Antigen-specific murine T-cell lymphomas: functional heterogenicity

Two ovalbumin (OVA)-specific helper lymphomas (designated ROT/6.1 and ROT/6.2) were established by transformation of enriched, OVA-immune T cells with the radiation leukemia virus (RadLV). Shortly after establishment these lymphomas provided carrier (OVA)-specific help for anti-hapten antibody response. However, 5 months later ROT/6.1 lost its OVA specificity and could augment anti-hapten antibody response in the presence of an unrelated carrier. ROT/6.2 retained its antigen-specific helper function over 10 months of repeated passaging. This OVA-specific helper line inhibited anti-hapten antibody response when given together with an unrelated carrier. Cloning of ROT/6.2 by limiting dilution revealed that only 3 of 10 clones tested had OVA-specific helper activity. None of the clones could induce antigen-specific DTH reaction. The interrelationship between the functional heterogenicity, specificity, and stability of the helper lines is discussed.     

The male antigen. II. Regulation of the primary and secondary responses to H-Y by H-2 associated genes

The primary response of females of ten inbred mouse strains to the male antigen (H-Y) was investigated by transfer of peritoneal exudate cells (PEC). Three distinct classes of reactivity were seen. Early primary response to H-Y was associated with H-2 haplotypes b and s; intermediate response with H-2 haplotypes, k, d, i, and h; and late or absent response with H-2 haplotypes a and f. The failure of A.CA (H-2^f) females to mount a detectable primary response against syngeneic male cells was not due to the lack of the antigen from A.CA male cells. The ability of (A.CA × B10)F₁ hybrid females to respond to the male antigen demonstrated the dominant nature of the B10 (H-2^b) response and excluded the possibility that A.CA females possess a dominant self-antigen cross-reactive with H-Y. The secondary response of eight inbred strains was investigated; at least three distinct levels of reactivity were apparent. The speed of the secondary response was associated with the various H-2 haplotypes in the same way as the primary response. The implications of differential strain reactivity, background effect, and the association of Ir-1 with response to H-Y are discussed.     

Antigen-specific murine T-cell proliferation: role of macrophage surface Ia and factors.

The proliferation of Mycobacterium-primed murine lymph node T cells to purified protein derivative of tuberculin (PPD), as measured by the uptake of tritiated thymidine, requires the obligatory participiation of macrophages which stimulate the T cells either directly with antigen in association with cell surface Ia (I region-defined antigens), or indirectly by means of soluble factors. We have examined the possibility that this functional dichotomy is due to heterogeneity within the macrophage population. Since the maturation of macrophages from the precursor monocytes is associated with cell enlargement, macrophage subpopulations differing in developmental stage are obtained by cell fractionation according to size by velocity sedimentation. Nylon-wool-purified T cells which have been depleted of macrophages and B cells are stimulated with PPD either in a free form or bound to macrophages which have been incubated for a short time (i.e., pulsed) with PPD. We found that for PPD-pulsed macrophages, only the smallest (and probably the most immature) are capable of inducing T-cell proliferation. This antigen presentation function is mediated by cell surface Ia since it is abolished by pretreatment of the macrophages with anti-Ia serum and complement. On the other hand, all macrophages, irrespective of sensitivity to anti-Ia serum, secrete factors which will stimulate T-cell proliferation in the presence of free PPD. Thus the maturation of macrophages is accompanied by a shift from Ia-dependent to Ia-independent mechanisms of immunostimulation.     

Functional interactions of human and murine lymphoid cells. I. Analysis of primary and secondary responses

In this study human T-cell responses against murine alloantigens were analyzed. The results show that optimal primary responses are obtained from peripheral blood mononuclear cells only when murine splenic adherent cells (SAC) were used as antigen. Further analysis revealed that human T cells were able to respond directly to murine cells without the need for antigen reprocessing; however, human interleukin 1 (IL-1) was required for optimal stimulation. In contrast, secondary proliferative responses to murine cellular antigens could be induced from primed T cells even in the absence of SAC and/or IL-1. These proliferative responses, and in addition, cytotoxic T-cell responses, were specific for the priming antigen. Long-term human T-cell lines specific for murine alloantigens were found to replace the need for murine T cells in antigen-specific murine B-cell responses to sheep red blood cells. The mechanism of help delivered by the human T cells appeared to be by the release of nonspecific helper-T-cell factors. The evidence presented for this is the inability of these cells to stimulate cells from mice that express the X chromosome B-cell defect xid.     

Elicitation of antigen-induced primary and secondary murine IgE antibody responses in vitro

Described herein are methods for eliciting and quantitating primary and secondary murine IgE antibody responses in vitro, and the important role of antigen concentration in determining the level of IgE produced during an immune response. The methods for quantitating IgE antibody levels in culture supernatant fluids and in serum by ELISA are presented in detail. The specificity of such methods was confirmed in that (1) no other isotype of antibody registered in the IgE-ELISA, and (2) parallel determinations of IgE antibody concentrations could be obtained by independent analysis using Fc epsilon RI-dependent basophil degranulation. We examined various parameters of cell donor immunization and lymphoid cell culture which allow for optimal in vitro primary and secondary IgE responses. High relative antigen doses result in diminished IgE antibody responses in experimental animals, a finding confirmed herein. High antigen concentrations in vitro also result in relative suppression of IgE antibody synthesis. This was also true for in vitro production of IgG1 and IgA antibodies. Conversely, IgM and IgG2a responses were elicited at both low and high antigen concentrations; IgG2b and IgG3 were not produced under the conditions of priming and culture used herein. Finally, production of IgE in vitro depended on the presence of carrier-primed CD4+ T cells and hapten-specific B cells. Generation of maximal IgE antibody secretion, and hence elicitation of an allergic reaction, is thus dependent on the amount of antigen acting as stimulant for the immune response.     

Antigen-specific inhibition of ongoing murine IgE responses. II. Inhibition of IgE responses induced by treatment with glutaraldehyde-modified allergens is paralleled by reciprocal increases in IgG2a synthesis.

Administration of high m.w. glutaraldehyde-polymerized OVA (termed OVA-POL) before OVA-[A1(OH)3] immunization of C57BL/6 mice markedly impairs their capacity to generate OVA-specific IgE responses, while simultaneously resulting in striking enhancement of Ag-specific IgG2a responses. We demonstrate here that treatment with this class of chemically modified allergen also results in pronounced inhibition of ongoing IgE responses in vivo. The abrogation of well established murine IgE responses that is elicited after treatment with OVA-POL (i) is potent (97%), (ii) is long lived, and (iii) reflects reciprocal regulation of Ag-specific IgE and IgG2a responses in vivo. Moreover, the capacity of OVA-POL-treated mice to generate secondary IgE responses remains strongly decreased for at least 260 days and six subsequent immunizations with native allergen, despite there being no further treatment with modified allergen. These changes in IgE and IgG2a responsiveness are Ag specific and T cell dependent.     

Distinct helper virus requirements for Abelson murine leukemia virus-induced pre-B- and T-cell lymphomas.

Abelson murine leukemia virus (A-MuLV) can induce pre-B- or T-cell lymphomas (thymomas) in mice depending on the route and time of injection. Previous studies have shown that the choice of the helper virus used to rescue A-MuLV greatly influences its ability to induce pre-B-cell lymphomas. In this study, we investigated the role of the helper virus in A-MuLV-induced thymomas. A-MuLV rescued with the helper Moloney MuLV, BALB/c endogenous N-tropic MuLV, and two chimeric MuLVs derived from these two parents were injected intrathymically in young adult NIH Swiss mice. All four A-MuLV pseudotypes were found to be equally efficient in the induction of thymomas, whereas drastic differences were observed in their pre-B-cell lymphomagenic potential. Thymoma induction by A-MuLV was independent of the replication potential of the helper virus in the thymus, and no helper proviral sequences could be detected in the majority of thymomas induced by A-MuLV rescued with parental BALB/c endogenous or chimeric MuLVs. In the thymomas in which helper proviruses were present, none of them were found integrated in the Ahi-1 region, a common proviral integration site found in A-MuLV-induced pre-B-cell lymphomas (Y. Poirer, C. Kozak, and P. Jolicoeur, J. Virol. 62:3985-3992, 1988). In addition, helper-free stocks of A-MuLV were found to be as lymphomagneic as other pseudotypes in inducing thymomas after intrathymic inoculation, in contrast to their inability to induce pre-B-cell lymphomas when injected intraperitoneally in newborn mice. Restriction enzyme analysis revealed one to three A-MuLV proviruses in each thymoma, indicating the oligoclonality of these tumors. Analysis of the immunoglobulin and T-cell receptor loci confirmed that the major population of cells of these primary thymomas belongs to the T-cell lineage. Together, these results indicate that the helper virus has no effect in the induction of A-MuLV-induced T-cell lymphomas, in contrast to its important role in the induction of A-MuLV-induced pre-B-cell lymphomas. Our data also revealed distinct biological requirements for transformation of these two target cells by v-abl.     

Genetic analysis of chemoresistance in primary murine lymphomas

Understanding the basis of chemoresistance is a principal goal of molecular oncology. We have exploited a murine lymphoma model and retroviral gene transfer to rapidly generate a series of spontaneous tumors differing only in a gene of interest, and subsequently studied the impact of the test gene on the treatment sensitivity of tumors at their natural site. We demonstrate that the Bcl-2 oncoprotein produces multi-drug resistance when assessed in primary lymphomas in vivo. In contrast, this effect was dramatically reduced when the primary lymphomas were subjected to long-term culture, and completely missed in the standard clonogenic survival assay. This model highlights the importance of physiological test systems to address the complexity of clinical drug resistance and provides a novel strategy to evaluate compounds targeting specific genetic lesions.     

Apparent lack of H-2 restriction of allograft rejection.

BALB/c ByJ (H-2d) mice immunized with tail skin grafts of either B10.D2 (H-2d) or B10 (H-2b) demonstrated similar second set rejection of B10.D2 tail skin. This apparent lack of H-2 restriction was not due to the induction of a new population of cytotoxic T lymphocytes (Tc) since 450R given 24 hr before the challenge graft did not abrogate the second set reactivity. Host macrophage processing or anti-Qa-2 reactivity was also not the explanation for the lack of H-2 restriction since immunization of BALB/c Li mice with either B10.D2 or B10 tail skin grafts resulted in second set rejection of B10 tail skin. Shared public H-2 specificities of H-2d and H-2b may result in cross-reactive Tc, thus causing the apparent lack of H-2 restriction. However, no H-2 restriction of allograft rejection is observed when only one public H-2 specificity is shared between the recipient and the allogeneic challenge graft (H-2f and H-2k combination). These results suggest that H-2 restriction of T cell cytotoxicity has little relevance in allograft rejection because 1) one public H-2 specificity is sufficient to cause cross-reactivity or 2) Tc are not the major effectors of allograft rejection.     

Murine responses to (Tyr-Glu-Ala-Gly)n: II. H-2 and non-H-2 gene effects

Mice of the H-2^b haplotype responded to the sequential polymer poly(Tyr-Glu-Ala-Gly) in the in vitro T-cell proliferative assay, irrespective of whether they were homozygous or heterozygous at the H-2^b locus. The antibody responses of the H-2^b congenic mice to this polymer were variable, with A.BY and BALB.B showing responses better than those of C57BL/6 and C57BL/10 strains. The antibody responses of the F₁ progeny of (responder × nonresponder) strains of mice to this polymer are generally lower than the responder parents. F₁ mice with C57BL/10 background were the poorest responders. Studies with F₂ mice and backcross progenies of selective breeding of high and low antibody responder (C57BL/6 × BALB/c) F₁ to high responder C57BL/6 mice indicated that both non-H-2 genes and H-2 gene dosage effects influenced the magnitude of the humoral antibody responses. Animals having low responder non-H-2 background and only half the dosage of the responder immune response genes has greatly diminished antibody responses.     

Antigen-specific helper T cells recognize determinants encoded in the H-2D region of the H-2 gene complex

Observations have frequently been interpreted as showing that the helper T cells which collaborate with alloantigen-specific cytotoxic T-cell precursors can only recognize antigens encoded in the I region of the H-2 gene complex. An experimental system is described here that allows analysis of the recognition repertoire of these helper cells. CBA helper T-cell precursors can be primed in vitro to antigens encoded in the H-2 ^b gene complex. These helpers can then be tested for the existence of a subset of helper cells which recognize antigens encoded in the D region of H-2 ^b haplotype. CBA thymocytes were used as a source of cytotoxic T-cell precursors that respond poorly in the absence of exogeneous helper activity. The source of alloantigen was varied by using irradiated spleen cells from various (BALB/c × recombinant)F₁ hybrid mice as stimulator cells. When the stimulator cell bears BALB/c determinants recognized by the cytotoxic T-cell precursor and also bears only the D region antigens of the H-2 ^b haplotype, an anti-BALB/c cytotoxic response is generated only if the anti-H-2^b helper population contains cells able to recognize H-2D^b. A positive cytotoxic response was obtained, indicating that helper cells are not limited to recognition of I region antigens and can efficiently recognize antigens encoded in the D region of the H-2 gene complex. This was confirmed by the demonstration of helpers specific for H-2D^d. We were unable to detect any evidence for Ia-restricted recognition of the H-2D alloantigens, suggesting that, as for cytotoxic T lymphocytes (CTL), helper cell recognition of class I alloantigens is an unrestricted event.     

Synthesis of surface H-2 alloantigens in murine splenocytes.

The synthesis and turnover of cell surface H-2 alloantigens were studied in murine splenocytes by the anti-H-2-bind method to separate precursor-labeled surface from intracellular molecules. Results indicate that newly synthesized H-2 antigen, labeled in either its peptide or carbohydrate portion enters a relatively small pool of intracellular H-2 antigen and then is rapidly transported to the plasma membrane which represents a larger compartment. The simplest interpretation of these findings is that H-2 antigen is synthesized and transported along a conventional secretory pathway. Pulse-chase experiments indicate that H-2 antigens are not readily chased from the plasma membrane and are not shed or internalized during short-term culture. The aforementioned observations are discussed in terms of a cellular heterogeneity.     

T-cell inhibition of humoral responsiveness. I. Experimental evidence for restriction by the K- and/or D-end of the H-2 gene complex.

The inhibition (“suppression”) of an in vitro antibody response to SRBC by T-cells with specificity for histocompatibility antigens, is H-2 K/D-restricted whether or not the target determinant is H-2 I-region or non-H-2 encoded. By contrast, cytotoxic T-cells specific for histocompatibility antigens mediate a lysis of targets that is K/D-restricted in all cases except one: when the target determinant is H-2 I-region encoded, lysis is unrestricted. Further, it is shown that the target determinant and the restricting element must be on the same cell for effector function to be mediated. A spleen population immunized with histocompatibility antigens contains inhibitory and cytotoxic T-cells separable when I-region encoded determinants are the target. Thus, we conclude that the cytotoxic T-cell does not function as an inhibitory T-cell and vice versa. If the findings with this model experimental system may be extrapolated to the regulation of normal in vivo immune responsiveness then two K/D-restricted separable T-cell populations must mediate cytotoxicity and inhibition of humoral responsiveness. This finding, that with I-region encoded targets only, inhibitory T-cells are K/D-restricted whereas cytotoxic T-cells are not, raises questions about the mechanism of restrictive recognition which are dealt with in the context of the “two signal” model.     

Major histocompatibility complex restriction of T-cell responses to varicella-zoster virus in guinea pigs.

Varicella-zoster virus (VZV), adapted to grow in guinea pig fibroblasts, was injected subcutaneously into Hartley, strain 2, and strain 13 guinea pigs. Serum immunoglobulin G antibodies were detected 2 weeks later, and T-cell proliferative responses by blood lymphocytes were found 3 weeks after injection. The proliferating cells bound the 155 antibody, which defines a CD4-like subset of guinea pig T lymphocytes. VZV-infected fibroblasts of human, Hartley, and strain 13 origin elicited equivalent amounts of proliferation, which was quantitatively greater than that obtained with an extracted VZV antigen. Uninfected (control) human or guinea pig fibroblasts did not elicit T-cell proliferation. The proliferative response to VZV required the presence of autologous (strain 2 or 13) antigen-presenting cells and was blocked by the addition of an anti-class II major histocompatibility complex antibody. Effector cells obtained from in vitro cultures mediated class II-restricted cytotoxicity to L2C cells incubated with VZV. Class I-restricted responses were obtained only by cross-priming strain 2 animals with strain 13 peritoneal exudate cells which had been preincubated with VZV. The data indicate that guinea pigs resemble humans in that class II-restricted T cells with specificity for VZV are more readily cultured from blood than are class I-restricted cells.