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P Marrack

Publications and source records attributed to P Marrack.

At least 217 records · Page 12Linked to original sources

Antigen-specific, H-2-restricted helper T cell hybridomas.

We have examined the carrier-specific helper activity of a number of antigen-specific, I region-restricted T cell hybridomas prepared in our laboratory. The hybridomas were assayed for helper activity in the presence or absence of exogenously added nonspecific factors found in the concanavalin A-activated supernatants of normal mouse spleen cells. Of six hybridomas tested, all six could stimulate the IgM anti-hapten response of hapten-primed B cells in the presence of the appropriate hapten-carrier conjugates. At low or moderate carrier doses, the response was dependent upon hapten-carrier linkage and the ability of the hybridoma cells to interact with carrier in association with H-2 products of the responding B cells themselves. Plaque-forming cell responses stimulated by some of the hybridomas were absolutely dependent upon the addition of nonspecific factors, suggesting that anti hapten-protein responses require both an antigen specific I region restricted signal from the T cell hybridomas and nonspecific helper factors, made either by the T cell hybridomas or added exogenously. Under two sets of circumstances, B cells were stimulated in the absence of a simultaneous signal delivered through their immunoglobulin receptor. This occurred either when hapten-primed B cells were stimulated with an ovalbumin/I-Ak-specific hybridoma in the presence of very high concentrations of ovalbumin, or when H-2b B cells were incubated with a hybridoma specific for I-Ab alone. This was interpreted to mean that B cells can be stimulated by reaction of T cells with surface I molecules.

Animals↗

Antigen presentation by Ia+ B cell hybridomas to H-2-restricted T cell hybridomas.

The Ia+, H-2d BALB/c lymphoma cell line L10.A 2J was fused to T cell-depleted spleen cells from mouse strains bearing other H-2 haplotypes. A portion of the selected hybrids expressed Ia antigens of the normal spleen cell partner in the fusion as evidenced by their presentation of various antigens to a set of antigen-specific I-region-restricted T cell hybridomas. Three cloned hybrids were studied in detail. Antigen presentation was shown to be inhibited specifically by monoclonal anti-Ia antibodies. Both I-A and I-E molecules were expressed, including in the one case examined hybrid I-A and I-E molecules between the H-2d and H-2b haplotypes. These Ia+ B cell hybridomas provide a useful set of tools for studying the role of I-region-encoded molecules in antigen presentation to T cells.

Antibody Formation↗

Antigen-specific. I region-restricted interactions in vitro between tumor cell lines and T cell hybridomas.

A series of H-2d B cell tumor lines and one monocytic tumor cell line were shown to be capable of I region-restricted antigen presentation to I-A-d- and I-Ed- restricted, antigen-specific cloned T cell hybridomas. For the most part, antigen presentation correlated with the present of Ia antigens on the presenting cells, although in a few interesting cases Ia-expression lines failed to present antigen. These T cell hybridomas, together with the B cell and to monocyte tumor cell lines, offer a unique set of tools to study the phenomenon of I region-restricted antigen presentation.

Animals↗

A role for T cell helper factors in antibody responses to "T-independent" antigens.

Plaque-forming cell responses were assessed in unprimed mouse spleen cells after stimulation in vitro with SRBC or "T-independent" type 2 (DNP-Ficoll or DNP-liposomes) or type 1 (TNP-LPS or DNP-liposomes containing lipid A) antigens. Rigorously T cell depleted, Sephadex G-10 passed spleen cells were almost completely unresponsive to all these antigens. In every case, responsiveness could be restored by adding supernates from concanavalin A stimulated spleen cells (Con A SN). Experiments in which supernates containing interleukin 1 (IL-1) from the P388D1 cell line, interleukin 2 (IL-2), and a non-IL-2 T cell replacing factor (IL-X) were mixed showed that optimal responses to all the above antigens could only be obtained if both IL-2 and IL-X were present. IL-2 was the only factor that had any effect alone. IL-2 restored responsiveness to different degrees depending on the antigen. This ranged from about 3% (for SRBC) to about 70% (for TNP-LPS) of the optimal response. The P388D1 supernate enhanced responsiveness only if IL-2 and IL-X were present, and only with the T-independent type 2 antigens. These results demonstrate that at least some examples of both type 1 and 2 "T-independent" antigens actually require nonspecific factors one of which, IL-2, is clearly T cell derived, to stimulate antibody production by B cells.

Animals↗

B cell helper factors. II. Synergy among three helper factors in the response of T cell- and macrophage-depleted B cells.

The concanavalin A- (Con A) stimulated supernatant of normal spleen cells (normal Con A SN) was shown to contain a set of helper factors sufficient to allow T cell- and macrophage- (M phi) depleted murine splenic B cells to produce a plaque-forming cell response to the antigen sheep red blood cells (SRBC). The activity of normal Con A SN could be reconstituted by a mixture of three helper factor preparations. The first was the interleukin 2- (IL 2) containing Con A SN of the T cell hybridoma, FS6-14.13. The second was a normal Con A SN depleted of IL 2 by extended culture with T cell blasts from which the 30,000 to 50,000 m.w. factors were isolated (interleukin X, IL X). The third was a SN either from the M phi tumor cell line P388D1 or from normal M phi taken from Corynebacterium parvum-immune mice. The combination of all three helper factor preparations was required to equal the activity of normal Con A SN; however, the M phi SN had the least overall effect. The M phi SN and IL 2 had to be added at the initiation of the culture period for a maximal effect, but the IL X preparation was most effective when added 24 hr after the initiation of culture. These results indicate that at least three nonspecific helper factors contribute to the helper activity in normal Con A SN.

Animals↗

Antigen presentation to T cell hybridomas by a macrophage cell line: an inducible function.

We have investigated the ability of an Ia-, nonantigen-presenting macrophage tumor cell line, P388D, (H-2d), to present antigen to T cell hybridomas after incubation in a lymphokine-containing preparation. P388D, cells were incubated in microtiter wells with various concentrations of Con A-stimulated spleen cell supernatants. Antigen-specific stimulation of H-2d-restricted, KLH-specific T cell hybridomas was observed by P388D1 incubated with SUP.P388D1 cells incubated for 3 days in medium or control SUP did not present antigen. In addition, no stimulation of T hybridomas was seen by P388D1 in the inhibited by the appropriate monoclonal anti-Ia reagents. These results demonstrate that a macrophage tumor cell line can be induced to present antigen and provides for large numbers of readily available, homogeneous macrophages for studying the cellular biochemical requirements for antigen processing and presentation.

Antigens↗

B cell helper factors. I. Requirement for both interleukin 2 and another 40,000 mol wt factor.

A helper factor(s) distinct from interleukin 2 (IL-2) was shown to be present in the concanavalin A-stimulated supernatant of normal mouse spleen cells (normal Con A Sn). Spleen cells thoroughly depleted of T cells required both IL-2 and this factor to produce antibody-secreting cells in response to sheep erythrocytes, although in the presence of IL-2 and a few T cells the requirement for the factor was less apparent. The factor had an apparent approximately 40,000 mol wt. The factor was found in normal Con A Sn that had been depleted of IL-2 by absorption with IL-2-dependent T cells and was absent from Con A-stimulated supernatants of the IL-2-producing T cell hybridoma, FS6-14.13. These results indicate that multiple helper factors control the B cell response to antigen and that IL-2, in addition to its T cell growth promoting activity, plays a direct role in B cell responses.

Animals↗

Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas. Lack of independent antigen and H-2 recognition.

We developed a method for production of antigen-specific, H-2-restricted T cell hybrids. The tumor cell partner in the fusions was itself a T cell hybrid, FS6-14.13.AG2 (or its derivatives), which could be induced to produce the growth factor, interleukin-2 (IL-2), in response to a challenge with concanavalin A, but had no known antigen specificity. The normal T cell partner in the fusions was a population of lymph node T cell blasts that had been highly enriched in antigen-specific, H-2-restricted T cells by in vivo immunization, followed by in vitro challenge with antigen and clonal expansion in IL-2-containing medium. These fusions produced hybrids that grew constitutively in culture. A sizable proportion of the hybrids demonstrated the ability to produce IL-2 in response to a challenge with specific antigen presented by irradiated spleen cells of the appropriate H-2 type. Four cloned antigen/H-2-specific hybrid lines were produced. AO-40.10 responded to chicken ovalbumin (OVA) when presented by I-A(k)-bearing cells. DC1.18.3 responded to the apo form of beef cytochrome c when presented with I-A(d). AODK-10.4 responded to keyhole limpet hemocyanin (KLH) presented with I-A (d). AODK-1.16 also responded to KLH presented by a product of the I region of H-2(d), but the data were consistent with either a product of the I-J-I-E(d) region or a combinatorial molecule with elements from both I-A(d) and I-E(d)/I-C(d). Coincidentally, AO-40.10 was shown to have an unexpected alloreactivity with a product of H-2(b) mapping to the K-I-A region. These hybrids should prove invaluable as sources of monoclonal material for the study of the receptor(s) on T cells with H-2-restricted antigen specificities. We also generated T cell hybrids with two antigen/H-2 specificities by fusing an azaguanine-resistant clone of AO-40.10 to normal T cells with a different antigen/H-2 specificity. Many of the hybrids retained reactivity to OVA plus H-2(a) and to the second antigen/H-2 combination. None reacted to either OVA plus the second H-2 type or to the second antigen plus H-2(a). One of these hybrids was successfully cloned to produce the line AOFK- 11.11.1. It retained the ability to recognize OVA plus I-A(k) inherited from one parent, and KLH plus IA(f) inherited from the other. It did not recognize OVA plus IA(f) or KLH plus I-A(k). These results have some bearing on models describing the nature of T cell receptors for antigen recognized in association with H-2 products. They do not support models in which antigen and H-2 are recognized separately by two independent T cell receptors.

Animals↗

Functional subsets of B cells defined by quantitative differences in surface I-A.

The quantitative expression of surface I-A determinants has been used to define functional B cell heterogeneity based on responsiveness to the trinitrophenyl (TNP) hapten on various classes of carrier molecules. Unprimed, anti-Thy 1 and complement-treated B10.A (I-Ak) B cells were stained with a monoclonal anti-I-Ak reagent that had been fluorescein (FL) conjugated. By using a fluorescence-activated cell sorter these cells were then sorted into 2 populations of cells staining heavily or lightly with this reagent. These populations of cells were then assayed for anti-TNP precursor frequency, in responses to 3 TNP-antigens, by limiting dilution analysis. B cells responsive to TNP-lipopolysaccharide (LPS) and TNP-sheep red blood cells (SRBC) were found in both the B cells that expressed a low amount of surface I-A and the B cells that expressed a high amount of I-A. However, B cells with the ability to respond to TNP-Ficoll were found only in the B cells that expressed a low amount of surface I-A. These experiments suggest that the quantitative expression of surface I-A can be used to define B cell populations with different abilities to respond to hapten coupled to Ficoll.

Animals↗

The role of H-2-linked genes in helper T cell function. VII. Expression of I region and immune response genes by B cells in bystander help assays.

The mode of action by bystander helper T cells was investigated by priming (responder X nonresponder) (B6A)F1 T cells with poly-L-(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys [(TG)-A--L] and titrating the ability of these cells to stimulate an anti-sheep red blood cell (SRBC) response of parental B cells and macrophages in the presence of (TG)-A--L. Under limiting T cell conditions, and in the presence of (TG)-A--L, (TG)-A--L-responsive T cells were able to drive anti-SRBC responses of high-responder C57BL/10.SgSn (B10) B cells and macrophages (M0), but not of low-responder (B10.A) B cells and M0. Surprisingly, the (TG)-A--L-driven anti-SRBC response of B10.A B cells was not restored by addition of high-responder acessory cells, in the form of (B6A)F1 peritoneal or irradiated T cell-depleted spleen cells, or in the form of B10 nonirradiated T cell-depleted spleen cells. These results suggested that (TG)-A--L-specific Ir genes expressed by B cells controlled the ability of these cells to be induced to respond to SRBC by (TG)-A--L-responding T cells, implying that direct contact between the SRBC-binding B cell precursor and the (TG)-A--L-responsive helper T cells was required. Analogous results were obtained for keyhold limpet hemocyanin (KLH)-driven bystander help using KLH-primed F1 T cells restricted to interact with cells on only one of the parental haplotypes by maturing them in parental bone marrow chimeras. It was hypothesized that bystander help was mediated by nonspecific uptake of antigen [(TG)-A--L or KLH] by SRBC-specific b cells and subsequent display of the antigen on the B cell surface in association with Ir of I-region gene products, in a fashion similar to the M0, where it was then recognized by helper T cells. Such an explanation was supported by the observation that high concentrations of antigen were required to elicit bystander help. This hypothesis raises the possibility of B cell processing of antigen bound to its immunoglobulin receptor and subsequent presentation of antigen to helper T cells.

Animals↗

Concanavalin A-inducible, interleukin-2-producing T cell hybridoma.

The fusion of an AKR T cell tumor line to normal B6D2F1, T cells resulted in the production of a cloned T cell hybridoma (FS6-14.13) inducible with the mitogen concanavalin A (Con A). The supernate from Con A-stimulated hybridoma cells was active both in the stimulation of an anti-sheep red blood cell response by partially T cell-depleted B cells and in the stimulation of the growth of antigen-specific T cell blasts. The active principle in both assays had a molecular weight of approximately 30-40,000. These results indicated the presence of interleukin 2 (IL2) in the hybridoma supernate. The activity of the hybridoma supernate in B cell responses was dependent on the presence of adherent cells and a few contaminating T cells. On the other hand, Con A-stimulated supernates from normal spleen cells were active after either adherent cell removal or severe T cell depletion. These results suggested that IL2 was the only active helper factor in the hybridoma supernate, but that additional helper factors were present in supernates from Con A-stimulated normal spleen cells.

Animals↗

Flow sorting of antigen-binding B cell subsets.

Antigen binding was used as a probe in the definition of functional B cell heterogeneity. Unprimed, anti-Thy 1 and complement-treated spleen cells were stained with fluorescent trinitrophenylated bovine serum albumin (FL-TNP-BSA). These cells were sorted, fluorescence negative from fluorescence positive, by using a multiparameter cell sorter and assayed for precursor frequency in antibody responses to TNP by limiting dilution analysis. The cells that bound FL-TNP-BSA were demonstrated to be enriched for antibody-forming precursors to the antigens TNP-lipopolysaccharide (LPS), TNP-sheep red blood cells (SRBC), and TNP- or DNP-Ficoll, whereas the fluorescence negative cells were depleted for these responses. B cells that bound FL-TNP-BSA were then sorted into populations that bound a moderate or high amount of FL-TNP-BSA. The B cells responsive to TNP-LPS and TNP-SRBC were present in both the moderate and high binding populations. In contrast, the B cells responsive to TNP- or DNP-Ficoll were present only in the cells that bound a moderate amount of FL-TNP-BSA. These experiments suggest that there is a population of B cells in adult mouse spleen that binds large amounts of antigen, and that can respond to antigen carried on LPS or SRBC but not carried on Ficoll.

Animals↗

Functional analysis of T cells expressing Ia antigens. I. Demonstration of helper T-cell heterogeneity.

We have examined the expression of I-region antigens on functional subpopulations of murine T cells. A.TH anti-A.TL (anti-Ik, Sk, Gk) alloantiserum was raised by immunization of recipients with concanavalin A (Con A) stimulated thymic and peripheral T-cell blasts. In contrast to similar antisera made by conventional methods, the anti-Ia blast serum was highly cytotoxic for purified T lymphocytes. Moreover, it reacted in a specific fashion with T cells having particular functions. Treatment of keyhole limpet hemocyanin (KLH)-primed B10.A (H-2 alpha) T cells with this antiserum plus complement resulted in the elimination of helper activity for B-cell responses to trinitrophenyl-KLH. Inhibition was shown to be a result of the selective killing of one type of helper T cell whose activity could be replaced by a factor(s) found in the supernate of Con A-activated spleen cells. A second type of helper cell required for responses to protein-bound antigens appeared to be Ia-. By absorption and analysis on H-2 recombinants, at least two specificities were detectable on helper T cells; one mapping in the I-A subregion and a second in a region(s) to the right of I-J. In addition, the helper T cell(s) involved in the generation of alloreactive cytotoxic lymphocytes was shown to be Ia+, whereas cytotoxic effector cells and their precursors were Ia- with this antiserum. These results provide strong evidence for the selective expression of I-region determinants on T-cell subsets and suggest that T-cell-associated Ia antigens may play an important role in T-lymphocyte function.

Animals↗

The role of H-2-linked genes in helper T-cell function. VI. Expression of Ir genes by helper T cells.

We examined the expression of (TG)-A--L specific Ir genes in helper T cells using T cells from low responder leads to (B10, high responder x low responder) F1 chimeric mice. In this paper, the low responder strain studied was B10.M, H-2f. B10.M T cells from these chimeric animals do not help anti-TNP-(TG)-A--L responses, even though they have matured in a high responder thymus and been primed and challenged with antigen on high responder Mphi and B cells. These findings indicate that in the H-2f haplotype an Ir-gene controlling anti-(TG)-A--L activity is expressed in helper T cells. The findings are in contrast to those we have obtained and previously reported with T cells of another low responder haplotype, H-2a. Taken together with our previous findings that (TG)-A--L specific Ir genes are expressed by B cells and Mphi of both the H-2a and H-2f haplotypes, the results indicate two sites of action for Ir genes, and suggest two different gene products acting at different stages of the response, both of which are defective in H-2f cells, and only one of which is defective in H-2a cells.

Animals↗