Antigenic promotion. Increase in hapten-specific plaque-forming cells after pre-injection with structurally unrelated macromolecules.

Rabbits were immunized with p-azobenzene arsonic acid derivatives of human serum albumin (HA-As) or of dissociated keyhole limpet hemocyanin. The IgM response to the hapten was evaluated in terms of the number of hapten-specific plaque-forming cells in the lymph node draining the injection site. In some experiments, antibody was measured by agglutination of tanned and sensitized erythrocytes. The hapten response of animals immunized with HA-As was increased (promoting effect) when the animals were injected with one of several structurally unrelated macromolecules: keyhole limpet hemocyanin (KLH), horse spleen ferritin (HSF), lysozyme (Lys), alum-precipitated human gamma globulin (alum-precipitated HGG). Different macromolecules differed in the magnitude of the promoting effect they induced, e.g., promotion by the associated form of KLH was greater than that by the dissociated form; alum-precipitated HGG was a better promoter than was soluble HGG. The relative magnitude of promotion by different macromolecules (associated vs. dissociated KLH, alum-precipitated vs. soluble HGG) correlated with the relative magnitude of the carrier effect, as judged by the hapten response induced by p-azobenzene arsonic acid conjugated to various proteins. Promotion was detected by agglutination assay of circulating antibody, by plaque assay of cells from the popliteal lymph node draining the site of preinjection, but not by plaque assay of cells from the contralateral lymph node. Promotion was dependent on the dose of the promoting macromolecule and on the dose of the hapten-protein conjugate. It was not observed in animals tolerant to the promoting macromolecule. Inhibition (i.e. antigenic competition), rather than promotion, was observed upon a secondary response to the preinjected macromolecule or when the hapten-protein conjugate was incorporated in Freund's adjuvant.

The effect of cross-reacting antigens on the tolerant state.

Rabbits were rendered tolerant to human albumin (HA) and were then injected with azo and oxazolonated derivatives of human albumin. These injections were continued to a time at which all animals would have lost tolerance if they had not been injected. Injection of cross-reacting antigens prolonged the duration of tolerance, as judged by the mode of elimination of lightly iodinated human albumin (HA.(131)I). Different derivatives of HA differed in their capacity to prolong tolerance. Those neonatally injected rabbits which were immunized with cross-reacting antigens and lost tolerance, responded much more promptly to HA.(131)I than animals which were not immunized. Animals immunized with cross-reacting antigen which went on to eliminate HA.(131)I triphasically, usually had responded earlier by making antibodies. These antibodies contained a fraction which was reactive with HA, and which was usually equally well adapted to determinants on HA and on the cross-reacting antigen.

The effect of tolerance on the specificity of the antibody response and on immunogenicity. Antibody response to conformationally and chemically altered antigens.

Animals were rendered tolerant to human albumin and were then immunized with azo derivatives of human albumin which differed in the number of hapten groups per molecule and in the extent of conformational change. The incidence and specificity of the resulting antibody response was studied and the presence of antibody to azo groups and to conformationally altered protein determinants was demonstrated. Reactivity with the tolerance-inducing antigen was shown to be due to antibodies directed against conformationally altered protein determinants. The difference in the response of tolerant animals to hapten-poor and hapten-rich derivatives was attributed to the extent of conformational alteration. A genetic factor appeared to be implicated in the capacity of tolerant animals to respond to an antigen which cross-reacts with tolerance-inducing macromolecules.

Cells as antigen carriers and as immunoglobulin producers. Synthesis of antibody and allogeneic immunoglobulin after transfer of antigen-treated cells to newborn rabbits.

The injection into newborn rabbits of a small quantity of human albumin, associated with red blood corpuscles or nucleated rabbit cells, induces an antibody response in the majority of animals, whereas the same quantity of antigen in solution fails to stimulate antibody formation or induces tolerance. The promoting capacity of the cells depends on attachment of antigen to them. The antibody produced after the injection of albumin, associated with nucleated cells, is of recipient origin. However, immunoglobulin carrying the marker of donor cells can be demonstrated in the recipient animals, and may reach serum concentrations similar to those normally present in animals which are heterozygous with respect to the marker. It appears that the antibody-promoting function and the synthetic capacity for allotype are quite distinct and that the period required for allotype formation is very short with mononuclear peritoneal exudate cells and is very much longer with cells from the thymus. The capacity of cells from lymph nodes for sustained allotype formation is less than that of thymus cells but greater than that of mononuclear peritoneal exudate cells.

Immunosuppressive and graft-rejecting anti--bodies in heterologous anti-lymphocytic serum.

Skin allografts survived longer on ALS-treated, complement-deficient (C5 negative) recipients than on ALS-treated, complement-competent (C5 positive) recipients. Administration of C5-positive serum to C5-negative, ALS-treated recipients resulted in reduced graft survival. A percentage of grafts from ALS-treated, C5-positive donors was rejected when transferred to untreated syngeneic recipients; this was not observed when C5-negative, syngeneic animals served as ALS-treated donors and untreated recipients. It was concluded that ALS has graft-rejecting properties which are promoted by late acting complement components. Unlike ALS-mediated graft rejection, ALS-mediated immunosuppression appeared to be independent of the late acting complement components. The effect of ALS on the humoral response to sheep erythrocytes was examined in complement-deficient and complement-competent mice. Immune-suppression was determined by ALS treatment of C5-competent and C5-deficient mice and also by transfer of in vitro ALS-treated spleen cells from C5-negative and C5-positive donors to cyclophosphamide-treated recipients. The ability of ALS to depress the humoral response to sheep cells and to decrease immunological competence of spleen cells was the same in the presence as in the absence of C5.

Membrane lesions in immune lysis: surface rings, globule aggregates and transient openings.

It is known that there are 100 A-wide circular structures associated with the erythrocyte membrane in immune lysis. To determine whether these structures were functional holes extending through the membrane, freeze-etch electron microscopy was carried out. Sheep erythrocytes incubated with either rabbit complement or rabbit antibody (anti-sheep erythrocyte antibody) did not hemolyze and did not reveal any abnormalities in freeze-etch or negative-stain electron microscopy. Erythrocytes incubated with both complement and antibody revealed rings on the extracellular surface (etch face) of the cell membrane. Allowing for the 30 A-thick Pt/C replica, the dimensions of the surface rings were similar to those seen by negative staining. The ring's central depression was level with the plane of the membrane; some rings were closed circles, others were crescent shaped. The cleavage face of the extracellular leaflet revealed globule aggregates, each aggregate appearing to be composed of about four fused globules. The cleavage face of the cytoplasmic leaflet was normal. When immune lysis was carried out in the presence of ferritin, ferritin was subsequently detected in all lysed erythrocytes. If ferritin was added after immune lysis was complete, only 15% of the cells were permeated by ferritin, indicating that transient openings exist in the cell membrane during immune lysis. No abnormal structures were detected when C6-deficient rabbit serum was used as a source of complement. It is concluded that antibody and complement produce surface rings, prelytic leakage of K(+), colloid osmotic swelling, membrane disruption, and membrane resealing; the surface rings persist after these events.

Tumor colony formation by Friend virus-infected cells in immunosuppressed mice.

We have investigated the role of host immunological factors in the formation of "tumor colonies" in the spleens of unirradiated C57BL/6 X C3Hf/Bi FI mice 9 days after i.v. injection of spleen cells from Friend virus (FV)-infected C3Hf/Bi donors. Pretreatment of hosts with antilymphocyte serum (ATS) increased the number of tumor colonies. Pretreatment with formalinized FV-infected cells had the opposite effect, and ATS diminished the inhibitory effect of preimmunization. Cell suspensions from 11 individual FV-infected donors were examined. The suspensions differed with respect to their behavior on transplantation into untreated and ATS-pretreated F1 hybrid hosts. With several suspensions, the number of tumor colonies produced was approximately proportional to the number of cells injected; in all of these, ATS increased the slope of the line relating colony number to cell number. With most of the suspensions, tumor colony-forming efficiencies in untreated hosts strikingly decreased with increasing number of cells injected; ATS induced an increase in the number of tumor colonies and rendered the colony-forming response more nearly proportional to cell number. With two suspensions, few or no colonies developed; pretreatment with ATS had no significant effect. When the 11 cell suspensions were considered together, a proportional relation was found between the magnitude of the ATS effect (i.e., colony number in the presence of ATS minus colony number in the absence of ATS) and the colony-forming efficiency in ATS-treated mice. The ATS effect on the average was equivalent to a 2-fold increase in tumor colony-forming efficiency. We interpret these findings to indicate that two factors interact to determine the number of tumor colonies produced by spleen cells from FV-infected C3H donors in untreated F1 hybrid hosts. One is a property of the FV-infected cell population and includes its frequency of tumor colony-forming units; this factor varies widely among different cell suspensions. The other is a property of the tumor colony-forming units-host interrelationship and includes the vulnerability of tumor colony-forming units to the host immune response elicited by the injected cells; this factor appears to be constant with different cell suspensions. The present results show that the two factors can be dissociated in immunosuppressed hosts.

Cross-reaction of a monoclonal antibody to human MHC class II molecules with rabbit B cells.

Monoclonal antibodies, 21w4 and 44H10, against human MHC class II determinants, were analysed for their reactivity with rabbit lymphoid cells. Both MAb bind to all human B lymphoblastoid lines, irrespective of their HLA-DR phenotype. HLA-DR antigens, purified by affinity to 21W4 IgG Sepharose, can be precipitated with 44H10 MAb, indicating that both antibodies react with the same molecules. Competitive inhibition studies with purified MAb IgG show that 44H10 and 21w4 recognize different epitopes of HLA-DR molecules. The 21w4 MAb, previously shown to cross-react with cells of pig, mouse and sheep, does not react with rabbit lymphoid cells. The 44H10 MAb binds to lymphoid cell suspensions prepared from rabbit appendix, spleen and mesenteric lymph nodes. Its reactivity correlates with that of RABELA, a polyclonal antibody specific for rabbit B cells. Mesenteric lymph node and spleen cell suspensions, depleted of sIg+ cells, are devoid of 44H10+ cells, while similarly-treated appendix B cells still contain a subset of B cells which are sIg-, RABELA+ and 44H10+. These studies thus report on the presence of MHC class II determinants on rabbit B cells, cross-reacting with human HLA-DR.

Immune response, tolerance circumvention and autoantibodies in aging MRL/Mp-lpr and MRL/Mp-+ mice.

Isotype distribution was analyzed, as a function of age in MRL/Mp-lpr and MRL/Mp-+ mice. The mice were tested for: (1) "spontaneous" response to nucleic acid (2) induced response to alum-precipitated phosphorylcholine-rabbit gamma globulin (PC-RGG) (immunized animals) and (3) induced response to alum-precipitated PC-RGG after pretreatment with aggregate-free RGG (tolerized-immunized animals). "spontaneous" nucleic acid antibodies of isotypes, other than IgM, increased as animals became older. The quantity of RGG antibody declined as a function of the age at which animals were immunized. Young tolerized-immunized animals made less antibody of all isotypes than did immunized animals. In later life, resistance against tolerance induction developed. Aggregate-free RGG sensitized older animals and, thus, augmented the response to alum-precipitated PC-RGG. Up to the age of 20 weeks, spontaneous antibody and antibody of tolerized-immunized animals showed striking similarities in age- and strain-dependent changes of IgG2b and IgA isotypes. Results were discussed in terms of: (1) a defect in down regulation of immune responsiveness, which contributes to the initiation of autoimmunity and age-dependent resistance to tolerance induction; (2) regulatory mechanisms for isotype switching, which contribute to resistance to tolerance induction, whether naturally occurring or experimentally induced; and (3) age-related immunological changes which are inherent in the MRL/Mp genome, the mutant gene, lpr/lpr, accelerating the changes.

Polymorphism of age-related changes in stimulatory capacity of murine dendritic cells.

All through life, regulatory and executive components of the immune system undergo changes, differing in rate and extent, dependent on the genetic background. We have, here, examined age-dependent changes in stimulatory capacity of dendritic cells (DC) in allogeneic mixed leukocyte reaction (MLR). DC of mice of five strains showed very little change as they aged. DC from mice of two other strains showed a significant age-related decrease of stimulatory activity and those of one strain showed an increase. The capacity of DC to stimulate syngeneic MLR was examined in three strains, as a function of age, and was found to decrease in one and to slightly increase in two. The underlying cause for this extensive polymorphism remains to be determined. We could not find supporting evidence for the view that the observed changes were related to changes in Ia density on dendritic cells.

Effects of aging on neuronal electrical membrane properties.

The electric membrane properties (EMP) of dorsal root ganglion (DRG) neurons in cell cultures prepared from control mice (8-14 weeks) and old mice (90-92 weeks) were compared. The old neurons had a number of significant alterations in EMP compared to controls including decreased electrical excitability, increased action potential duration and more pronounced biphasicity of the repolarization phase. The old neurons also had larger action potential overshoot and afterhyperpolarization. The pattern of altered electric membrane properties was consistent with an age-induced shift from voltage-sensitive sodium channels to less excitable voltage-sensitive calcium channels and also a decrease in potassium permeability during the repolarizing phase of the action potential.

Variations in age-related decline in striatal D2-dopamine receptors in a variety of mouse strains.

To determine the importance of inheritance on the age-associated decline in D2-dopamine receptor number, the binding of [3H]spiperone to mouse striatal membranes was measured in animals ranging from 7 to 104 weeks of age from 5 murine strains (C57BL/6J, C3/HeJ, A/J, SJL/J and DBA/1J). In young mice, receptor number (Bmax) was influenced by genetic background such that C57BL/6J less than SJL/J less than A/J = DBA/1J = C3H/HeJ. A 50-60% decline in Bmax with age was found in all strains except for C57BL/6J. Bmax in the C57BL/6J mice were lower than in the other strains of young animals (7-15 weeks) but remained relatively constant throughout life (measured up to 104 weeks of age). Furthermore, the maximal decline in receptor number was observed relatively early in life (16-30 weeks) and remained constant thereafter. Neither age nor genetic background influenced ligand affinity (Kd). Thus the results of this study suggest that the maximal decline in Bmax for the dopamine receptor occurs before the second half of life and that the magnitude of this decline is polymorphic.

Age-related decrease in ultraviolet induced DNA repair in neurons but not in lymph node cells of inbred mice.

DNA repair capacity was measured, as UV induced, unscheduled DNA synthesis (UDS), in cells of the nervous and immune system of three mouse strains, as a function of age. The strains examined were DBA/1J, C57B1/6J and SJL/J. For dorsal root ganglion neurons of strain DBA/1J, aged 97-98 weeks, a significant decline in UDS of 53% at 20 J/m2 and 73% at 40 J/m2, respectively, was measured, when compared to mice aged 17-18 weeks. Similarly, neurons from C57B1/6J mice, aged 115-116 weeks, showed significant, age dependent decreases of 31% at 20 J/m2 and 40% at 40 J/m2, respectively, compared to mice aged 7-8 weeks. In lymph node cells of all three strains employed, no significant age related decreases in UDS were detected. While the complexity of processes involved in DNA repair makes conclusive interpretation of the results precarious, the results obtained for post-mitotic cells of the nervous system, may be viewed as compatible with the DNA repair hypothesis of ageing.

Economic correction; quantity and age-related rate of change.

Correlation between the relative magnitude of activity or concentration in youth and the relative magnitude of age-related decrease occurs in several systems. We have observed this correlation in density of D2-dopamine receptors of the striatal membrane, activity of lymphocyte activated killer cells, relative density of CD8 on thymocytes, augmenting activity of T splenocytes and mRNA, coding for IL-1 in Langerhans cells. We have suggested that this correlation should be considered in the context of balanced investment in lifespan and reproductive efficiency, that it may be the result of feedback regulation and have designated it as "economic correction".

Strain differences of age-dependent changes in the responsiveness to a T-independent type-2 antigen in mice.

We have assessed age-associated early changes in antibody response to a T-independent type-2 (TI-2) antigen, dinitrophenylated ficoll (DNP-Ficoll). Mice of most strains, including long-lived and autoimmune-prone strains, give a high response when approximately 2 months old; thereafter the response declines sharply to the 3rd-4th month of age and continues to do so, more gradually, up to the age of 6 months. Age-related changes in the response of C57BL/6 mice follows a different course: the response remains unchanged up to the first year of life, i.e. to middle age. The in vitro anti-DNP-Ficoll antibody response of B cells could be increased by the addition of young syngeneic T cells. The augmenting activity of splenic T cells of C3H/He mice declines clearly as a function of age. In contrast, splenic T cells of C57BL/6 mice have low augmenting activity whether the T cells are obtained from young or middle-aged donors. Unlike the augmenting capacity of T cells, B-cell responsiveness to DNP-Ficoll increases until middle age in all strains examined. We conclude that early age-associated changes in antibody response to the TI-2 antigen is polymorphic and that the early age-related decline in in vivo responsiveness is attributable to an age-associated decline in augmenting T helper cell activity.

Immunosuppression after injection of Friend virus into C57BL/6 mice of different ages.

Injection with Friend virus (FV) causes immunosuppression in young and old C57BL/6 mice, i.e. it occurs whether or not the virus replicates very briefly or for a long period. There are only minor age-related differences in the extent of immunosuppression, except that suppression appears to persist somewhat longer in old than in young animals.

Age-related changes in contact photosensitivity differ among mouse strains.

There are differences among mouse strains in the age-related changes in reactivity to the contact photosensitizer tetrachlorosalicylanilide (TCSA). We found a tendency to lower reactions in older mice, with some strains showing declines from an early age (BALB/cJ, MRL/MpJ +/+, MRL/MpJ lpr/lpr and SJL/J). Others had increasing reactions until about 30-50 weeks of age before declining (DBA/1J, C3H/HeJ, and A/J) and one strain (C57BL/6J) had increased reactivity with age. There are also differences in the role of cyclophosphamide-sensitive T-suppressor cells in these age-related changes. In some mouse strains, BALB/cJ, C57BL/6J, A/J, DBA/1J and C3H/HeJ, age-related changes in reactivity to TCSA are independent of changes in cyclophosphamide-sensitive suppressor cells. In other strains, MRL/MpJ +/+, MRL/MpJ lpr/lpr and SJL/J, the development of cyclophosphamide-sensitive suppressor cells is responsible for the initial, though not later, stages of the age-related decline in reactivity.

Friend virus replication as a function of age.

We have investigated the effect of age on the replication of Friend spleen focus-forming virus (SFFV). Recovery of SFFV from the spleens of four strains of mice was determined following intravenous infection with NB-tropic Friend virus (FV) complex at ages ranging from 6 to 134 weeks. In C57BL/6 mice, the virus did not replicate in adults up to 40 weeks of age, but beyond that there was a steep exponential increase with age in the amounts of SFFV recoverable. In C3H/He mice, which replicate the virus as young adults, the amount of SFFV recovered was 6-fold greater in old than in young mice. Recovery of virus was biphasic with age in SJL mice; in A strain mice no consistent change with age was noted. In C57BL/6 mice, reconstitution of lethally irradiated recipients with syngeneic marrow cells, followed by i.v. infection with FV, showed that the amounts of SFFV recovered depended on the age of the recipient. The present work shows that Friend SFFV replication is a sensitive indicator and can be used as a tool for the investigation of aging processes. The mechanisms responsible for the age-dependent change in regulation of virus replication and for the polymorphism remain to be determined.

Purification of rabbit T-derived (Ig-) lymphocytes.

Rabbit peripheral blood cells were rosetted by means of mixed agglutination technique described by Coombs et al. and the unrosetted Ig-, T-derived lymphocytes were separated in Ficoll-Triosil gradient. The cells which did not rosette in mixed agglutination reaction represented highly purified T-derived (Ig-) lymphocytes, as evidence by their response to T and B rabbit cell mitogens, their cytotoxic reactivity versus rabbit thymus lymphocyte antiserum (RTLA), and by failure to be stained with labelled anti-rabbit immunoglobulins antibodies and antibodies against allotypic specificities of the a and b series.

Purification of rabbit B spleen cells by removal of adherent and of T cells.

B lymphocytes from the rabbit spleen were freed of T cells by removal of cells which formed rosettes with papain-treated rabbit erythrocytes. Additional purification could be achieved if fractionation by rosette removal was preceded by removal with a magnet of cells which adhered to or ingested poly L-lysine coated iron core particles. Cell yield and purification were assessed by complement mediated cytotoxic kill of B and T cells with antibody directed against RABELA and RTLA, respectively. Other criteria depended on determination of the number of Fc receptor bearing cells and of thymidine uptake by cells which were stimulated with concanavalin A, PHA or with antibody directed against the allotypic specificity of receptor Ig light chains. Purified preparations of B cells were obtained in a yield of about 20% of the B cells in the original spleen and contained less than 10% of cells which were not B cells. This method allows purification which does not interfere with the membrane of the isolated cells.