are frequently observed. As a consequence, AT patients are predisposed to life-threatening sinopulmonary infections. The pathogenic mechanisms remain unknown, but a role for ATM in signal transduction from membrane receptors has been proposed. We have explored the effects of a defective ATMgene on isolated human T-lineage cells from 13 AT patients with proven T cell dysfunction by transforming their CD4(+) and CD8(+) T lymphocytes with Herpesvirus saimiri, and analyzing their signaling behavior as compared to normal controls. Several functional parameters were assayed in response to both membrane (anti-CD3 and IL-2) and transmembrane (phorbol myristate acetate plus the calcium ionophore ionomycin) stimuli: (i) calcium mobilization, (ii) induction of activation molecules (CD25, CD40 ligand, CD69 and CD71), (iii) cytokine synthesis (IL-2 and tumor necrosis factor-alpha) and (iv) proliferation. All these early and late activation events were found to be normal in the transformed ATM-/-T cells, indicating that ATM is not necessary for their induction. As expected, ATM-/- transformed T cells showed an increased radiosensitivity by both radioresistant DNA synthesis and cell survival assays. In contrast to an earlier report testing transformed B lymphocytes, our results indicate that transformed mature peripheral T lymphocytes from AT patients do not have intrinsic immune function defects. Rather, the described T-lineage signaling impairments observed in patients may be secondary in vivo to extrinsic ATM-dependent suppressive factors and/or to a developmental defect. These transformed T cells may help to understand the distinct biological role of ATM in different cell types and to develop rational therapies for the immunological dysfunction of AT patients.