Differential effects of poly (Glu60, Phe40) (GPhe) on murine TH1 and TH2 cells.

The random amino acid copolymer (Glu60, Phe40)n (GPhe) was previously shown to augment antigen-dependent proliferation of the murine TH2 cell lines DCL-2 and D10.G4.1. In the present study, the addition of GPhe to (Glu36, Lys24, Ala40)n (GLA)-primed BALB/c primary lymph node (1 degree LN) T cell cultures, the source of DCL-2, resulted in significant suppression of both the proliferative and lymphokine response to GLA. Suppression by GPhe of the 1 degree LN response was subsequently shown to be neither antigen- nor haplotype restricted, and was inhibitable by polyclonal anti-GPhe antibodies. Studies were extended to a GLA-reactive T cell hybridoma clone (DL.4G6.1). where significant suppression by GPhe of GLA-stimulated lymphokine production was observed as measured by markedly decreased HT-2 stimulatory activity of the collected supernatants. Subsequent antibody blocking experiments employing the monoclonal anti-murine IL-4 antibody 11B11 revealed that BALB/c GLA-reactive 1 degree LN T cells and DL.4G6.1 did not produce detectable levels of IL-4 in their culture fluids when stimulated by GLA, which suggested that these cells, unlike DCL-2, were TH1-like in nature. The addition of GPhe to the TH1 clones 5.2 and 5.9 resulted in significant suppression of proliferation to homologous antigen (ovalbumin), in contrast to the augmentation observed with the TH2 cell lines DCL-2 and D10.G4.1. It was concluded from these data, that the addition of GPhe to various T cell cultures lead to unusual suppressive and augmenting activities specific for TH1 and TH2 cells, respectively. Although the mechanism for these dichotomous effects of GPhe is as yet undetermined, several possibilities are considered.

Enhanced proliferation of murine T cell lines following interaction of Poly(Glu60, Phe40) (GPhe) and antigen-presenting accessory cells. III. Possible mechanisms responsible for activity of GPhe.

The random copolymers (Glu80, Phe20)n (GPhe20), (Glu60, Phe40)n (GPhe), and (Glu50, Phe50)n (GPhe50) were compared for the capacity to augment proliferation of antigen-reactive murine T cell lines. GPhe20, GPhe, and GPhe50 showed "augmenting" activity in order of increasing potency. Phenylalanyl residues constituted a significant portion of the "active" determinant(s) in the GPhe polymers tested. High titer murine anti-GPhe (ascites fluid) inhibited augmentation by GPhe of exogenous (IL-1 + rat-conditioned media (RCM] driven T cell proliferation, indicating that (a) the antibodies by binding to specific active determinant(s) in GPhe may have prevented critical GPhe-APC membrane interaction, and/or (b) "GPhe-anti-GPhe" complexes interfered with necessary "processing" of GPhe by APCs. Time course studies demonstrated that the appearance of increased T cell proliferation after GPhe addition occurred after proliferation to (a) nominal antigen or (b) exogenous (IL-1 + RCM) had reached peak [3H]thymidine incorporation ([3HT]). This suggested that more than GPhe-APC membrane interaction was necessary for GPhe activity. Leupeptin, a lysosomal protease inhibitor, inhibited the augmentation of T cell proliferation by GPhe, which led to the conclusion that GPhe must be "processed" by APCs to exhibit activity.

Enhanced proliferation of murine T cell lines following interaction of poly(Glu60,Phe40) (GPhe) and antigen-presenting accessory cells. I. GPhe-stimulated enhancement of antigen-dependent proliferative responses.

Following interaction of the random polymer (Glu60,Phe40)n (GPhe) with antigen-presenting accessory cells (APC), unusual costimulatory activities were noted in several murine T cell systems. When GPhe, in contrast with other random copolymers (GT,GL), was added during "inhibition" and T cell "repertoire" studies as a (negative) control to GLA-reactive nonclonal T cell lines of haplotypes H-2d (DCL-2) or H-2bm12, augmentation of T cell proliferation ([3H]thymidine incorporation ([3HT]) to homologous antigen was observed. Augmentation by GPhe was also observed in the response of a GLPhe-reactive (H-2s X H-2d)F1 T cell line and the allogeneic response of the clonal T cell line D10.G4.1. This augmentation was critically dependent on the concentration of adherent accessory cells. Although the mechanism of action of GPhe remains, as yet, undefined, the GPhe-mediated enhancement of DCL-2 (a TH2, H-2d anti-GLA, T cell line) proliferation was not dependent upon the production of either IL-1 or IL-6 by accessory cells. In addition, enhanced DCL-2 proliferation was not accompanied by a significant increase in detectable IL-4 release.

Enhanced proliferation of murine T cell lines following interaction of poly(Glu60,Phe40) (GPhe) and antigen-presenting accessory cells. II. The role of accessory cells and cytokines in the activity of GPhe on antigen-independent proliferation.

Our previous study (1) demonstrated the "cytokine-like" activity of poly(Glu60,Phe40)(GPhe) in augmenting the antigen-dependent proliferation of a variety of long-term murine T cell lines, particularly the bulk, BALB/c anti-poly (Glu36,Lys24,Ala40) (GLA), interleukin-4-producing, DCL-2 T cell line. GPhe was found to also augment the antigen-independent proliferation of DCL-2 in response to exogenous cytokines ([interleukin(IL)-2 +/- IL-1] in most experiments). Such exogenous cytokine-driven proliferative responses of DCL-2 were used to investigate further the role of accessory cells and of various soluble factors in the action of GPhe. GPhe did not act as a direct mitogen for T cells, rather it acted in a costimulatory fashion, requiring the presence of plastic-adherent accessory cells and a T cell growth factor (either IL-2 or IL-4). In the presence of accessory cells and exogenous IL-2, augmentation of antigen-independent DCL-2 proliferation by GPhe or by IL-1 depended upon the induction of autocrine IL-4 production. However, GPhe also augmented the response of these cells in the presence of exogenous IL-4 (+/- IL-2, +/- IL-1), and exogenous IL-4 added in combination with exogenous IL-2 (+/- IL-1) failed to mimic the GPhe effect, suggesting that another signal was involved in the mechanism of action of GPhe. The ability of allogeneic accessory cells to interact with GPhe to augment proliferative responses suggested that either a soluble factor or an unusual non-MHC-restricted cell-cell interaction provided this signal. In the presence of uv-irradiated accessory cells, DCL-2 proliferation was enhanced over that observed in the presence of non-uv-treated accessory cells, mimicking the GPhe effect, and interaction of GPhe with uv-irradiated accessory cells did not result in further enhancement of DCL-2 cytokine-driven proliferation. Using monoclonal antibodies which could block the function of IA or CD4 molecules, these cell-surface "adhesion" molecules were shown not to participate in the activity of GPhe. By the addition of recombinant cytokines, neutralizing antibodies, or indomethacin, the mechanism of action of GPhe was also shown not to be dependent upon IL-1, IL-6, IL-7, TNF alpha, or prostaglandin production by accessory cells. However, the presence, individually, of some of these factors (IL-1, IL-7, or prostaglandins) could influence to a variable degree the magnitude of the GPhe effect.

Different T helper cell subsets elicited in mice utilizing two different adjuvant vehicles: the role of endogenous interleukin 1 in proliferative responses.

Mice were immunized with either poly(Glu, Arg, Ala) or poly(Glu, Lys, Phe) contained in two different adjuvant preparations, alum (A1K(SO4)2) or complete Freund's adjuvant (CFA), and in vitro antigen-driven proliferative responses of lymph node cells were assayed 4-16 days later. After immunization with antigens on alum, endogenous interleukin 1 (IL-1) as well as interleukin 4 (IL-4) production was required for the proliferation of the elicited T cells as inclusion of either polyclonal goat anti-mouse IL-1 alpha or monoclonal anti-mouse IL-4 (11B11) in the cultures inhibited proliferative responses to antigen. In contrast, proliferative responses of cells elicited by antigen in CFA were not inhibited by either anti-IL-1 or anti-IL-4. Monoclonal antimouse CD4 (GK 1.5) inhibited proliferative responses regardless of which adjuvant was used to elicit antigen-reactive cells. These data indicated that phenotypically different subpopulations of CD4+ cells were elicited by the same antigen administered in different adjuvant preparations, Th2-like cells after immunization with polymers on alum and Th1-like cells after immunization with antigens in CFA. An examination of the isotypes of polymer-specific antibodies present in the sera of immunized mice also supported this conclusion.

Unusual "cytokine" activities of the random polymer (Glu60, Phe40) in murine immune systems.

When the random polymer (Glu, Phe)n was added to primary lymph node T cell cultures from mice immunized with (Glu, Lys, Ala) and other immunogens, significant inhibition of [3H]thymidine incorporation was noted. A similar addition of (Glu, Phe)n to murine T cell lines (Fathman) from the above reacting lymph nodes led to augmentation of maximal incorporation of [3H]thymidine. The importance of the antigen presenting cells in these phenomena and the possible role of IL-1 in these observations will be documented.

Monoclonal antibodies for specific cell labeling: considerations, preparations and preliminary evaluation.

Two types of monoclonal antibodies (MAb), B79.7 and B59.2 determined to be specific for human platelet glycoprotein III.a and III.a.II.b respectively, were labeled with 125I and 111In via diethylenetriaminepentaacetic acid (DTPA) bifunctional chelation technique and evaluated as agents for labeling platelets. Under the chemical conditions chosen one DTPA molecule was bound to one MAb molecule. The specific activities of 111In and 125I preparation averaged 5 and 2.5 Ci/mumol respectively. Both agents showed saturable binding to human platelets. Scatchard analysis showed that the Kd values for 111In-B79.7 and 125I-B79.7 were 83.3 X 10(-9) M and 113.3 X 10(-9) M respectively. The corresponding protein molecules bound per human platelet were 16.75 X 10(3) and 29.5 X 10(3). The Mab B59.2 also reacted with canine platelets and at 50% antigen saturation 49.5% of the added 111In-DTPA-B59.2 was bound to platelets. Using these platelets experimental thrombi were imaged. At 2 h post injection, the thrombi to blood ratios were 15.8. These radiolabeled MAbs are worthy of further evaluation.

Murine Gc globulin binds to a synthetic sequential polypeptide that is a murine B lymphocyte mitogen.

A non-immunoglobulin component in mouse serum was found to compete with antibody for the binding of a polyanionic antigen, poly (Tyr-Glu-Ala-Gly). The percent of the polymer (44 ng/ml) bound at physiological pH by the serum component in normal mouse serum was low (18%), but was much higher at pH 6.0 (85%). As a result, the binding of the polyanionic peptide antigen by antibody in immune serum was considerably lower at pH 6.0 than it was at physiological pH. These results should be taken into account when dealing with similar antigens that have been frequently studied in immunology, such as poly (Glu60 Ala40) and poly (Glu60 Ala30 Tyr10). The non-immunoglobulin serum component has been identified as Gc globulin, a vitamin D-binding protein that has been shown to be present on the surface of B lymphocytes. This binding might be responsible for the murine B cell mitogenicity of poly (Tyr-Glu-Ala-Gly) that has been previously reported.

Monoclonal T cell responses to two epitopes on a single immunogen controlled by two distinct genes.

The fine specificities of immune T cells were studied in a system in which the response to the antigen can involve two immune response (Ir) genes and two epitopes on a single synthetic polypeptide immunogen. The (BALB/c X SJL)F1 (H-2d X H-2s) mice can respond to the random terpolymer poly(Glu55, Lys36, Phe9) (GLPhe) through the H-2d-linked Ir gene (Ir-d) or through the complementing Ir gene (Ir-dxs), which controls the immune response to poly(Glu, Phe), epitopes that are present in GLPhe. Nine groups of monoclonal T cells were obtained from (H-2d X H-2s)F1 mice immunized with GLPhe. These groups were delineated by the differences in major histocompatibility (MHC) restriction on antigen-presenting cells (APC) and the cross-reactions with GPhe or GLT. A unique T cell line was discovered that can react to the three polymers (GLPhe, GLT, GPhe) even though GLT and GPhe immune T cells do not normally show reciprocal cross-reactions. The monoclonal T cells retain helper activities in the Mishell-Dutton culture. Although the activation of T cells is antigen specific and MHC restricted, the subsequent B cell response is nonspecific.

Complementation of class II A alleles in the immune response to (GluLysTyr) polymers.

The proliferative T cell responses to poly(GluLysTyr) (GLT) and poly(GLULysPhe) (GLPhe) are restricted by the E alpha E beta class II MHC molecule (E) in most responded strains. Some nonresponder strains that carry responder E beta, but cannot express cell surface E molecules, can complement with other nonresponder strains that provide the missing E alpha chain needed for the expression of E molecules and for responsiveness to GLT and GLPhe. Here another type of complementation is described between two E-nonexpressor haplotypes, H-2f and H-2s, which result in E-nonexpressor F1 hybrids, which are responders to GLT. The restriction element involved in this response is an Af/As hybrid molecule. The data support the hypothesis that conformational determinants resulting from the free association of alpha and beta chains in heterozygotes can increase the immune potential of the individual.

Immunogenicity in mice of the sequential polypeptides (Ala-Tyr-Glu-Gly)n, (Ala-Glu-Tyr-Gly)n, (Glu-Ala-Tyr-Gly)n and (Glu-Tyr-Ala-Gly)n and their linkage with the major histocompatibility complex.

The immunogenicity of the four sequential polymers of alpha-L-amino acids referred to below was studied in inbred strains of mice. The responses were linked to the H-2 haplotype, and the Ir genes controlling the responses were mapped to the IA region. The responses were restricted to two haplotypes as follows: (Ala-Tyr-Glu-Gly)n and (Ala-Glu-Tyr-Gly)n - H-2k; (Glu-Ala-Tyr-Gly)n - H-2b; (Glu-Tyr-Ala-Gly)n - H-2k and b. The absence of reciprocal crossreactions among all of these polymers, plus the sequential polymers (Tyr-Ala-Glu-Gly)n and (Tyr-Glu-Ala-Gly)n at both the antibody and T-cell levels, indicated that each of these six polymers was indeed structurally unique.

Murine responses to poly (Phe-Glu-Ala-Gly) and poly (Phe-Ala-Glu-Gly): comparisons between the specificities of the T-cell and B-cell responses.

The murine immune response patterns to (Phe-Glu-Ala-Gly)n and (Phe-Ala-Glu-Gly)n differ from those of the tyrosine analogues (Tyr-Glu-Ala-Gly)n and (Tyr-Ala-Glu-Gly)n. (Phe-Glu-Ala-Gly)n was not immunogenetic in inbred, congenic or recombinant strains of mice. (Phe-Ala-Glu-Gly)n was immunogenic only in mice having f alleles in the IA subregion of the H-2 complex. Reciprocal in vitro cross reactions were noted with T cells from mice of H-2f that respond to either (Phe-Ala-Glu-Gly)n or (Tyr-Ala-Glu-Gly)n. T cells from mice of H-2b, which are responders to (Tyr-Glu-Ala-Gly)n, could not be stimulated with the nonimmunogenic (Phe-Glu-Ala-Gly)n. These results support other studies showing that for any polymer to elicit cross T-cell proliferative responses it must be (a) structurally related to the homologous polymer and (b) immunogenic in the mouse strain whose T cells are being challenged in vitro. Explanations are offered for these statements.

Fine specificities of the murine T cell responses to (Glu Lys Ala) terpolymers.

The T cell proliferative responses to GLA terpolymers among mice of H-2 a, b, d, k, q, r and s haplotypes are heterogeneous. Following immunization with GLA20, GLA40 and GLA60, mouse T cells reacted well with the three terpolymers. The cross-reactions of the above T cells to GLA5 decreased as the alanine content in the terpolymers (immunogens) increased, whereas the trend of the cross-reactions to GA40 did not change. The RIII strain showed no cross-reaction with GLA5. Only mice of H-2 haplotypes q and s failed to show cross-reactions to GA40. Mice of the H-2q haplotype are genetic non-responders to GA40, whereas those of the H-2s haplotype are responders. Immunization of SJL mice with GA40 mixed with GLA40 led to reduction of the specific T cell response to GA40. This reduction of GA40 specific T cell responses in SJL mice by GLA40 occurred during the early stage of the immune response. The kinetic study showed that it was necessary to inject the GLA40 intraperitoneally at least 7 days before GA40 in order for the reduction to take place. The PETLES and lymph node T cells from DBA/1 mice were found to respond to GLA40 differently, and these cells from SJL mice responded differently to GA40.

Changes in the Ir gene controlled response phenotypes in mice of the Ap and Ak family of alleles expressing naturally occurring variant molecules.

Several wild-derived H-2 haplotype mice were recently shown by serology and tryptic peptide fingerprinting to express I-region A molecules closely related to the Ap and Ak molecules of the laboratory strains. To determine if such naturally occurring minor structural variations in the A molecule alter Ir gene-controlled responsiveness, we examined the immune responses of these strains after primary immunizations to three synthetic polypeptide antigens: G60Phe40, GLPhe9, and GAT10. Inbred strains carrying the Ap (or Aq) allele are known to be high responders to G60Phe40 and GLPhe9 but low responders to GAT10. Strains expressing the Ak allele are classified as low responders to G60Phe40 and GLPhe9, but high responders to GAT10. Of seven strains examined belonging to the Ap family, one (B10.CAS2) failed to respond to either G60Phe40 or GLPhe9 as measured by antibody production and T cell proliferation. In addition, two strains (B10.STC90 and W12A) of the Ak family were found to be of responder phenotype to GLPhe9. Both GLPhe9 responses resulted from the introduction of new E beta genes into the I region through naturally occurring intergenic recombination between A beta A alpha and E beta. All strains of mice in the Ak family proved to be of the high responder phenotype in their responses toward GAT10. These results contrast strongly with known patterns of alloreactivity against the variant Ap and Ak molecules.

An unusual suppression of immune response: pretreatment with Poly(Glu60,Phe40) suppresses response to poly(Glu60,Phe40).

The synthetic random copolymer poly( Glu60 , Phe40 ) ( GPhe ) is an excellent immunogen in SWR/J (H-2q) mice. The intravenous injection of soluble GPhe by itself led to antibody production and GPhe -specific T-lymphocyte proliferation. The proliferating lymphocyte was sensitive to anti-Thy 1.2 and anti-Ly 1 antisera. A subsequent immunization with GPhe should therefore lead to an enhanced response. Yet, a single intravenous injection of an aqueous solution of GPhe suppressed the immune response to a subsequent immunization with GPhe in CFA as measured by GPhe -specific plaque-forming colonies, ELISA, T-cell proliferation, and delayed-type hyper-sensitivity. The suppression was not transferable from pretreated mice into normal or irradiated syngeneic recipients with either sera or lymphoid cells. The antibody generated from the pretreatment could not be responsible for the suppression as injection of SWR anti- GPhe antibody into SWR/J mice enhanced, rather than suppressed, the response to the subsequent immunization with GPhe . Pretreatment of mice with a rabbit anti-idiotypic antiserum produced against (SWR anti- GPhe antisera) had no effect on the immune response to GPhe . Thus, the suppression cannot be explained by a simple B-cell tolerance mechanism. This type of unusual suppression was observed only with mice of H-2q haplotype and not with mice of H-2 haplotypes a and k which are also responders to GPhe .

Ir gene control of T and B cell responses to determinants in (Glu Lys Ala) terpolymers.

All mice responding to the terpolymer GLA40 make GL, GA and GLA specific antibodies irrespective of their response to GL or GA alone. The mice displayed positive T cell proliferative responses against the homologous terpolymer, but no T cell responses were obtained with GL, which is non-immunogenic in mice. T cells from GLA immune mice, which are also responders to GA, such as mice of H-2 haplotypes a, b, d, k and r, could be stimulated by GA. T cells from GLA immune mice of H-2 haplotypes p and q which are non-responders to GA could not be stimulated by GA. On the other hand, T cells from H-2s mice immune to GLA and which are also responders to GA alone could not be stimulated by GA. Thus mice of H-2 haplotypes p, q and s recognize the terpolymer via 'GLA' determinants alone, whereas mice of H-2 haplotypes a, b, d, k and r may recognize both GA and GLA determinants in GLA terpolymer.