The predominance of CD8+ T cells after infection with measles virus suggests a role for CD8+ class I MHC-restricted cytotoxic T lymphocytes (CTL) in recovery from measles. Clonal analyses of human CD8+ class I MHC-restricted CTL

Stimulation of PBMC, in children recovering from acute measles, with autologous EBV-transformed and measles virus (MV)-infected lymphoblastoid cell lines (B-LCL) expanded primarily MV-specific CD8+ T cells. A large number of CD8+ T cell clones were obtained either by passaging of bulk cultures at limiting dilutions or by direct cloning of PBMC without previous stimulation in bulk culture. The MV-specific CD8+ T cell clones responding in a proliferative and a CTL assay were found to be class I MHC restricted. In contrast, CD4+ MV-specific T cell clones, which were generated by the same protocol, recognized MV in association with class II MHC molecules. Analysis of processing requirements for Ag presentation to CD8+ and CD4+ T cell clones, measured by the effect of chloroquine in a proliferative T cell response, revealed that both types of T cells recognized MV Ag processed via the endogenous/cytoplasmic pathway. Thus, these studies indicate that, as in most other viral infections and in contrast to previous suggestions, the class I MHC-restricted CTL response by CD8+ T cells may be an important factor in the control and elimination of MV infection. Therefore, the role proposed for CD4+ class II-restricted T cells in recovery from measles needs to be reevaluated.     

Measles virus-specific murine T cell clones: characterization of fine specificity and function

Measles virus (MV)-specific murine helper T cell clones (Thy-1.2+, CD4+, CD8-) were generated from mice immunized with MV-infected mouse brain homogenate by limiting dilution and in vitro stimulation of spleen cells with UV-inactivated MV Ag. The protein specificity of 7 out of 37 stable T cell clones, which displayed MHC-restricted MV Ag recognition, could be assessed by using purified MV proteins. Two fusion (F) protein-specific, two hemagglutinin-specific, and three nucleoprotein- or matrix protein-specific clones were shown to be established. The F protein-specific T cell clones together with a panel of previously generated F protein-specific T cell clones were characterized for their fine specificity by using beta-galactosidase fusion products, which contained different parts of the F protein. It was shown that at least two epitopes on the major part of the F protein (amino acid 2-513) can be recognized by mouse T cells. Functional characterization of three T cell clones showed that they were able to assist MV-specific B cells and bystander B cells for antibody production. Furthermore, they were shown to produce the lymphokines IL-2 and IFN-gamma. It was also shown that these T cell clones induced a MV-specific delayed type hypersensitivity response. These observations suggest that all of the T cell clones characterized belong to the TH1 helper subset.     

Differences in surface phenotype and mechanism of action between alloantigen-specific CD8+ cytotoxic and suppressor T cell clones

We have previously demonstrated that fresh CD8+ T cells proliferate in response to autologous, alloantigen-primed CD4+ T cells, and differentiate into Ts cells, which inhibit the response of fresh T cells to the primary allogeneic stimulator cell but not irrelevant stimulators. Although such Ts do not have discernible cytolytic activity, like classical cytotoxic T cells (Tc) they express CD3 and CD8 on their surface and function in a class I MHC-restricted manner. Our study was an attempt to compare the surface phenotype and mechanism of action of Ts and Tc clones derived from the same individual. Ts clones were generated from donor JK by repeated stimulation of CD8+ T cells with an autologous CD4+ T inducer line specific for an allogeneic lymphoblastoid cell line (LCL). These clones were noncytolytic for either the inducer line or the allogeneic stimulator LCL. Tc clones, generated by direct stimulation of JK CD8+ T cells with the same allogeneic LCL, mediated potent, alloantigen-specific cytolysis. All Tc clones were alpha, beta TCR+, CD3+, CD4-, CD8+, CD11b-, and CD28+. Ts clones were also alpha, beta TCR+, CD3+, and CD8+, but in contrast to Tc clones, Ts clones were CD11b+ and CD28-. When added to MLR both Ts and Tc clones inhibited the response of fresh JK CD4+ T cells to the original but not irrelevant allogeneic LCL. However, Ts inhibited the response of only those CD4+ T cells that shared class I)MHC determinants with the Ts donor, whereas Tc inhibited the response of CD4+ T cells from all responders, regardless of HLA type. Pretreatment of Ts clones with mAb to CD2, CD3, or CD8 blocked suppression, whereas similar pretreatment of Tc clones blocked cytotoxicity in 4-h 51Cr release assays but had no effect on Tc-mediated suppression of the MLR. These results suggest that both Ts and Tc clones can inhibit the MLR but they do so through different mechanisms. Moreover, the maintenance of distinct surface phenotypes on these long term clones suggests that Ts may be a distinct sublineage of CD8+ T cells rather than a variant of CD8+ Tc.     

Helper activity in antigen-specific antibody production mediated by CD4+ human cytotoxic T cell clones directed against herpes simplex virus

Three HSV type 1 (HSV-1) and HSV type 2 (HSV-2) common ("HSV-type common") and three HSV-1 specific CTL clones, which were CD3+, CD4+, CD8-, 4B4+, and 2H4-, were established. These clones proliferated in response to stimulation with HSV in the presence of autologous APC. The HSV type specificity of the proliferative response was identical with that of the cytotoxic activity of the clones. The cytotoxic activity and the proliferative response were both inhibited by addition of anti-HLA-DR mAb to the culture. After culture of these CTL clones with autologous B cells and macrophages followed by HSV Ag stimulation, anti-HSV antibody was detected in the culture supernatant. The HSV type specificity of the helper function for antibody production was identical with that of the cytotoxicity, i.e., HSV-type common clones, upon stimulation with either HSV-1, or HSV-2, and HSV-1-specific clones, upon stimulation with HSV-1 but not with HSV-2, showed helper activity for anti-HSV antibody production by autologous B cells. Moreover, it was found that these clones produced humoral factors which help autologous B cells to produce antibody. The helper factors were produced by T cell clones in an HSV-type-specific manner. These data suggest that some CD4+ T cells can simultaneously manifest both specific cytotoxicity and helper activity for Ag-specific antibody production by B cells, and that these multifunctional T cells might play an important role in protection against viral infection.     

CD40-ligated dendritic cells effectively expand melanoma-specific CD8+ CTLs and CD4+ IFN-gamma-producing T cells from tumor-infiltrating lymphocytes

Professional APC, notably dendritic cells (DC), are necessary for stimulation and expansion of naive T cells. By means of murine models, the interaction between CD40 on DC and its ligand CD154 has been recognized as an important element for conditioning of DC to prime and expand CTL. We translated these findings into the human system, scrutinizing the ability of DC to initiate clonal expansion of single T cells. DC generated under completely autologous conditions from peripheral blood monocytes were cocultured at a rate of 0.3 cell/well with melanoma-infiltrating T cells; this procedure guaranteed that either a CD4+ or a CD8+ cell interacted with the DC, thus avoiding the contact of more than one T cell to the DC. In the absence of further stimulation, this cloning protocol yielded almost exclusively CD4+ T cell clones that predominantly exhibited a Th2 phenotype. However, cross-linking of CD40 on DC resulted in the induction of IFN-gamma-producing Th1 CD4+ T cell clones. In addition, CD40-activated DC were capable of expanding CD8+ CTL clones. The ratio of CD4 to CD8 T cell clones corresponded to the ratio present in the initial tumor-infiltrating lymphocyte preparation. The CTL clones efficiently lysed autologous tumor cells whereas autologous fibroblasts or MHC-mismatched melanoma cells were not killed. Our findings support the critical role of CD40/CD154 interactions for the induction of cellular immune responses.     

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.     

Composite response of naive T cells to stimulation with the autologous lymphoblastoid cell line is mediated by CD4 cytotoxic T cell clones and includes an Epstein-Barr virus-specific component.

We have approached the challenge of generating a primary T cell response to Epstein-Barr virus (EBV) in vitro by stimulating naive T cells with the autologous EBV-transformed lymphoblastoid cell line (LCL), a rich source of EBV-associated cytotoxic T lymphocyte (CTL) epitopes. Responsive T cells from three EBV-seronegative donors were cloned in agarose, phenotyped for T cell markers by flow cytometry, and their cytotoxic properties analyzed in the 51Cr release assay. Most clones (greater than 95%) expressed the CD4 phenotype and 59% of these clones showed cytotoxic properties. The dominant CTL response was specific for FCS-associated epitopes presented by FCS-grown autologous LCL target cells and was restricted by class II HLA antigens. Other clonal components included: (i) an EBV-specific response by HLA-restricted CD4 CTL clones that did not discriminate between A- and B-type EBV transformants; (ii) an EBV-specific response by an HLA-restricted CD4 CTL clone that discriminated between A- and B-type transformants, and (iii) a nonspecific cytotoxic response by CD3+,4+,8-, CD3+,4-,8-, and CD3-,4-,8- clones that were broadly allotypic or restricted to the lysis of K562 target cells. The EBV-specific CTL clones did not lyse the autologous EBV-negative B or T cell blasts and their specificity patterns of lysis were supported by the cold target competition data. These studies highlight the role of CD4 CTL in the establishment in vitro of a primary immune response to a human virus.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

CD4+ cytolytic T cell clones restricted to HLA class II, DR beta I, and DR beta III chains

We have investigated the functional polymorphism of HLA class II antigens using CD4+ CTL clones. Seven CD4+ CTL clones were isolated from a healthy donor (HLA A2 A24; B8 B27; DRw17 DRw52a) by repeated stimulation with irradiated autologous EBV-transformed B cell lines (EBV-B). According to the HLA restriction specificity we divided CD4+ CTL clones into three subgroups: (i) DRw17-restricted CD4+ CTL clones; (ii) DRw52a-restricted CD4+ CTL clones; and (iii) the CD4+ CTL clones, of which the restriction specificity could not be assigned to products of a single HLA locus. Interestingly, DRw17-restricted CD4+ CTL clones distinguished between DRw17 and DRw18. Similarly, DRw52a-restricted CD4+ CTL clones distinguished between DRw52a, w52b, and w52c. There are four amino acids which differ between DRw17 and DRw18, whereas five differ between DRw52a and the other two alleles (DRw52b and DRw52c). The recent elucidation of the crystal structure of a human class I MHC molecule has identified the probable peptide binding site to be a cleft on the outer surface of the molecule, between two alpha-helices. On the basis of the theoretical model for HLA class II molecules, amino acid positions 26 and 28 (DRw17 vs DRw18) and amino acid positions 26, 28, and 74 (DRw52a vs the other two alleles) lie within the "cleft." We propose that amino acid positions 26 and 28 are very important sites with regard to the recognition of antigen-MHC complex by the TCR.     

Vaccinia virus-specific human CD4+ cytotoxic T-lymphocyte clones.

Vaccinia virus-specific cytotoxic T-lymphocyte (CTL) clones were established from a healthy donor, who had been immunized with vaccinia virus vaccine, by stimulation of peripheral blood lymphocytes with UV-inactivated vaccinia virus antigen. The phenotype of all of the clones established was CD3+ CD4+ CD8- Leu11-. We used a panel of allogenic vaccinia virus-infected B-lymphoblastoid cell lines and demonstrated that some of the clones recognized vaccinia virus epitopes presented by human leukocyte antigen (HLA) class II molecules. Monoclonal antibodies specific for either HLA-DP or HLA-DR determinant reduced the cytotoxicity of specific clones. The HLA-restricted cytotoxicity of the clones is vaccinia virus specific, because vaccinia virus-infected but not influenza virus-infected autologous target cells were lysed. Using vaccinia virus deletion mutants, we found that some of the CTL clones recognize an epitope(s) that lies within the HindIII KF regions of the vaccinia virus genome. These results indicate that heterogeneous CD4+ CTL clones specific for vaccinia virus are induced in response to infection and may be important in recovery from and protection against poxvirus infections.     

Vaccinia virus-specific CD8+ cytotoxic T lymphocytes in humans.

Stimulation of human vaccinia virus immune peripheral blood mononuclear cells in vitro from vaccinia virus-immune donors with live vaccinia virus-infected autologous cells generated vaccinia virus-specific cytotoxic T lymphocytes (CTL) capable of lysing vaccinia virus-infected cells. We generated vaccinia virus-specific CD8+ clones and CD4+ CTL lines by limiting dilution from two donors by using peripheral blood mononuclear cells obtained 2 months or 4 years postrevaccination with vaccinia virus. These results demonstrate that vaccinia virus-specific CTL are generated as a result of immunization of humans with vaccinia virus and that both CD8(+)- and CD4(+)-specific T cells are maintained as memory cells.     

Regulation of myelin basic protein-specific helper T cells in multiple sclerosis: generation of suppressor T cell lines.

Suppressor T cell (Ts) lines specific for myelin basic protein (MBP)-reactive helper T cell (Th) clones were generated from two patients with multiple sclerosis (MS) following a primary culture of peripheral blood mononuclear cells (PBMC) with MBP and cyclosporine A (CsA). These suppressor T cell lines were maintained in culture by alternate stimulation with MBP and antigen-presenting cells (APC). The Ts lines expressed preferentially the CD4 phenotype (5/6 Ts lines tested) and exhibited potent antigen-specific suppressor activity on the proliferation of MBP-specific Th clones and not on the T cell lines with other antigen specificity. For some Ts lines, a Ts-to-Th ratio of 1 was sufficient to inhibit the proliferation of MBP-specific T cells by 90%. The suppressor T cells obtained were weakly responsive to MBP and required the presence of the autologous PBMC for proliferation. Furthermore, proliferation of these suppressor T cell lines was restricted by HLA-DR molecules (for CD4+ Ts lines) and HLA class I (for a CD8+ Ts line). The suppressor T cell lines generated and the techniques described in this study may be helpful in our understanding of the events involved in the immune regulation in MS and other autoimmune diseases.     

Requirement of cognate CD4+ T-cell recognition for the regulation of allospecific CTL by human CD4+ CD127- CD25+ FOXP3+ cells generated in MLR

Although immunoregulation of alloreactive human CTLs has been described, the direct influence of CD4(+) Tregs on CD8(+) cytotoxicity and the interactive mechanisms have not been well clarified. Therefore, human CD4(+)CD127(-)CD25(+)FOXP3(+) Tregs were generated in MLR, immunoselected and their allospecific regulatory functions and associated mechanisms were then tested using modified (51)Chromium release assays (Micro-CML), MLRs and CFSE-based multi-fluorochrome flow cytometry proliferation assays. It was observed that increased numbers of CD4(+)CD127(-)CD25(+)FOXP3(+) cells were generated after a 7 day MLR. After immunoselection for CD4(+)CD127(-)CD25(+) cells, they were designated as MLR-Tregs. When added as third component modulators, MLR-Tregs inhibited the alloreactive proliferation of autologous PBMC in a concentration dependent manner. The inhibition was quasi-antigen specific, in that the inhibition was non-specific at higher MLR-Treg modulator doses, but non-specificity disappeared with lower numbers at which specific inhibition was still significant. When tested in micro-CML assays CTL inhibition occurred with PBMC and purified CD8(+) responders. However, antigen specificity of CTL inhibition was observed only with unpurified PBMC responders and not with purified CD8(+) responders or even with CD8(+) responders plus Non-T "APC". However, allospecificity of CTL regulation was restored when autologous purified CD4(+) T cells were added to the CD8(+) responders. Proliferation of CD8(+) cells was suppressed by MLR-Tregs in the presence or absence of IL-2. Inhibition by MLR-Tregs was mediated through down-regulation of intracellular perforin, granzyme B and membrane-bound CD25 molecules on the responding CD8(+) cells. Therefore, it was concluded that human CD4(+)CD127(-)CD25(+)FOXP3(+) MLR-Tregs down-regulate alloreactive cytotoxic responses. Regulatory allospecificity, however, requires the presence of cognate responding CD4(+) T cells. CD8(+) CTL regulatory mechanisms include impaired proliferation, reduced expression of cytolytic molecules and CD25(+) activation epitopes.     

Activation of HLA-restricted EBV-specific cytotoxic T cells does not require CD4+ (helper) T cells or exogenous cytokines

MHC-restricted, viral Ag-specific "memory" CTL are thought to play a decisive role in the defense against pathogenic viruses. However, the requirements for activating such CTL remain controversial. In particular, the role of CD4+ helper cells and their soluble products (e.g., IL-2) are uncertain. To approach these questions as they relate to EBV-specific CTL, highly purified CD8+ T cells from healthy EBV-seropositive individuals were cultured with autologous irradiated EBV-transformed B lymphoblastoid cell lines (LCL), in the presence or absence of autologous CD4+ cells or 1 to 10 U/ml purified rIL-2. The results indicate that the induction of CTL requires neither Th cells nor exogenous IL-2. The CTL generated from isolated CD8+ cells were HLA class I restricted as demonstrated by their ability to lyse targets sharing at least one HLA-A or -B Ag with the stimulating autologous LCL. Furthermore, a mAb (W6/32) to a common determinant on HLA class I Ag blocked both the generation and effector phases of killing, whereas an HLA class II directed mAb had no effect. Addition of an IL-2R-specific antibody (anti-Tac) to the culture medium blocked induction of CTL, suggesting that endogenously produced IL-2 plays an obligatory role in this system. Paraformaldehyde fixation of LCL abrogated their ability to function as stimulator cells; however, addition of 2 U/ml exogenous IL-2 to fixed LCL cultured with CD8+ cells allowed for the induction of highly specific CTL. These results indicate that EBV-specific memory CTL can be activated in the absence of CD4+ helper cells or their soluble products, but nonetheless require Ag and IL-2.     

Antigen specific and MHC nonrestricted cytotoxicity of T cell receptor alpha beta+ and gamma delta+ human T cell clones isolated in IL-4

IL-4 has been shown to act as a growth factor for human T cells. In addition, IL-4 can enhance CTL activity in MLC, but blocks IL-2 induced lymphokine activated killer cell activity in PBL. In our study, the cloning efficiencies, Ag-specific CTL activity and non-MHC-restricted cytotoxicity of CTL clones generated in IL-2 were compared to those generated in IL-4. In a first experiment, T cells were stimulated with the EBV-transformed B cell line JY and cloned 7 days later with feeder cells and either IL-2 or IL-4. In a second experiment, stimulation of the T cells was carried out in the presence of IL-2 plus anti-IL-4 antibodies or IL-4 plus anti-IL-2 antibodies in order to block the effects of IL-4 and IL-2, respectively, produced by the feeder cells. Although the cloning efficiencies in the second experiment were lower than those obtained in the first experiment, the cloning efficiencies obtained with IL-2 or IL-4 were similar in both experiments. The overall proportion of TCR alpha beta+ T cell clones cytotoxic for the stimulator cell JY established in IL-2 or IL-4 were comparable. A striking difference between the clones obtained in IL-2 or IL-4 was that a large proportion of the clones obtained in IL-4 expressed CD4 and CD8 simultaneously, whereas none of the clones isolated in IL-2 were double positive. Also gamma delta+ T cell clones could be established with IL-4 as a growth factor. TCR gamma delta+ T cell clones isolated in either IL-2 or IL-4 were CD4-CD8- or CD4-CD8+, but the proportion of CD4-CD8+ clones isolated in IL-4 was higher. Interestingly, one TCR gamma delta+ clone isolated in IL-2 was CD4+CD8-. Most of the TCR alpha beta+ and TCR gamma delta+ CTL-clones isolated in IL-2 lysed the NK cell sensitive target cell K562. In contrast, only a small proportion of the TCR alpha beta+ or TCR gamma delta+ CTL clones isolated in IL-4, lysed K562. One TCR gamma delta+ T cell clone (CD-124) isolated in IL-4 and subsequently incubated in IL-2 acquired lytic activity against K562.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alloantigen-specific cytotoxic clones bearing the alpha,beta T cell antigen receptor but not CD4 or CD8 molecules

The vast majority of circulating lymphocytes that express the alpha,beta TCR in association with CD3 also express either CD4 or CD8 molecules, which are thought to act as important accessory structures in HLA class II- and I-restricted T cell functions, respectively. In the current study alpha,beta TCR+ clones devoid of detectable CD4 or CD8 were generated by repeated stimulation of fresh CD3+,CD4-,CD8- cells with an allogeneic lymphoblastoid cell line in the presence of conditioned medium containing IL-2. Except for the absence of CD4 and CD8, which was associated with undetectable levels of CD4 and CD8 mRNA, the clones were phenotypically indistinguishable from classical CD3+,alpha,beta TCR+ cells. Furthermore, they mediated potent cytolysis of their specific stimulator line but did not kill irrelevant LCL or NK-sensitive targets. mAb to CD3 and the alpha,beta TCR inhibited cytolysis, suggesting that the clones use the TCR/CD3 complex to recognize and respond to their targets. mAbs to CD2 and CD11a also inhibited cytolysis, indicating that the clones use these accessory molecules to interact with their targets. Finally, cytolysis was inhibited by an HLA-A,B,C framework-specific mAb (W6/32) as well as a mAb (MA2.1) specific for an HLA-A2 epitope. These results demonstrate that CD3+,alpha,beta TCR+,CD4-,CD8- cytotoxic clones can be generated from the peripheral blood of healthy adults, and use their TCR/CD3 complexes to function in an HLA class I-restricted manner.     

Differential in vitro activation of CD4+CD8- and CD8+CD4- herpes simplex virus-specific human cytotoxic T cells

In order to clarify the differential activation of CD4+ and CD8+ HSV-specific CTL, we compared the characteristics of CTL generated by different methods of in vitro HSV stimulation by treatment of effectors with anti-CD4 and anti-CD8 mAb and C after the elimination of nonspecific cytotoxic effector cells. Cell-free HSV mainly activated CD4+ CTL precursors, whereas HSV-infected fibroblasts were more effective in activating CD8+ CTL precursors than CD4+ CTL precursors. In addition, limiting dilution analyses with enriched T cells from two HSV-seropositive donors revealed that the frequency of HSV-specific CD4+ CTL precursors responsive to stimulation with free HSV was approximately 1/4,000 to 6,000 CD4+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/19,000 to 22,000 CD4+ T cells. Conversely, the frequency of CD8+ CTL precursors in peripheral blood responsive to stimulation with free HSV was approximately 1/28,000 to 30,000 CD8+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/10,000 to 11,000 CD8+ T cells. The present data suggest that generalized viral infection due to cell-free viruses is fought mainly by CD4+ CTL, which have previously been reported to possess both cytotoxicity and helper function, and that localized viral infection on HLA class II-negative fibroblasts is prevented from spreading to adjacent cells mainly by CD8+ CTL. Such differential activation of CD4+ and CD8+ CTL seems probable when considering the protective mechanisms against viral infection.     

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.