Activation of MHC class I-restricted CD8+ CTL by microbial T cell mitogens. Dependence upon MHC class II expression of the target cells and V beta usage of the responder T cells

The T cell response to microbial T cell mitogens (MTM) such as enterotoxins from Staphylococcus aureus (SE) and the soluble mitogen from Mycoplasma arthritidis, resemble the minor lymphocyte stimulatory locus (Mls) response in several aspects. An important feature of the Mls response is it restriction to CD4+ cells. This study demonstrates that in contrast to Mls, the MTM response includes both CD4+ and CD8+ subsets. Both CD4+ and CD8+ cells expanded in IL-2 after stimulation with SEB showed preferential expression of T cell receptors bearing V beta 8 domains. Mouse and human target cells could be lysed in the presence of MTM both by MTM-stimulated CD8+ lymphocytes and by MHC class I-restricted CTL clones of defined Ag specificity. MTM-induced lysis required the expression of MHC class II, but not class I Ag, on the target cells. Inhibition studies of SEB and Ag-dependent cytolysis by CTL clones underlined the crucial role of CD3 and LFA-1 in both instances, but showed CD8 dependence only for AG-dependent cytolysis. Together these findings suggest important differences between the putative MTM-mediated interaction of TCR with MHC molecules and the classical TCR/MHC interaction involved in MHC-restricted Ag recognition.     

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)     

IL-15 promotes survival but not effector function differentiation of CD8+ TCRalphabeta+ intestinal intraepithelial lymphocytes

CD8 single-positive cells, including CD8alphaalpha+ and CD8alphabeta+ subsets, constitute the majority of TCRalphabeta+ intestinal intraepithelial lymphocytes (alphabeta iIEL) in mice. CD8+ alphabeta iIEL show significantly weaker responses to TCR stimulation in the presence of exogenous IL-2 than do CD8+ T cells of the central immune system. IL-15 is a T cell growth factor likely expressed in the intestine mucosa. To understand the role of IL-15 in CD8+ alphabeta iIEL biology, we compared the effects of exogenous IL-15 and IL-2 on the survival and primary responses of the two CD8+ alphabeta iIEL subsets in vitro. In contrast to the death of approximately 60% of both CD8alphaalpha+ and CD8alphabeta+ iIEL cultured in IL-2 with or without TCR stimulation, IL-15 promoted survival of the CD8alphaalpha+ subset in the presence of TCR stimulation and promoted survival of both subsets in the absence of TCR stimulation. The higher proliferation level of TCR stimulated CD8alphaalpha+ alphabeta iIEL cultured in IL-15 compared with those cultured in IL-2 is likely due to IL-15's prosurvival effects. In addition, unlike exogenous IL-2, exogenous IL-15 did not support the effector functions of either iIEL subsets, including IFN-gamma production, IL-4-induced Th2 cytokine production, and anti-TCR mAb-redirected cytotoxicity. These findings demonstrate that IL-15 and IL-2 are functionally distinct and suggest that IL-15 plays a unique role in the maintenance of the CD8+ alphabeta iIEL pool in the absence of Ag stimulation and in the survival and expansion of CD8alphaalpha+ alphabeta iIEL upon Ag stimulation.     

Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.     

Unrestricted recognition of a nonpeptide antigen by CD8+ cytolytic T lymphocytes

CD8+ murine CTL that are specific for an unusual nonpeptide Ag, the heme moiety of hemoglobin, have been derived by in vitro stimulation of spleen cells with hemin. Such CTL demonstrate a requirement for the expression of class I Ag on target cells, yet appear to be unrestricted to the extent that both syngeneic and allogeneic targets precoated with hemin are sensitive to lysis. A series of CTL clones with specificity for hemin was derived from C57BL/6 mice. They exhibited the same type of promiscuous recognition that was observed in CTL populations from a number of different strains. The possibility that hemin acts as a nonspecific mediator of lysis by CTL was ruled out by the fact that a variety of CTL populations and clones specific for different Ag did not exhibit hemin-specific lysis. Some explanations offered to explain these results include 1) the possibility that hemin is recognized by binding to a site on the MHC other than the Ag-binding groove, and 2) the possibility that TCR recognition of a rigid molecule, such as hemin, may be less sensitive to polymorphic variation in the MHC than is recognition of a conventional peptide Ag whose conformation may differ significantly when bound to MHC molecules whose sequences differ within the Ag-binding groove.     

Helper-independent CD8+ cytotoxic T lymphocytes express IL-1 receptors and require IL-1 for secretion of IL-2

The purpose of this study was to examine the role of IL-1 on the activation of CD8+/CD4- class I-restricted helper cell-independent cytolytic T cell (HITc) clones known to produce IL-2 and proliferate in vitro after Ag stimulation with a Friend retrovirus-induced leukemia (FBL). The functional role of IL-1 in Ag-specific proliferation and IL-2 secretion was assessed by stimulating the T cell clones with FBL either in the presence or absence of macrophages (M phi), rIL-1, or rIL-2. Resting cloned HITc cells, purified from residual accessory cells, failed to proliferate in response to FBL alone, but proliferated in response to FBL plus M phi, rIL-1 or rIL-2. Stimulation with FBL alone in the absence of M phi or IL-1 was sufficient for induction of IL-2R expression, and rendered cells responsive to IL-2, but M phi or IL-1 were also required to induce production of IL-2. The activity of IL-1 was further examined by measuring the binding of [125I]rIL-1 alpha, which demonstrated that resting cloned HITc cells expressed IL-1R that increased in number after activation with Ag. This expression of IL-1R and requirement for IL-1 by CD8+ HITc was surprising because previous studies examining T cell populations after mitogen stimulation have not detected IL-1R on the CD8+ population. Therefore, the role of IL-1 in the activation of CD8+ CTL that do not secrete IL-2 after activation was assessed. By contrast to HITc, CD8+ CTL required exogenous IL-2 to proliferate in vitro and did not express IL-1R. These data demonstrate that the subset of CD8+ T cells responsible for IL-2 production express IL-1R and that triggering this receptor with IL-1 after Ag stimulation results in the production of IL-2 and subsequent proliferation.     

Human antitumor CD8+ T cells producing Th1 polycytokines show superior antigen sensitivity and tumor recognition

Adoptive transfer of T cells expressing transgenic TCR with antitumor specificity provides a hopeful new therapy for patients with advanced cancer. To fulfill a large need for TCR with high affinity and specificity for various tumor entities, we sought to identify parameters for rapid selection of CTL clones with suitable characteristics. Twelve CTL clones displaying different Ag sensitivities for the same peptide-MHC epitope of the melanoma-associated Ag tyrosinase were analyzed in detail. Better MHC-multimer binding and slower multimer release are thought to reflect stronger TCR-peptide-MHC interactions; thus, these parameters would seem well suited to identify higher avidity CTL. However, large disparities were found comparing CTL multimer binding with peptide sensitivity. In contrast, CD8(+) CTL with superior Ag sensitivity mediated good tumor cytotoxicity and also secreted the triple combination of IFN-γ, IL-2, and TNF-α, representing a Th1 pattern often missing in lower avidity CTL. Furthermore, recipient lymphocytes were imbued with high Ag sensitivity, superior tumor recognition, as well as capacity for Th1 polycytokine secretion after transduction with the TCR of a high-avidity CTL. Thus, Th1 polycytokine secretion served as a suitable parameter to rapidly demark cytotoxic CD8(+) T cell clones for further TCR evaluation.     

Priming of a novel subset of CD28+ rapidly expanding high-avidity effector memory CTL by post maturation electroporation-CD40L dendritic cells is IL-12 dependent

Dendritic cell (DC)-based immunotherapeutics must induce robust CTL capable of killing tumor or virally infected cells in vivo. In this study, we show that RNA electroporated post maturation and coelectroporated with CD40L mRNA (post maturation electroporation (PME)-CD40L DC) generate high-avidity CTL in vitro that lyse naturally processed and presented tumor Ag. Unlike cytokine mixture-matured DC which induce predominantly nonproliferative effector memory CD45RA(+) CTL, PME-CD40L DC prime a novel subset of Ag-specific CTL that can be expanded to large numbers upon sequential DC stimulation in vitro. We have defined these cells as rapidly expanding high-avidity (REHA) CTL based on: 1) the maintenance of CD28 expression, 2) production of high levels of IFN-gamma and IL-2 in response to Ag, and 3) the demonstration of high-avidity TCR that exhibit strong cytolytic activity toward limiting amounts of native Ag. We demonstrate that induction of REHA CTL is dependent at least in part on the production of IL-12. Interestingly, neutralization of IL-12 did not effect cytolytic activity of REHA CTL when Ag is not limiting, but did result in lower TCR avidity of Ag-reactive CTL. These results suggest that PME-CD40L DC are uniquely capable of delivering the complex array of signals needed to generate stable CD28(+) REHA CTL, which if generated in vivo may have significant clinical benefit for the treatment of infectious disease and cancer.     

The poststimulation program of CD4 versus CD8 T cells (death versus activation-induced nonresponsiveness)

Both CD8 and CD4 T cells undergo autocrine IL-2-induced proliferation and clonal expansion following stimulation with Ag and costimulation. The CD8 T cell response is transient because the cells rapidly become activation-induced nonresponsive (AINR) and exhibit split anergy. In these cells, the capacity for IL-2 production is lost, but TCR-mediated IFN-gamma production and cytotoxicity are maintained. At this point, the CTL become dependent on IL-2 provided by CD4 Th cells for continued expansion. If IL-2 is available to support expansion for a brief period, AINR is reversed and the cells regain the ability to produce IL-2. In this study, we show that CD4 T cells do not become AINR, but instead are rendered susceptible to Fas-mediated activation-induced cell death following stimulation through TCR and CD28. Using z-VAD-fmk or anti-Fas ligand mAb to inhibit cell death, we demonstrate that previously activated CD4 T cells retain the ability to up-regulate c-Jun N-terminal kinase activity and IL-2 mRNA levels upon TCR engagement and no longer require costimulation. This rewiring of signaling pathways is similar to that seen following reversal of AINR in CD8 T cells. Thus, CD8 and CD4 T cells appear to use distinct mechanisms, AINR and activation-induced cell death, respectively, to limit excessive clonal expansion following a productive response, while permitting important effector functions to be expressed.     

CD25-expressing CD8+ T cells are potent memory cells in old age

We have recently described an IL-2/IL-4-producing CD8+CD25+ non-regulatory memory T cell population that occurs in a subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We demonstrate that CD8+CD25+ memory T cells frequently exhibit a CD4+CD8+ double-positive phenotype. The expression of the CD8 alphabeta molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells. They also have longer telomeres than their CD8+CD25- memory counterparts, thus indicating a shorter replicative history. CD8+CD25+ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags, whereas the CD8+CD25- memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same clones occur in CD8+CD25+ and CD8+CD25- memory T cell populations, demonstrating a lineage relationship between CD25+ and CD25- memory CD8+ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the differentiation of CD8+ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune responsiveness in the absence of naive T cells in old age.     

Characterization of T cell clones from an athymic mouse.

Although Thy-1+ lymphocytes have been observed in lymphoid tissues of athymic mice, attempts to analyze these cells on the clonal level have previously yielded only populations of CD4-CD8+ cytolytic T cells. Furthermore, studies of responses of these cells to various mitogenic stimuli have demonstrated significant defects in the ability of these cells to proliferate in culture. We report here on the cloning and maintenance in long term culture of T cells from an athymic mouse stimulated in vitro with allogeneic spleen cells. Of 10 Thy-1+ clones, 7 CD4+CD8- and 3 CD4-CD8+ Ag-specific cells were obtained. Among the CD4+ T cells, we observed a variety of specificities, including an autoreactive I-Aq specific clone, a minor lymphocyte stimulating determinant (Mls)-reactive clone, and five allo-I-Ad-specific CD4+ clones; a class II-specific CD4-CD8+ clone was also obtained. In addition, we observed two Thy-1-CD3+ clones (one of which is also CD4+ and expresses V beta 8) which are constitutively responsive to the lymphokines IL-2 and IL-4. Of 11 clones tested, 7 produce IL-2 and/or IL-4 lymphokines after stimulation through the TCR, whereas 4 do not, requiring exogenous lymphokines for optimal responses to Ag. Of 10 clones tested for IL-2R expression, 3 had notably low levels, correlating with low proliferative responses to IL-2. The results reveal the spectrum of T cells available to a mouse which is congenitally athymic and describe the heterogeneity of immune defects expressed in such cells at the clonal level.     

The requirements for triggering of lysis by cytolytic T lymphocyte clones. II. Cyclosporin A inhibits TCR-mediated exocytosis by only selectively inhibits TCR-mediated lytic activity by cloned CTL

TCR-mediated granule exocytosis, as measured by the release of serine esterase activity, has been implicated in the lytic process of Ag-specific CTL. Exocytosis appears to be the mechanism of release of other lysis-relevant molecules including cytotoxic lymphokines and proteins that have the capacity to induce membrane lesions as measured by the hemolysis of non-nucleated SRBC. In the studies presented here, we assessed the contribution of exocytosis and lymphokine production in CTL lysis of nucleated and non-nucleated target cells by using a panel of murine CTL clones. Ag-mediated activation of cytolysis, lymphokine production, and exocytosis could be mimicked by mAb against the TCR/CD3 complex, or by stimulation with the combination of PMA + calcium ionophore, which appear to bypass the TCR (neither PMA nor calcium ionophore alone induced these functions efficiently in our CD8+ CTL clones). Although lysis, IFN-gamma production and exocytosis of N-alpha-benzyloxycarbonyl-L-lysin esterase (BLTE) activity were induced by either stimulus, we were able to identify distinct activation requirements for each of these functions. We found that lymphokine production, exocytosis, and cytolysis could be selectively inhibited. Cycloheximide inhibited IFN-gamma production, but did not inhibit exocytosis of BLTE activity or cytolysis. In addition we showed that cyclosporine A (CsA) profoundly inhibited IFN-gamma production as well as exocytosis induced by stimulation through the Ag receptor or by PMA + calcium ionophore. In contrast, CsA had little or no effect on lysis of nucleated target cells that bear the relevant Ag. These findings indicate that our CTL clones can lyse target cells by a mechanism independent of exocytosis or (de novo) lymphokine production. To directly assess the capacity of our CTL clones to lyse target cells without inducing nuclear damage we developed a system of coating non-nucleated SRBC with anti-CD3 mAb for use as stimuli and as targets for lysis. We found that our cloned CTL were indeed activated to produce IFN-gamma by SRBC that were coated with anti-CD3 mAb, and, furthermore, they were able to lyse the SRBC in a short term cytolytic assay. Thus our CD8+ CTL are capable of lysing certain target cells by a mechanism independent of DNA degradation, presumably by inducing a membrane lesion. In addition, CsA did inhibit lysis of the non-nucleated SRBC targets as well as exocytosis of BLTE activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein kinase C-dependent and -independent mechanisms of cloned murine T cell proliferation. The role of protein kinase C translocation and protein kinase C activity

PMA can induce the proliferation of several CTL clones but not of several Th clones derived and tested in our laboratory. The PMA-stimulated proliferation of our CTL clones (which do not make IL-2 mRNA or protein) occurs independently of IL-2 and is not accompanied by lymphokine release. We now report, however, that protein kinase C (PKC) translocation is induced by PMA in CTL clones as well as in Th clones, which lack a proliferative response to PMA. These results suggest that PKC translocation itself is not a sufficient regulatory mechanism to account for cloned T cell proliferation. Moreover, IL-2 did not induce PKC translocation in a CTL clone, which proliferates when stimulated with IL-2. Thus, PKC translocation may not be necessary for activation of CTL proliferation. Nonetheless, cellular PKC activity appears to be required for the proliferative response of T cell clones after stimulation by PMA/PMA + calcium ionophore (A23187) or by triggering through the TCR: chronic PMA treatment, which depletes intracellular PKC activity, abrogates the proliferative response of T cell clones stimulated by PMA/PMA + A23187 or triggered through the TCR. T cell clones depleted of PKC activity, however, retain the ability to proliferate when challenged with IL-2. Murine T cell clones, therefore, possess PKC-dependent and PKC-independent pathways of proliferation that are not regulated by PKC translocation alone.     

A partially agonistic peptide acts as a selective inducer of apoptosis in CD8+ CTLs.

We have analyzed the effect of partially agonistic peptides on the activation and survival of CTL clones specific for a highly immunogenic HLA A11-restricted peptide epitope derived from the EBV nuclear Ag-4. Several analogues with substitutions of TCR contact residues were able to trigger cytotoxic activity without induction of IL-2 mRNA and protein or T cell proliferation. Triggering with these partial agonists in the absence of exogenous IL-2 resulted in down-regulation of the cytotoxic potential of the specific CTLs. One analogue selectively triggered apoptosis as efficiently as the original epitope, subdividing the partial agonists into apoptosis-inducing and noninducing ligands. Analysis of early T cell activation events, induction of Ca2+ influx, and acid release did not reveal significant differences between the two types of analogue peptides. These results demonstrate that some partial agonists can dissociate the induction of CTL death from CTL activation. Peptides with such properties may serve as useful tools to study signal transduction pathways in CD8+ lymphocytes and as therapeutic agents modulating natural immune responses.     

Selection of CD8+ T cells with highly focused specificity during viral persistence in the central nervous system

The relationships between T cell populations during primary viral infection and persistence are poorly understood. Mice infected with the neurotropic JHMV strain of mouse hepatitis virus mount potent regional CTL responses that effectively reduce infectious virus; nevertheless, viral RNA persists in the central nervous system (CNS). To evaluate whether persistence influences Ag-specific CD8+ T cells, functional TCR diversity was studied in spleen and CNS-derived CTL populations based on differential recognition of variant peptides for the dominant nucleocapsid epitope. Increased specificity of peripheral CTL from persistently infected mice for the index epitope compared with immunized mice suggested T cell selection during persistence. This was confirmed with CD8+ T cell clones derived from the CNS of either acutely (CTLac) or persistently (CTLper) infected mice. Whereas CTLac clones recognized a broad diversity of amino acid substitutions, CTLper clones exhibited exquisite specificity for the wild-type sequence. Highly focused specificity was CD8 independent but correlated with longer complementarity-determining regions 3 characteristic of CTLper clonotypes despite limited TCR alpha/beta-chain heterogeneity. Direct ex vivo analysis of CNS-derived mononuclear cells by IFN-gamma enzyme-linked immunospot assay confirmed the selection of T cells with narrow Ag specificity during persistence at the population level. These data suggest that broadly reactive CTL during primary infection are capable of controlling potentially emerging mutations. By contrast, the predominance of CD8+ T cells with dramatically focused specificity during persistence at the site of infection and in the periphery supports selective pressure driven by persisting Ag.     

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.     

IL-2 and a contact-mediated signal provided by TCR alpha beta + or TCR gamma delta + CD4+ T cells induce polyclonal Ig production by committed human B cells. Enhancement by IL-5, specific inhibition of IgA synthesis by IL-4.

To study the role of T cells in T-B cell interactions resulting in isotype production, autologous purified human splenic B and T cells were cocultured in the presence of IL-2 and Con A. Under these conditions high amounts of IgM, IgG, and IgA were secreted. B cell help was provided by autologous CD4+ T cells whereas autologous CD8+ T cells were ineffective. Moreover, CD8+ T cells suppressed Ig production when added to B cells cocultured with CD4+ T cells. Autologous CD4+ T cells could be replaced by allogeneic activated TCR gamma delta,CD4+ or TCR alpha beta,CD4+ T cell clones with nonrelevant specificities, indicating that the TCR is not involved in these T-B cell interactions. In contrast, resting CD4+ T cell clones, activated CD8+, or TCR gamma delta,CD4-,CD8- T cell clones failed to induce IL-2-dependent Ig synthesis. CD4+ T-B cell interaction required cell-cell contact. Separation of the CD4+ T and B cells by semiporous membranes or replacement of the CD4+ T cells by their culture supernatants did not result in Ig synthesis. However, intact activated TCR alpha beta or TCR gamma delta,CD4+ T cell clones could be replaced by plasma membrane preparations of these cells. Ig synthesis was blocked by mAb against class II MHC and CD4. These data indicate that in addition to CD4 and class II MHC Ag a membrane-associated determinant expressed on both TCR alpha beta or TCR gamma delta,CD4+ T cells after activation is required for productive T-B cell interactions resulting in Ig synthesis. Ig production was also blocked by mAb against IL-2 and the IL-2R molecules Tac and p75 but not by anti-IL-4 or anti-IL-5 mAb. The CD4+ T cell clones and IL-2 stimulated surface IgM-IgG+ and IgM-IgA+, but not IgM+IgG- or IgM+IgA- B cells to secrete IgG and IgA, respectively, indicating that they induced a selective expansion of IgG- and IgA-committed B cells rather than isotype switching in Ig noncommitted B cells. Induction of Ig production by CD4+ T cell clones and IL-2 was modulated by other cytokines. IL-5 and transforming growth factor-beta enhanced, or blocked, respectively, the production of all isotypes in a dose-dependent fashion. Interestingly, IL-4 specifically blocked IgA production in this culture system, indicating that IL-4 inhibits only antibody production by IgA-committed B cells.     

Human T cell leukemia/lymphoma virus type I infection of a CD4+ proliferative/cytotoxic T cell clone progresses in at least two distinct phases based on changes in function and phenotype of the infected cells

The effect of human T cell leukemia/lymphoma virus type I (HTLV-I) infection on the function and the phenotype of a human proliferating/cytotoxic T cell clone, specific for tetanus toxin, was investigated. During the period after infection, two distinct phases were observed, based on growth properties, phenotype, and functional activity of the infected cells. Phase I HTLV-I infected cells (0 to about 150 days after infection) proliferated in an IL-2-dependent way, but without the requirement for repetitive antigenic stimulation. No differences in expression of the CD2, CD3, CD4, Tp103, and CD28 Ag between these cells and the parental cells could be demonstrated, with the exception of the expression of IL-R p55 and HLA-DR Ag, which were constitutively expressed on the phase I cells. The phase I HTLV-I-infected cells, as well as the parental 827 cells reacted with a mAb specific for an epitope on the variable part of the TCR beta-chain, indicating that the TCR was not altered after HTLV-I infection. Like the parental clone, the phase I cells proliferated in response to tetanus toxin, but the tetanus toxin-specific response of the phase I cells did not require the presence of APC. Results of experiments, in which the levels of intracellular Ca2+ were measured, indicated that HTLV-I cells can acquire the capability to process Ag and present that to themselves. Phase I HTLV-I-infected T cells had lost their cytotoxic activity which was likely to be due to an effect on the lytic machinery rather than on Ag recognition by the TCR, inasmuch as it was found that phase I HTLV-I-infected T cells did no longer contain N-alpha-benzyloxy-L-lysine thiobenzylester-serine esterase activity. Furthermore, it was found that phase I HTLV-I-infected T cells had a diminished capacity to form conjugates with target cells. From a period of about 200 days after HTLV-I infection, phase II cells emerged that proliferated strongly in the absence of IL-2 and that had lost all functional activity. These cells did not express the CD3/T cell receptor complex on their surface. Phase I as well as phase II HTLV-I-infected cells were targets for CTL raised in the autologous donor.     

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.     

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.