Intrinsic autoimmune capacities of hematopoietic cells from female New Zealand hybrid mice.

Most systemic autoimmune diseases occur more frequently in females than in males. This is particularly evident in Sjögren's syndrome, systemic lupus erythromatosis (SLE) and thyroid autoimmunity, where the ratio of females to males ranges from 20:1 to 8:1. Our understanding of the etiology of SLE implies important roles for genetics, environmental factors and sex hormones, but the relative significance of each remains unknown. Using the New Zealand hybrid mouse model system of SLE, we present here a new fetal liver chimera-based system in which we can segregate effects of immune system genes from that of sex hormones in vivo. We show that female hematopoietic cells express an intrinsic capacity to drive lupus-like disease in both male and female recipient mice, suggesting that this capacity is hormone independent. Particularly, only chimeric mice with a female hematopoietic system showed significantly increased numbers of germinal center B cells, memory B cells and plasma cells followed by a spontaneous loss of tolerance to nuclear components and hence elevated serum antinuclear autoantibodies. A protective effect of testosterone was noted with regard to disease onset, but not disease incidence. Thus, genetic factors encoded within the female hematopoietic system can effectively drive lupus-like disease even in male recipients.

Autoimmune disease: why and where it occurs.

Autoimmune disease is controlled by genetic and environmental factors. Both of these affect susceptibility to autoimmunity at three levels: the overall reactivity of the immune system, the specific antigen and its presentation, and the target issue.

Use of somatic cell genetics to study chromosomes contributing to antigen plus I recognition by T cell hybridomas.

Keyhole limpet hemocyanin (KLH)/I region-specific T cell hybridomas have been prepared by fusing KLH/I-specific T cell blasts from mice with single pairs of metacentric chromosomes to the inducible, interleukin 2 (IL-2)-secreting T cell hybridoma FS6-14.13.AG2.1. T cell hybridomas with KLH/I receptors were identified by their ability to secrete IL-2 in response to KLH and the appropriate antigen-presenting cells. After cloning and subcloning, KLH/I reactivity was correlated with the presence or absence of metacentric chromosomes derived from the KLH/I-specific T cell blast parent. Hybridomas were identified that had lost all chromosomes 4 and 6 or 16 and 17 derived from their normal T cell parent, but retained the ability to respond to KLH/I. This suggested that products of genes on these chromosomes did not contribute to the specific portions of T cell Ag/I receptors. These gene products would include, of course, kappa and lambda chains and H-2. We did not obtain any T cell hybridomas that had lost both metacentric (8.12) chromosomes derived from T cells of the Robertsonian mouse strain Rb(8.12)5, so we could not draw any conclusions about the contributions of products of genes on these chromosomes. T cell hybridomas with KLH/I reactivity were found that contained only one metacentric (8.12) chromosome derived from this strain. Moreover, a T cell hybridoma was found that retained both metacentric (8.12) chromosomes from its normal T cell parent, but had lost KLH/I reactivity. These results suggested that neither two chromosomes 8 nor two chromosomes 12 were required for antigen/I reactivity in normal T cells and that antigen/I reactivity was controlled, at least in part, by genes mapping on chromosomes other than 8 or 12.

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.

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.

The role of H-2-linked genes in helper T-cell function. III. Expression of immune response genes for trinitrophenyl conjugates of poly-L(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys in B cells and macrophages.

Using lymph node T cells from poly-L(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys[(TG)-A--L]-primed animals and B cells from animals primed with trinitrophenylated (TNP) protein or lipopolysaccharide, we have obtained anti-TNP-(TG)-A--L direct plaque-forming responses in vitro. Response to this antigen was shown to be controlled by the H-2 haplotype of the animal studied. The strain distribution of in vitro response was very similar to that previously reported by others for in vivo secondary IgG responses to (TG)-A--L. We investigated the cell types expressing the Ir gene(s) for (TG)-A--L in our cultures. F1, high responder x low responder mice were primed with (TG)-A--L. Their T cells were active in stimulating anti-TNP-(TG)-A--L responses of high responder but not low responder B cells and macrophages (MPHI), even though both preparations of B cells and Mphi were obtained from mice congenic at H-2 with one of the parents of the F1. For three low responder strains tested, of the H-2h2, H-2k, and H-2f haplotypes, the anti-TNP-(TG)-A--L response of low responder B cells and Mphis in the presence of high responder, F1 T cells could not be improved by the addition of high responder, antigen-bearing Mphis to the cultures. In one strain of the H-2a haplotype, it was shown that neither the B cells nor Mphis could be functional in anti-TNP-(TG)-A--L responses. Our results therefore suggested the Ir genes for anti-TNP-(TG)-A--L responses were expressed at least in B cells in all the low responder strains we studied, and, in mice of the H-2a haplotype, in Mphis too.

The role of H-2 linked genes in helper T-cell function. IV. Importance of T-cell genotype and host environment in I-region and Ir gene expression.

We have studied the properties of helper T cells specific for sheep erythrocytes (SRBC), keyhole limpet hemocyanin (KLH), or poly-L-(Tyr,Glu)-poly-DL-Ala-poly-L-Lys [(T,G)-A--L]. These T cells differentiated and were primed in vivo in irradiation chimeras constructed of various combinations of F1 and parental bone marrow donors and irradiated recipients. Primed T cells were then tested for helper activity in the in vitro response of B cells and macrophages (Mphi) of parental or F1 origin to the hapten trinitrophenol coupled to the priming antigen. When testing either SRBC or KLH-specific T cells of parental H-2 type which had differentiated in F1 hosts, we found that they cooperated equally well with B cells and Mphi of either parental H-2 type. On the other hand, when testing F1 T cells which had differentiated in parental hosts, we found that they cooperated well only with B cells and Mphi which had the K-IA region type of the parental host. In similar experiments we found that (T,G)-A--L-specific T cells of low responder H-2 type which had differentiated in (high responder X low responder) F1 hosts induced high responses in high responder B cells and Mphi (T,G)-A--L-specific F1 T cells which differentiated in high responder but not those which differentiated in low responder hosts induced high responses in high responder B cells and Mphi. Low responder B cells and Mphi yielded low responses in all cases regardless of the source of (T,G)-A--L-specific T cells with what they were tested. Our results support the conclusion that I-region and Ir genes function via their expression in B cells and Mphi and in the host environment during helper T-cell differentiation, but not, at least under the conditions of these experiments, via their expression in the helper T cell itself. These findings place constraints upon models which attempt to explain the apparent dual recognition of antigen and I-region gene products by helper T cells.

The role of H-2-linked genes in helper T-cell function. I. In vitro expression in B cells of immune response genes controlling helper T-cell activity.

The ability of murine helper T cells primed to the antigen, sheep erythrocytes (SRBC) to cross-react with burro erythrocytes (BRBC) in the in vitro anti-trinitrophenol (TNP) response to TNP-RBC was shown to be under genetic control. Although non-H-2 genes were shown to influence the absolute level of helper activity assayed after SRBC priming, the extent of cross-reaction of SRBC-primed helpers with BRBC was shown to be controlled by an H-2-1inked Ir gene(s). H-2 haplotypes were identified which determined high, intermediate, or low response to the cross- reacting determinants and the gene(s) controlling the cross-reaction tentatively mapped to the K through I-E end of the H-2 complex. Helpers primed in F(1) mice of high x intermediate or high x low responder parents were tested for cross-reaction using B cells and macrophages (Mphi) of parental haplotypes. In each case the extent of cross-reaction was predicted by the H-2 haplotype of the B cells and Mphi, establishing the expression of the Ir gene(s) in B cells and/or Mphi a t least, but not ruling out its expression in T cells as well. The low cross-reaction seen when T cells from F(1) mice of high x low responder parents were tested on low responder B cells and Mphi was not increased by the presence of high responder Mphi, indicating the Ir gene(s) is expressed in the B cell a t least although it may be expressed in Mphi as well. These and our previously reported experiments are consistent with the hypothesis that helper T cells recognize antigen bound to the surface of B cells and Mphi in association with the product(s) of Ir gene(s) expressed on the B cell and Mphi.

Analysis of V beta 17a expression in new mouse strains bearing the V beta a haplotype.

A set of new mouse strains were produced that carry the V beta a haplotype of the TCR-alpha/beta and any of a number of different H-2 haplotypes on backgrounds derived from related C57BL, C57L, and C57BR mice. Study of V beta 17a expression in these mice confirms the association between the presence of IE and the deletion of V beta 17a+ T cells. A second H-2 gene causing deletion of V beta 17a+ T cells was mapped in these mice to the K end of H-2k, and H-2 influences on the level of selection of CD4+ V beta 17a+ T cells were indicated.

Structural analysis of a mouse mammary tumor virus superantigen.

It has recently been shown that the minor lymphocyte stimulating-like products expressed by some mice are actually encoded by open reading frames in the 3' long terminal repeats of mouse mammary tumor viruses. These products act as viral superantigens (vSAGs). That is, they stimulate most T cells bearing particular V beta s almost regardless of the rest of the variable components of the T cell receptors expressed by those cells. To find out more about the structure of these vSAGs, a set of truncated vSAG genes was used in transfection and in vitro translation experiments to show that the functional vSAG is a type II integral membrane protein with a large glycosylated extracellular COOH-terminal domain and a small, nonessential, intracellular NH2-terminal cytoplasmic domain. These results are consistent with the fact that the vSAGs must be expressed on the cell surface in order to interact with T cells and class II major histocompatibility complex proteins. They also account for the finding that much of the V beta specificity of the vSAGs is controlled by amino acids at the COOH-terminal end of the vSAG proteins, amino acids that will be extracellular in type II proteins.

The effects of chronic infection with a superantigen-producing virus.

C3H/HeJ mice transmit a mouse mammary tumor virus from mother to pup in milk. The retrovirus infects mice shortly after birth and, when expressed in recipient mice, produces a V beta 14-specific superantigen. The consequences of such expression on V beta 14-bearing T cells are examined in this paper. Most cells bearing V beta 14 and either CD4 or CD8 are eliminated in the thymus. Some V beta 14-bearing cells escape to the periphery, however. Those bearing CD8 are unaffected by expression of the viral superantigen. The percentage of peripheral CD4+ T cells bearing V beta 14 drops with time after birth. In large part this seems to be due to the fact that many of these cells become anergic because of exposure to the viral superantigen. Unlike normal T cells, these anergic cells cannot undergo peripheral postthymic expansion. Consequently, they drop in percentage even during a time when their total numbers are constant.

Thymocytes with the predicted properties of pre-T cells.

T cell receptor synthesis in thymocytes was examined by the differential immunoprecipitation of receptors from the surfaces and interiors of metabolically labeled newborn and adult thymocytes. Precipitated molecules were then analyzed for size, charge, and state of glycosylation. Our experiments identified cells within the thymic cortex that contained a large pool of cytoplasmic-free receptor beta chain. The beta chain in this pool was synthesized and degraded rapidly and bore only high-mannose N-linked oligosaccharides. This pool was found predominantly in cells that lacked surface alpha/beta receptors and appeared in ontogeny before cells expressing surface alpha/beta. These results are consistent with a model in which the progenitor of cells with surface alpha/beta expression is the T cell equivalent of the pre-B cell, which has rearranged and expressed beta chain, but not alpha chain.

Thymocytes can become mature T cells without passing through the CD4+ CD8+, double-positive stage.

T cells bearing the class II-restricted, DO-T cell receptor (TCR) are CD4+ if their thymocyte precursors are positively selected on the class II protein, IAd, but they are almost all CD4- after positive selection on a class II for which they have higher avidity, IAb. DO-TCR+ T cells mature in H-2b mice lacking CD4. CD4- DO-TCR+ T cells appear in H-2b mice at the same rate as their CD4+ counterparts appear in H-2d animals, suggesting that the CD4- cells are not the product of some minor pathway of thymocyte development and selection. In H-2b CD4 knock out mice expressing human CD2 under the control of the mouse CD4 promoter, mature DO-TCR+ cells did not express human CD2. These results suggest that the CD4-CD8-, DO-TCR+ mature T cells have developed without ever passing through the equivalent of a CD4+,CD8+ stage. The early expression of alpha/beta receptors (TCRs) on thymocytes in TCR transgenic mice may allow maturation of this type. Passage through the equivalent of the CD4+ CD8+, double-positive stage is not essential for differentiation of thymocytes into mature T cells.

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.

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.

CD4+ T cell division in irradiated mice requires peptides distinct from those responsible for thymic selection.

We investigated the mechanism by which alpha/beta T cells expand upon transfer to T cell-deficient host mice by injecting carboxyfluorescein diacetate succinimidyl ester-labeled T cells into mice depleted of T cells by sublethal irradiation. We found that CD4+ T cells divided when transferred to irradiated hosts and that the division of more than half of these cells required class II expression. However, division of transferred CD4+ T cells did not occur in irradiated hosts that expressed class II molecules occupied solely by the peptide responsible for thymic selection, indicating that peptides distinct from those involved in thymic selection cause the division of CD4+ T cells in irradiated mice. These data establish that class II-bound peptides control the expansion of CD4+ T cells transferred to T cell-deficient hosts and suggest that the same peptides contribute to the maintenance of T cell numbers in normal mice.

Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing.

We examined the ability of a set of cloned chicken ovalbumin (cOVA)-specific, Id-restricted, T cell hybridomas to produce interleukin-2 in response to cOVA presented by the Ia+ B cell lymphoma line, A20-2J. Although viable A20-2J cells presented native, denatured, and fragmented cOVA more or less equally well, A20-2J cells that were glutaraldehyde-fixed could present only enzymatically or chemically fragmented cOVA. These results suggest that antigen fragmentation may be both necessary and sufficient to define accessory cell processing of soluble antigens so that they may be recognized in association with I-region molecules by T cells.

Helper signals in the plaque-forming cell response to protein-bound haptens.

We have demonstrated the ability of a series of murine T cell hybridomas to deliver an antigen-specific, B cell I-region-restricted helper signal in the generation of specific PFC responses to protein-bound haptens. With some hybridomas the elicitation of optimal PFC responses required the addition of nonspecific factors provided by culture supernatants of concanavalin A-stimulated (Con A SN) spleen cells. Using hapten-primed B cells depleted of both T cells and macrophages (Mphi) we have now demonstrated a requirement for three nonspecific factor preparations to substitute for spleen Con A SN in the elicitation of optimal PFC responses. The first preparation was the interleukin 1 containing culture supernatant of the Mphi tumor cell line P388D1, the second the interleukin 2 (IL-2) and B cell growth factor containing Con A SN of the T cell hybridoma FS6-14.13, and the third, the gamma interferon containing Con A SN of the T cell hybridoma FS7-20.6.18. The P388D1 and FS6-14.13 factor preparations were most effective when added at the initiation of culture, while the FS7-20.6.18 factor preparation was most effective when added at 24 h of culture. The activity of FS6-14.13 Con A SN was depleted by incubation with the IL-2-dependent T cell line HT-2. The activity of FS7-20.6.18 Con A SN was abrogated by incubation at pH 2. The results suggest that the generation of PFC responses to protein-bound haptens require at least three nonspecific factors in addition to an antigen/Ia specific helper signal.

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.

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.