Identification of naive or antigen-experienced human CD8(+) T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8(+) T cell response

Human CMV (HCMV) infection provides an informative model of how long term human CD8(+) T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8(+) T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8(+) T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8(+) clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45RO(high) cells and the subpopulation of CD45RA(high) cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8(+) T cell memory, we found that in healthy virus carriers highly purified CD28(-)CD45RA(high)CCR7(-) cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8(+) T cells are predominantly CD28(-)CD45RO(high)CCR7(-). During convalescence, an increasing proportion of pp65-specific CD8(+) T cells were CD28(-)CD45RA(high)CCR7(-). We conclude that naive human CD8(+) T cells are CD28(+)CD45RA(high), express CCR7 but not CCR6, and are predominantly CD27(+) and L-selectin CD62 ligand-positive. The phenotype CD27(+)CD45RA(high) should not be used to identify naive human CD8(+) T cells, because CD27(+)CD45RA(high) cells also contain a significant subpopulation of CD28(-)CD27(+) Ag-experienced expanded clones. Thus CD8(+) T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45RO(high) and CD28(-)CD45RA(high) T cell pools.     

Functional heterogeneity among herpes simplex virus-specific human CD4+ T cells.

One hundred thirteen HSV-specific CD4+ T cell clones were established from the PBL of a healthy person and their functional heterogeneity was investigated. All clones proliferated in response to stimulation with HSV in the presence of autologous APC. Among those, 48 clones showed cytotoxic activity to HSV-infected autologous EBV-transformed lymphoblastoid cell line, but not to HSV-infected autologous fibroblasts, HSV-infected allogeneic cells, or K562 cells (group 1). Five clones showed cytotoxicity against HSV-infected autologous cells as well as HSV-infected allogeneic cells and K562 cells (group 2). The cytotoxicity of these clones was found to be mediated by the direct killing but not by the "innocent bystander" killing of target cells. Sixty clones showed no cytotoxic activity, however, among these, 23 revealed HLA-unrestricted and nonspecific cytotoxicity in the presence of PHA in culture (group 3), and the remaining 37 did not show any cytotoxic activity even in the presence of PHA (group 4). The cytotoxic patterns of these clones did not change in activated and resting phases, suggesting that the difference in cytotoxic ability does not depend on cell cycles. The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR or anti-CD3 mAb to the culture, whereas these mAb had no effect on the cytotoxicity of group 2. All four groups of clones had helper activity for anti-HSV antibody production by autologous B cells. Moreover it was found that all groups of clones simultaneously produced IL-2, IL-4, and IFN-gamma after culture with APC followed by HSV Ag stimulation. The surface phenotype of all clones was uniformly CD2+, CD3+, CD4+, CD8-, CD29+, CD45RA-, but expression of Leu 8 was varied. These data therefore indicate that HSV-specific human CD4+ T cells are classified into at least four groups according to the presence and specificity of cytotoxicity, i.e., Th cells with HSV-specific and HLA-class II-restricted cytotoxicity, Th cells with HLA-unrestricted and nonspecific cytotoxicity, Th cells with lectin-dependent cytotoxicity, and Th cells without cytotoxic activity. The present finding of functional heterogeneity among virus-specific human CD4+ T cells might shed light on the pathogenesis of CD4+ T cell immunodeficiency, such as human retrovirus infections.     

Epstein-Barr virus-transformed lymphoblastoid cell lines as antigen-presenting cells and "augmenting" cells for human CMV-specific Th clones

The ability of Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) to present human cytomegalovirus (HCMV) antigen to a panel of HCMV-specific T helper (Th) clones was evaluated. Among the seven Th clones studied, only one clone (SP-CN/T3-16) proliferated well to HCMV presented by both autologous mononuclear cells (MNC) and LCL, and one clone (SP-CN/T3-9) proliferated significantly better to HCMV presented by autologous LCL than by autologous MNC. The majority of the HCMV-specific Th clones tested (five out of seven) responded much better to HCMV presented by MNC than to HCMV presented by LCL. The mechanism(s) responsible for the inefficiency of LCL to present HCMV to certain clones was studied. Our results suggested that the defect of LCL is not due to insufficient interleukin 1 production, insufficient MHC class II molecule expression, nor an inhibitory mechanism or factor. In this report, we also demonstrate that by adding a minimum amount of LCL along with MNC as antigen-presenting cells (APC), one can restimulate and expand Th clones much more efficiently than by using MNC alone as APC.     

Bovine insulin and porcine or human insulin prime distinct CD4(+) T cell subsets in C57BL/6 mice.

H-2(b) mice produce insulin-specific antibody when injected with bovine but not porcine or human insulin. Nevertheless, CD4(+) T cells have been cloned from C57BL/6 mice primed with porcine, human, and bovine insulin. Here we tested the hypothesis that CD4(+) T cells from C57BL/6 mice primed with porcine or human insulin are functionally distinct from those primed with bovine insulin. Our results show that variants of insulin that stimulate antibody responses induced Th2 clones, whereas variants of insulin that fail to stimulate antibody induced Th0 clones. Th0 clones triggered delayed-type hypersensitivity (DTH) in adoptive recipients, whereas Th2 clones did not. Insulin variants that primed Th0 clones also directly primed for DTH responses, while variants that activated Th2 clones did not. Thus, induction of Th2 clones correlated with the ability of mice to make antibody responses to insulin while development of Th0 clones correlated with DTH responses and the failure to produce antibody.     

CD1-restricted recognition of exogenous and self-lipid antigens by duodenal gammadelta+ T lymphocytes

Gammadelta T cells are present in the mucosal intestinal epithelia and secrete factors necessary to maintain tissue integrity. Ags recognized by these cells are poorly defined, although in mice non-classical MHC class I molecules have been implicated. Since MHC class I-like CD1 receptors are widely expressed at the surface of epithelial and dendritic intestinal cells and have the capacity to present lipid Ags to T cells, we hypothesized that these molecules might present autologous and/or exogenous phospholipids to intestinal gammadelta T lymphocytes. Intraepithelial T lymphocytes from normal human duodenal mucosal biopsies were cloned and exposed to natural and synthetic phospholipids using CD1a-, CD1b-, CD1c- or CD1d-transfected C1R lymphoblastoid or HeLa cell lines as APCs. Their cytolytic properties and regulatory cytokine secretion were also examined. Most clones obtained from duodenal mucosa (up to 70%) were TCRalphabeta+, and either CD4+ or CD8+, whereas 20% were CD4-CD8- (6 clones) or TCRgammadelta+ (12 clones). A relevant percentage (up to 66%) of TCRgammadelta+ but few (<5%) TCRalphabeta+ T cell clones responded to synthetic and/or natural phospholipids presented by CD1 molecules, as measured by both [(3)H]thymidine incorporation and IL-4 release assays. A Th1-like cytolytic and functional activity along with the ability to secrete regulatory cytokines was observed in most phospholipid-specific gammadelta T cell clones. Thus, a substantial percentage of TCRgammadelta+ but few TCRalphabeta+ from human duodenal mucosa recognize exogenous phospholipids in a CD1-restricted fashion. This adaptive response could contribute to mucosal homeostasis, but could also favor the emergence of inflammatory or allergic intestinal diseases.     

Human CD28-CD8+ T cells contain greatly expanded functional virus-specific memory CTL clones.

At birth, almost all human peripheral blood CD8+ T cells express the costimulatory molecule CD28. With increasing age, the proportion of CD8+ T cells that lack CD28 increases. Because the Ag specificity of CD28-CD8+ T cells has not previously been defined, we studied the contribution of CD28-CD8+ T cells to the memory CD8+ CTL response against two human persistent viruses, human CMV (HCMV) and HIV. From PBMC of healthy virus carriers we generated multiple independent CTL clones specific for defined viral peptides and sequenced their TCR beta-chains. We designed clonotypic oligonucleotides complementary to each beta-chain hypervariable sequence and quantified the size of individual immunodominant CTL clones in PBMC. Some individual CTL clones were very large, comprising up to 3.1% of all CD8+ T cells in PBMC, and were generally maintained at a stable level for months. Individual virus-specific CTL clones were consistently more abundant in purified CD28- cells than in the CD8+ population as a whole. Because CD28-CD8+ cells as a population have been reported to proliferate poorly in response to mitogen, we studied the function of these virus-specific CD28- CTL clones by quantifying the frequency of peptide-specific CTL precursors using limiting dilution analysis. CD28-CD8+ T cells contained high frequencies of functional memory CTL precursors specific for peptides of HCMV or HIV, generally higher than in the CD8+ T cell population as a whole. We conclude that in asymptomatic HCMV and HIV infection, human CD28-CD8+ T cells contain high frequencies of functional virus-specific memory CTL clones.     

Long-term stable expanded human CD4+ T cell clones specific for human cytomegalovirus are distributed in both CD45RAhigh and CD45ROhigh populations

T cells play an important role in the control of human CMV (HCMV) infection. Peripheral blood CD4+ T cell proliferative responses to the HCMV lower tegument protein pp65 have been detected in most healthy HCMV carriers. To analyze the clonal composition of the CD4+ T cell response against HCMV pp65, we characterized three MHC class II-restricted peptide epitopes within pp65 in virus carriers. In limiting dilution analysis, we observed high frequencies of pp65 peptide-specific CD4+ T cells, many of which expressed peptide-specific cytotoxicity in addition to IFN-gamma secretion. We analyzed the clonal composition of CD4+ T cells specific for defined HCMV peptides by generating multiple independent peptide-specific CD4+ clones and sequencing the TCR beta-chain. In a given carrier, most of the CD4+ clones specific for a defined pp65 peptide had identical TCR nucleotide sequences. We used clonotype oligonucleotide probing to quantify the size of individual peptide-specific CD4+ clones in whole PBMC and in purified subpopulations of CD45RAhighCD45ROlow and CD45RAlowCD45ROhigh cells. Individual CD4+ T cell clones could be large (0.3-1.5% of all CD4+ T cells in PBMC) and were stable over time. Cells of a single clone were distributed in both the CD45RAhigh and CD45ROhigh subpopulations. In one carrier, the virus-specific clone was especially abundant in the small CD28-CD45RAhigh CD4+ T cell subpopulation. Our study demonstrates marked clonal expansion and phenotypic heterogeneity within daughter cells of a single virus-specific CD4+ T cell clone, which resembles that seen in the CD8+ T cell response against HCMV pp65.     

Modulation of HLA-A*0201-restricted T cell responses by natural polymorphism in the IE1(315-324) epitope of human cytomegalovirus

Cytotoxic T lymphocytes play a central role in the control of persistent human CMV (HCMV) infection and reactivation. In healthy virus carriers, the specific CD8(+) CTL response is almost entirely directed against the virion tegument protein pp65 and/or the 72-kDa major immediate early protein, IE1. Studies that included a large panel of HCMV(+) donors suggested that immunorelevance of pp65 and IE1 was directly related with individual HLA haplotype difference. Nevertheless, there are no data on the incidence of HCMV natural polymorphism on virus-specific CTL responses. To assess the impact of IE1 polymorphism on CTL response, we have sequenced in 103 clinical isolates the DNA region corresponding to IE1(315-324), an immunodominant epitope presented by HLA-A*0201 molecules. Seven peptidic variants were found with extensive difference in their frequencies. The response of four HLA-A*0201-restricted anti-IE1 T lymphocyte clones, which were previously generated from one donor against autologous B lymphoblastoid cells expressing a recombinant clinical variant of IE1, was then evaluated using target cells loaded with mutant synthetic peptides or expressing rIE1 variants. One of four clones, which have been sorted 19 times among 22 clones targeted against IE1(315-324), recognized six of the seven tested variant epitopes. All three other clones showed distinct reactivity patterns to target cells loaded with the different mutant peptides or expressing IE1 variants. Therefore, in the HLA-A2 context, clonal expansions of anti-IE1 memory CTLs may confer a protection against HCMV successive infections and reactivations by killing cells presenting most of the naturally occurring IE1(315-324) epitope variants.     

Human breast carcinoma patients develop clonable oncofetal antigen-specific effector and regulatory T lymphocytes.

Oncofetal Ag (OFA) is a 44-kDa glycoprotein expressed during early to mid-gestation fetal development and re-expressed as a surface Ag by tumor cells soon after transformation. The Ag is detectable on all types of human and rodent tumors tested, but is undetectable on normal cells. In experimental animals it is autoimmunogenic and induces potentially protective T cell responses both after experimental immunization and during tumor development subsequent to carcinogenic insult. To determine whether this tumor-associated Ag is also immunogenic for human T lymphocytes, breast carcinoma patients' peripheral blood mononuclear leucocytes were stimulated in vitro with autologous tumor cells in the presence of IL-2, gamma-IFN, and IL-6 for 2 wk. The tumor-reactive cells were then restimulated and cloned by limiting dilution, and the clones were analyzed. We established 24, 19, 11, and 16 tumor-reactive clones from the four respective patients. Of those, 4, 6, 4, and 7, respectively, proliferated specifically to purified OFA. Both CD4 and CD8 OFA-specific clones were established, which responded equally well to purified OFA or 32- to 44-kDa immature laminin receptor protein. All were CD3+, TCR-alpha beta+. All CD4 clones secreted gamma-IFN, but neither secreted IL-4 nor IL-10. Both IFN-gamma-secreting cytotoxic CD8 clones and IL-10-secreting inhibitory CD8 clones were established. Thus, during human cancer development, the same types of OFA-specific effector and regulatory T cells are induced as during murine T lymphomagenesis.     

Major histocompatibility complex-unrestricted cytolytic activity of human T cells. Analysis of precursor frequency and effector phenotype

The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. These conditions were shown to expand a mean of 96% of cells cultured. All of the 198 clones generated by this method were T cells (CD2+, CD3+, CD4+ or CD2+, CD3+, CD8+) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. In addition, the activity was not inhibited by monoclonal antibodies directed against class I or class II nonpolymorphic MHC determinants. Killing, however, was inhibited by soluble monoclonal antibodies against the CD3 complex. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur or K562 was not mediated by a soluble factor secreted by the clones. Some of the clones retained their cytotoxic activity when grown in rIL-2 alone for 4 to 6 wk, whereas others exhibited markedly diminished cytotoxicity after maintenance in this manner. Clones that exhibited diminished or no cytotoxic activity after prolonged maintenance in rIL-2 could be induced to kill by stimulation with immobilized but not soluble monoclonal antibodies to CD3 in the absence of lectin. All of the clones examined expressed NKH1 and CD11b but none were CD16 positive. The degree of cytotoxicity of resting or activated clones could not be correlated with expression of these markers. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.     

Rapid CD8+ T cell repertoire focusing and selection of high-affinity clones into memory following primary infection with a persistent human virus: human cytomegalovirus

To investigate the mechanism of selection of individual human CD8+ T cell clones into long-term memory following primary infection with a persistent human virus (human CMV (HCMV)), we undertook a longitudinal analysis of the diversity of T cell clones directed toward an immunodominant viral epitope: we followed this longitudinally from early T cell expansion through the contraction phase and selection into the memory pool. We show that following initial HCMV infection, the early primary response against a defined epitope was composed of diverse clones possessing many different TCR Vbeta segments. Longitudinal analysis showed that this usage rapidly focused predominantly on a single TCR Vbeta segment within which dominant clones frequently had public TCR usage, in contrast to subdominant or contracted clones. Longitudinal clonotypic analysis showed evidence of disproportionate contraction of certain clones that were abundant in the primary response, and late expansion of clones that were subdominant in the primary response. All dominant clones selected into memory showed similar high functional avidity of their TCR, whereas two clones that greatly contracted showed substantially lower avidity. Expression of the IL-7R is required for survival of murine effector CD8+ T cells into memory, but in primary HCMV infection IL-7R was not detected on circulating Ag-specific cells until memory had been established. Thus, the oligoclonal T cell repertoire against an immunodominant persistent viral epitope is established early in primary infection by the rapid selection of public clonotypes, rather than being a stochastic process.     

Specificity analysis of human CD4+ T-cell clones directed against human herpesvirus 6 (HHV-6), HHV-7, and human cytomegalovirus.

In order to clarify antigenic variations among various isolates of human herpesvirus 6 (HHV-6) and cross-reactivity among HHV-6, HHV-7, and human cytomegalovirus (HCMV) in the T-cell immune response, the antigenic specificity of the proliferative response mediated by 232 CD4+ human T-cell clones directed against HHV-6, HHV-7, or HCMV was examined. The results obtained were as follows. (i) Although the majority of T-cell clones directed against HHV-6 proliferated in response to stimulation with all strains of HHV-6 used (U1102, Z29, SF, and HST), 7% (8 of 122) of the T-cell clones showed distinct patterns of proliferative response against strain U1102 (group A) and other strains of HHV-6 (group B). (ii) Of 99 T-cell clones, 71 showed a distinct proliferative response to HHV-6 and HHV-7, whereas 28 proliferated in response to stimulation with both HHV-6 and HHV-7. (iii) A small number of T-cell clones (9 of 232) showed cross-reactivity against HHV-6 and HCMV, and 2 of the 232 clones were reactive with HCMV as well as with HHV-6 and HHV-7. (iv) The specificity of gamma interferon production by T-cell clones following the stimulation with virus antigen was identical to that of their proliferative response. These data thus indicate the presence of antigenic variations among isolates of HHV-6 and also epitopes common to HHV-6 and HHV-7 and to HHV-6, HHV-7, and HCMV which are recognized by CD4+ T cells.     

IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones.

The cytolytic potential of a total number of 118 CD4+ human T cell clones specific for purified protein derivative (PPD) from Mycobacterium tuberculosis, tetanus toxoid, Lolium perenne group I allergen (Lol p I), Poa pratensis group IX allergen (Poa p IX), or Toxocara canis excretory/secretory antigen(s) (TES) was assessed by both a lectin (PHA)-dependent and a MHC-restricted lytic assay and compared with their profile of cytokine secretion. The majority of clones with Th1 or Th0 cytokine profile exhibited cytolytic activity in both assays, whereas Th2 clones usually did not. There was an association between the cytolytic potential of T cell clones and their ability to produce IFN-gamma, even though IFN-gamma produced by T cell clones was not responsible for their cytolytic activity. IL-4 added in bulk culture before cloning inhibited not only the differentiation of PPD-specific T cells into Th1-like cell lines and clones, but also the development of their cytolytic potential. The depressive effect of IL-4 on the development of PPD-specific T cell lines with both Th1 cytokine profile and cytolytic potential was dependent on early addition of IL-4 in bulk cultures. In contrast, the addition in bulk culture of IFN-gamma enhanced both the cytolytic activity of PPD-specific T cell lines, as well as the proportion of PPD-specific T cell clones with cytolytic activity. The addition in bulk cultures before cloning of IFN-gamma or IFN-alpha favored the development of TES-specific and Poa p IX-specific T cells into T cell clones showing a Th0 or even a Th1, rather than a Th2, cytokine profile. Accordingly, most of TES- and Poa p IX-specific T cell clones derived from cultures containing IFN-gamma or IFN-alpha displayed strong cytolytic activity. These data indicate that the majority of human T cell clones that produce IFN-gamma, but not IL-4 (Th1-like), as well as of T cell clones that produce IFN-gamma in combination with IL-4 (Th0-like) are cytolytic. More importantly, they demonstrate that the addition of IFN (alpha and gamma) or IL-4 in bulk cultures before cloning may influence not only the cytokine profile of human CD4+ T cell clones but also their cytolytic potential.     

c-myc gene expression and interleukin-2 receptor levels in cloned human CD2+,CD3+ and CD2+,CD3- lymphocytes

The levels of c-myc mRNA and interleukin-2 receptors (IL-2 Rec) were studied in human peripheral blood lymphocytes (PBL); mature CD2+,CD3+ T cell clones and CD2+,CD3- natural killer (NK) cell clones, and CD2+,CD3+ and CD2-,CD3- T lymphoma cell lines. A transient induction of the expression of c-myc and IL-2 Rec was observed in PBL after activation with phytohemagglutinin (PHA). Expression of c-myc and IL-2 Rec was also found in the CD2+,CD3+ and CD2+,CD3- clones. The CD2+,CD3+ showed higher levels of c-myc mRNA and IL-2 Rec than the CD2+,CD3- clones. In three T lymphoma cell lines constitutively high levels of c-myc mRNA but no IL-2 Rec were found. Only in JURKAT (CD2+,CD3+), c-myc mRNA levels could be further enhanced by PHA. These results suggest that in the presence of PHA, expression of c-myc and IL-2 Rec is induced via the CD3 receptor, and in the absence of PHA and/or the CD3 receptor alternative routes of induction are involved.     

Generation of cytomegalovirus-specific human T-lymphocyte clones by using autologous B-lymphoblastoid cells with stable expression of pp65 or IE1 proteins: a tool to study the fine specificity of the antiviral response

Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent human cytomegalovirus (HCMV) infection in healthy virus carriers. Previous analyses of the specificity of HCMV-reactive CD8(+) CTLs drawn from in vitro models in which antigen-presenting cells were autologous fibroblasts infected with laboratory HCMV strains have shown focusing of CTL responses against the major tegument protein, pp65. By contrast, the 72-kDa major immediate-early protein (IE1) was identified as a minor target for this response. Here we have studied the fine specificity and T-cell-receptor features of T-cell clones generated against autologous B lymphoblastoid cell lines stably transfected with HCMV cDNA coding for either pp65 or a natural variant of IE1. This strategy allowed efficient generation of T-cell clones against IE1 and pp65 and led to the identification of several new IE1 and pp65 epitopes, including some located in polymorphic regions of IE1. Such an approach may provide relevant information about the characteristics of the CTL response to IE1 and the effect of viral polymorphism on the immune response against HCMV.     

CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation. Expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production.

Ag-presenting cells provide at least two distinct signals for T cell activation. T cell receptor-dependent stimulation is provided by presentation of a specific peptide Ag in association with MHC molecules. In addition, APC also supply costimulatory signals required for T cell activation that are neither Ag- nor MHC restricted. One such costimulatory signal is mediated via the interaction of B7 on APC with the CD28 receptor on T cells. Recently, CTLA-4 has been shown to be a second B7 receptor on T cells. In the present report, we have examined the expression of CD28 and CTLA-4 on a panel of resting and activated normal T cell subsets and T cell clones by RNA blot analysis in an attempt to determine whether their expression defines reciprocal or overlapping subsets. CD28 was detected in resting T cells, whereas CTLA-4 was not. After stimulation with PHA and PMA for 24 h, CTLA-4 mRNA was expressed in both the CD4+ and CD8+ subsets as well as in CD28+ T cells. We examined 37 human and six murine T cell clones that had been previously characterized for their cytokine production. After activation, CTLA-4 and CD28 mRNA were coexpressed in 36 of 37 human T cell clones and all six murine T cell clones. These included T cells of CD4+8-, CD4-8+, and CD4-8- phenotypes as well as clones with Th1 and Th2 cytokine profiles. In contrast, CD28 but not CTLA-4 mRNA was detected in leukemic T cell lines and myelomas. CTLA-4 and B7 mRNA but not CD28 mRNA was detected in two long term HTLV-I-transformed T cell lines. These data demonstrate that CD28 and CTLA-4 mRNA are coexpressed in most activated T cells and T cell clones, providing evidence that they do not define reciprocal subsets. Moreover, they are consistent with the hypothesis that B7 transmits its signal through a single receptor, CD28, on resting T cells, and multiple receptors, CD28 and CTLA-4, on activated T cells.     

T cell clones for antigen selection and lymphokine production in murine Schistosomiasis mansoni.

The role of T cells in immunity to murine schistosomiasis was examined through the use of T cell clones that recognize the live schistosomulum stage of Schistosoma mansoni. T cell clones of three different phenotypes were isolated and expanded into long term culture from lymph nodes of C57B1/6J mice vaccinated with irradiated S. mansoni larvae. They were characterized by surface markers, lymphokine production, and functional assays. The m.w. range of the Ag recognized by one clone was identified through nitrocellulose blotting and confirmed with a preparation of the putative protein made by immunoaffinity purification. All but one of the clones were CD4+, CD5+, Th cells. One clone, 35, produced Il-2 and IFN-gamma and was designated a TH1 clone. The others were designated TH2 clones because of Il-4 production. One clone was CD8+ and failed to show help. Clone 35 recognized live schistosomula and produced Il-2 when presented a 27-kDa protein from nitrocellulose. It proliferated in response to purified Ag. Clone 35 participated along with macrophages to induce up to 98% killing of live schistosomula in vitro. IFN-gamma and TNF-alpha were essential to the killing mechanism whereas Il-1, Il-2, and Il-4 were not required. This study has approached Ag identification for vaccine development from the point of view of T cells and showed that TH1 cells are essential to in vitro macrophage killing of schistosomula in murine schistosomiasis.     

Functional heterogeneity among human inducer T cell clones

Analysis of mouse CD4+ inducer T cells at the clonal level has established that a dichotomy among CD4+ T cell clones exists with regard to types of lymphokines secreted. Mouse T cell clones designated Th1 have been shown to secrete IL-2 and IFN-gamma, whereas T cell clones designated Th2 have been shown to produce IL-4 but not IL-2 or IFN-gamma. To determine if such a dichotomy in the helper inducer T cell subset occurred in man, we examined a panel of human CD4+ helper/inducer T cell clones for patterns of lymphokine secretion and for functional activity. We identified human T cell clones which secrete IL-4 but not IL-2 or IFN-gamma, and which appeared to correspond to murine Th2 clones. In marked contrast to murine IL-2 secreting Th1 clones which do not produce IL-4 or IFN-gamma, we observed that some human T cell clones secrete IL-2, and IFN-gamma as well as IL-4. Southern blot analysis indicated that these multi-lymphokine-secreting clones represented the progeny of a single T cell. IL-4 secretion did not always correlated with enhanced ability to induce Ig synthesis. Although one T cell clone which secreted IL-2, IL-4, and IFN-gamma could efficiently induce Ig synthesis, another expressed potent cytolytic and growth inhibitory activity for B cells, and was ineffective or inhibitory in inducing Ig synthesis. These results indicate that although the equivalent of murine Th2 type cells appears to be present in man, the simple division of T cells into a Th1 and Th2 dichotomy may not hold true for human T cells.     

Production of lymphokine mRNA by CD45R+ and CD45R- helper T cells from human peripheral blood and by human CD4+ T cell clones

The expression of lymphokine mRNA by human CD4+CD45R+ and CD4+CD45R- Th cells was assessed after mitogen stimulation. These Ag have previously been shown to relate closely to virgin and primed T cells, respectively. CD4+CD45R+ (virgin) and CD4+CD45R- (primed) cell fractions were isolated by sorting double-labeled cells with a fluorescence-activated cell sorter. CD4+CD45R+ cells produced high levels of IL-2 mRNA when stimulated with either PMA together with calcium ionophore, or with PHA, but they expressed only trace quantities of mRNA for IL-4 or IFN-gamma. In contrast, CD4+CD45R- cells produced high levels of mRNA for IL-2, IL-4, and IFN-gamma. After 14 days of continuous culture, CD4+CD45R+ Th cells lost expression of the CD45R Ag, but gained high level expression of CDw29, such that they were indistinguishable from the cell population which originally expressed this Ag. At the same time, they acquired the ability to synthesize IL-4 mRNA. It seemed likely that the broad lymphokine profile of primed Th cells might mask clonal heterogeneity. Analysis of 122 CD4+ T cell clones showed that all of them synthesized IL-2 mRNA. One clone failed to express IL-4 mRNA, but did produce those for IL-2 and IFN-gamma. A total of 34 of the clones was investigated to determine expression of IFN-gamma mRNA; two of these clones were negative for IFN-gamma mRNA, and both expressed IL-2 and IL-4 message. These data suggest that while fresh virgin and primed peripheral blood T cells show a clear resolution of lymphokine production, a simple subdivision of human CD4+ T cell clones on the basis of their lymphokine production (such as that reported for mouse Th cell clones) is not possible.     

Epitopes of the Mycobacterium tuberculosis 65-kilodalton protein antigen as recognized by human T cells

A synthetic peptide approach has been used to identify the epitopes recognized by clonal and polyclonal human T cells reactive to the recombinant mycobacterial 65-kDa protein Ag. Three of the four epitopes identified were recognized as cross-reactive between Mycobacterium tuberculosis and Mycobacterium leprae, although their amino acid sequence in two of three cases was not identical. The peptide (231-245) defining an epitope recognized as specific to the M. tuberculosis complex contains two substitutions compared with the homologous M. leprae region of which one or both are critical to T cell recognition. The reactive T cell clones showed helper/inducer phenotype (CD4+, CD8-), and secrete IL-2, granulocyte-macrophage-CSF, and IFN-gamma upon Ag stimulation. The same clones display cytotoxicity against macrophages pulsed with the relevant peptides or mycobacteria.