Serologic and immunochemical characterization of HLA-A9 xenoantisera

There was a pronounced quantitative difference between the helper activities of B6C3F1 splenic T cells sensitized with unmodified vs modified antigens of SRBC. Modified SRBC induced the greater helper activity which was measured by the magnitude of an anti-TNP response (IgM and IgG) elicited in vivo by virgin B lymphocytes. Antigen modification was produced by conjugating SRBC with the hapten or simply by incubating SRBC in cacodylate buffer. There were restrictions with respect to both erythrocyte species and mouse strains for this differential priming to occur. The relatively poor performance of SRBC-primed T lymphocytes was apparently not due to suppressor T cells, but rather to activation of only one of two identified T cell subpopulations required for full helper activity. Unmodified SRBC activated a subpopulation of "helper" cells characterized as sensitive to elimination by ATS and long-lived after ATx, but failed to activate in B6C3F1 mice a second subpopulation of "amplifier" cells resistant to elimination by ATS and short-lived after ATx. In contrast, modified SRBC activated both helper and amplifier cells. Under appropriate conditions these subsets of T cells were strongly synergistic in promoting antihapten antibody formation especially of the IgG class. The involvement of two distinct types of T lymphocytes in the positive regulation of antibody responses raises interesting and novel questions concerning the sequence of events in the triggering of B cells and the subsequent development of the response.     

Functional heterogeneity among the T-derived lymphocytes of the mouse. VII. Conversion of T1 cells to T2 cells by antigen.

The T1 subpopulation of peripheral T cells was defined in mice by its short half life, insensitivity to anti-thymocyte sera (ATS) in vivo, and slow kinetics of response to antigen. The T2 subpopulation was defined by its long life time, elimination by ATS in vivo, and rapid response to antigen. Mice containing only T1-type T cells were constructed by adult thymectomy (ATx) followed immediately by the elimination of T2 cells by ATS treatment. Immunization of these mice with SRBC led to the production of memory helper cells in the T2 subpopulation. This process depended on the presence of T1 cells and for the most part required SRBC immunization, although a few SRBC-specific T2 cells reappeared in the mice in the absence of antigen. We conclude that T1 cells can give rise to T2 cells in an antigen-driven step and that the two populations correspond to virgin and memory T cells, respectively.     

Immunosuppressive ATS. V. Analysis of the effect of anti-thymocyte serum on T lymphocyte subsets

The effect of anti-thymocyte serum (ATS) on functional T cell subsets within the immunoregulatory network was examined. We found that low doses of ATS depleted the IL 2-producing subset of T helper cells and higher doses of antiserum also eliminated cytotoxic precursors. It was estimated that a standard low dose of ATS, which reduced T helper cells 80-fold, depleted cytotoxic precursors only about four-fold. The data indicate that ATS acts on different T cell subsets in a dose-dependent fashion and suggest that monoclonal antibodies might be used to preferentially deplete specific T cell subpopulations. Two monoclonal rat anti-mouse Lyt sera, however, proved ineffective in inducing functional changes in vivo, although an alloantiserum to Lyt1.1 was able to reduce cell-mediated cytotoxicity in a dose-dependent fashion. The results support the feasibility of specific depletion of T cell subsets with anti-lymphoid antibodies.     

Functional heterogeneity among the T-derived lymphocytes of the mouse: III. Helper and suppressor T-cells activated by concanavalin A

We have studied the properties of T-cells which when activated by concanavalin A (Con A) either suppress or help the in vitro humoral response of mouse spleen cells. Previously established criteria for the T-cell populations, T₁ and T₂ were applied. T₁ cells were defined by their short half-life (2–3 wk) after adult thymectomy (ATx) and their resistance to small doses of antithymocyte serum (ATS). T₂ cells were defined by their long half-life (~15 wk) and their high sensitivity to ATS. T-cells which could be activated by Con A to help the response to the thymus-dependent antigen, sheep red blood cells, were found mainly in the T₂ subpopulation. T-cells which could be activated by Con A to suppress the response to the thymus-independent antigen, trinitrophenyl-lipopolysaccharide (TNP-LPS), were found within both the T₁ and T₂ subpopulations.These results, our previous results, and those of others suggest that the T-cell responses to phytomitogens distinguish precursors committed to different functions, while the T₁ and T₂ classifications distinguish T-cells at different stages of maturation.     

Immunoregulation in the rat: characteristics of a suppressor T cell that inhibits antigen-dependent cell proliferation

We have examined the characteristics of a rat suppressor T cell (Ts) that inhibited the antigen-dependent proliferative response of antigen-primed T cells. The kinetics of in vitro induction of Ts from lymph node T cells obtained from antigen-primed rats indicated that Ts were induced in the presence of the priming antigen within 48 hr of culturing. The Ts produced during the first 48 hr of in vitro cultures were radiosensitive (2000 rad) but became partially radioresistant within the next 48 hr of culturing. In the presence but not the absence of priming antigen, Ts inhibited the antigen-dependent proliferative response to the priming antigen as well as to heterologous antigens. Suppression appeared to be mediated via a nondialyzable suppressor factor (TsF). The induction of Ts in cultures required the presence of OX-6-/OX-8- T cells, antigen-presenting cells, and the antigen. Although a majority of cells recovered from the induced cultures were OX-8+, there was no evidence that OX-8+ antigen expression per se was related to Ts activity. Addition of highly purified IL 2 augmented the Ts-mediated suppression. The immunoregulatory implications of these findings are discussed.     

CD4+ T cell subsets. Lymphokine secretion of memory cells and of effector cells that develop from precursors in vitro

We have studied the properties of several developmentally defined subpopulations of CD4+ T cells from normal animals which can be stimulated to secrete lymphokines. We find that the Th cells responsible for direct secretion of lymphokines after stimulation are from a resting, very long lived subpopulation of CD4+ T cells which persists for over 25 wk after adult thymectomy. These T cells are depleted by in vivo administration of antithymocyte serum and they are enriched among T cells which express high levels of Pgp-1. This phenotype suggests that the T cells responsible are most likely memory T cells which have resulted from antigen exposure in vivo. T cells in this subset secrete predominantly IL-2 with small quantities of IL-3, granulocyte/macrophage CSF, and IFN-gamma. In contrast, the CD4+ T cells which require in vitro culture and restimulation before they develop into an effector population with the ability to secrete lymphokines after restimulation, differ dramatically by most of these criteria. The precursors we study are resting Th cells which are considerably shorter lived after adult thymectomy (5 to 10 wk) and resistant to the same doses of antithymocyte serum which deplete the putative memory population. We hypothesize that this precursor population represents naive helper cells which have not yet encountered Ag. The effectors derived from such precursors can be stimulated to secrete high levels of both Th cell types 1 and 2 lymphokines (IFN-gamma, IL-4, IL-5, granulocyte/macrophage CSF, and IL-3). Generation of effectors requires proliferation and differentiation events which occur during a mandatory culture with lymphokines and antigen presenting cells for 3 to 4 days. We discuss the striking phenotypic and functional differences among these subpopulations of helper cells--the precursor population and the two types--memory and cultured effector Th which secrete lymphokines. We also discuss the relationship of these populations to CD4+ T cell subsets defined by other studies of patterns of lymphokine secretion and by cell surface phenotype.     

Functional heterongeneity among the T-derived lymphocytes of the mouse. VI. Memory T cells stored in the T2 subpopulation.

Memory T cells were demonstrated in mice which were the precursors for cells causing sheep red blood cell-(SRBC) specific helper activity in the in vitro response of spleen cells to trinitrophenyl (TNP)-SRBC. These memory cells were distinguished from the effectors of helper activity generated during the primary response and were shown to belong to the T2 subpopulation of peripheral T cells on the basis of 1) their rapid response to a challenge with SRBC, 2) their long lifetime, and 3) their sensitivity to small in vivo doses of anti-thymocyte serum.     

T cell priming in vivo: a major role for B cells in presenting antigen to T cells in lymph nodes

Previous studies have shown that lymph node (LN) T cells from mice given repeated injections of anti-mu antisera from birth (mu sm) fail to mount secondary T proliferative responses to antigen in vitro after s.c. priming in vivo. This finding raised the possibility that priming of T cells in LN depends on the presence of B cells, Ig+ B lymphocytes being absent in mu sm. In support of this idea, the present paper shows that the priming defect in LN of mu sm can be largely overcome by injecting B cell populations s.c. 1 day before s.c. priming with antigen. Restoration of LN priming was observed with s.c. injection of highly purified populations of small B cells but not with heat-killed or lightly irradiated B cells. Homing studies indicated that approximately 10% of s.c.-injected B cells reached the draining LN. In other studies, irradiated mice injected i.v. with purified T cells manifested poor priming in LN after s.c. injection of antigen. It was reasoned that the LN priming defect in this situation reflected the lack of B cells in irradiated mice, B cells being highly radiosensitive. In support of this notion, it was found that s.c. injection of B cells into irradiated recipients of T cells led to high priming of T cells in LN after s.c. injection of antigen. Although T cells exposed to antigen in B-depleted LN of mu sm and irradiated mice gave negligible T proliferative responses in vitro, low but significant levels of primed T helper function were detected in a sensitive T helper assay in vivo. In light of this finding, our working hypothesis is that the initial induction of T cells to antigen in LN is controlled by resident dendritic cells (or other non-B antigen-presenting cells), the main role of B cells being to control the clonal expansion of activated T cells.     

Antigen-specific and nonspecific mediators of T-cell/B-cell cooperation. VI. Distinct patterns of cross-reactivity by two human gamma-globulin-primed helper T-cell subpopulations.

We have previously characterized the activities, in vitro, of two different helper T-cell subpopulations, primed with human γ-globulin (HGG). One T-cell subpopulation helps the response of B cells to determinants (e.g., haptens) bound to the same antigen to which the T cells are primed (specific help); the other helper T-cell subpopulation responds to the same priming antigen by secreting a nonspecific molecule which helps B-cell responses to erythrocyte antigens co-cultured with the priming antigen (nonspecific help). These subpopulations also differ in their frequency and dose response to antigen, both in vivo and in vitro. They are similarly susceptible to the induction of unresponsiveness to HGG. In order to determine whether these T-cell subpopulations share or differ in their ranges of antigen recognition, we have compared the reaction of these two HGG-primed helper T-cell subpopulations to a number of γ-globulins (γG's) from other species. Plaque-forming cells generated in response to HGG shared little or no cross-reactivity with any of the heterologous (γG's) tested. In contrast, HGG-primed nonspecific helper T cells responded with significant cross-reactivity when challenged in vitro with dog γG, but HGG-primed specific helper T cells did not respond with any such cross-reactivity. No other heterologous γG tested stimulated any significant cross-reactivity from either HGG-primed T-cell subpopulation. Thus, these two T-cell subpopulations differ in their antigenic recognition. Possible explanations of these data include: (i) a difference in receptor specificity; (ii) a difference in the receptor affinity; (iii) a difference in Ia determinants of the two subpopulations.     

Repertoires of T cells directed against a large protein antigen, beta-galactosidase. I. Helper cells have a more restricted specificity repertoire than proliferative cells

The proliferative and helper T cell repertoires were compared in the CBA/J mouse for the response to the large protein antigen, tetrameric beta-galactosidase (GZ = 1021 a.a/monomer). The systems assessed the ability of cyanogen bromide (CB) peptides of GZ to: 1) prime for a T cell proliferative response to GZ; or 2) generate T cell help, measured by the production of anti-FITC PFC in the in vitro response to GZ-FITC. Priming for in vitro proliferation was attempted with 11 CB peptides comprising 70% of the GZ molecule. Strong priming was found with five peptides and intermediate priming was found with four other peptides; two peptides were without effect (CB-20 = a.a 767-862, and CB-4 = a.a. 188-202). Despite this indication of generally dispersed recognition of GZ epitopes, only two CB peptides, CB-2 (a.a. 3-92) and CB-10 (a.a. 378-418) were able to induce a T helper cell response. The surprising dearth of helper T cell-inducing epitopes may be peculiar to the limited fluorescein (FITC) substitution on GZ-FITC (17-25 FITC residues per tetramer) or it may reflect the constraints involved in T cell recognition required for T-B collaboration. Also considered was the possibility that the helper T cell repertoire might be distinct from the proliferative repertoire, the latter reflecting DNA synthesis and recruitment by other functional T cell subpopulations.     

Maintenance of hyporesponsiveness to antigen by a distinct subclass of T lymphocytes.

Immunization with increasing doses of SRBC, in excess of 10(8), results in a progressive decline in the anti-SRBC PFC response. This hyporesponsive state is antigen specific and is reflected in a decrease of both T helper and B antibody-forming activity. We asked whether the apparent defect of T helper activity reflected a) an absence of alphaSRBC helper T cell activity, or b) the presence of SRBC-specific suppressor T cells within the hyporesponsive population. Our results indicate that at least a portion of hyporesponsiveness noted after antigen exposure to large doses of antigen can be ascribed to specific suppressor T cell activation. Fractionation of the suppressive T cell population using Ly antiserum showed that specific suppressive activity was mediated by a subclass of T cells (Ly2+), distinct from that committed to express helper function (Ly1).     

Effects of antithymocyte serum on lymph node cells participating in the graft-vs-host reaction.

BALB/c mice were injected with 0.1 ml of antithymocyte serum (ATS) and tested at various times thereafter for graft-versus-host (GVH) reactivity of lymph node cells (LNC) and spleen cells (SC). Splenomegaly produced by LNC or SC injected alone was used as a measure of precursor T cell function and the capacity of such cells to produce synergy when combined with normal thymocytes was used to evaluate amplifier T cell activity. In lymph node, both precursor and amplifier function were strikingly depressed 2 days after ATS administration; by day 11, precursor function showed slight recovery while amplifier activity had recovered to supranormal levels. In spleen, precursor activity recovered two fold between 2 and 11 days after ATS inoculation but no amplifier activity could be detected at day 11. Since donors had not been deliberately stimulated with alloantigens prior to testing of LN and SC GVH acivity, these studies demonstrate (a) that precursors and amplifiers are disinct T cell populations that are committed to express unique functional activities before antigen exposure and (b) that, following depletion with ATS, these two populations recover independently at different rates in separate lymphoid compartments.     

Immune maturation of T lymphocytes: sequential changes in the functional specificity and apparent affinity of hapten-reactive helper T cells during an immune response.

The maturation of helper T lymphocytes during an immune response was studied with respect to sequential changes in the functional specificity and affinity toward certain antigens. Protein-carrier (BαA)-reactive helper T cells obtained after a relatively long priming period were effectively stimulated by relatively lower doses of antigen than shortly primed helper T lymphocytes. When hapten (PAB)-reactive helper T lymphocytes were utilized as a model of helper T cells, reactivity also increased progressively to smaller concentrations of PAB-conjugates at successive intervals after primary immunization. Concomitantly, the cross-reactivity of PAB-reactive helper T cells to structurally related MAB- or OAB-determinants also decreased. Moreover, the PAB-reactive helper T cells of the relatively longer priming period were very susceptible to tolerance induction upon treatment with PAB-d-GL, whereas the reactivity of those helper T cells of the relatively shorter priming period was not abolished by this treatment. These results clearly indicate that there are qualitative changes in the helper T lymphocyte population during an immune response, and that this represents the sequential development or selection of helper T lymphocytes of higher specificity and apparent affinity to a corresponding antigenic determinant.     

In vitro studies of the rabbit immune system: III. T-cell requirement for initiation but not maintenance of anti-sheep erythrocyte plaque-forming cells

The in vitro anti-sheep erythrocyte plaque-forming-cell (anti-SRBC PFC) response of primed rabbit spleen cells can be abrogated by prior treatment with anti-thymocyte serum plus complement (ATS + C). The direct addition of ATS without C to stimulated cultures also reduces the response 60–90%, if the antiserum is added before the initiation of antigen-stimulated PFC production. However, the PFC response becomes refractory to ATS inhibition by 48 hr. This loss of ATS sensitivity is not caused by T cells themselves becoming refractory to ATS for the following reasons: (i) ATS + C treatment or ATS addition at 48 hr causes cytotoxic effects comparable to similar treatments at 0 hr; (ii) The responsiveness of cells incubated 48 hr before the addition of SRBC remains sensitive to ATS inhibition at that time. The loss of ATS sensitivity is also not the result of soluble T-cell factors which have accumulated in the medium, because the number of PFC in stimulated cultures continues to increase after treating with ATS + C and reculturing in fresh medium at 48 hr. We conclude that T cells are required to initiate the production of antibody by B cells but are not required to maintain the later proliferative events.     

Induction and properties of DNP-specific T cells in mice sensitized to DNP-coupled mycobacterium.

Splenic T lymphocytes from mice sensitized to 100 microgram of DNP-coupled mycobacterium (DNP-Tbc) showed in vitro proliferative response against DNP- or TNP-conjugated protein antigens. The increased uptake of 3H-thymidine induced by DNP-HSA was partially inhibited by the addition of 10(-4)M DNP-EACA. DNP-AECM-Ficoll did not induce any significant proliferative responses in DNP-Tbc-primed T cell population. However, priming with DNP-Tbc augmented anti-DNP IgG antibody response induced with DNP-Ficoll. The augmentation of IgG response was not due to the presence of DNP-primed B cells or anti-DNP antibody. The results showed that the priming with DNP-Tbc induced DNP-reactive T helper cells which could be triggered with DNP-Ficoll. The possible role of mycobacterium in the induction of hapten-specific T helper cells is discussed.     

T lymphocyte responses to non-H-2 histocompatibility antigens. I. Role of Ly-1+2+ T cells as cytotoxic effectors and requirement for Ly-1+2+ T cells for optimal generation of cytotoxic effectors

Cytotoxic effector T cells specific for non-H-2 histocompatibility (H) antigens were examined for phenotypic expression of lymphocyte differentiation (Ly) antigens. Virtually all H-Y-specific cytotoxic effectors generated in mixed lymphocyte culture were Ly-1+2+ T cells. H-3-specific effectors comprised both Ly-1+2+ and Ly-1-2+ T cells. However, cytotoxic effectors specific for multiple non-H-2 H antigens were predominantly Ly-1-2+ T cells. The optimal generation of H-Y- and H-3-specific effectors required Ly-1+2+ T cells; optimal generation of multiple non-H-2 H antigen-specific effectors required an interaction between Ly-1+2- and Ly-1-2+ T cells. These observations suggest that the identity of the target H antigen in part determines the Ly type of responsive T cells. Our observations suggest that 2 alternative pathways of T cell response exist for non-H-2 H antigens. The first pathway involves an interaction between Ly-1+2- helper T cells and Ly-1-2+ cytotoxic effector precursors. The 2nd pathway simply involves the response of Ly-1+2+ T cells proliferating and generating H antigen-specific cytotoxic effectors.     

Regulation of anti-hapten antibody response by chemically modified carrier antigen preferentially provoking delayed-type hypersensitivity (DTH). II. DTH-reactivity and carrier-specific suppression of anti-DNP antibody response induced by priming with dodecanoyl-BSA are mediated by functionally distinct T cell subpopulations.

Experiments were carried out to determine whether or not the cell populations involved in DTH and in the suppression of antibody response are identical. The effects of four treatments, i.e., adult thymectomy (ATx), X-irradiation, anti-mouse thymocyte serum (ATS) and hydrocortisone (HC) on the induction of DTH and on the carrier-specific suppression of antibody response were observed in mice immunized with chemically modified antigen, dodecanoyl-BSA (d-BSA), emulsified with complete Freund's adjuvant (CFA), with the following results: 1) DTH induced by immunization with D -BSA remained constant in adult thymectomized mice, whereas the suppression of antibody response was not inducible in these animals. 2) Injection of low doses of ATS caused the depression of DTH in mice primed with D -BSA, but did not affect the suppressive activities of their spleen cells. 3) Sublethal X-irradiation 1 week prior to D -BSA priming inhibited the generation of suppressor cells but did not affect the generation of cells mediating DTH. The suppressive effect was also abrogated by sublethal X-irradiation given 2 days after immunization with DNP-BSA (14 days after priming with D -BSA). 4) The treatment of animals with HC 2 days before the footpad challenge or immunization with DNP-BSA depressed the ability of animals to induce both DTH and the suppression of antibody response. However, the latter was more sensitive to HC than the former. In addition to these results, it was also found that D -BSA-primed spleen cells were capable of suppressing anti-DNP response, but not of inducing DTH-reactivity upon transfer to recipient mice. These results suggest that DTH-reactivity and the carrier-specific suppression of anti-hapten antibody response induced by injection of D -BSA are mediated by different cell populations.     

Hapten-specific T cell response to azobenzenearsonate-N-acetyl-L-tyrosine in the Lewis rat. II. Induction of helper activity demonstrated by TNP-haptenated ABA-peptide-Ficoll

In an effort to study T cell functions in Lewis rats immunized with ABA-N-acetyl-L-tyrosine (ABA-tyr), we developed an antigen that provides a sensitive assay of ABA-specific helper function that is read as an increase in TNP-specific plaque-forming cells (PFC). This antigen has ABA coupled to AECM-Ficoll by virtue of a tripeptide (tyr-ala-ala) spacer and TNP coupled to the AECM side chains. At subimmunogenic doses, this antigen induced 400 anti-TNP PFC/10(6) spleen cells in ABA-tyr-immunized rats. As many as 8000 PFC/10(6) spleen cells were induced with larger doses of antigen (200 micrograms). By contrast, only 490 PFC/10(6) spleen cells could be induced with 1 mg of the conventional doubly haptenated protein carriers such as ABA-BSA-TNP. Both direct and indirect PFC were induced by this antigen in primed rats. The use of this antigen and passive transfer techniques to study ABA-specific helper activity revealed some differences from ABA-specific delayed-type hypersensitivity (DTH) and in vitro proliferation, which were studied previously. Cells responsible for helper activity appeared sooner after immunization and were found most prominently in peritoneal exudates but also significantly in spleen where the cells responsible for DTH or in vitro proliferative responses were never found. By contrast, helper cells were not seen in lymph nodes, where some proliferative activity could be found. Of these three ABA-specific T cell functions, helper activity was least easily suppressed by the previously used regimens of ABA-tyr in incomplete freunds adjuvant (IFA). Moreover, helper activity appears after injection of ABA-tyr in IFA, a method that has never in our hands yielded detectable DTH or in vitro proliferative responses. Despite these differences, phenotyping with monoclonal antibodies indicated that cells responsible for helper and proliferative activities were both W3/25+ and OX8-.     

Reactivity to SV40 T antigen in athymic (nude), anti-thymocyte serum-treated, and normal mice.

Antisera prepared in syngeneic mice by hyperimmunization with intact SV40-transformed mouse cells or with somatic cell hybrids between SV40-transformed human and normal mouse cells exhibit anti-SV40 tumor (T) antigen reactivity. Athymic mice bearing tumors formed by SV40-transformed mouse, human or mouse-human hybrids were not reactive with SV40 T antigen. Anti-thymocyte serum (ATS)-treated mice also lacked T antigen reactivity during suppressive treatment but developed antibody to T antigen after discontinuing ATS treatment and tumor regression. We conclude that that presence of growing tumors in the mouse is not necessary for the production of anti-SV40 T antigen antibodies but that helper thymus-derived cells are essential for the humoral response.     

Antigen-specific suppressor cell activity in man: I. Temporal, antigenic, and proliferative requirements

The temporal, antigenic, and proliferative requirements of antigen-specific suppression of the in vitro plaque-forming cell (PFC) response of human peripheral blood mononuclear cells (PBM) were studied. Suppressor cell activity (SCA) was generated by priming PBM with high doses of the T-cell-dependent antigen ovalbumin (OA) and measured by adding washed primed cells to target PFC cultures. Priming with high doses of OA was shown to induce a population of antigen-specific T lymphocytes which interfered with the anti-OA PFC response of optimally stimulated target cultures. The generation of SCA was demonstrated following as little as 24 hr of high-dose OA priming and could be abrogated by prolonged priming (72 hr) or by pretreatment with mitomycin prior to priming. The expression of optimal SCA required the addition of primed PBM at the initiation of the target culture and could be directly correlated to both the OA concentration used for priming and the number of primed cells added. Higher priming doses of OA (up to 100 μg) generated increasing numbers of cells capable of antigen-specific SCA as measured via a cell dilution protocol. Our data suggest that an antigen-driven and dose-dependent expansion of an antigen-specific T-suppressor cell pool is an early regulative event limiting the in vitro PFC response of human lymphocytes to OA.