Clonal analysis of B and T cell responses to Ia antigens. IV. Proliferative T cell clones recognizing E beta and/or E alpha allodeterminants

The allospecific T cell recognition of the I-Ek molecule was assessed by using eight A. TH anti-A. TL proliferative T cell clones, all of which expressed the Thy-1-2+, Lyt-1+, Lyt-2-, Ia-, and p94,180+ cell surface phenotype. The use of panels of stimulating cells from homozygous of F1 hybrid strains indicated each T cell clone exhibited specificity for distinct alloactivating determinants including: i) a private E beta k-controlled determinant expressed in cis- or trans-complementing E beta kE alpha strains; ii) an apparently nonpolymorphic E alpha determinant resembling the serologic specificity Ia.7, i.e., present in all strains carrying E alpha and E beta expressor alleles; and iii) a series of conformational I-E determinants, the expression of which required a precisely defined combinatorial association of E beta plus E alpha chains. Two clones were found to be reactivated by cis- but not trans-complementing E beta k E alpha k strains, and another recognized an allodeterminant shared by the I-Ab molecule. Various I-Ek-reactive monoclonal antibodies (mAb) directed to epitopes presumably expressed on either E alpha (epitope clusters I and II) or E beta (epitope cluster III) chains inhibited the proliferative responses of seven clones recognizing private E beta k or unique E beta E alpha conformational activating determinants. By contrast, the restimulation of the clone directed to a nonpolymorphic E alpha determinant was selectively blocked by anti-Ia.7 mAb defining epitopes on the E alpha chains but not by those directed to the E beta chain. On the basis of these data, it was concluded that the recognition sites of most anti-I-Ek proliferative T cells were expressed on the E beta chain or the E beta plus E alpha interaction products, and that a minority of such alloreactive T cells could be activated through recognition of the E alpha chain per se.     

Receptor specificity of Ia-restricted T lymphoblasts activated against trinitrobenzene sulfonate-coupled spleen cells: recognition of distinct trinitrophenyl and Ia moieties

The receptor specificity of H-2-restricted T lymphoblasts activated against trinitrobenzene sulfonate (TNBS)-coupled spleen cells was examined using an antigen binding assay. A population of Lyt-1+,2-T lymphoblasts acquired syngeneic Ia determinants during 4 days of primary culture with hapten-coupled stimulator cells. Syngeneic Ia was not reexpressed after trypsin treatment of the T cells, but was found after incubation with soluble Ia shed from lipopolysaccharide-activated blasts. Self-Ia binding was specific in that Lyt-1+,2- but not Lyt-1-,2+ cells acquired the antigen, and in that self-Ia bound more effectively than allogeneic Ia material. To determine the relationship of self-Ia binding to the recognition of foreign antigen, the binding of trinitrophenyl (TNP)-coupled plasma membrane vesicles by TNP-specific T cells was studied. TNP-vesicle binding occurred via TNP and H-2(Ia) molecules on the vesicles in that binding was inhibited with antibodies against TNP or H-2(Ia) molecules but not non-major histocompatibility complex (e.g., Ly-6.2) molecules on the vesicles. Complete inhibition of TNP-vesicle binding by an Iak-restricted TNP-specific T-cell line occurred with soluble TNP-lysine, but not an unrelated hapten, N-iodoacetyl-N-(5-sulfonic-1-naphthyl)ethylenediamine (I-AED)-cysteine. Conversely, I-AED-cysteine, but not TNP-lysine, inhibited binding of I-AED-coupled B6 vesicles by B6 anti-I-AED T cells. Significant, but weak inhibition of TNP-vesicle binding by the anti-TNP line was observed with glycoprotein preparations containing partially purified self-Ia molecules. However, inhibition was specific for I-Ak molecules, in that inhibition was lost after removal of I-Ak molecules from the glycoprotein preparation, and very little inhibition occurred with soluble glycoproteins prepared from thymocytes which contained very little Ia material or from LPS blasts of an unrelated H-2 haplotype. These results suggest a recognition model in which TNP and Ia determinants are recognized by neighboring receptor combining sites.     

Dissection of the poly(Glu60 Ala30 Tyr10) (GAT)-specific T-cell repertoire in H-2I k mice

Twenty-five allospecific monoclonal antibodies (mAb), produced in the A. TH. A.BY, or B10.S (7R) anti-A.TL combinations, were shown to recognize determinants organized in four spatially distinct polymorphic regions on the same I-Ak-encoded molecule(s). These reagents were used to assess the recognition of the class II major histocompatibility complex (MHC) determinants in a series of GAT-reactive A.TL T-cell clones exhibiting various restriction specificity or alloreactivity patterns. Of the proliferative responses of 13 cloned T cells, 12 responses were found to be inhibited similarly by the same set of mAbs.A hierarchy in the blocking effects of these reagents that could be correlated with the spatial organization of their determinants was observed. (i) All the mAbs defining the epitope region I (i.e., recognizing public Ia.1- or Ia.17-like determinants, presumably expressed on the A beta subunit) and some of those identifying new public determinants in the epitope region II profoundly inhibited these T-cell responses. (ii) Intermediate blocking was observed when mAbs recognizing public determinants in the epitope region III were used. (iii) Finally, among the mAbs that identified the epitope group IV, the Ia.19-specific mAb 39.J was inhibitory, whereas mAbs directed against private Ia.2-like determinants were not. By contrast, the GAT-specific proliferative response of the T-cell clone AT-20.1, which recognized its nominal antigen in an extensively cross-reactive MHC-restricted fashion, could only be inhibited by a subset of the mAbs recognizing epitopes in groups I and II, but not by those recognizing epitopes in groups III and IV. It was also shown that the same subset of I-Ak-and I-Au-reactive mAbs displayed similar blocking effects on the proliferation of two T-cell clones exhibiting dual specificity for I-Ak- and I-Au-restricting and/or I-Ak- and I-Au-alloactivating determinants. Finally, all the cloned T-cell responses examined were found to be inhibited by rat mAbs against the LFA.1 molecule or the murine equivalent of the human OKT4 differentiation antigen. These studies suggest that class II specific mAbs can impair proliferation of cloned T-cells by a mechanism(s) other than the masking of the T-cells' restriction determinants per se.     

T cell subsets in (responder x nonresponder)F1 mice regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine (GAT)

Immune responses to GAT are controlled by H-2-linked Ir genes; soluble GAT stimulates antibody responses in responder mice (H-2b) but not in nonresponder mice (H-2q). In nonresponder mice, soluble GAT stimulates suppressor T cells that preempt function of helper T cells. After immunization with soluble GAT, spleen cells from (responder x nonresponder: H-2b X H-2q)F1 mice develop antibody responses to responder H-2b GAT-M phi but not to nonresponder H-2q GAT-M phi. This failure of immune F1 spleen cells to respond is due to an active suppressor T cell mechanism that is activated by H-2q, but not H-2b, GAT-M phi and involves two regulatory T cell subsets. Suppressor-inducer T cells are immune radiosensitive Lyt-1 +2-, I-A-, I-J+, Qa-1+ cells. Suppressor-effector T cells can be derived from virgin or immune spleens and are radiosensitive Lyt-1-2+, I-A-, I-J+, Qa-1+ cells. This suppressor mechanism can suppress responses of virgin or immune F1 helper T cells and B cells. Helper T cells specific for H-2b GAT-M phi are easily detected in F1 mice after immunization with soluble GAT; helper T cells specific for H-2q GAT-M phi are demonstrated after elimination of the suppressor-inducer and -effector cells. These helper T cells are radioresistant Lyt-1+2-, I-A+, I-J-, Qa-1- cells. These data indicate that the Ir gene defect in responses to GAT is not due to a failure of nonresponder M phi to present GAT and most likely is not due to a defective T cell repertoire, because the relevant helper T cells are primed in F1 mice by soluble GAT and can be demonstrated when suppressor cells are removed. These data are discussed in the context of mechanisms for expression of Ir gene function in responses to GAT, especially the balance between stimulation of helper vs suppressor T cells.     

Specificity repertoire of splenic Lyt-2+/F23+ cytotoxic lymphocyte precursors from B6 mice

As revealed by flow cytometric analysis, about 30% of nylon wool nonadherent Lyt-2+ B6 spleen cells were F23+, i.e., were stained with the monoclonal antibody F23.1 directed against an allotypic T-cell receptor determinant. The specificity repertoire of splenic Lyt-2+/F23+ cytotoxic lymphocyte precursors (CLP) from B6 mice was investigated in a limiting dilution (LD) system designed to support clonal expansion in vitro of a representative fraction of this T-cell subset: in highly purified Lyt-2+ responder cells cocultured with mitomycin-treated F23 hybridoma cells in the presence of (recombinant) interleukin 2 under LD conditions, one out of three Lyt-2+/F23+ CLP gave rise to a functional cytotoxic T lymphocyte (CTL) clone. The split-well analysis of individual CTL populations demonstrated a clear-cut segregation of the lytic reactivities toward different allogeneic Con A blast targets. A large fraction of B6-derived CTL clones (3-10%) specifically lysed fully H-2 allogeneic (H-2k, H-2d), or H-2K mutant (bm1) targets. Self-reactive and allorestricted lytic patterns were not found.     

Murine T-cell clones specific for chicken erythrocyte alloantigens

We have established murine T-cell clones which respond to allotypic and species-specific determinants found on chicken erythrocytes (cRBC). Their relative antigen specificities were determined by assessing lymphokine production and proliferation in response to syngeneic spleen cells and cRBC obtained from chickens homozygous for major histocompatibility complex (MHC) antigens. The specificity pattern suggested that the T-cell clones recognized a more restricted set of cRBC MHC-associated allodeterminants than do antibody-producing cells. The antigen-specific responses required antigen processing, and were MHC restricted and antigen dose dependent. Approximately 20% of T-cell clones from appropriate strains of mice were also Mls alloreactive. This second reactivity showed no correlation with nominal cRBC specificity. The induction-specific lymphokine activities of T-cell growth factor, mast cell growth factor, and Ia induction factor were identified as interleukin 2 (IL-2), interleukin 3 (IL-3), and interferon-gamma respectively.     

Recognition of Rous sarcoma virus-induced tumor antigens by cytotoxic T lymphocytes (CTL): studies on specificity of killing by CTL employing H-2 congenic and recombinant mouse tumor cells

The specificities of cytotoxic T lymphocytes (CTL) were studied for the analysis of CTL against tumor-specific cell surface antigen(s) (TSSA) of non-virus-producing tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. Eight CTL clones were established from immune spleen cells of B10.A(5R) mice. These clones demonstrated six patterns of cytotoxic reactivity in vitro: Two clones showed H-2 restriction in tumor cell lysis. Two other clones had the capacity to lyse syngeneic, H-2K-compatible B10 and H-2-incompatible B10.A(4R) tumor cells, but not YAC-1 cells. One clone had cytotoxic activity against syngeneic, H-2D-compatible B10.D2 tumor cells and YAC-1 cells, but not against H-2-incompatible tumor cells. One clone had cytotoxic activity against syngeneic and YAC-1 tumor cells, but not against either H-2-compatible or H-2-incompatible tumor cells. One clone had lytic activity to syngeneic, H-2-compatible, H-2-incompatible, and YAC-1 tumor cells. Another clone killed H-2-incompatible B10.A(4R) tumor and YAC-1 cells, but not syngeneic or H-2-compatible tumor cells. All these clones strongly expressed surface Thy-1.2 antigens, whereas the expression of Lyt-1.2 and Lyt-2.2 antigens was different from clone to clone. These results demonstrate heterogeneity of both lytic specificity and phenotype of CTL against RSV-induced mouse tumor cells, suggesting the existence of multiple antigenic sites on the RSV TSSA recognized by CTL populations.     

Clonal heterogeneity in colony stimulating factor production by murine T lymphocytes

A panel of 55 alloreactive murine T-lymphocyte clones was screened for the production of granulocyte-macrophage colony stimulating factor (GM-CSF), multilineage CSF (multi-CSF), human-active eosinophil CSF (human-active EO-CSF), and interleukin 2 (IL-2) in response to stimulation with the lectin concanavalin A. Many clones were also characterized for cytolytic specificity and expression of the T-cell antigen receptor-associated surface markers Lyt-2 and L3T4, which reflect their specificity for Class I (H-2K, H-2D) or Class II (H-2l, Mls) histocompatibility antigens, respectively. Eighty percent of the clones secreted detectable quantities of at least one of the four factors measured. Of the factor-producing clones, all appeared to secrete GM-CSF and half also secreted multi-CSF. A subpopulation of multi-CSF producers also released human-active EO-CSF. More than half of the factor-producing clones secreted detectable IL-2; whereas the IL-2-producing clones included some that did not secrete multi-CSF, IL-2 production was always associated with concomitant synthesis of GM-CSF. Comparison of the range and quantities of factors secreted by Lyt-2+ and L3T4+ clones indicated that more L3T4+ clones produced measurable titers of the four factors; on average, this group also secreted 10- to 100-fold higher titers of both the hemopoietic regulators and IL-2 than Lyt-2+ clones. Cells of the L3T4+ phenotype would therefore be expected to account for the majority of CSF and IL-2 secretion by polyclonal populations of activated T lymphocytes.     

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.     

Characterization of the fine specificity and antigen markers associated with the receptor of autoreactive T-cell hybridomas

In this study we attempted to define the determinants on Ia molecules recognized by autoreactive hybridomas obtained from (C57BL/6 X BALB/c)F1 mice. The epitopes recognized by the T cells were characterized (a) using stimulating cells from various congenic and H-2 recombinant inbred strains and (b) by inhibition of activation with anti-Ia antibodies. Our hybridomas were strictly autoreactive and did not exhibit any alloreactivity, as is often observed for such cells. Our results show that more epitopes than previously believed are recognized by autoreactive T cells. One T-cell hybridoma (QW27.1) is unique in that it recognizes a hybrid F1 Ia determinant. Antigenic markers associated with the receptor of the T-cell hybridomas were studied with monoclonal antibodies (mAbs) specific for L3T4 and a V beta "idiotype". The results indicate that all Lyt 1.2 autoreactive T cells express L3T4 antigen in association with their receptor. One clone (QW64.14) expresses the V beta idiotype recognized by F23.1 monoclonal antibody. Moreover, this clone is activated by F23.1, linked to Sepharose 4B beads, which was believed previously to activate only Lyt 2+, L3T4 T cells. The supernatant of one clone (QW17.5) helps B cells to differentiate into antibody-producing cells without requiring direct contact with the autoreactive clone.     

In vivo activation of autoreactive cytotoxic T lymphocytes after bone marrow transplantation: evidence for the prethymic specificity of T cells?

The light density fraction (A + B, i.e., remaining above the 26% concentration in the discontinuous BSA gradient) of BCF1 (H-2b X H-2k) mouse bone marrow contains cells that after injection into irradiated syngeneic recipients give rise to autoreactive Lyt-2+, Thy-1+ CTL. After injection of unfractionated bone marrow cells, the levels of these CTL were low or undetectable, suggesting that either the precursors were highly enriched in the A + B fraction or that bone marrow cells with higher density have a suppressive function. The specificity of the killing was not directed toward all the available class I MHC antigens: only targets carrying H-2Kb-coded determinants were killed. There was no overlapping between the autoreactive and alloreactive precursors: cells from the A + B fraction could not respond to an alloantigen in vitro, not even in the presence of an interleukin 2-containing supernatant, and the autoreactive CTL activated in vivo could not kill allogeneic targets. The induction of the autoreactive CTL did not require the presence of the appropriate MHC antigen in the maturation environment, thus differing from the activation of mature T cells. The observed CTL specificity, together with the previous findings showing that prethymic T cells are locating in the same BSA fraction as the precursors for these autoreactive cells, support the idea that the prethymic T cell repertoire is, at least partially, directed to recognize self-MHC antigens.     

T cell subsets regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in virgin and immunized nonresponder mice

T cell subsets from virgin and immunized mice, which are Ir gene controlled nonresponders to GAT, which regulate antibody responses to GAT have been characterized. Virgin nonresponder B10.Q B cells develop GAT-specific antibody responses to GAT, B10.Q GAT-M phi, and GAT-MBSA when cultured with virgin or GAT-primed Lyt-1+, I-J-, Qa1- B10.Q helper T cells. Virgin T cells are radiosensitive, whereas immune T cells are radioresistant (750 R); qualitatively identical helper activity is obtained with T cells from mice immunized with soluble GAT, B10.Q GAT-M phi, and GAT-MBSA. Responses to GAT and GAT-M phi are not observed when virgin or GAT-primed Lyt-1+, I-J+, Qal+ T cells are added to culture of virgin or GAT-primed Lyt-1+, I-J-, Qa1- helper T cells and virgin B cells; the GAT-specific response to GAT-MBSA is intact. The Lyt-1+, I-J+, Qa1+ T cells from mice primed with GAT, GAT-M phi, and GAT-MBSA were qualitatively identical in mediating this suppression. Virgin Lyt-2+ T cells have no suppressive activity alone or with virgin Lyt-1+, I-J+, Qa1+ T cells, whereas responses to GAT, GAT-M phi, and GAT-MBSA are suppressed in cultures of GAT-primed helper T cells containing GAT-primed Lyt-2+ T cells (with or without GAT-primed Lyt-1+, I-J+, Qa1+ T cells). Suppression of responses to GAT-MBSA in cultures of GAT-M phi-primed helper T cells requires both GAT-M phi-primed Lyt-1+, I-J+, Qa1+ T cells and Lyt-2+ T cells; the Lyt-1+, I-J+, Qa1+ T cells appear to function as inducer cells in this case. In cultures containing GAT-MBSA-primed helper T cells, either GAT-MBSA-primed Lyt-1+, I-J+, Qa1+ or Lyt-2+ T cells suppress responses to GAT and GAT-M phi; under no circumstances are responses to GAT-MBSA suppressed by GAT-MBSA-primed regulatory T cells. This regulation of antibody responses to GAT by suppressor T cells is discussed in the context of the involvement of suppressor T cells in responses to antigens under Ir control, and of the evidence that nonresponsiveness to GAT is not due to a defect in the T cell repertoire, but rather is due to an imbalance in the activation of suppressor vs helper T cells.     

Characteristics and functions of Sendai virus-specific T-cell clones

Several murine Sendai virus-specific T-cell clones were characterized in vitro and in vivo. All T-cell clones were phenotypically Thy-1.2+, and most clones were Lyt-1+,2-; one T-cell clone was Lyt-1-,2-. Some of the clones proliferated in response to antigen presented on I region-compatible stimulator cells. Proliferation could be inhibited by monoclonal antibodies directed against class II antigens. Clones which proliferated in response to antigen secreted lymphokines which could be identified as Interleukin 2 and Interleukin 3. All of the clones tested in vivo induced a delayed-type hypersensitivity response in syngeneic mice challenged with antigens. Depending on the experimental conditions chosen, Interleukin 2-producing clones as well as non-Interleukin 2-producing clones mediated help for stimulation of cytolytic T lymphocytes.     

Activation requirements of cloned inducer T cells. III. Need for two stimulator cells in the response of a cloned line to Mls determinants

To gain insight into the nature of Mls determinants, we examined the stimulator cells responsible for the activation of inducer T cell clones by Mls determinants. Two types of clones responding to Mls determinants were identified. One type responded to purified B cells, but not to splenic adherent cells (SAC), from mice bearing Mls stimulatory determinants. The other type of Mls-reactive T cell clone, including the representative clone Ly1-N5, demonstrated a vigorous response to unfractionated spleen cells, but showed little or no response to B cells alone or to SAC alone from mice bearing the Mlsa or Mlsd stimulatory determinant. The response of these clones to Mls determinants required stimulation by two cell types. The failure of clone Ly1-N5 to respond to Mlsa-bearing B cells was reversed by the addition of SAC taken from mice bearing the Mlsa allele. In addition, SAC from mice bearing the nonstimulatory Mlsb allele could synergize with B cells from Mlsa-bearing animals. B cells were required to provide the Mlsa determinant, because the combination of Mlsa-bearing SAC and Mlsb-bearing B cells did not activate the clone. The response of clone Ly1-N5 to Mls is restricted by Ia determinants (shared by H-2b, H-2d, and H-2k haplotypes but not by the H-2q haplotype). The permissive H-2 alleles can be present either on the stimulator B cell or on the SAC. The optimal response of the clone was obtained by using B cells bearing Mlsa and the permissive Ia epitopes. However, a significant response of the clone to B cells bearing Mlsa but an inappropriate Ia (Iaq) was also seen in the presence of SAC bearing the nonstimulatory Mlsb allele but the permissive Ia epitopes.     

Interaction between MHC-encoded products and cloned T cells

The interaction between class I major histocompatibility complex (MHC) products and T cells was studied using H-2K^b-specific alloreactive T-cell lines and clones obtained by repeated in vitro stimulation with allogeneic cells. Induction of proliferation of these T cells appeared to involve two signals: the H-2K^b alloantigen and interleukins. Immunopurified liposome-inserted H-2K^b, which stimulates specific secondary in vitro cytotoxic T lymphocyte (CTL) responses, could not replace cell-associated H-2K^b in the stimulation of these T-cell lines, even in the presence of feeder cells and interleukins. When T-cell lines were initiated in vitro and repeatedly stimulated with H-2K^b liposomes and feeder cells, it was possible to obtain T cells that could proliferate in response to H-2K^b liposomes in the presence of feeder cells and interleukin-2-containing supernatants or on H-2K ^b -expressing cells. Only stimulation with cells permitted maintenance of these T cells in culture for more than 12 weeks. Analyses of cell surface markers and of patterns of inhibition of proliferation by monoclonal antibodies (mAb) of T-cell lines induced in vitro with cell- or liposome-associated H-2K^b indicated that T-cell stimulation by class I antigen can occur in at least two ways. In the first, the H-2K^b-induced proliferation of Lyt-1^- Lyt-2⁺ T4^- T cells is inhibited by H-2K^b- and by Lyt-2-specific mAb, but not by Ia or T4-specific mAb. In the second, both Lyt-2⁺ and T4⁺ T cells are involved and the H-2K^b-induced proliferation is inhibited by H-2K^b- and Lyt-2-specific mAb and by Ia- and T4-specific mAb.Abbreviations used in this paper Ab antibody - mAb monoclonal antibody - C complement - i.p. intraperitoneally - PBS phosphate-buffered saline - PBS-B-N PBS containing bovine serum albumin and NaN₃ - CTL cytotoxic T lymphocyte - Th T helper cell - MHC major histocompatibility complex - PMA 4-phorbol 12-myristate 13-acetate - SCA concanavalin A-stimulated rat spleen cell supernatant - SC16 EL4 clone 16 supernatant - IL-1 interleukin-1 - IL-2 interleukin-2 (T-cell growth factor) - FCS fetal calf serum - H-2K^b-lip. H-2K^b inserted in liposomes - C. E. cell equivalents     

Isolation and characterization of an I-A-restricted T cell clone with dual specificity for poly(Glu60Ala30Tyr10) (GAT) and Mlsa,dl

We have isolated a BALB/c (H-2d, Mlsb) T cell clone (JTL-G12) specific for the synthetic polypeptide antigen poly(Glu60Ala30Tyr10) (GAT) in the context of self I-A determinants and for Mlsa,d antigens in the absence of GAT. JTL-G12 proliferation in response to GAT was mapped to the Kd, I-Ad subregions by using inbred H-2 congenic and recombinant strains. In addition, monoclonal antibody directed against I-Ad but not Kd or I-As determinants blocked JTL-G12 proliferation in response to GAT presented by syngeneic splenocytes, indicating I-A restriction. The Mls cross-reactivity of this clone was verified by using a panel of inbred strains bearing the Mlsa,b,c,d alleles and by using BXD recombinant inbred strains bearing the Mlsa allele or the Mlsb allele. All of the Mlsa BXD strains of the H-2d or H-2b haplotypes stimulated JTL-G12 in the absence of GAT, whereas all of the Mlsb BXD strains were nonstimulatory. This response pattern is in complete accordance with recognition of the Mlsa determinant encoded by Mls or closely linked loci on chromosome 1. JTL-G12 proliferation in response to GAT/I-Ad and Mlsa,d determinants could be blocked with a monoclonal antibody (GK1.5) directed against L3T4, a structure involved in class II major histocompatibility complex antigen recognition. These results suggest that antigen/class II responsiveness, Mls reactivity, and expression of L3T4 can be properties of a single T cell population.     

Antigenic sequences of poliovirus recognized by T cells: serotype-specific epitopes on VP1 and VP3 and cross-reactive epitopes on VP4 defined by using CD4+ T-cell clones.

A panel of poliovirus-specific murine CD4+ T-cell clones has been established from both BALB/c (H-2d) and CBA (H-2k) mice immunized with Sabin vaccine strains of poliovirus serotype 1, 2, or 3. T-cell clones were found to be either serotype specific or cross-reactive between two or all three serotypes. Specificity analysis against purified poliovirus proteins demonstrated that T-cell clones recognized determinants on the surface capsid proteins VP1, VP2, and VP3 and the internal capsid protein VP4. Panels of overlapping synthetic peptides were used to identify eight distinct T-cell epitopes. One type 3-specific T-cell clone recognized an epitope within amino acids 257 and 264 of VP1. Three T-cell epitopes corresponding to residues 14 to 28, 189 to 203, and 196 to 210 were identified on VP3 of poliovirus type 2. The remaining four T-cell epitopes were mapped to an immunodominant region of VP4, encompassed within residues 6 and 35 and recognized by both H-2d and H-2k mice. The epitopes on VP4 were conserved between serotypes, and this may account for the predominantly cross-reactive poliovirus-specific T-cell response observed with polyclonal T-cell populations. In contrast, T-cell clones that recognize epitopes on VP1 or VP3 were largely serotype specific; single or multiple amino acid substitutions were found to be critical for T-cell recognition.     

Detection of HLA-DR associated PLT determinants by alloreactive lymphocyte clones

This study deals with alloreactive T-cell clones which recognize cellular determinants associated with HLA-DR antigens. Two clones, CB55 and DS56, exhibited a PLT specificity that was perfectly associated with DR5. On the other hand, clones CB7, DS1 and HS1 showed PLT reactivity with approximately one-half of the DR5 positive cells and none of the DR5 negative cells, whereas clone MD4 largely reacted with the other half of DR5 positive cells. Another MLR culture generated two alloreactive clones DS6 and DS9 with PLT specificity for DR2. However, these clones did not respond to DR2 cells, which were also positive for the DR2-associated HLA-B7 and B18 antigens. Monoclonal antibody (mAb) inhibition studies showed heterogenous patterns, whereby monomorphic non-DR mAbs inhibited the DR2-associated PLT clones while the DR5-associated PLT clones were inhibited by different groups of anti-DR and non-DR mAbs. These observations suggest the existence of several lymphocyte-activating determinants associated with HLA-DR antigens. This diversity may be an important consideration in studies of the role of HLA-DR in immune mechanisms and transplant compatibility.     

Role of endogenous peptides in murine allogenic cytotoxic T cell responses assessed using transfectants of the antigen-processing mutant 174xCEM.T2.

One model to explain the high frequency of alloreactive T cells proposes that allogeneic MHC molecules are recognized together with host cell-derived peptides. A model system was developed to investigate the relevance of this mechanism by expression of H-2Dd or H-2Ld in 174xCEM.T2 (T2) cells. This human cell line contains a mutation in its Ag-processing pathway that should restrict the association of endogenous peptides with cell surface class I molecules. CTL generated by stimulating C57BL/6 (H-2b) responder cells with H-2Dd or H-2Ld transfectants of the human B cell line C1R or the murine T cell lymphoma EL4 were assayed for their ability to recognize alloantigenic determinants on these transfectants. The major fraction of the H-2Dd-specific allogeneic CTL response, generated in a MLC or under clonal limiting dilution conditions, was composed of T cells that recognized H-2Dd expressed on C1R or EL4 cells, but failed to recognize this molecule on T2 cells. Clonal analysis indicated that approximately one-third of these CTL recognized determinants that were unique to H-2Dd expressed on C1R stimulator cells whereas the remainder recognized determinants that were also found on EL4 transfectants. Less than 10% of H-2Dd-reactive CTL recognized the T2 transfectant, and these clones also killed C1R-Dd and EL4-Dd. This result suggests that the great majority of H-2Dd-specific alloreactive CTL recognize determinants that are formed by a complex of H-2Dd with endogenous peptides that are absent or significantly reduced in T2 cells. Based on recognition of human or murine transfectants, these CTL exhibit some level of specificity for the structure or composition of the bound peptides. Examination of allogeneic CTL specific for H-2Ld revealed populations similar to those described for H-2Dd. In addition, a major new population was present that recognized determinants shared between C1R-Ld and T2-Ld but not present on EL4-Ld. These results are consistent with the idea that the alloreactive response to H-2Ld is also largely dependent on the presence of bound peptide. However, they also may indicate that the H-2Ld molecule expressed on T2 cells is occupied by one or more peptides that are shared with other human, but not murine, cells. The significance of these results to current models of alloreactivity is discussed.     

Establishment of T-cell clones recognizing difference in H-2K antigen and inducing graft-versus-host disease

From the spleen cells of C57BL/6 (B6) mice, T-cell clones were established which responded to mutant H-2K antigen of B6. B-H-2bml (Hz1) mice. One clone and its subclones were maintained in culture medium containing lower concentration (2.5%) of IL-2 (culture supernatant of rat spleen cells stimulated with concanavalin A). They were shown to have a capability to induce graft-versus-host reaction in (B6 X Hz1)F1 recipients as well as to exert cytotoxicity against concanavalin-A-activated spleen cells of Hz1 mice and also their kidney fibroblasts. They lacked cytotoxicity against syngeneic and also third-party Con-A-stimulated spleen cells. These reactive clones were Thy-1.2 and Lyt-2 positive and Lyt-1 negative. On the other hand, clones which were maintained in culture medium containing higher concentration (20%) of IL-2 were devoid of activities in vivo as well as in vitro. These clones were Thy-1.2 positive but Lyt-1.2 and Lyt-2.2 negative.