Relationship among function, phenotype, and specificity in primary allospecific T cell populations: identification of phenotypically identical but functionally distinct primary T cell subsets that differ in their recognition of MHC class I and class II allodeterminants

The goal of the present study was to evaluate the relationship among function, Lyt phenotype, and MHC recognition specificity in primary allospecific T cell populations. By using Lyt-2+ and L3T4+ T cells obtained from the same responder populations, we assessed the ability of T cells of each phenotype to generate cytotoxic effector cells (CTL) and IL 2-secreting helper T cells in response to either class I or class II MHC allodeterminants. It was found that a discordance between Lyt phenotype and MHC recognition specificity does exist in primary allospecific T cells, but only in one T cell subpopulation with limited functional potential: namely, Lyt-2+ T cells with cytotoxic, but not helper, function that recognize class II MHC alloantigens. Target cell lysis by these Lyt-2+ class II-allospecific CTL was inhibited by anti-Ia monoclonal antibodies (mAb), but not anti-Lyt-2 mAb, indicating that they recognized class II MHC determinants as their "restriction" specificity and not as their "nominal" specificity even though they were Lyt-2+. A second allospecific T cell subset with limited functional potential was also identified but whose Lyt phenotype and MHC restriction specificity were not discordant: namely, an L3T4+ T cell subset with helper, but not cytotoxic, function specific for class I MHC allodeterminants presented in the context of self-Ia. Thus, the present study demonstrates that primary allospecific T cell populations contain phenotypically identical subpopulations of helper and effector cells that express fundamentally different MHC recognition specificities. Because the recognition specificities expressed by mature T cells reflect the selection pressures they encountered during their differentiation into functional competence, these findings suggest that functionally distinct but phenotypically identical T cell subsets may be selected independently of one another during ontogeny. Thus, the existence of Lyt-2+ CTL specific for class II allodeterminants can be explained by the hypothesis that the association of Lyt phenotype with MHC recognition specificity results from the process of thymic selection that these Lyt-2+ effector cells avoid.     

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     

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.     

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)     

Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule

Monoclonal antibody GK1.5 recognizes a previously undescribed murine T cell surface molecule, designated L3T4, which migrates on SDS-PAGE under reducing conditions as a single band with an apparent m.w. of 52,000. L3T4 is expressed by approximately 80% of thymocytes and by approximately 20% of spleen cells. There appears to be poor correlation between expression of L3T4 by functional T cell clones and expression of Lyt-2, expression of the cytolytic phenotype, and class I MHC antigen reactivity. On the other hand, both a class II MHC antigen-reactive HTL clone and an Lyt-1- Mls-reactive HTL clone express L3T4. Analysis of the effect of mAb GK1.5 on PFC responses in adoptive transfer suggests that L3T4 is expressed by the helper/inducer subset of murine T cells. Expression of L3T4 by murine T cells, however, may correlate primarily with class II MHC antigen reactivity rather than with functional phenotype; mAb GK1.5 profoundly blocks antigen-specific cytolysis by the cloned class II MHC antigen-reactive CTL line A15-1.17. Antigen-specific cytolysis by A15-1.17 is blocked by mAb GK1.5 at a step before the lethal hit. Collectively, the flow cytometric, functional, and biochemical data indicate that L3T4 is similar to the human Leu-3/T4 molecule.     

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.     

Role of L3T4+ and Lyt-2+ donor cells in graft-versus-host immune deficiency induced across a class I, class II, or whole H-2 difference

The i.v. injection of parental T cells into F1 hybrid mice can result in a graft-vs-host (GVH)-induced immune deficiency that is Ag nonspecific and of long duration. The effect of the GVH reaction (GVHR) on the host's immune system depends on the class of F1 MHC Ag recognized by the donor cells. To determine the role of different subsets of donor-derived T cells in the induction of GVHR, donor spleen cells were negatively selected by anti-T cell mAb and C, and the cells were injected into F1 mice that differed from the donor by both class I and II MHC Ag or by class I or class II MHC only. The induction of GVHR across class I + II differences was found to require both L3T4+ and Lyt-2+ parental cells. Induction of GVHR across a class II difference required only L3T4+ parental T cells in the combination tested [B6-into-(B6 x bm12)F1]. In contrast, B6 Lyt-2+ cells were sufficient to induce GVHR across a class I difference in (B6 x bm1)F1 recipients. In addition, a direct correlation was observed between the cell types required for GVH induction and the parental T cell phenotypes detected in the spleens of the GVH mice. The number of parental cells detected in the unirradiated F1 hosts was dependent upon the H-2 differences involved in the GVHR. Induction of a class I + class II GVHR resulted in abrogation of both TNP-self and allogeneic CTL responses. In contrast, induction of a class II GVHR resulted in only a selective loss of TNP-self but not of allogeneic CTL function. Unexpectedly, the induction of a class I GVHR also resulted in the selective loss of the TNP-self CTL response. Thus, these class I and class II examples of GVH both result in the selective abrogation of L3T4+ Th cell function. The data are discussed in terms of respective roles of killer cells and/or suppressor cells in the induction of host immune deficiency by a GVHR, and of the selective deficiency in host Th cell function induced by different classes of GVHR.     

Expression of H-2I region-restricted cytolytic activity by an Lyt-2+ influenza virus-specific T lymphocyte clone

The expression of Lyt-2 on T lymphocytes has been postulated to correlate closely with restriction by, or alloreactivity to, class I MHC gene products, whereas I region-restricted or alloreactive populations appear to be associated with Lyt-1 and L3T4 expression. However, exceptions to this axiom among alloreactive T cells have been shown to exist. In this report we describe a clonal population of influenza virus-specific T lymphocytes that bears the Lyt-2+, L3T4- phenotype. Notably, this clone is restricted in influenza virus recognition by class II MHC molecules and is cytolytic for virus-infected target cells expressing the appropriate class II molecules. Antibody directed to the Lyt-2 molecule does not inhibit cytolysis.     

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.     

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.     

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.     

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.     

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.     

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.     

Antigen form influences induction and frequency of influenza-specific class I and class II MHC-restricted cytolytic T lymphocytes

The induction of class I and class II MHC-restricted CTL in response to different forms of A/JAP/57 influenza virus was compared. Splenocytes removed from influenza-immune BALB/c mice and stimulated in vitro with infected syngeneic splenocytes are mainly CD8+ (Lyt-2+) and specifically lyse infected Ia- and Ia+ target cells. To a lesser extent they also lyse non-infectious virus-pulsed Ia+ but not Ia- target cells. In contrast, syngeneic stimulators pulsed with non-infectious virus (exogenous Ag) induce effector T cells that specifically lyse both infected and non-infectious virus-pulsed Ia+ target cells. The cells present in this heterogeneous culture predominantly express the CD4 (L3T4) cell surface marker. Frequency analysis by limiting dilution of splenocytes derived directly from influenza-immune mice revealed a similar pattern of precursor induction: In vitro stimulation with infected splenocytes yielded primarily class I MHC-restricted CTL, whereas stimulation with non-infectious virus reciprocally induced primarily class II MHC-restricted CTL. Thus, the Ag form and consequently the intracellular route of viral Ag presentation profoundly influence the MHC restriction of CTL precursors induced.     

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.     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). I. Analysis of bulk populations and establishment of Lyt-2+L3T4- and Lyt-2-L3T4+ bulk CTL lines

Murine allogeneic cytolytic T lymphocytes (CTLs), including long-term bulk CTL lines, were induced in I-region-incompatible combinations of strains in vitro in order to study the phenotypes of class II major histocompatibility complex (MHC) antigen-specific CTLs, as well as the possible functional involvement of accessory cell interaction molecules such as Lyt-2 and L3T4. This report shows that class II-specific allogeneic CTL populations consist of two types of T cells. Lyt-2+L3T4- (2+4-) and Lyt-2-L3T4+ (2-4+), in variable proportions depending on the strain combination, that in vitro bulk CTL lines with each of these phenotypes can be established, that the killing function of 2-4+ CTL is sensitive to the blocking effect of anti-L3T4 antibodies, suggesting functional involvement of this molecule in the CTL-target interaction, that anti-Lyt-2 antibodies fail to block killing by 2+4- cells, suggesting that such CTLs do not utilize this molecule in their killing function, and that while I-A-specific CTLs of both phenotypes are detectable, 2-4+ cells could not be detected among I-E-specific CTL populations.     

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.     

Role of accessory cell processing and presentation of shed H-2 alloantigens in allospecific cytotoxic T lymphocyte responses

The present study was undertaken to evaluate the role of accessory cell processing of MHC alloantigens in the initiation of primary allospecific CTL responses. To first determine whether antigen processing by accessory cells is involved in the initiation of allospecific CTL responses, accessory cells were retreated with the lysosomotropic drug chloroquine before their addition to culture. It was found that chloroquine pretreatment abrogated their ability to function as accessory cells only when they were of responder haplotype and had no effect when the accessory cells were of stimulator haplotype. Although accessory cells of either responder or stimulator haplotype can initiate allospecific CTL responses, we have previously demonstrated that they do so by activating distinct classes of T helper TH) cells. Indeed, the differential effects of chloroquine on accessory cells of responder or stimulator haplotypes were shown to reflect the fact that chloroquine pretreatment markedly impaired the ability of accessory cells to activate self-Ia-restricted TH cells, but had little effect on the ability of the same accessory cells to activate either allo-class I- or allo-class II-specific TH cells. We next examined the possibility that accessory cells of responder haplotype mediate alloresponses by acquiring and processing shed MHC alloantigens derived from the stimulator cell population. In these experiments, accessory cell-depleted stimulator cells were fixed with paraformaldehyde to inhibit shedding of their surface MHC alloantigens. It was observed that even though mixed stimulator cells were recognized normally by allospecific CTL precursors, they completely failed to stimulate CTL responses mediated by responder haplotype accessory cells, indicating that the function of such accessory cells is dependent upon their acquisition of shed MHC alloantigens. Taken together, the data presented in this report demonstrate that accessory cells of responder haplotype function in allospecific CTL responses by acquiring and processing shed class I MHC alloantigens, and by then presenting the processed alloantigens in association with self-Ia determinants to self-Ia-restricted TH cells. Thus, these data indicate that the self-Ia-restricted TH cells that are involved in allospecific CTL responses recognize processed class I alloantigens in association with self-Ia determinants.