The physiology of germinal centers.

GCs contain Ag-induced rapidly proliferating B cell blasts called centroblasts and centrocytes and are located in primary follicles of peripheral lymphoid tissue. Their peak development is at 7 to 10 d after Ag. The generation of memory B cells occurs in GCs, accompanied by frequent Ig isotype switching, Ig gene V region somatic hypermutation, and/or (in rabbits) gene conversion. The nature and function of the cell types that make up the microenvironment of the GC B cells, the CD4+ T cells and FDCs, are discussed in detail. The high rate of apoptosis that occurs in GCs is thought to be the result of processes of positive and negative selection ongoing in different compartments of GCs. The rescue of cells through high-affinity interaction with Ag localized on FDCs and subsequent presentation of Ag by GC B cells to T cells may represent the positive selection with apoptosis as the default pathway. Negative selection may occur, aimed at the prevention of autoimmunity caused by cells with mutated sIg binding to autoAgs. Some aspects of GC-derived lymphomas in humans and mice are also reviewed.

B cell lymphomas of C57L/J mice; the role of natural killer cells and T helper cells in lymphoma development and growth.

The Hodgkin's-like Type B neoplasms which arise spontaneously in aging C57L mice (25% incidence at 21 months of age) were first reported over 40 years ago, but since then relatively little has been published about these lymphomas. Based on previous studies in SJL mice, we investigated the phenotypic and functional properties of C57L-derived lymphomas in relation to Mtv29-encoded vSAg expression by the tumor cells, and their ability to stimulate TCR Vbeta-restricted T cells. The cell surface phenotype of the C57L lymphomas indicates a B cell origin (sIg(+), MHC II(+)). These B lymphoma cells also express co-stimulatory molecules [B7-1 (CD80) and HSA (CD24)], and stimulate marked proliferation of syngeneic CD4(+) T cells. C57L B lymphoma cells exhibit Mtv-encoded mRNA by northern analysis, and also stimulate IL-2 production from Vbeta16(+) T cell hybrids, suggesting a role for Mtv 29 in this syngeneic T cell response. After transfer to syngeneic recipients, primary C57L lymphomas grow slowly, if at all. However, tumor growth is greatly accelerated by pretreatment of C57L recipients with anti-asialo GM1 antibody (but not anti-CD8 mAb), suggesting that NK cells play a major role in inhibiting lymphoma growth. If, in addition to anti-asialo GM1, the mice are also pretreated with anti-CD4 mAb, tumor growth is markedly inhibited, indicating that the lymphoma-responsive syngeneic CD4(+) T cells promote tumor growth. Therefore, although the vSAg-induced response stimulated by vSAg29 expressing lymphoma cells in syngeneic TCR Vbeta-restricted CD4(+) T cells is an important etiologic factor in this type of B cell neoplasm both in C57L and in SJL mice, the final outcome of the spontaneous neoplastic process appears strongly influenced by endogenous NK activity in aging mice.

IL-5 responsive subsets among normal and lymphomatous murine B cells.

Two normal murine B-cell subpopulations, germinal center and coelomic B cells, and at least some of the lymphomas derived from them, respond to IL-5. In the case of normal B cells, a comitogen (DxS) is required. IFN-gamma is strongly inhibitory to proliferation of the coelomic B-cell subset but not for germinal center cells or the SJL lymphomas derived from them.

A feather-sexed strain of laying hens was more responsive to dietary supplements of choline and methionine than a vent-sexed strain.

A response surface design was used to study Cho and Met interactions with corn and soybean diets, using two strains of hens. The strains were a feather-sexed line (FS strain), and a vent-sexed line (SS strain). The diets contained 3% meat and bone meal and, on chemical analysis, 15.1% crude protein, .29% Met, .225% Cys, and 1,041 ppm of Cho. Nine diets were fed from 20 to 68 wk of age, using added Met levels ranging from 0 to 500 ppm and added Cho levels ranging from 0 to 1,500 ppm, to fix the design points. The FS strain consumed significantly more feed per day (117 versus 108 g) than the SS strain, but there were no significant differences for the 24 to 68 wk period in egg production, egg weight, or feed per dozen eggs. Three and five combinations of Met and Cho were significant in improving egg production (P less than .05) out of the eight combinations for the SS and FS strains, respectively. The best egg production for the FS strain for the period 24 to 68 wk was observed at 250 ppm Met and 1,500 ppm Cho, or 427 ppm Met and 220 ppm added Cho. The SS strain showed no significant (P greater than .05) dietary responses in egg production between 250 ppm Met and no Cho, or 427 ppm Met and either 220 or 1,280 ppm Cho. The SS strain showed no significant (P greater than .05) dietary response in egg weight to either Cho or Met.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of choline supplementation in growing pullet and laying hen diets.

Two experiments were conducted to determine the effect of choline supplementation on corn-soy-meat-based grower and laying hen diets. Diets contained 2.5% and 3% meat and bone meal in the growing and laying diets, respectively, and on chemical analysis contained 1005 and 1041 ppm of choline respectively. In the first experiment, 1000 ppm of choline were added to the basal growing and laying diets, and in the second experiment the laying diet was supplemented with 550 ppm or 1000 ppm of choline. In both trials, choline supplementation did not increase gains or feed efficiency for pullets from 8 to 20 weeks. However, choline supplementation during the laying period resulted in a statistically significant improvement of egg production and egg size. Supplementation of choline in the growing phase did not affect the laying performance. Laying performance was not improved by 2 micrograms/kg of supplementary vitamin B12 in a 1000 ppm choline supplement diet (78% vs. 76% hen-day production). In the second trial, added levels of choline (0, 500, and 1000 ppm) resulted in egg production from 24 to 64 weeks of 73, 76, and 76% hen-day production, respectively. Egg weights were 59, 61, and 61 g, respectively. This suggests that the total choline requirement of laying hens on a corn-soy-meat diet, and in absence of supplementary methionine, is greater than 1000 ppm but no more than 1500 ppm.

Efficacy of cysteine in replacing methionine in the immune responses of broiler chicks.

Two trials were conducted to compare the effects of supplements of methionine and cysteine on the growth and immune responses of broiler chicks fed corn-soy diets. The basal diet contained 21% crude protein, 3,255 kcal metabolizable energy/kg diet, .35% methionine, .37% cysteine, and .13% choline. Additions to the basal diet were methionine (.063, .25, .85, and 1.45%), or cysteine (.203%), or a combination of methionine (.063%) and cysteine (.153%). Total antibody and 2-mercaptoethanol-resistant antibodies, immunoglobulin G (IgG), were determined in chicks inoculated intraperitoneally at 14 days of age and serially bled at 4, 7, and 10 days postinoculation. Thymus-derived (T)-cell-dependent in vivo mitogen response to phytohemagglutinin-P (PHA-P) was assessed via wing web swelling. The methionine requirement for growth (0 to 3 wk of age) was found to be no more than .413% of the diet (.35% in the basal diet plus .063% added). Addition of 1.45% methionine to the basal diet resulted in significant depression (P less than .05) in growth. The antibody responses generally peaked at 7 days postprimary inoculation. Both methionine and cystine supplementation at low levels resulted in improvement in the cell mediated PHA-P responses as well as in the IgG (T-cell-dependent) responses. High supplemental methionine (1.45%), however, caused significant (P less than .05) depressions in both responses. Equimolar additions of methionine and cysteine (16.8 mmol/kg diet) showed that cysteine was about 84 and 70% as efficacious as methionine in the IgG and the PHA-P stimulation (PHA-I), respectively, in healthy chicks.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced immune responses in broiler chicks fed methionine-supplemented diets.

Effects of feeding supplementary methionine and choline on broiler growth and immunity were examined by supplementing a corn-soybean diet that contained 21% crude protein, 3,255 kcal metabolizable energy/kg diet, .35% methionine, .37% cystine, and .13% choline. Methionine (.063, .125, .25%) and choline (.125, .25%) were dietary variables. Sulfate (.055%) was added either alone or along with methionine (.125 or .25%) and choline (.125%). In one study, the .25% methionine diet was supplemented with .121% betaine. Sodium and chloride levels were constant in all the diets. Feed and distilled water were supplied ad libitum. Total antibodies, immunoglobulin (Ig) G (2-mercaptoethanol-resistant antibodies) and IgM (2-mercaptoethanol-sensitive antibodies) were determined in 3-wk-old chicks inoculated intraperitoneally with sheep red blood cells. The thymus-derived (T)-cell-dependent in vivo mitogen response to phytohemagglutinin-P (PHA-P) was assessed via wing web swelling. The methionine requirement for growth (0 to 3 wk of age) was approximately .413% of the diet (.35% in the basal diet plus .063% added). Supplementation of the basal diet with .125% choline stimulated growth to the same extent as did the extra .063% of methionine. Addition of .055% sulfate with .125% choline did not improve the ability of the latter to spare methionine. Supplemental methionine resulted in significant (P less than .05) dose-related increases in total antibody, IgG, and response to the mitogen PHA-P, but not in IgM. There were no effects of choline on the immune variables studied. These results suggest that methionine is required for select components of the antibody response, which effect might be related to T-cell help.

Alterations in phospholipid composition of egg yolks from laying hens fed choline and methionine-supplemented diets.

A trial was conducted to assess the effects of choline and methionine supplementation of the diets of hens on phospholipid composition of egg yolk. By chemical analysis, the basal diet contained 16.6% protein, 1,041 parts per million (ppm) of choline, and .53% TSAA. The energy level was 2,899 kcal ME/kg diet, with no added fat. A rapid isocratic HPLC procedure using a Waters mu Bond-NH2 column (Waters Associates, Milford, MA) was developed to quantify phospholipid components. Analysis of egg samples at peak production (36 wk of age) showed significant (P less than .05) elevations in total phospholipids, phosphatidylcholine (PC), and the ratio of PC to phosphatidylethanolamine (PE), when choline was added at 500 or 1,000 ppm. Phosphatidylethanolamine, in contrast, showed a decline with choline supplementation. Methionine supplementation (125 to 250 ppm) also caused elevations in the total phospholipid composition of the egg yolk but had only modest effects on PC and PE. There was a significant (P less than .05) correlation between the yolk weight and total phospholipid concentration, PC, and the PC:PE ratio. There was a relationship between phospholipid content and weight of eggs when diets of laying hens were supplemented with choline.

Critical vitamin supplementation of broiler diets high in alfalfa juice protein.

Three trials were conducted to identify the critical vitamins in the diets of broiler-strain chicks fed alfalfa juice protein concentrate (AJPC) corn-soy diets from 0 to 3 wk of age. Vitamin supplements were added to AJPC diets. Diets were formulated to contain, parts 30/121, 40/128, 50/135, and 60/142 parts AJPC/parts total diet. Parts were used to permit usage of wet materials and still maintain about 90% dry matter. All diets were formulated to contain 20% crude protein, .93% total sulfur-containing amino acids, and 2,940 kcal metabolizable energy/kg diet. Propionic acid was added at .2% to all diets. Feeds were refrigerated (7 C) and fed out daily. The addition of choline, riboflavin, vitamin B12, vitamin A or E, folic acid, or biotin did not increase weight gains. Addition of 3 mg/kg vitamin B6 completely overcame the growth depression caused by the 50-parts AJPC diet and significant (P less than .05) growth increases (13 to 29%) were achieved with vitamin-B6 supplementation to the 60-parts AJPC diet. Depressed immune responses were completely prevented by the addition of 3 mg/kg of vitamin B6. The significant (P less than .05) increases in leg deformities observed in birds fed the 60-parts AJPC diet were also brought back to more typical values in birds fed the diet supplemented with 3 mg/kg vitamin B6. Vitamin K supplementation (.53 mg/kg) to the 60-parts AJPC diet resulted in significant decreases from 15 min in blood clotting times to 3 to 5 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of softness properties of feathers.

An instrument to be called the "Crimp Meter" was designed and used with a conventional balance to enable the plotting of a force-displacement curve for individual feathers. A comparison of the curves described by different feathers was made by regression analysis. The type of curve described by a feather is characteristic of its tensile properties and its degree of softness. This procedure was employed to objectively confirm the observation that chicks fed toxic levels of methionine (1.45% added) in corn-soybean diets had significantly softer (P less than .001) feathers than those fed nontoxic levels of cysteine (.203% added). The basal diet was analyzed and found to contain .35% methionine and .37% cystine. This device had potential for the down-feather, wool, and fabric industries.

T-lymphocytopaenia, opportunistic infections and pathological findings in Ghanaian AIDS patients and their sexual partners.

Ninety-nine patients at Center for Disease Control (CDC) clinical stage IV were studied. Twelve (12.12%) of these patients turned out to be HIV seronegative. Ten out of the 12 HIV negative patients were immunocompetent whereas the other two had proportional decreases in both CD4+ and CD8+ T-lymphocytes. HIV-1, HIV-2, and dual infection, were detected in 51.5%, 2%, and 22.2% respectively of clinical AIDS patients. The other 12.12% of clinical AIDS patients were indeterminate for HIV antibodies. All HIV positive patients with the exception of two, were immunocompromised with respect to CD4+ and CD8+ T-lymphocyte counts. Two healthy spouses and three children of patients who died from the disease were seronegative for HIV antibodies. Herpes simplex virus type 2 (HSV-2) and cytomegalovirus (CMV) antibody titres were higher in HIV infected than uninfected blood. Patients with chronic diarrhoea, lymphadenopathy, pneumonia, and tuberculosis, either alone or in combination of two or more of such symptoms, were found to be more likely to be confirmed by serology and immunology as definitive AIDS patients in Ghana. In postmortem studies on 20 patients, pneumonia due to tuberculosis constituted the major cause of death. Toxoplasmosis, cytomegaloviral eosophagitis and enteritis, and cryptococcosis were the major opportunistic infections detected. Programmed cell death (apoptosis) was found by the DNA gel electrophoresis method to be an unlikely major mechanism of accelerated culture induced death of PBMCs from CDC stage IV AIDS patients.

PIP: Since HIV infection was first diagnosed in Ghana in 1986, the incidence of HIV infection has increased steadily in the country over the years. Until 1990, most people infected with HIV in Ghana were infected with HIV-2. However, in 1990, most people tested were found to be dually infected with HIV-1 and HIV-2, and recently, most HIV-infected people in Ghana are only HIV-1 positive. Findings are presented from the study of 99 US Centers for Disease Control and Prevention (CDC) clinical stage IV AIDS patients. Polymerase chain reaction assay identified 12 of these patients as HIV-seronegative. HIV-1, HIV-2, and dual infection were identified in 51.5%, 2%, and 22.2% of clinical AIDS patients, respectively, with the remaining patients being indeterminate for HIV antibodies. All but 2 HIV-positive patients were immunocompromised with regard to CD4+ and CD8+ T-lymphocyte counts. 2 healthy spouses and 3 children of patients who died from AIDS were seronegative for HIV antibodies. Herpes simplex virus type 2 and cytomegalovirus antibody titers were higher in HIV-infected than in uninfected blood. Patients with chronic diarrhea, lymphadenopathy, pneumonia, and tuberculosis (TB), either alone or in combination of 2 or more such symptoms, were more likely to be confirmed by serology and immunology as definitive AIDS patients in this study. Pneumonia due to TB was the major cause of death identified through postmortem studies conducted upon 20 patients. Toxoplasmosis, cytomegaloviral esophagitis and enteritis, and cryptococcosis were the major opportunistic infections detected. Programmed cell death was probably not a major mechanism of accelerated culture-induced death of peripheral blood mononuclear cells.

Adaptive immune response in osteoclastic bone resorption induced by orally administered Aggregatibacter actinomycetemcomitans in a rat model of periodontal disease.

There is mounting evidence that innate and adaptive immunity are critical for periodontal disease-mediated bone resorption. These studies examined the role of B and CD4 T cells in adaptive immunity of rats infected with Aggregatibacter actinomycetemcomitans (Aa). Sprague-Dawley male rats were fed Aa-containing mash or control-mash for 2 weeks. B and CD4 T cells were obtained from draining lymph nodes at 2, 4 and 12 weeks, postinoculation. Quantitative polymerase chain reaction-based messenger RNA expression was conducted for 89 cytokine family genes. Disease-relevance of the differentially expressed genes was assessed using a biological interaction pathway analysis software. B and CD4 T cells of Aa-infected rats increased and were activated, resulting in enhanced isotype-switched serum immunoglobulin G by 2 weeks postinoculation. Bone resorption was evident 12 weeks after Aa-feeding. In B cells, interleukin-2 (IL-2), macrophage-inhibiting factor, IL-19, IL-21, tumor necrosis factor (TNF), CD40 ligand (CD40L), CD70, bone morphogenetic protein 2 (BMP2), BMP3, and BMP10 were upregulated early; while IL-7, Fas ligand (FasL), small inducible cytokine subfamily E1, and growth differentiation factor 11 (GDF11; BMP11) were upregulated late (12 weeks). BMP10 was sustained throughout. In CD4 T cells, IL-10, IL-16, TNF, lymphotoxin-beta (LTbeta), APRIL, CD40L, FasL, RANKL and osteoprotegerin were upregulated early, whereas IL-1beta, IL-1RN, IL-1F8, IL-24, interferon-alpha1, GDF11 (BMP11), and GDF15 were upregulated late (12 weeks). Adaptive immunity appears crucial for bone resorption. Several of the deregulated genes are, for the first time, shown to be associated with bone resorption, and the results indicate that activated B cells produce BMP10. The study provides a rationale for a link between periodontal disease and other systemic diseases.

Paraproteins and primary lymphoma in SJL mice. I. Individuality of idiotypes on paraproteins.

The isotype distribution and idiotypic determinants of paraproteins (PP) found in sera from aging SJL mice were studied. One or more PP were found in 79% of 9-11 month old mice; the isotypes were gamma 1 greater than or equal to alpha greater than or equal to gamma 2a much greater than gamma 3 much greater than gamma 2b. There was a significant tendency for the PP to occur in pairs or triplets, multiple PP being found in 35% of the mice without an evident association between any two isotypes. Rabbit anti-Id was prepared against four isolated PP, two gamma 1 and two gamma 2a. Inhibition of anti-Id-Id interaction was specific for the immunizing PP only and was absent with all other PP studied (8 gamma 1 and 10 gamma 2 PP) and with normal SJL Ig. It is concluded that SJL PP fail to exhibit cross-reactive idiotypes and show a wider range of isotype distribution than was previously suspected, although PP of mu, delta, and epsilon isotypes are absent.

Protection against apoptosis in chicken bursa cells by phorbol ester in vitro.

Like the thymus, the bursa of Fabricius is a site of massive lymphopoiesis accompanied by cell death in vivo. In the present study we have, therefore, examined whether chicken bursa and thymus cells exhibit apoptosis. Bursa and thymus cells from SC chickens, 4-10 weeks of age, were incubated for 8-24 hr with various reagents. Genomic DNA was isolated, electrophoresed in 3% Nusieve agarose gels, and examined for patterns of DNA fragmentation. A laddering of DNA in multiples of 200 base pairs, indicative of apoptosis, was observed particularly with bursa and, to a much smaller extent, with thymus or spleen cells. These patterns of DNA fragmentation from bursa cells could be prevented by adding phorbol myristate acetate (PMA) but not by its inactive analogue 4 alpha-PMA during culture. Ionomycin is not required for this effect and, alone, appears to be slightly toxic for bursa cells, although it does not inhibit the effect of PMA. PMA did not affect the degree of DNA fragmentation in spleen or thymus cells. The addition of the protein kinase C (PKC) inhibitor staurosporine abolished both the preventive effect of PMA on apoptosis and its protective effect on bursa cells, as assayed by [3H]thymidine incorporation 24-48 hr after the initiation of cultures. PKC inhibitors also prevented the proliferation-inducing effect of PMA + ionomycin on spleen cells. It is concluded that the activation of protein kinase C and perhaps other kinases protects against apoptosis in cultured bursa cells.

Biology of germinal centers in lymphoid tissue.

Germinal centers in lymphoid tissue are the sites of generation of memory B cells undergoing isotype switching and somatic mutation in their Ig genes. Their formation cannot be induced by stimuli other than immunogenic ones. It seems likely that in the function and possibly also in the formation of germinal centers, one important factor is the localization of immune complexes with fixed complement on the surface of follicular dendritic cells. CD4+ T cells, located primarily in the "apical light zones" of the centers, are necessary for germinal center formation. However, their exact role in the process needs clarification, as both cell to cell contact and cytokine production could be involved at different stages of the germinal center generation. These T cells are usually specific for the antigen inducing the germinal center, but they may sometimes respond to other surface components on the B cell surface. In view of the possible stimulatory role of CD4+ T cells in follicular center-derived lymphomas, the functional significance of these T cells in germinal center proliferation is important to unravel. The B cells in germinal centers proliferate extremely rapidly, especially those located in the "dark zones." Many of them undergo apoptosis, particularly in the "basal light zones." The microenvironment of these centers is well suited to the task of expanding and selecting memory B cells of high affinity for the inducing antigen. The interactions of the proliferating B cells with dendritic cells and T cells, unevenly distributed in the various zones of the germinal center, are thought to determine which cells deserve rescue from apoptosis and induction to differentiation into small resting memory B cells. The memory B cells that emerge from the germinal center bear sIg, usually of "switched" isotype, and exhibit somatic mutations in the variable regions of their rearranged Ig genes.

I-E expression does not by itself influence growth of or T cell unresponsiveness to SJL lymphomas.

The nature of the antigen on SJL lymphoma (reticulum cell sarcomas, RCS) cells that is strongly stimulatory to syngeneic CD4+ T cells is still elusive. Previously, we showed that the response to RCS of T cells from F1 hybrids of SJL by strains expressing I-Ak,d and/or I-Ek,d was much lower than that of T cells from SJL mice or from F1 hybrids of SJL by H2b- or H2s-bearing strains. We now show that removal of CD8+T cells from the responding cell population of (SJL x BALB/c)F1 or (SJL x A.TL)F1 mice does not enhance their responses, suggesting that the negative effect of H2k,d is not due to suppressor cells. Moreover, repeated injections of RCS cells into such F1 mice also fail to enhance the response, suggesting that these mice lack responder cells. T cells from I-E alpha transgenic (C57BL x SJL)F1 mice backcrossed to SJL respond to RCS as do T cells from I-E alpha- littermates or SJL mice. Similarly, I-E alpha+ SJL mice support RCS growth in vivo to the same (LN + spleen)/body weight ratio as do I-E- littermates. Thus, while I-E appears to have a negative influence on T cell responsiveness and RCS growth in F1 mice, it does not have such an effect when present, by itself, on a SJL background. The role of V beta 17 a+ T cells in the response of SJL T cells to RCS was also examined, because such cells are known to be responsive to I-E. The responses of V beta 17a(+)-depleted (0.3% V beta 17 a+) and V beta 17 a(+)-enriched (25.3% V beta 17a+) SJL T cell populations to RCS were examined by limiting dilution. We found the incidence of responding cells to be slightly higher in the depleted (0.016%) than in the (0.006%) enriched population. Furthermore, lymph node blast cell populations responding to RCS do not exhibit a higher percentage of cells staining for V beta 17a than do blast cells responding to Con A or unstimulated lymph node cells.

Proliferative response of T cells from tumor-immune chickens to carcinogen-induced fibrosarcoma cells.

Immunity to carcinogen-induced transplantable fibrosarcomas (CHCT-NYU-4, -97, -36 and -20) in SC chickens was studied by the ability of spleen cells from NYU-4 or -97 immune chickens to proliferate in response to tumor cells in vitro. Spleen, but not peripheral blood cells, from NYU-4 immune chickens proliferated significantly more vigorously to gamma-irradiated NYU-4 cells than did cells from normal chickens. The proliferative response was not much affected by addition of indomethacin. Spleen cells from NYU-4-immune agammaglobulinemic (A gamma) chickens exhibit the same ability to proliferate in presence of gamma-irradiated NYU-4 tumor cells. Analysis of the phenotype of the T-cell component involved in proliferation showed that the proliferative response was significantly decreased by removal of CT4+ cells through indirect panning. Removal of CT8+ cells enhanced background proliferation without affecting the total thymidine incorporation in the presence of tumor cells. Immune spleen cells usually gave highest responses to the immunizing tumor, but also exhibited cross-reactivity to cells from other individual tumors induced by the same carcinogen.

Control of endogenous mouse mammary tumor virus superantigen expression in SJL lymphomas by a promoter within the env region.

SJL mouse lymphomas (reticulum cell sarcomas, or RCSs) of germinal center B cell origin express an endogenous mouse mammary tumor virus (mtv-29) superantigen (vSAg) that stimulates Vbeta16+ T cells to produce cytokines essential for RCS growth. Normal or LPS-activated SJL/J B cells contain two to three larger mRNAs for mouse mammary tumor virus-long terminal repeat (LTR) but not the 1.8-kb mRNA, which is prominent in RCS cells and encodes the vSAg-29. mRNAs from RCS and normal lymphoid cells were characterized by Northern hybridization using DNA probes from various regions of mtv-29, as well as by reverse transcription PCR, RNase protection, and primer extension. The larger mtv-29 transcripts, coding for envelope protein, are initiated in the 5' LTR, as expected. Surprisingly, the 1.8-kb mRNA, encoding the open reading frame of the LTR, is initiated in the middle of the env region and spliced in the 3' env. This is the first report of an mtv-vSAg transcript that is not controlled by promoter(s) located in the 5' LTR. The env initiation site appears identical to that of the mouse mammary tumor virus env transcriptional activator-directed PMA-induced defective LTR transcript in the C57BL6 T cell lymphoma, EL-4. The stimulus independence, B lymphoma specificity, and absence of deletions within either the 5' or 3' LTR regions of mtv-29 in RCS distinguish the situation in RCS cells from that in EL-4. These findings suggest that the novel mtv-29-vSAg transcript reflects an RCS-cell-specific regulation of transcription.

Evaluation of presence and functional activity of potentially self-reactive T cells in aged mice.

Autoimmunity is known to increase in aging. A possible factor could be an alteration in the T cell repertoire with advancing age. Antibodies to the variable region of the beta chain of the TCR activate T cells and can serve as probes for analysis of the T cell repertoire. We have used V beta 3 and V beta 17a antibodies to determine the presence and functionality of normally deleted T cells bearing potentially self-reactive TCR in peripheral lymphoid tissue and blood from aged (SJL/J x BALB/c) F1, LAF1 and BALB/c mice. Although an occasional 20- to 24-month-old mouse exhibited V beta 3+ or V beta 17a+ T cells in their lymph nodes or peripheral blood lymphocytes (PBL) slightly above the range for normal young mice of these I-E+ strains, there was no striking 'escape' from the normal thymic deletion process. However, responsiveness to anti-V beta 3 and anti-V beta 17a was slightly higher in aged, and particularly in aged thymectomized (TX), than in young mice. This was in contrast to proliferative responses to stimulation with antibody to the normally expressed V beta 8, which were lower in the lymph nodes from aged than from young mice. The PBL of some 30- to 36-month-old mice were also examined. Enhanced numbers of 'forbidden' V beta bearing T cells were seen more frequently at this age. In spite of the age-related decrease in overall CD4/CD8 T cell ratios in all organs, the mice with relatively high V beta 17a + T cells exhibited proportionally more CD4+ cells in that V beta population. We conclude that the 'forbidden' T cells that respond to anti-V beta stimulation in the 20- to 24-month-old mice are most likely to extra-thymic origin, since they were more readily detectable in aged TX mice. Potentially self-reactive Cd4 (and CD8) single-positive T cells were detectable in PBL only in very aged (30-36 months old) euthymic mice.