Contribution of bone marrow cells and lack of expression of thymocytes in genetic controls of immune responses for two immunopotent regions within poly-(Phe,Glu)-poly-Pro--poly-Lys in inbred mouse strains.

Previous cellular studies on the genetic regulation of immunological responsiveness for two immunopotent regions within the branched chain synthetic polypeptide (Phe, G)-Pro--L demonstrated a direct correlation between the number of detectable immunocompetent splenic precursor cells and the response patterns of SJL, DBA/1, and F(1) mice (21). In order to establish the cellular origin(s) of the genetic defect, the present study first demonstrated that thymus and bone marrow cell cooperation was required for (Phe, G)- and Pro--L-specific immune responses. Secondly, limiting dilution experiments, in which several graded and limiting inocula of marrow cells were mixed with a non-limiting number of 10(8) thymocytes and injected into irradiated, syngeneic recipients, indicated that the low responsiveness of the SJL and DBA/1 strains to the (Phe, G) and Pro--L specificities, respectively, could be attributed to a reduced number of precursor cells found in bone marrow. About five times more marrow precursors were detected in SJL mice for Pro--L than for (Phe, G), whereas about five times as many precursor cells were estimated for (Phe, G) as for Pro--L in the DBA/1 strain. These differences are similar to those obtained using spleen cells from unimmunized SJL and DBA/1 donors (21), and indicate that these genetically determined variations in responsiveness can be accounted for by differences in the frequencies of monospecific populations of immunocompetent cells present in bone marrow. In contrast, limiting dilution transfers of thymocytes or thymus-derived cells with an excess of syngeneic marrow cells resulted in equally frequent (Phe, G) and Pro--L responses for both SJL ad DBA/1 strains. This finding in conjunction with the observation that the generation of (Phe, G)- and Pro--L-specific responses were associated in individual recipients injected with limiting inocula of thymocytes indicated that a single population of thymocytes was stimulated by (Phe,G)-Pro--L. Therefore, it is improbable that the thymic population of immunocompetent cells contributes to expression of these genetically controlled defects.

Elicitation of delayed-type hypersensitivity responses to poly(L-Tyr,LGlu)-poly(DLAla)--poly(LLys) by anti-idiotypic antibodies.

The in vivo effect of murine anti-idiotypic serum against C3H.SW anti-poly(LTyr,LGlu)-poly(DLAla)-(LLys) [(T,G)-A--L] antibodies on delayed type hypersensitivity responses to (T,G)-A--L was studied. Anti-idiotypic serum could challenge DTH responses in C3H.SW mice transferred with antigen-sensitized T cells. The elicitation activity was shown to be antigen and strain specific. With H-2-compatible (but allotype different) strain combinations of (T,G)-A--L-educated T cells and recipients, we were able to show that the biological effect of the anti-idiotypic serum is expressed on the first antigen-sensitized idiotype-positive radioresistant T cell, but not on the proliferating normal cells of recipient origin that participate in the efferent phase of delayed-type hypersensitivity responses to (T,G)-A--L.

Genetic regulation of delayed-type hypersensitivity responses to poly(LTyr,LGu)-poly(DLAla)--poly(LLys). I. Expression of the genetic defect at two phases of the immune process.

Delayed-type hypersensitivity (DTH) responses served in this study as an experimental model for the analysis of genetic regulations of T-cell responses. Educated irradiated cells from H-2b mice mediated responses in syngeneic recipients, whereas mice of the a, d, f, k, and s haplotypes were nonresponders to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys)[(T,G)-A--L]. These results suggest that cell-mediated immune responsiveness to (T,G)-A--L is linked to the H-2 complex, as was shown for humoral responses. Educated irradiated T cells of F1 hybrids between high and low responders mediated DTH responses, which indicates that the gene(s) controlling the DTH responses is dominant. To analyze the genetic defect in DTH responses to (T,G)-A--L, we separated the T-cell activation phase from the effector phase that was determined in recipient mice. Two types of nonresponders were observed: (a) When lymphocytes of the a or k haplotypes were educated in a syngeneic environment and then transferred into hybrids between the parental (nonresponder x responder) F1 recipients, DTH responses could have been manifested. (b) On the other hand, no DTH responses could be mediated by transferring educated cells of the H-2s or H-2f origin into the appropriate F1 recipients. In addition, irradiated F1 cells that had been activated to (T,G)-A--L could not mediate DTH responses in both types of nonresponder recipients. These results suggest that T cells of H-2k or H-2a mice can be activated to generate DTH responses to (T,G)-A--L and that the defect in these mouse strains is expressed in another cell population needed for the manifestation of the DTH reaction in the recipient mice. In contrast, T cells of H-2s and H-2f origin cannot be activated to (T,G)-A--L and, thus, fail to manifest DTH responses.

Genetic regulation of delayed-type hypersensitivity responses to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys). II. Evidence for a T-T-cell collaboration in delayed-type hypersensitivity responses and for a T-cell defect at the efferent phase in nonresponder H-2k mice.

The intercellular interactions and the site of the genetic defect in delayed-type hypersensitivity (DTH) response to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys) [(T,G)-A--L] has been studied in a system where the T-cell education phase was separated from the efferent phase. In the cellular response, T-T-cell collaboration is required, because T cell-depleted mice were unable to manifest DTH responses after they were transferred with educated and irradiated T cells. Reconstitution of adult thymectomized mice that were irradiated and supplemented with bone marrow cells after treatment with anti-Thy-1.2 serum and complement, with T cells but not with accessory cells gave rise to significant responses. Educated, radioresistant cells required the presence of normal radiosensitive T cells for successful DTH responses to (T,G)-A--L. The genetic defect of nonresponder H-2k and H-2a mice has been located in the above-mentioned, second T-cell population that participates in the efferent phase of this immune reaction. Further characterization revealed that the educated cells are of the Lyt1+ phenotype and that the second normal T cells are expressing the Lyt 1+,2+,3+ phenotype. Thus, the genetic defect of H-2k and H-2a mice in the DTH response to (T,G)-A--L is expressed on the non-antigen-stimulated Lyt 1+,2+,3+ T cells.

Genetic control of immune response. The dose of antigen given in aqueous solution is critical in determining which mouse strain is high responder to poly(LTyr, LGlu)-poly(LPro)--poly(LLys).

Antibody response to different doses of (T,G)-Pro--L, given in aqueous solution, was investigated in the high responder SJL and low responder DBA/1 strains by measuring hemolytic plaque-forming cells (PFC) in the spleens as well as hemagglutination titers in the sera. The gene responsible for the difference between the two strains in the response to this antigen, given in complete Freund's adjuvant, has been previously denoted Ir-3. This gene is not linked to the major histocompatibility locus. In the response to the optimal dose (1 mug) of antigen, no difference could be shown between the strains. The peak of the response and the numbers of direct and indirect PFC were similar in both strains in the primary and secondary response. After injection of higher doses (10-100 mug) of antigen, both the direct and indirect PFC responses were lower in the low responder than in the high responder strain. Moreover, the peak of the response occurred earlier in the high responder strain in the primary response to the 10 mu dose of antigen. After administration of a suboptimal dose (0.02 mug) of antigen, the low responder strain produced in the primary response 4-20 times more indirect plaques than the high responder strain. Also the number of direct plaques was higher in the low responder than in the high responder strain. The serum antibody responses to the optimal and higher doses of antigen were parallel to the PFC responses. From inhibition of PFC with free antigen, it was concluded that a similar proportion of cells was producing high and low affinity antibodies to (T,G)-Pro--L in both strains. High and low zone tolerance could be induced in the two strains with (T,G)-Pro--L, but no difference could be shown between the strains. It is suggested that the Ir-3 gene plays a role in the regulation of the balance stimulation and suppression according to the dose of antigen given.

Antigen-specific T-cell factors in the genetic control of the immune response to poly(Tyr,Glu)-polyDLAla--polyLys. Evidence for T- and B-cell defects in SJL mice.

The cellular basis of the genetic control of the immune response to poly(LTyr, LGlu)-polyDLAla--polyLLys [(T,G)-A--L] in SJL (H-2s, low responder) mice has been investigated using T-cell factors. Thymocytes of SJL origin were educated to (T,G)-A--L and tested for their ability to produce an antigen-specific factor capable of cooperating in vivo with bone marrow cells of either SJL or C3H.SW (high responder) origin. SJL T cells were found to be incapable of producing such a cooperative factor, in contrast with results previously obtained with C3H/HeJ (low responders) and C3H.SW strains. Moreover, SJL bone marrow cells did not produce an antibody response to (T,G)-A--L, even when combined with factor produced by high responder (C3H.SW) mice. Thus, both T and B cells appear to be defective in the SJL strain in the response to (T,G)-A--L.

Induction of acetylcholine receptor-specific suppression. An in vitro model of antigen-specific immunosuppression in myasthenia gravis.

This report describes the in vivo and in vitro induction of murine (AChR)-specific suppressor T cells (Ts) and T cell factors (TsF), and the development of an appropriate assay system for their measurement. The assay described is based on the in vitro Mishell-Dutton culture system. Using this assay, it was shown that the AChR-specific helper cell is an Lyt-2- radiosensitive T cell. Moreover, the proliferating cell measured in the lymphocyte transformation assay was shown to provide AChR-specific T cell help. In vivo induction of Ts cells is achieved by injection of soluble AChR; potent AChR-specific suppression is found in the spleen 1 wk later. In vitro induction of Ts cells involves the primary education of naive splenocytes by culturing them with high concentrations of AChR. Both the in vivo- and in vitro-induced Ts cells were shown to secrete AChR-specific factors that mediate their suppressive effects. The possibility of specifically suppressing the AChR-immune response may be of a particular clinical importance since the AChR is the target autoantigen in the neuromuscular autoimmune disease myasthenia gravis.

Cellular basis of the genetic control of immune responses to synthetic polypeptides. II. Frequency of immunocompetent precursors specific for two distinct regions within (Phe, G)-Pro--L, a synthetic polypeptide derived from multichain polyproline, in inbred mouse strains.

DBA/1 mice are high responders to the (Phe, G) determinant of the synthetic polypeptide (Phe, G)-Pro--L, whereas SJL mice respond well to the Pro--L region of this macromolecule (6). In order to determine whether the phenomenon described above is related to the number of antigen-sensitive units detected for both specificities, and whether responses to these determinants can be transferred independently, graded and limiting inocula of spleen cells from SJL, DBA/1, and F(1) donors were injected into X-irradiated, syngeneic, recipient mice with (Phe, G)-Pro--L. By this approach, one antigen-sensitive unit specific for (Phe, G) was detected in 1.7 x 10(6) and 8.5 x 10(6) spleen cells from immunized and nonimmunized DBA/1 donors, respectively. In contrast, one (Phe, G) relevant precursor was detected in 20 x 10(6) SJL spleen cells, irrespective of whether the donors had been immunized. On the other hand, for the Pro--L specificity, one limiting splenic precursor was found in 1.3 x 10(6) and in 3.4 x 10(6) cells for immunized and nonimmunized SJL donors, respectively; whereas one response unit was estimated for this determinant in 9.4 x 10(6) and in 38 x 10(6) spleen cells from immunized and nonimmunized DBA/1 mice. The findings reported here indicate that the phenotypic expression of the genetic control(s) for immune responsiveness to different immunopotent regions of (Phe, G)-Pro--L is directly correlated with the number of immunocompetent response units detected in two inbred mouse strains. In the spleens of immunized F(1) donors, similar frequencies of one limiting precursor in 3.0 x 10(6) and in 2.8 x 10(6) cells were detected for (Phe, G) and Pro--L, respectively. The results of a chi-square test for independence of (Phe, G) and Pro--L responses in F(1) animals is compatible with the hypothesis that the transferred spleen cells limiting the response to (Phe, G)-Pro--L are restricted to generate antibodies specific for only one of the two determinants of this macromolecule.

Cellular basis of the genetic control of immune responses to synthetic polypeptides. I. Differences in frequency of splenic precursor cells specific for a synthetic polypeptide derived from multichain polyproline ((T,G)-Pro--L) in high and low responder inbred mouse strains.

SJL mice are high responders to the synthetic multichain polypeptide antigen (T,G)-Pro--L, whereas DBA/1 mice are low responders (10, 11). In order to determine whether the genetic control of immune response can be correlated with the number of antigen-sensitive precursor cells, spleen cell suspensions from normal and immunized SJL and DBA/1 donor mice were transplanted into lethally X-irradiated syngeneic recipients (incapable of immune response) along with (T, G)-Pro--L. Anti-(T, G)-Pro--L responses (donor-derived) were assayed in the sera of the hosts 12-16 days later. By transplanting graded and limiting numbers of spleen cells, inocula were found which contained one or a few antigen-sensitive precursors reactive with the immunogen. Using this method to estimate the relative numbers of such cells for the high responder SJL strain, one precursor was detected in approximately 1.3 x 10(6) and approximately 7.2 x 10(6) spleen cells from immunized and normal donors, respectively. In contrast, one precursor was detected in about 30 x 10(6) spleen cells from low responder DBA/1 mice, irrespective of whether the donors had been immunized. These results indicate that the genetic control of immunity to the synthetic polypeptide antigen investigated is directly correlated to the relative number of precursor cells reactive with the immunogen in high and low responder strains.

Contribution of different cell types to the genetic control of immune responses as a function of the chemical nature of the polymeric side chains (poly-L-prolyl and poly-DL-alanyl) of synthetic immunogens.

Genetic regulation of immunological responsiveness was studied at the cellular level by comparing the limiting dilutions of immunocompetent cells from spleen, thymus, and bone marrow of high and low responders as a function of the poly-L-prolyl and poly-DL-alanyl side chains of two synthetic polypeptide immunogens. The spleens of immunized and unimmunized high responder DBA/1 mice were found to contain respectively, 18- and 7-fold more limiting precursor cells specific for (Phe, G)-A--L than the spleens of SJL low responder donors. These results, using a synthetic polypeptide built on multichain poly-DL-alanine, confirm the findings reported for polypeptides built on multichain poly-L-proline (1, 2), that there is a direct correlation between immune response potential and the relative number of immunocompetent precursors stimulated. Cell cooperation between thymocytes and bone marrow cells was demonstrated for both (T, G)-Pro--L and (Phe, G)-A--L. Limiting dilutions of thymus and bone marrow cells in the presence of an excess amount of the complementary cell type indicated an eightfold lower number of detected (T, G)-Pro--L-specific precursors in DBA/1 (low responder) marrow when compared with SJL (high responder) marrow. No differences were observed in the frequency of relevant high and low responder thymocytes for the (T, G)-Pro--L immunogen. These results are similar to those reported for the (Phe, G)-Pro--L (3). In contrast to the cellular studies reported for the Pro--L series of immunogens, the marrow and thymus cell dilution experiments for (Phe, G)-A--L revealed genetically associated differences in both the marrow and thymus populations of immunocytes from high (DBA/1) and low (SJL) responders. In addition to a fivefold difference in limiting marrow cell precursors (similar to that seen in the Pro--L studies), a striking difference was observed between the helper cell activity of high responder DBA/1 and low responder SJL thymocytes. This difference was indicated by the observation that low responder thymocyte dilutions followed the predictions of the Poisson model, whereas dilutions of high responder thymocytes did not conform to Poisson statistics. Transfers of allogeneic thymus and marrow cell mixtures from DBA/1 and SJL donors confirmed the syngeneic dilution studies showing that the genetic defect of immune responsiveness to (Phe, G)-A--L is expressed at both the thymus and marrow immunocompetent cell level. The parameters presently known for genetic control of immune responses specific for (Phe, G) (Ir-1 gene) and for Pro--L (Ir-3 gene) have been compared. The Ir-1 and Ir-3 genes are not only distinct by genetic linkage tests (to H-2) (5, 6, 9), but they are also seen to be different by cellular studies. Furthermore, expression of low responsiveness within a given cell population was shown to depend on the chemical structure of the whole immunogenic macromolecule.

Thymus independence of a collagen-like synthetic polypeptide and of collagen, and the need for thymus and bone marrow-cell cooperation in the immune response to gelatin.

Several inbred mouse strains were screened for their ability to respond to the ordered periodic collagen-like polymer (Pro-Gly-Pro)(n), to the random copolymer (Pro(66), Gly(34))(n), to the protein conjugate Pro-Gly-Pro-ovalbumin, to rat tail tendon collagen, rat tail tendon gelatin, and to Ascaris cuticle collagen. Differences were obtained in the magnitude of the antibody titers towards the above immunogens among the strains tested. The level of the response to the ordered polymer (Pro-Gly-Pro)(n) was not similar to that towards the random (Pro(66), Gly(34))(n), confirming differences in the antigenic determinants of the two immunogens. The role of the thymus in the immune response to (Pro-Gly-Pro)(n) and (Pro(66), Gly(34))(n) as well as to two collagens and gelatin, was studied in order to find out a possible correlation with the structural features of the immunogens. Heavily irradiated recipients were injected with syngeneic thymocytes, marrow cells, or a mixture of both cell populations and were immunized with the above-mentioned antigens. An efficient immune response to the ordered collagen-like (Pro-Gly-Pro)(n) was obtained in the absence of transferred thymocytes. The thymus independence of (Pro-Gly-Pro)(n) was confirmed when thymectomized irradiated mice were used as recipients. In contrast with these results, cooperation between thymus and marrow cells was necessary in order to elicit an immune response to (Pro(56), Gly(34))(n). Similarly, the immune response to the triple helical collagen was found to be independent of the thymus, whereas for an effective response to its denatured product, gelatin, thymus cells were required. These findings indicate that a unique three-dimensional structure of immunogens possessing repeating antigenic determinants plays an important role in determining the need for cell to cell interaction in order to elicit an antibody response.

Antigen-specific thymus cell factors in the genetic control of the immune response to poly-(tyrosyl, glutamyl)-poly-D, L-alanyl--poly-lysyl.

The genetic control of the antibody response to a synthetic polypeptide antigen designated poly-L(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys [(T, G)-A--L] has been studied in congenic high responder C3H.SW (H-2(b)) and low responder C3H/HeJ (H-2(k)) strains of mice. This response is controlled by the Ir-1 gene and is H-2 linked. The method employed was to study the ability of specifically primed or "educated" T cells of each strain to produce cooperative factors for (T, G)-A--L in vitro. Such factors have been shown to be capable of replacing the requirement for T cells in the thymus-dependent antibody response to (T, G)-A--L in vivo. The T-cell factors produced were tested for their ability to cooperate with B cells of either high or low responder origin by transfer together with bone marrow cells and (T, G)-A--L into heavily irradiated, syngeneic (for bone marrow donor) recipients. Direct anti-(T, G)-A--L plaque-forming cells were measured later in the spleens of the recipients. The results showed that (a) educated T cells of both high and low responder origin produced active cooperative factors to (T, G)-A--L, and no differences between the strains in respect to production of T-cell factors could be demonstrated; and (b) such factors, whether of high or low responder origin, cooperated efficiently with B cells of high responder origin only, and hardly at all with B cells of low responder origin. The conclusion was drawn that the cellular difference between the two strains lies in the responsiveness of their B cells to specific signals or stimuli received from T cells. As far as could be discerned by the methods used, no T-cell defect existed in low responder mice and the expression of the controlling Ir-1 gene was solely at the level of the B cells in this case.

Antibody response of inbred mouse strains to ordered tetrapeptides of tyrosine and glutamic acid attached to multichain polyalanine or polyproline. Tyr-Tyr-Glu-Glu is a major determinant of the random poly-(Tyr, Glu)-polyDLAla--polyLys.

Five inbred mouse strains which represent high and low responders to the random synthetic polypeptide poly(LTyr,LGlu)-polyDLAla--polyLLys, designated (T, G)-A--L, to which the immune response is controlled by an H-2-linked gene, were immunized with three ordered tetrapeptides composed of tyrosine and glutamic acid attached either to multichain poly-DL-alanine or to polyproline. Only one of the three antigenic determinants, namely tyrosyl-tyrosyl-glytamyl-glutamic acid (T-T-G-G), resembled the random peptide (T, G) in the pattern of immune responses elicited against it, and in the cross-reactivity of the specific antibodies with (T, G)-A--L. The immune response pattern to the other two ordered tetrapeptides, T-G-T-G and G-T-T-G, was different from that obtained with (T, G)-A--L, and no cross-reactivity was detected between the antibodies provoked with these peptides and (T, G)-A--L. Thus, it is suggested that T-T-G-G is a major determinant in the random (T, G)-A--L.

The genetic control of antibody specificity.

The immune response to a synthetic polypeptide built on multichain polyproline, poly-L-(Tyr,Glu)-poly-L-Pro-poly-L-Lys [(T,G)-Pro--L], in the offspring of a cross between DBA/1 and SJL mice is under a genetic control superficially similar to the one operating for the immune response to a similar synthetic polypeptide built on multichain polyalanine, poly-L-(Tyr,Glu)-poly-D,L-Ala-poly-L-Lys [(T,G)-A--L], in the offspring of a cross between CBA and C57 mice. In both cases, the genetic control is a quantitative trait in which the major gene(s) is (are) dominant and the trait is not linked to any of the known structural genes coding for mouse immunoglobulin heavy chains. However, the genetic control of response to (T, G)-Pro--L, designated immune response-3 (Ir-3), is qualitatively different from the one operating for (T,G)-A--L [immune response-1 (Ir-1)] in that it is not linked to the histocompatibility-2 (H-2) locus. A study of the immune response to a related polypeptide built on multichain polyproline, poly-L-(Phe,Glu)-poly-L-Pro-poly-L--Lys [(Phe, G)-Pro--L], in the DBA/1 x SJL cross has shown a genetic control of antibody specificity. F(1) x DBA/1 backcross anti-(Phe, G)-Pro--L sera segregate in their ability to bind (T,G)-Pro--L, and there is no linkage of anti-(T,G)-Pro--L binding capacity with the H-2(s) allele of the SJL grandparent. F(1) x SJL anti-(Phe, G)-Pro-L sera segregate in their capacity to bind poly-L-(Phe,Glu)-poly-D,L-Ala-poly-L-Lys [(Phe, G)-A--L] and the ability to bind (Phe, G)-A--L is clearly linked to the H-2(q) allele from the DBA/1 grandparent. Thus, in mice all responding well to a given antigen [(Phe, G)-Pro--L], the specificity of the antibodies produced [i.e., anti-(Phe,G) or anti-prolyl] is genetically determined. Cross-inhibition of binding m (DBA/1 x SJL)F(1) anti-(Phe,G)-Pro--L antisera indicates that the anti-(Phe,G) and anti-prolyl specificities are a function of two separate and largely non-crossreacting antibody populations.

The nature of the antigenic determinant in a genetic control of the antibody response.

The response of inbred mouse strains to two polypeptides derived from multichain polyprolines, (T,G)-Pro--L and (Phe,G)-Pro--L, is different from the response of the same mouse strains to a similar series of polymers built on multi-poly-D,L-alanyl--poly-L-lysine, although the same short sequences of amino acids are attached to the side chains of the polypeptides in the two series. These results indicate that a portion of the side chain (e.g. polyalanine or polyproline) participates in the antigenic determinant. This was confirmed by studying the response of different mouse strains to two kinds of polypeptides: (T,G)-Pro-A--L 717 and 718 and (T,G)-A-Pro--L 719 and 721. Antibody assay of antisera to (Phe,G)-Pro--L with the cross-reacting antigens (T,G)-Pro--L and (Phe,G)-A-L indicates that different inbred mouse strains make antibodies specific for different parts of the same polypeptide. Thus, antibody from DBA/1 mice reacts almost exclusively with the (Phe,G) sequence, while SJL antisera bind only (T,G)-Pro--L and fail to bind (Phe,G)-A-L. The immune responses to the same amino acids on two different polypeptides (i.e. A--L and Pro--L) appear to be under separate genetic control.

The role of thymocytes and bone marrow cells in defining the response to the dinitrophenyl hapten attached to positively and negatively charged synthetic polypeptide carriers. Cell fractionation over charged columns.

An inverse relationship exists between the net electrical charge of immunogens and the antibodies they elicit (1). Results of an earlier study have demonstrated that the net charge phenomenon has a cellular basis, since the immune response potential of murine spleen cells to 2,4-dinitrophenyl (DNP) on a negatively charged synthetic polypeptide carrier was reduced by cell fractionation over negatively charged glass beads, whereas the response to the same hapten on a positively charged carrier was unaffected (14). To verify that the net charge correlation is expressed at the cellular level, spleen cells were fractionated over positively charged poly-L-lysine-coated glass bead columns, and their immunocompetence to DNP on positively and negatively charged carriers was tested by cell transfers in irradiated recipient mice. In this case, the fractionated cells showed reduced response potential to DNP on the positively charged carrier only. Thus, the cellular basis of the net charge phenomenon has been demonstrated for both positively and negatively charged immunogens (for the same specificity) by cell separation techniques over columns of opposite charge. In order to establish whether the cell population relevant for the charge properties of immunogens was of thymus or marrow origin, thymocytes and bone marrow cells were selectively passed over positively or negatively charged columns and mixed with unfractionated cells of the complementary type. Transfers of the filtered and unfiltered cell mixtures in irradiated recipient mice immunized with DNP on either a positive or a negative synthetic polypeptide carrier indicated that fractionation of thymocytes, but not of marrow cells, correlated with the spleen population. Thus, thymocytes fractionated over negatively charged columns and mixed with unfractionated marrow cells exhibited reduced response to DNP on the negative carrier, but normal responses to DNP on the positive carrier. The opposite result was obtained when thymocytes were passed over positively charged columns. No effect on the anti-DNP response was detected by filtration of bone marrow cells over columns of either charge. These findings indicate that it is possible to distinguish between thymocytes on the basis of their capacity to react with more acidic or more basic surfaces and that a population of thymus-derived cells may recognize immunogens on the basis of their overall electrical charge. No evidence was found by these techniques that marrow-derived cells contribute to the net charge phenomenon.

Resistance of MHC class I-deficient mice to experimental systemic lupus erythematosus.

Experimental systemic lupus erythematosus (SLE) can be induced in mice by immunization with a human monoclonal antibody to DNA that bears a common idiotype (16/6Id). These mice generate antibodies to 16/6Id, antibodies to DNA, and antibodies directed against nuclear antigens. Subsequently, manifestations of SLE develop, including leukopenia, proteinuria, and immune complex deposits in the kidney. In contrast, after immunization with 16/6Id, mice lacking major histocompatibility complex (MHC) class I molecules generated antibodies to 16/6Id but did not generate antibodies to DNA or to nuclear antigen. Furthermore, they did not develop any of the above clinical manifestations. These results reveal an unexpected function of MHC class I in the induction of autoimmune SLE.

In vitro proliferative responses and antibody titers specific to human acetylcholine receptor synthetic peptides in patients with myasthenia gravis and relation to HLA class II genes.

To investigate which parts of the acetylcholine receptor are involved in the initiation and development of myasthenia gravis (MG), peptides representing different sequences of the human acetylcholine receptor alpha-subunit were synthesized. These peptides were tested for their ability to stimulate T cells of myasthenic patients and healthy control patients in proliferation assays and to bind to sera antibodies. Three of eight peptides discriminated significantly between the two groups in the proliferation assay, as well as in their ability to bind to serum antibodies. HLA-DR3 and DR5 were associated with proliferative responses to specific AChR peptides in the group of myasthenics. Acetylcholine receptor epitopes that might play a specific role in myasthenia gravis thus were demonstrated.

Role of MHC class I molecules in autoimmune disease.

The MHC class I molecules play a pivotal role in triggering cellular immune responses, binding and presenting intracellularly derived peptide antigens. Studies of MHC class I expression revealed a complex regulatory mechanism that integrates tissue-specific and hormonal modulation. Dynamic regulation occurs in the thyroid, in response to hormonal repression by TSH and stimulation by thyroid hormone. This dynamic cycle provides the basis for proposing the model that such regulation is important to maintain tolerance to self-antigens in tissues synthesizing large amounts of secretory proteins. Failure to appropriately regulate class I levels is predicted to result in autoimmunity. In support of this model, we found that class I-deficient mice are resistant to the experimentally induced autoimmune diseases, SLE, and blepharitis. Furthermore, pharmacological treatment with an agent that reduces class I expression also reduces the incidence and severity of both experimental and spontaneous autoimmune SLE.

Effects of aging on the induction of experimental systemic lupus erythematosus (SLE) in mice.

The study was designed to determine whether manifestations of autoimmunity are altered with age, using an experimental model in which systemic lupus erythematosus (SLE) is induced in mice. Young (2-month-old), and aging (18-month-old) BALB/c female mice were immunized with a human monoclonal anti-DNA antibody that bears a common idiotype (16/6 Id). Control groups were either left untreated or were injected with human IgM (HIgM). Anti-16/6 Id levels were found to be significantly lower in the old mice than in the young. Similarly, anti-anti-16/6 Id (murine 16/6 Id+) values were lower in the old. Mice injected with the 16/6 Id also produced various autoantibodies, including anti-dsDNA, anti-RNP, anti-Sm and anti-histones antibodies. The levels of these antibodies were lower in the old mice than in the young, yet the differences were not statistically significant. Levels of autoantibodies examined in control animals were either similar in both age groups (anti-RNP and histones) or lower in the old (anti-dsDNA and Sm). Four months after a booster injection of 16/6 Id, the young mice developed clinical manifestations of SLE, including proteinuria and leukopenia, which were seen, in milder form, in the aged mice. Immune complex depositions examined by immunohistology on kidney sections suggested similar differences based on the age of the animals. Our results suggest that aging might actually be associated with a decline in the capacity to produce autoimmune responses.