Specificity, phenotype, and precursor frequency of primary cytolytic T lymphocytes specific for class II major histocompatibility antigens

Most cytolytic T lymphocytes (CTL) recognize class I rather than class II MHC determinants, and relatively little is known about those CTL that do recognize class II MHC determinants. The present study was undertaken to document the specificity, phenotype, and precursor frequency of primary class II allospecific CTL. It was found that class II-allospecific CTL could be consistently generated in vitro from unprimed spleen or thymus populations in the presence of exogenously added helper factors. The class II MHC specificity of both the precursor and CTL effectors activated in primary cultures by Ia-disparate stimulator cells was documented both by blocking experiments with anti-Ia mAb and by the use of L cell transfectants. The mechanism by which primary allospecific CTL effectors lysed their targets appeared to involve direct cell-cell contact, because they failed to lyse bystander target cells. The frequency in unprimed spleen populations of precursor CTL specific for class II alloantigens was examined by limiting dilution analysis and was found to be as high as 1/15,000 splenocytes and approximately 10% of the frequency reported for primary class I allospecific CTL. Finally, the Lyt phenotype of primary class II allospecific CTL precursors and effectors was determined. It was found that anti-class II CTL derive from at least two distinct precursor subpopulations that are either L3T4+Lyt-2- or L3T4-Lyt-2+, and that the Lyt phenotype expressed by the CTL effectors are concordant with that of their precursors. No correlation was found between the I subregion gene products recognized by CTL effectors and the Lyt phenotype they expressed in that both I-A- and I-E-specific CTL were both L3T4+Lyt-2- and L3T4-Lyt-2+.     

Recognition of MHC class I allodeterminants regulates the generation of MHC class II-specific CTL

We have analyzed the signals influencing the generation of major histocompatibility complex (MHC) class II allospecific cytolytic T lymphocytes (CTL) and have found that the development of these CTL is actively regulated in primary in vitro cultures by Lyt-2+ T cells triggered in response to MHC class I alloantigens. Class II allospecific CTL can be readily stimulated in primary cultures, but the presence of a simultaneous class I MHC stimulus in these cultures causes a marked reduction of class II-specific CTL activation. This reduction can be prevented by adding to culture a dose of monoclonal anti-Lyt-2 antibody (in the absence of complement) that does not block the generation of class I-specific CTL. The role of MHC class I alloantigens in the regulation of class II allospecific responses illustrates that T cells recognizing class I and class II MHC antigens in mixed leukocyte cultures interact in a complex and nonreciprocal manner to influence the final effector T cell repertoire elicited by this complex immunogenic challenge.     

T cell-accessory cell interactions that initiate allospecific cytotoxic T lymphocyte responses: existence of both Ia-restricted and Ia-unrestricted cellular interaction pathways

The specificity of the T-accessory cell interactions that initiate primary allospecific cytotoxic T lymphocyte (CTL) responses were found to be surprisingly diverse and of three distinct major histocompatibility complex (MHC) specificities, involving responder T cell recognition of: a) self-Ia accessory cell determinants, b) allo-Ia accessory cell determinants, or c) allo-K/D accessory cell determinants. Any one of these T-accessory cell interactions was sufficient to initiate allospecific CTL responses. It was observed that when accessory cells did not express foreign class I MHC determinants, primary allospecific CTL responses were invariably initiated by Ia-restricted T-accessory cell interactions. In contrast, it was observed that when accessory cells did express foreign class I MHC determinants, primary allospecific CTL responses could be initiated by Ia-independent T-accessory cell interactions that were specific for allogeneic, but not self, K/D determinants and that did not involve recognition of polymorphic Ia determinants. The MHC specificities of the T-accessory cell interactions that initiate primary allospecific and primary trinitrophenyl (TNP)-self CTL responses were also compared. It was observed that primary allospecific and primary TNP-self CTL responses could be initiated by self-Ia-restricted T-accessory cell interactions, and that in both responses the Ia determinants that the responding T cells recognized as self-specificities on the accessory cell surface were those that their precursors had encountered on radiation-resistant thymic elements in their differentiation environment. In contrast to the initiation of primary TNP-self CTL responses that required the activation by accessory cells of Ia-restricted T helper (TH) cells, allospecific CTL responses could also be initiated by class I-restricted T cells specific for accessory cell K/D determinants. Interestingly, such class I-restricted T cells present in primary responder cell populations were triggered only by recognition of allogeneic, but not self, K/D accessory cell determinants, even when the accessory cells were modified with TNP. Thus, the present study demonstrates that primary allospecific CTL responses, but not TNP-self CTL responses, are initiated by Ia-restricted or Ia-independent cellular interaction pathways. These results raise the possibility that unprimed class I-restricted TH cells that mediate the Ia-independent cellular interaction pathway may predominantly express an allospecific, but not a self + X-specific, receptor repertoire. Possible mechanisms by which these distinct T-accessory cell interactions initiate primary allospecific CTL responses are discuss     

Differential helper and effector responses of Lyt-2+ T cells to H-2Kb mutant (Kbm) determinants and the appearance of thymic influence on anti-Kbm CTL responsiveness

The goal of this study was to assess and compare the allorecognition requirements for eliciting Lyt-2+ helper and effector functions from primary T cell populations. By using interleukin 2 (IL 2) secretion as a measure of T helper (Th) function, and cytolytic T lymphocyte (CTL) generation as a measure of effector function, this study compared the responses of Lyt-2+ T cells from wild-type B6 mice against a series of H-2Kb mutant determinants. Although all Kbm determinants stimulated B6 Lyt-2+ T cells to become cytolytic effector cells, the various Kbm determinants differed dramatically in their ability to stimulate Lyt-2+ T cells to function as IL 2-secreting helper cells. For example, in contrast to Kbm1 determinants that stimulated both helper and effector functions, Kbm6 determinants only stimulated B6 Lyt-2+ T cells to become cytolytic and failed to stimulate them to secrete IL 2. The distinct functional responses of Lyt-2+ T cells to Kbm6 determinants was documented by precursor frequency determinations, and was not due to an inability of the Kbm6 molecule to stimulate Lyt-2+ Th cells to secrete IL 2. Rather, it was the specific recognition and response of Lyt-2+ T cells to novel mutant epitopes on the Kbm6 molecule that was defective, such that anti-Kbm6 Lyt-2+ T cells only functioned as CTL effectors and did not function as IL 2-secreting Th cells. The failure of Lyt-2+ anti-Kbm6 T cells to function as IL 2-secreting Th cells was a characteristic of all Lyt-2+ T cell populations examined in which the response to novel mutant epitopes could be distinguished from the response to other epitopes expressed on the Kbm6 molecule. The absence of significant numbers of anti-Kbm6 Th cells in Lyt-2+ T cell populations was examined for its functional consequences on anti-Kbm6 CTL responsiveness. It was found that primary anti-Kbm6 CTL responses could be readily generated in vitro, but unlike responses to most class I alloantigens that can be mediated by Lyt-2+ Th cells, anti-Kbm6 CTL responses were strictly dependent upon self-Ia-restricted L3T4+ Th cells. Because the restriction specificity of L3T4+ Th cells is determined by the thymus, in which their precursors had differentiated, anti-Kbm6 CTL responsiveness, unlike responsiveness to most class I alloantigens, was significantly influenced by the Ia phenotype of the thymus in which the responder cells had differentiated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression and function of asialo GM1 in alloreactive cytotoxic T lymphocytes

In the present study we examined asialo GM1 (AsGM1) expression and its function in alloreactive cytotoxic T lymphocytes (CTL). We consistently found that the cytotoxic activity of bulk culture-derived allo-CTL was susceptible to the treatment of anti-AsGM1 (alpha AsGM1) plus complement. To further determine whether the expression of AsGM1 was maintained in CTL, we examined cloned T cells. The expression of AsGM1 in the T cell clones was assessed by their susceptibility to lysis by alpha AsGM1 plus complement and the reduction or abrogation of their cytotoxic activity by this treatment. It was found that, with one exception, all Lyt-2+, Thy-1+ CTL clones were AsGM1+ (seven out of eight), independent of their class specificity (class I or class II). In contrast, all Thy-1+, L3T4+ CTL (2) or helper T cell (4) clones AsGM1-. These findings suggested that there was a close association between the expression of AsGM1 and the expression of Lyt-2. The cytotoxic reaction of the anti-class I MHC CTL clones that expressed AsGM1 was blocked by alpha AsGM1 or alpha Lyt-2 antibody. The Lyt-2+, AsGM1+ anti-class II MHC CTL clone-mediated lysis was inhibited by alpha AsGM1. Addition of AsGM1 in micelle form (AsGM1-M) alone also blocked the cytotoxic reactions. Addition of other structurally similar but antigenically different glycolipids or other non-AsGM1-containing liposome preparations did not affect CTL-mediated cytotoxicity. Furthermore, adding both alpha AsGM1 and AsGM1-M together at proper doses inhibited the blocking effect (deblocking) of either alone, and other structurally similar glycolipids did not inhibit the blocking. The deblocking was specific, since AsGM1-M did not affect the blocking by alpha Lyt-2. These findings indicate that not only is AsGM1 expressed in a majority of Lyt-2+ CTL clones, but it may also be involved in the CTL- target interaction to mediate lytic reaction.     

Veto cell H-2 antigens: veto cell activity is restricted by determinants encoded by K, D, and I MHC regions

Responder cells from primary syngeneic and allogeneic one-way mixed-lymphocyte cultures (MLC) specifically inhibit the development of cytotoxic T lymphocytes (CTL) directed against the major histocompatibility complex (MHC) antigens of the MLC responder cells. This special kind of suppressor activity is known as veto suppression. Ia+ cells with veto activity obtained from H-2 recombinant mouse strains were shown to downregulate alloantigen (class II)-specific helper activity for class I-specific CTL development in a primary MLC provided that the veto cells expressed the same I-E alpha subregion as the MLC stimulator cells. The veto-induced suppression of allo-help was prevented by the addition of supernatant from concanavalin A-stimulated spleen cells (Con A-SN) and was inhibited considerably by very high amounts of recombinant interleukin-2 (IL-2). In the presence of Con A-SN, CTL precursors recognizing either the K end or the D end of the veto cell MHC were found to be inactivated. Thus, our results indicate that MLC responder cells include active veto cells expressing Ia region-encoded restriction elements for allospecific T helper cells, as well as K- or D-encoded restriction elements for allospecific T cytotoxic cells.     

Graft-vs-host reaction limited to a class II MHC difference results in a selective deficiency in L3T4+ but not in Lyt-2+ T helper cell function

Hybrid mice of the (B6 X bm12)F1 combination were inoculated i.v. with parental B6 spleen cells to induce a class II graft-vs-host disease (GVH). Such mice failed to generate in vitro cytotoxic T lymphocyte (CTL) responses that were dependent upon L3T4+ T helper cell (Th) function (e.g., anti-B6-TNP) but were capable of generating in vitro CTL responses that could be mediated by Lyt-2+ Th cells (anti-allo class I). When Th function was assayed directly by interleukin 2 (IL 2) secretion, class II GVH animals were found to be deficient in L3T4+ but not Lyt-2+ IL 2-secreting Th cells. This selective deficiency in L3T4+ Th function correlates with a selective decrease in class II GVH mice of host-derived derived L3T4+ T cells. In addition, it was found that the spleens of class II GVH mice contained cells capable of selectively suppressing L3T4+ Th function. In contrast, mice in which a class I + II GVH occurred were depleted of both L3T4+ and Lyt-2+ Th function as assessed by IL 2 production. The findings that class II GVH selectively depletes L3T4+ T cells and T cell functions are discussed with respect to the immune function of distinct T cell subsets in normal and diseased states.     

Intrathymic differentiation of cytotoxic T lymphocyte (CTL) precursors. I. The CTL immunocompetence of peanut agglutinin-positive (cortical) and negative (medullary) Lyt 123 thymocytes

To analyze the developmental and functional interrelationship between cortical and medullary thymocytes, the peanut agglutinin-(PNA) binding capacity was used to separate thymocytes into PNA+ (cortical) and PNA- (medullary) thymocytes. Virtually, all positively selected PNA+ thymocytes (90% of the overall thymocyte population) expressed the Lyt 123 phenotype, whereas 90% of negatively selected PNA- thymocytes expressed Lyt 1 alloantigens, about 10% being Lyt 123 thymocytes. Provided, the requirement of Lyt 1 T helper cells was bypassed by Interleukin 2, a nonspecific mediator of T help, PNA+ Lyt 123 thymocytes mounted cytotoxic T cell responses comparable in magnitude to that of peripheral T cells. Their repertoire included antigenic disparities coded for by the complete MHC complex, H-2K, I-A, H-2D, mutational events at H-2K, as well as antigenic disparities expressed on TNP conjugated- and Sendai virus-infected syngeneic cells. PNA- Lyt 123 thymocytes represent a highly reactive pool of primary cytotoxic T lymphocyte (CTL) precursors for both alloreactive and H-2-restricted CTL responses. Since PNA- thymocytes include also Lyt 1 T helper cells, PNA- responder thymocytes are able to mount autonomously (CTL responses. Our data are first to provide direct evidence that Lyt 123 cells represent a common source of alloreactive and H-2-restricted CTL precursors in unprimed lymphocyte populations. Moreover, the apparent immunocompetence of cortical PNA+ thymocytes is now explained by their lack of T helper cells.     

The role of T8 in the cytotoxic activity of cloned cytotoxic T lymphocyte lines specific for class II and class I major histocompatibility complex antigens

It is reported here that most cytotoxic T lymphocytes (CTL), which recognize class I major histocompatibility complex (MHC) loci, express the T cell differentiation antigen T8. However, a minority of T8+ CTL clones was found to recognize class II MHC antigens. To test the hypothesis that T8 is involved only in T cell recognition of class I MHC antigens, we studied the role of T8 in the cytotoxic activity of class II MHC-specific CTL. Monoclonal antibodies specific for T8 blocked the activity of most class I MHC-specific CTL clones but did not affect the activity of class II MHC-specific CTL clones. Moreover, a mild trypsin treatment of the clones, which removed and T8 determinant, affected the activity of class I MHC but not that of class II MHC-specific CTL clones. These findings indicate that the class II-specific MHC CTL clones described here did not require T8 for their cytolytic activity. The activity of one T8+ class I MHC-specific (HLA-B27) CTL clone (HG-61) against the B cell line JY, which was used to raise this CTL clone, was not blocked by trypsin treatment of this clone. However, the activity of CTL clone HG-61 against target cells different from JY but carrying the appropriate HLA specificity was blocked by anti-T8 antibodies and trypsin treatment. The implications of these findings for the hypothesis that T8 is involved only in the activity of CTL with a relatively low avidity for class I MHC antigens are discussed.     

Mouse alloantibodies capable of blocking cytotoxic T cell function. V. The majority of I region-specific CTL are Lyt-2+ but are relatively resistant to anti-Lyt-2 blocking

In an attempt to solve the conflict concerning the correlation between the Lyt-2 phenotype of T cells subsets and the type of the MHC antigens involved in the recognition by T cells, class 2 (I region) antigen-specific CTL were studied for their Lyt phenotypes and the sensitivity to the blocking effects of anti-Lyt-2,3 antibodies. To avoid contamination by CTL to class 1 antigens such as Qa antigens, A.TH anti-A. TL attackers and A.TH anti-A attackers were tested on LPS blasts of the A strain and the A.TL stain, respectively. By using these combinations, it was shown that the majority of I region-specific killers were Thy-1+, Lyt-1+23+. Specific target cell lysis by these cells were, however, found to be far less sensitive to the blocking effects of various monoclonal antibodies to the Lyt-2,3 antigens than conventional class 1-specific CTL. This conclusion was drawn by directly comparing the sensitivity of the I region-specific and K region-specific killing by identical numbers of the same attacker cells (A.TH anti-A). No significant difference was seen between the primary and the hyperimmune CTL. Lyt-2-, Thy-1+ killer cells with I region specificity could be induced when Lyt-2-depleted A.TH responder cells were stimulated in vitro. Such Lyt-2- killer cells were not induced to the H-2K alloantigen.     

Role of cytotoxic T lymphocytes in the prevention of lupus-like disease occurring in a murine model of graft-vs-host disease

The inoculation of B6D2F1 mice with T lymphocytes from the C57BL/6 parental strain induces an "immunosuppressive" graft-vs-host reaction (B6 GVH), whereas inoculation of T cells from the other, DBA/2 parental strain induces an "immunostimulatory" GVH reaction and a lupus-like disease (DBA GVH). The present study compares cytotoxic T lymphocyte (CTL) function in the spleens of these GVH mice as well as differences in the donor inoculum that could account for these different types of GVH. We observed that the B6 GVH induces an immunodeficiency that encompasses CTL precursors (and possibly T helper cells) and results in suppressor cells that abrogate responses to both trinitrophenyl (TNP)-modified self and third party alloantigens. In contrast, the DBA GVH induces only a T helper cell immunodeficiency and results in suppressor cells selective for class II restricted L3T4+ T helper cells. Chimeric T cells were detected in both types of GVH. In the B6 GVH both L3T4+ and Lyt-2+ donor cells were observed, although Lyt-2+ cells predominated. In the DBA GVH, donor T cells were almost exclusively of the L3T4+ phenotype. The lack of appreciable donor Lyt-2+ cells in the DBA GVH can be explained by a defect in the DBA donor inoculum manifested by a naturally occurring two-fold reduction in Lyt-2+ cell numbers as well as a nine-fold reduction in CTL precursors with anti-F1 specificity. T cells in the DBA inoculum, therefore, are predominantly L3T4+. A similar defect induced in B6 donor cells by anti-Lyt2 antibody and complement not only converted the suppressive GVH to a stimulatory GVH, as measured by anti-DNA antibodies, but also resulted in a T cell immune deficiency characteristic of the DBA GVH, i.e., a selective loss of the TNP-self CTL response. Thus the presence or absence of adequate numbers of functioning Lyt-2+ cells in the donor inoculum is correlated with the development of either a suppressive or stimulatory GVH, respectively. That donor Lyt-2+ cells mediate a suppressive GVH through cytolytic mechanisms is evidenced by greater than 70% reduction in B6 GVH spleen cell numbers and readily demonstrable anti-F1 CTL activity by these spleen cells despite an inability to generate anti-allogeneic or anti-TNP self CTL activity even in the presence of added T helper factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

A rat anti-mouse T4 monoclonal antibody (H129.19) inhibits the proliferation of Ia-reactive T cell clones and delineates two phenotypically distinct (T4+, Lyt-2,3-, and T4-, Lyt-2,3+) subsets among anti-Ia cytolytic T cell clones

Hybridoma H129 .19 was derived by fusion between spleen cells of a Lou / Ws1 rat immunized with an Lyt-1+,2- anti-I-Ak cytolytic T lymphocyte (CTL) clone and the nonsecreting myeloma X63-Ag8.653. The monoclonal antibody (mAb) H129 .19 (IgG2a, kappa) was selected for its capacity to inhibit the lytic potential of the immunizing clone. H129 .19 identified a monomorphic determinant on a 55 m.w. murine T cell differentiation antigen, which appeared to be homologous to the human T4 molecule in that: 1) H129 .19 reacted with 80% adult thymocytes, with a subset of splenic T cells, and with the interleukin 2 (IL 2)-producing EL4 thymoma; 2) The mAb bound to and inhibited the IL 2 production and the proliferation of various allo- or soluble antigen-reactive T cell clones that recognized restriction or activating determinants on the I-A or I-E molecules, respectively; 3) H129 .19 did not inhibit the proliferation and/or cytolysis of Lyt-2,3+ T cells specific for class I MHC antigen; and 4) Among six anti-Iak CTL clones examined in this study, the mAb H129 .19 reacted with two I-Ak-specific, Lyt-2,3- clones on which it exerted strong cytolysis inhibiting effect at the effector cell level. By contrast, two other anti-I-Ak and two anti-I-Ek CTL clones were found to express the Lyt-2,3+,T4- cell surface phenotype. The cytolytic potential of the latter clones was not inhibited by anti-Lyt-2,3 mAb. These studies strongly suggest that the mouse T4 molecule facilitates the recognition of class II MHC antigen by most but not all T cells.     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). II. Genuine class II specificity of Lyt-2+ CTL clones

Class II-specific allogeneic cytolytic T lymphocytes (CTL) consist of two types of cells, i.e., Lyt-2+L3T4- and Lyt-2-L3T4 T cells. The Lyt-2+L3T4- class II-specific CTL population constitutes a conspicuous exception to the general correlation observed between the class of major histocompatibility complex antigen recognized and the type of accessory molecules expressed by T cells. In order to examine the specificity of such an exceptional T cell population, CTL clones were established by limiting dilution of a bulk CTL line developed in an I region incompatible combination of mouse strains, B10.QBR anti-B10.MBR. These CTL lines showed single genetic specificity indicating their clonal nature with respect to CTL activities. Lyt-2+L3T4- (2+4-), Lyt-2-L3T4+ (2-4+) and Lyt-2-L3T4- (2-4-) clones were obtained. Among many CTL clones showing a spectrum of genetic specificities, 2+4- and 2-4+ clones with apparent I-Ak-specificity, were studied further and four lines of evidence confirmed their class II specificity: 1) genes encoding the target antigen for these CTL clones were mapped within the I-A subregion by simple genetics; 2) an I-Ak-specific monoclonal antibody readily blocked specific cytolysis by these clones; 3) the clones failed to react with cells expressing mutated I-Ak antigens; and 4) a B cell tumor transfected with alpha- and beta-chain genes of I-Ak was specifically lysed by these CTL clones. These data therefore establish the existence of Lyt-2+ CTL with genuine class II specificity. All 2-4+ CTL were sensitive to the blocking effect of an antibody to L3T4, whereas none of the 2+4- class II-specific CTL were sensitive to blocking by an anti-Lyt-2 antibody, indicating that class II-specific CTL with "wrong phenotype" is not dependent on the function of the accessory molecule. Besides true class II-specific CTL clones, 2+4- clones with a spectrum of genetic specificities were obtained, including clones recognizing a combination of an I-Ak product and the Kb molecule. Two 2-4- clones were also specific for the combination of Kb + I-Ak. These clones most likely recognize an allogeneic class II antigen in the context of a class I antigen and therefore would more appropriately be included in the class I-restricted T cell population.     

Frequency analysis of class I MHC-reactive Lyt-2+ and class II MHC-reactive L3T4+ IL 2-secreting T lymphocytes

The reactivity of Lyt-2+ or L3T4+ T cells stimulated with either mutant class I or class II MHC alloantigens was studied. Whereas stimulation with class I MHC antigens induced only Lyt-2+ T cells to proliferate and to secrete IL 2, stimulation with class II MHC alloantigens induced L3T4+ but not Lyt-2+ T cells. When the frequencies of precursors of IL 2-secreting T lymphocytes (IL 2TL-p) were determined by limiting dilution analyses, class I MHC-reactive Lyt-2+ T cells displayed frequencies (f = 1/200) as high in magnitude as those within class II MHC-reactive L3T4+ (f = 1/100). Clonally developing IL 2TL of either T cell subset were antigen-specific, as shown in split-culture experiments. Whereas L3T4+ helper TL could be induced to specific IL 2 secretion over a long time period (days 3 to 9), Lyt-2+ TL showed a marked time optimal on day 4; thereafter, the number of TL colonies inducible to secrete IL 2 decreased steadily. IL 2 production and IL 2TL-p frequencies of unseparated T responder cells were not the numerical superposition of the two individual T cell subsets (Lyt-2+ + L3T4+); the latter finding is likely to reflect regulatory influences of Lyt-2+ T cells on IL 2-secreting L3T4+ T cells.     

Evidence for an involvement of T4+ cytotoxic T cells in tumor immunity

Cell-mediated immunity against cancer cells primarily involves class I major histocompatibility complex (MHC)-restricted cytotoxic T cells and natural killer (NK) cells. To investigate whether T4+ cytotoxic T cells also have a role in tumor-specific immunity, mice were immunized with a B cell lymphoma. T cell hybridomas were constructed from the immune spleen cells and analyzed for their cytotoxic ability against the immunizing lymphoma. A T4+, Lyt-1+ hybridoma cell line was developed (103L2) which specifically killed the immunizing tumor cells but not normal B cells or a range of other tumor cells of B or non-B origin. This cytotoxic hybridoma cell line differed from Lyt-2+ cytotoxic T lymphocyte cells and NK cells, commonly identified with cytotoxicity, in a number of important ways. First, the cells were class II MHC restricted; second, interleukin-2 was released from activated effector cells; and finally but most importantly, innocent nonparticipating bystander cells were also killed. The significance of this observation was that normal cells were protected, although a broad range of tumor cell types, including tumor antigen-negative mutants, were killed. It is therefore conceivable that T4+ cytotoxic T cells might play an important role in tumor immunity through the direct recognition and lysis of tumor cells while any tumor variants, arising due to antigen loss, would remain susceptible through the bystander killing effect and normal cells would remain unaffected. These results strongly suggest that tumor-reactive T4+ cytotoxic T cells belong to a new category of effector cells with an important role in tumor-specific immunity.     

Induction and activity of class II-restricted, Lyt-2+ cytolytic T lymphocytes specific for the influenza H5 hemagglutinin

In influenza A virus infections, CTL are a significant component of the host immune response which limits viral replication and promotes recovery. To examine the CTL response to the influenza virus A/Ty/Ont/7732/66[H5N9], particularly the H5 hemagglutinin, a long term CTL line was generated from spleen cells of A/Ty/Ont-immune Balb/c [H-2d] mice secondarily stimulated in vitro with A/Ty/Cal/Hurst-2/71[H5N2]. This CTL line was highly specific for influenza viruses of the H5 subtype. From this line, clones were isolated by limiting dilution and shown to be H5 hemagglutinin-specific based on recognition of an H5 vaccinia virus recombinant (H5 Vac). The clones exhibited the classical CTL surface phenotype Lyt-1-2+L3T4-; however, unlike the typically class I-restricted Lyt-2+ CTL, they were restricted in antigen recognition by class II (I-E) MHC molecules based on target cell recognition and antibody blocking of cytotoxicity. The clones recognized both infectious and non-infectious A/Ty/Ont presented by class II+ target cells. In adoptive transfer studies to assess the biologic role of the clones in vivo, these class II-restricted clones did not appear to alter mortality. However, these cells significantly reduced both morbidity and virus titers in the lungs of infected animals at 5 days post-infection. Thus, in the immune response to this virus, class II-restricted Lyt-2+ CTL specific for the H5 hemagglutinin were readily generated and their biologic role in vivo involved viral clearance.     

L3T4+ T-cell-independent reactivity of Lyt2+ T cells in vivo

The aim of this study was to analyze in vivo the L3T4+ T-cell-subset-independent reactivity of Lyt2+ T cells toward transplantation alloantigens. To this end, we depleted normal mice of L3T4+ T cells by injection of monoclonal antibodies to the L3T4 antigen. This procedure not only led phenotypically to a disappearance of L3T4+ T cells, but also effectively abolished reactivity toward class II MHC antigens in vitro and in vivo. However, L3T4+ T-cell-depleted mice still reacted to class I MHC alloantigens in vivo: after immunization with class I MHC alloantigens Il-2 receptor-bearing T cells appeared in the draining lymph nodes, and developed antigen-specific cytolytic activity. Moreover, upon in vivo priming the frequencies of class I MHC-specific precursors of Il-2-producing and cytolytic Lyt2+ T lymphocytes increased up to 20-fold. L3T4+ T-cell-depleted mice rejected class I MHC-bearing skin grafts promptly. We conclude that not only in vitro but also in vivo Lyt2+ T cells remain reactive toward class I MHC antigens in the absence of L3T4+ T helper cells.     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

Lyt-2+ suppressor T cells are resistant to anti-Lyt-2 antibody blocking

Lyt-2 molecules play a role in antigen recognition by cytotoxic T lymphocytes (CTL). In an attempt to determine whether Lyt-2 molecules play a similar role in suppressor T cell (Ts) functions, the effect of anti-Lyt-2 antibodies on Ts generation and effector activity was studied. Allospecific Ts were induced in allogeneic mixed lymphocyte cultures (MLC). Anti-Lyt-2 antibodies added to MLC in the absence of complement abolished CTL generation, but had no effect on concomitant induction of Ts. In a different experimental system, allospecific Ts were induced in cultures treated with pyrilamine, which blocks generation of CTL but allows differentiation of Ts. The addition of anti-Lyt-2 antibodies to pyrilamine-treated MLC resulted in unaffected induction of Ts. It was further demonstrated that the effector activity of Ts was as resistant to anti-Lyt-2 antibodies as their induction, in contrast to the cytolytic activity of CTL, which was inhibited by the same antibodies. Ts in the present experimental system were Lyt-2+ antigen-specific cells. It therefore appears that Lyt-2 molecules, although expressed on both CTL and Ts, are involved in CTL activity, but do not play an essential role in Ts function.     

Human cytotoxic T cell clones directed at autologous virus-transformed targets: further evidence for linkage of genetic restriction to T4 and T8 surface glycoproteins

Human cytotoxic T cell clones were generated against autologous EBV-transformed B lymphocytes. Whereas the majority of the clones expressed the T8 surface glycoproteins and showed a specificity for class I MHC gene products on the target cell, a minority expressed the T4 surface glycoprotein and demonstrated a class II specificity. Monoclonal antibodies to T4 and T8 inhibited cytotoxic effector function of reactive clones in a fashion analogous to their effect on alloreactive CTL clones. Each autoreactive T cell clone was cytotoxic for EBV-transformed B lymphocytes but not pokeweed mitogen-activated or resting autologous lymphocytes, suggesting a dual specificity for an MHC gene product as well as an antigen induced and/or encoded by virus. Taken together, the present findings provide further support for the notion that T4 and T8 serve as associative recognition elements on T lymphocytes for MHC gene products.