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1.  Reversal of Paralysis and Reduced Inflammation from Peripheral Administration of Amyloid-β in Th1- and Th17-Versions of Experimental Autoimmune Encephalomyelitis 
Science translational medicine  2012;4(145):145ra105.
β-amyloid-42 (Aβ42) and β-amyloid-40 (Aβ40), major components of senile plaque deposits in Alzheimer’s disease (AD), are considered neurotoxic and pro-inflammatory. In multiple sclerosis (MS), Aβ42 is upregulated in brain lesions and damaged axons. Here we found, unexpectedly, that treatment with either Aβ42 or Aβ40 peptides reduced motor paralysis and brain inflammation in four different models of experimental autoimmune encephalomyelitis (EAE) with attenuation of motor paralysis, reduction of inflammatory lesions in the central nervous system (CNS), and suppression of lymphocyte activation. Aβ42 and Aβ40 treatments were effective in reducing ongoing paralysis induced with adoptive transfer of either autoreactive Th1 or Th17 cells. High-dimensional 14-parameter flow cytometry of peripheral immune cell populations after in vivo Aβ42 and Aβ40 treatment revealed substantial modulations in the percentage of lymphoid and myeloid subsets during EAE. Major pro-inflammatory cytokines and chemokines were reduced in the blood following Aβ peptide treatment. Protection conferred by Aβ treatment did not require its delivery to the brain: adoptive transfer with lymphocytes from donors treated with Aβ42 attenuated EAE in WT recipient mice and Aβ deposition in the brain was not detected in treated EAE mice by immunohistochemical analysis. In contrast to the improvement in EAE with Aβ-treatment, EAE was worse in mice with genetic deletion of the amyloid precursor protein. Therefore, in the absence of Aβ there is exacerbated clinical EAE disease progression. Since Aβ42 and Aβ40 ameliorate experimental autoimmune inflammation targeting the CNS, we might now consider its potential anti-inflammatory role in other neuropathological conditions.
PMCID: PMC3677069  PMID: 22855462
2.  Basophil CD203c Levels Are Increased at Baseline and Can Be Used to Monitor Omalizumab Treatment in Subjects with Nut Allergy 
Rationale: Basophils contribute to anaphylaxis and allergies. We examined the utility of assessing basophil-associated surface antigens (CD11b/CD63/CD123/CD203c/CD294) in characterizing and monitoring subjects with nut allergy. Methods: We used flow cytometry to analyze basophils at baseline (without any activation) and after ex vivo stimulation of whole blood by addition of nut or other allergens for 2, 10, and 30 min. We also evaluated whether basophil expression of CD11b/CD63/CD123/CD203c/CD294 was altered in subjects treated with anti-IgE monoclonal antibody (omalizumab) to reduce plasma levels of IgE. Results: We demonstrate that basophil CD203c levels are increased at baseline in subjects with nut allergy compared to healthy controls (13 subjects in each group, p < 0.0001). Furthermore, we confirm that significantly increased expression of CD203c occurs on subject basophils when stimulated with the allergen to which the subject is sensitive and can be detected rapidly (10 min of stimulation, n = 11, p < 0.0008). In 5 subjects with severe peanut allergy, basophil CD203c expression following stimulation with peanut allergen was significantly decreased (p < 0.05) after 4 and 8 weeks of omalizumab treatment but returned toward pretreatment levels after treatment cessation. Conclusions: Subjects with nut allergy show an increase of basophil CD203c levels at baseline and following rapid ex vivo stimulation with nut allergen. Both can be reduced by omalizumab therapy. These results highlight the potential of using basophil CD203c levels for baseline diagnosis and therapeutic monitoring in subjects with nut allergy.
PMCID: PMC3214954  PMID: 20975283
Anaphylaxis; Atopic; CD203c; Cockroach allergen; Flow cytometry; Human basophils; Omalizumab; Peanut allergy; Tree nut allergy
3.  Altered phosphorylated signal transducer and activator of transcription profile of CD4+CD161+ T cells in asthma: Modulation by allergic status and oral corticosteroids 
Asthma is a complex immunologic disorder linked to altered cytokine signaling.
We tested whether asthmatic patients showed any change in cytokine-dependent signal transducer and activator of transcription (STAT) levels, focusing on the central/effector-memory CD4+CD161+ subset, which represents 15% to 25% of circulating T cells.
We quantified intracellular levels of active phosphorylated STAT (phospho-STAT) 1, 3, 5, and 6 by means of flow cytometry, without any activation or expansion.
Baseline phospho-STAT1 and phospho-STAT6 levels were increased in CD4+CD161+ T cells from asthmatic patients compared with those from healthy control subjects (by 10- and 8-fold, respectively). This asthma-associated alteration was both subset specific because no change was seen in CD4+CD161−CD25+ (regulatory T cells) and CD4+CD161−CD25− subsets and isoform specific because phospho-STAT5 and phospho-STAT3 levels were unchanged. Among asthmatic patients, phospho-STAT1 and phospho-STAT6 levels correlated negatively with each other, suggesting antagonistic regulation. Oral corticosteroid (OCS) treatment significantly decreased phospho-STAT6 and IL-4 levels but not phospho-STAT1 levels. Disease parameters showing significant correlations with phospho-STAT1, phospho-STAT6, or both included age at onset, plasma IgE levels, and levels of the TH2 cytokines IL-4 and IL-10 and the TH1 cytokine IL-2. Overall, combined phospho-STAT1 and phospho-STAT6 measurements showed excellent predictive value for identifying (1) asthmatic patients versus healthy control subjects, (2) allergic versus nonallergic asthmatic patients, and (3) asthmatic patients taking versus those not taking OCSs.
Baseline changes in phospho-STAT1 and phospho-STAT6 levels in blood CD4+CD161+ T cells identify asthmatic patients and mirror their allergic status and response to OCSs.
Clinical implications
These results confirm the pathologic importance of activated STAT1 and STAT6 in asthma and suggest their potential use as clinical biomarkers.
PMCID: PMC2679255  PMID: 17919711
Allergy; atopy; fluorescence-activated cell sorting; immune polarization
4.  Modern Flow Cytometry: A Practical Approach 
Clinics in laboratory medicine  2007;27(3):453-v.
In this presentation, we outline ways in which current users of Fluorescence Activated Cell Sorting (FACS) can get more from their FACS work without undue effort. FACS technology development, and the emergence of new software support for various aspects of this technology are now cooperating in this effort.
The demonstration that CD4 T cell counts can be used to monitor HIV disease progression opened the way to the first clinical FACS application; the demonstration that stem cells can be sorted and transferred to appropriately pre-treated recipients now opens the way to new and constructive FACS uses in the future.
We hope that our readers join us in helping to achieve the goal of seeing more and better FACS data in the future.
PMCID: PMC1994577  PMID: 17658402
5.  B Lineage–specific Regulation of V(D)J Recombinase Activity Is Established in Common Lymphoid Progenitors 
Expression of V(D)J recombinase activity in developing lymphocytes is absolutely required for initiation of V(D)J recombination at antigen receptor loci. However, little is known about when during hematopoietic development the V(D)J recombinase is first active, nor is it known what elements activate the recombinase in multipotent hematopoietic progenitors. Using mice that express a fluorescent transgenic V(D)J recombination reporter, we show that the V(D)J recombinase is active as early as common lymphoid progenitors (CLPs) but not in the upstream progenitors that retain myeloid lineage potential. Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP. By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in ∼5% of splenic natural killer cells. Finally, we show that recombinase activity in CLPs is largely controlled by the Erag enhancer. As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.
PMCID: PMC2211824  PMID: 14769852
B lymphopoiesis; V(D)J recombination; lineage restriction; hematopoiesis; stem cell; transcription
6.  The regulation of CD5 expression in murine T cells 
CD5 is a pan-T cell surface marker that is also present on a subset of B cells, B-1a cells.Functional and developmental subsets of T cells express characteristic CD5 levels that vary over roughly a 30-fold range. Previous investigators have cloned a 1.7 Kb fragment containing the CD5 promoter and showed that it can confer similar lymphocyte-specific expression pattern as observed for endogenous CD5 expression.
We further characterize the CD5 promoter and identify minimal and regulatory regions on the CD5 promoter. Using a luciferase reporter system, we show that a 43 bp region on the CD5 promoter regulates CD5 expression in resting mouse thymoma EL4 T cells and that an Ets binding site within the 43 bp region mediates the CD5 expression. In addition, we show that Ets-1, a member of the Ets family of transcription factors, recognizes the Ets binding site in the electrophoretic mobility shift assay (EMSA). This Ets binding site is directly responsible for the increase in reporter activity when co-transfected with increasing amounts of Ets-1 expression plasmid.
We also identify two additional evolutionarily-conserved regions in the CD5 promoter (CD5X and CD5Y) and demonstrate the respective roles of the each region in the regulation of CD5 transcription.
Our studies define a minimal and regulatory promoter for CD5 and show that the CD5 expression level in T cells is at least partially dependent on the level of Ets-1 protein. Based on the findings in this report, we propose a model of CD5 transcriptional regulation in T cells.
PMCID: PMC32207  PMID: 11389772
7.  Thioredoxin, a Redox Enzyme Released in Infection and Inflammation, Is a Unique Chemoattractant for Neutrophils, Monocytes, and T Cells  
The Journal of Experimental Medicine  1999;189(11):1783-1789.
Thioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca2+ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1–infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.
PMCID: PMC2193090  PMID: 10359582
chemotaxis; thioredoxin; HTLV-1; migration
8.  Interleukin-1β Converting Enzyme–like Protease Involvement in Fas-induced and Activation-induced Peripheral Blood T Cell Apoptosis in HIV Infection. TNF-related Apoptosis-inducing Ligand Can Mediate Activation-induced T Cell Death in HIV Infection  
The Journal of Experimental Medicine  1997;186(8):1365-1372.
Apoptosis of peripheral blood T cells has been suggested to play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. Spontaneous, Fas (CD95)–induced and activation-induced T cell apoptosis have all been described in peripheral blood mononuclear cell cultures of HIV-infected individuals. We have previously shown that activation-induced T cell apoptosis is Fas independent in peripheral blood T cells from HIV+ individuals. In this study, we extend and confirm these observations by using an inhibitor of interleukin-1β converting enzyme (ICE) homologues. We show that z-VAD-fmk, a tripeptide inhibitor of ICE homologues, can inhibit Fas-induced apoptosis of peripheral blood CD4+ and CD8+ T cells from asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)– induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter flow cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas independent and could be inhibited with a blocking monoclonal antibody to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a recently described member of the TNF/nerve growth factor ligand family. The above findings show that Fas-induced T cell apoptosis is ICE dependent in HIV infection. AICD can be blocked by ICE inhibitors in some patients, and this AICD is mediated by TRAIL. These results show that TRAIL can be a mediator of AICD in T cells. These different mechanisms of peripheral blood T cell apoptosis may play different roles in the pathogenesis of HIV infection.
PMCID: PMC2199088  PMID: 9334376
The Journal of Experimental Medicine  1974;140(6):1660-1675.
In order to further delineate the mechanisms underlying genetic unresponsiveness, tetraparental mice were constructed from immune response-1A gene high responder and low responder parental genotypes, then were immunized with poly-L-(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys ((T,G)-A--L). An analysis of the total serum allotype mixture and of the antigen-binding capacity of the separated allotypes demonstrated that in the milieu of a tetraparental mouse, both high and low responder B cells could be stimulated equally to produce identical high titered anti-(T,G)-A--L responses. Furthermore, these studies show that effective stimulation could occur across a histocompatibility disparity.
PMCID: PMC2139758  PMID: 4139235
Cells binding DNP groups conjugated to fluoresceinated mouse gamma globulin (FDNP-MGG) were isolated from spleens of unprimed mice using a fluorescence-activated cell sorter (FACS). The isolated cells were specifically enriched at least 100-fold for anti-DNP precursor activity in an adoptive transfer assay as compared to unfractionated spleen. The fraction depleted of binding cells, although depleted of anti-DNP precursor activity, responded as well as unfractionated spleen when assayed for anticarrier (keyhole limpet hemocyanin [KLH]) precursor activity. High avidity binding cells were stained using low concentrations of FDNP-MGG. Medium and low avidity binding cells were stained using high concentrations of FDNP-MGG in the presence of free hapten which selectively blocked staining of the high avidity binding cells. Cells were supplemented with an excess of carrier-primed (KLH), nylon-purified splenic T cells and transferred to irradiated recipients. DNP-KLH was given at transfer and 5 days later. The anti-DNP plaque-forming cell (DNP-PFC) response and the avidities of the DNP-PFC in the irradiated recipients were measured by hapten inhibition of direct PFC plaque formation 12 days after transfer. At this time, very few indirect PFC were found. There was a positive correlation between the avidity of the DNP-binding cells and the avidity of the anti-DNP antibody secreted by their progeny. High avidity DNP-binding cells gave rise to predominantly high avidity anti-DNP-PFC. Medium and low avidity binding cells gave rise to medium and low avidity DNP-PFC.
PMCID: PMC2139630  PMID: 4139227
To determine whether or not B lymphocytes are committed to the synthesis of a single immunoglobulin heavy chain isotype during their differentiation into plasma cells, rabbit lymph node and Peyer's patch cells were separated into populations with and without membrane IgM, using a fluorescence-activated cell sorter (FACS). The potential of the µ-bearing (µ+) and non-µ-bearing (µ-) cells to give rise to plasma cells both in vivo after transfer into irradiated recipients and in vitro in the presence of pokeweed mitogen was assessed by immunofluorescence techniques, and the relative proportions of the cytoplasmic Ig-stained cells (CSC) synthesizing each class of heavy chains were determined. Most of the CSC arising in vitro from µ-bearing lymph node and Peyer's patch cells contained IgM; all IgM CSC appeared to be derived from µ+ cells. Peyer's patch lymphocytes, however, did not generate IgM CSC after cell transfer and thus may be functionally different from lymph node µ+ cells. It was found also that nearly all of the many IgA CSC generated by Peyer's patch lymphocytes either in culture or after transfer were derived from µ- cells. Further fractionation of these µ- cells with the FACS after they had been membrane stained with anti-b locus allotype reagents revealed that the precursors of IgA CSC belong to a minor population of cells which do have b locus light chain determinants on their membranes, although they do not have detectable µ-chains. These cells are not found in lymph nodes. Although the majority of Peyer's patch and lymph node cells were found to be precommitted to the synthesis of a single heavy chain isotype, a small proportion of cells may not be similarly restricted. Some of the CSC with membrane IgM were found to contain cytoplasmic IgA or IgG. In addition, µ+ populations did give rise to low numbers of IgA and IgG CSC. The implications of these results, obtained under experimental conditions, on the normal differentiation of B lymphocytes in situ are discussed.
PMCID: PMC2139601  PMID: 4603013
Lymphocytes from b5/b9 rabbits were stained in suspension with fluorescent antiallotype antibody reagents to selectively label with fluorescent molecules those cells bearing membrane immunoglobulin (Ig) of the b5 or b9 allotype. After staining, the cells were separated by the fluorescence-activated cell sorter into populations markedly enriched in cells bearing b5 or b9 membrane Ig or totally depleted of cells with detectable membrane Ig. The potential of these separated cells to give rise to Ig-synthesizing plasma cells either in vivo after transfer into irradiated recipients or in vitro during culture in the presence of phytohemagglutinin or pokeweed mitogen was assessed by immunofluorescence. The relative proportion of b5 and b9 cytoplasmic Ig-stained cells (CSC) arising from the separated cells was determined to test directly whether B lymphocytes and their progeny are committed to the synthesis of Ig of one allotype. It was found that b5- and b9-bearing cells gave rise almost exclusively to b5- and b9-producing plasma cells, respectively, in both the in vivo and in vitro assay systems. Most of these CSC were probably not derived from previously existing CSC but arose as the result of the differentiation of lymphocytes with membrane Ig. When cell populations totally depleted of Ig-bearing lymphocytes were cultured, very few CSC were found, indicating that the majority of immediate precursors of CSC have membrane Ig. These results suggest that individual B cell clones are phenotypically restricted to the expression of immunoglobulin genes on one chromosome; the significance of this clonal allelic exclusion is discussed.
PMCID: PMC2139550  PMID: 4591172
The Journal of Experimental Medicine  1973;137(6):1311-1324.
Thymus-derived cells (T cells) that actively suppress production of IgG2a immunoglobulins carrying the Ig-1b allotype have been found in adult (SJL x BALB/c)F1 mice exposed to anti-Ig-1b early in life. The suppression is specific for Ig-1b. The allelic product, Ig-1a, is unaffected. Spleen, lymph node, bone marrow, or thymus cells from suppressed mice suppress production of Ig-1b by syngeneic spleen cells from normal F1 mice. When a mixture of suppressed and normal cells is transferred into lethally irradiated BALB/c mice, there is a short burst of Ig-1b production after which Ig-1b levels in the recipient fall rapidly below detectability. Pretreatment of the cells from the suppressed mice with antiserum specific for T cells (anti-Thy-1b) plus complement before mixture destroys the suppressing activity. Similar results with suppressor cells were obtained in vitro using Mishell-Dutton cultures. Mixture of spleen cells from suppressed animals with sheep erythrocyte (SRBC)-primed syngeneic normal spleen before culture suppresses Ig-1b plaque-forming cell (PFC) formation while leaving Ig-1a PFC unaffected. Treatment of the suppressed spleen with anti-Thy-1b before transfer removes the suppressing activity.
PMCID: PMC2139347  PMID: 4541122
The Journal of Experimental Medicine  1970;131(6):1109-1120.
Congenic mice, differing genetically only at the loci coding for immunoglobulin H chain (or Fc) structures, have been used to study cell interactions in the 7S (γG2a) antibody response to sheep erythrocytes (SRBC), as detected by the Jerne plaque-forming cell (PFC) method. The interaction between thymus and bone marrow cells was studied in adult thymectomized irradiated recipients, protected with syngeneic bone marrow and injected with thymus cells from the partner congenic strain. All of the γG2a PFC detected in the spleens of these mice were of bone marrow allotype. Adoptive secondary immune responses were then studied to determine whether a similar interaction between memory cells and bone marrow derived cells could be detected. Primed spleen cells from the partner congenic strain, or a subpopulation of these cells obtained by BSA density gradient fractionation, were injected into irradiated recipients alone, or together with syngeneic nonimmune spleen or bone marrow cells. All γG2a PFC detected in these experiments were of primed cell allotype. There was no evidence that antibody forming cell precursors in normal spleen or bone marrow participate in the adoptive secondary immune response detected 7 days after transfer of primed spleen cells. This was true regardless of whether the bone marrow cells were injected at the time of transfer, or were injected 1–2 wk earlier and allowed to become established in the spleens of recipient mice. Although no specific cell interaction was seen, bone marrow (and, to a lesser degree, normal spleen) cells were found to have a nonspecific enhancing effect on the adoptive secondary response when they were injected together with the primed spleen cells. This enhancement was not evident if the bone marrow cells were injected 1 or 2 wk prior to primed cell transfer.
PMCID: PMC2138845  PMID: 4192568
The Journal of Experimental Medicine  1970;131(6):1093-1108.
Plaque forming cells (PFC) of different immunoglobulin classes producing antibodies against sheep erythrocytes were separated according to their buoyant densities by means of equilibrium centrifugation in a stepwise BSA gradient. In the period of 7–10 days after immunization γM PFC are markedly enriched in fractions of low density and relatively depleted in fractions of high density. The distribution of total γG PFC shows less enrichment in the lower density fractions and less depletion in the higher density fractions. The density profile for γG2a PFC is even flatter, with a significant difference (depletion) relative to the unseparated spleen cells only in the highest density fraction. The density gradient distributions of cells able to transfer an adoptive immune response of the various immunoglobulin classes are markedly different from the PFC distribution. Cells obtained 7–10 days after immunization able to transfer an IgM response are present in the same proportions across the density gradient, whereas memory cells for γG2a obtained at this time are markedly enriched in fractions of low density and virtually depleted from high density fractions. With increasing time after primary immunization, the γG2a memory cells increase progressively in density and by 6 weeks the higher and lower density fractions have the same proportions of γG2a memory cells. The total γG (mainly γG1) memory cells by 7–10 days show slight enrichment in low density fractions and no depletion in high density fractions. The conclusions were reached that (a) memory for γG1 develops earlier than memory for γG2a and (b) that memory for anti-SRBC antibodies of different classes is carried in separate cells. When gradient fractions enriched for PFC and memory cells for all classes were completely depleted of PFC using glass bead columns, the ability of this fraction to transfer memory for all classes was not diminished. This shows that memory cells are not identical with cells secreting antibodies.
PMCID: PMC2138841  PMID: 4192567
In the mouse, antibody directed against an immunoglobulin allotype, Ig-1b, passed from mother to offspring or injected into neonates, suppresses synthesis of immunoglobulin carrying Ig-1b. In allotype homozygotes as well as heterozygotes the allotype suppression is manifested both by a delay of several weeks in attaining initial detectable allotype levels and a reduction in allotype level continuing into adulthood. A possible mechanism for the differentiation of the immune system consistent with both the kinetics of suppression reported here for the mouse and the comparatively longer lived and more complete allotype suppression described for the rabbit is discussed. Evidence for a strong intralitter (as opposed to interlitter) correlation of age of onset of immunoglobulin allotype synthesis is presented.
PMCID: PMC2138387  PMID: 4168099
Further analysis of the isoantigens (allotypes) of 2 classes of normal mouse immunoglobulins, γG2a and γG2b, has shown a minimum of 10 specificities for the Ig-1 locus (controlling γG2a-antigens) and 3 specificities for the Ig-3 locus (controlling γG2b-antigens). Three γG2-myeloma proteins of plasma cell tumors induced in (NZB x BALB/c)F1 mice have been analyzed for the isoantigens they carry. NZB mice are genotypically Ig-1e Ig-3e, while BALB/c are Ig-1a Ig-3a. Two of the myeloma proteins are γG2a-globulins. One of these, GPC-7, carries all the isoantigenic specificities of the Ig-1e allele while the other, GPC-8, carries all the isoantigenic specificities of the Ig-1a allele. Thus only one of the parental alleles of the mouse in which the tumor arose is expressed in each of these myeloma proteins. The third myeloma protein GPC-5, also carries the antigens of only one parental strain (NZB). However GPC-5, a γG2b-globulin, carries only one of the Ig-3 specificities normally associated with γG2b-globulins of NZB. Most remarkably it also carries one Ig-1 specificity normally associated with γG2a-globulins of NZB. This is the first analyzed mouse myeloma shown (a) to express some but not all the antigenic specificities normally associated with an allele and (b) to carry antigenic specificities controlled by two distinct immunoglobulin loci. The implications of these findings are discussed in relation to the genetic control of immunoglobulins.
PMCID: PMC2180467  PMID: 4160400
Eight antigens of 7S γ2-immunoglobulins controlled by alleles at a single locus Ig-1, have been identified in mice. This locus has previously been shown to determine antigenic specificities on the F fragments of 7S γ2a-globulins. The reactions of these antigens with various isoantisera have shown that the antigens all cross react with one another. New methods for the analysis of antigenic specificities of soluble proteins are presented in detail. A sensitive method for detecting in the order of 0.01 µg of these isoantigens has been developed, based on the quantitative inhibition of precipitation of I125-labeled antigen. Cross-reactions of the antigens were analysed in inhibition assays and the data is compatible with the existence of a minimum of eight antigenic specificities. Each of the antigens is composed of different combinations of these specificities, with only one antigen having a specificity not present in any other. Sixty-eight mouse strains have been tested with specific isoantisera, and on the basis of the results, have been placed into the eight allele groups. Evidence for close genetic linkage of the Ig-1 locus and 11 chromosome markers has been sought and not found.
PMCID: PMC2137960  PMID: 14270242
The isoantigenic phenotype of the H-2 locus has been detected by isohemagglutinin absorption in a line of mouse lymphoma cells growing continuously in culture for 6 years and in two established lines of fibroblastic mouse cells growing continuously in culture for 1 year. Quantitative absorption studies suggest that the concentration of H-2 isoantigens is higher in the cultured lymphoma cells than in the other two fibroblastic cell lines.
PMCID: PMC2137608  PMID: 14018308
When long term cultures of mouse lymphoma cells, known to possess the isoantigenic phenotype determined by the H-2d allele, are incubated with anti H-2d isoantibody and guinea pig complement, slightly more than 99 per cent of cells are killed under optimal conditions. Growth in mass culture and colony formation by single cells after incubation with isoantibody and complement are employed to assess the cytotoxic effect. The cytotoxic action of isoantibody is complement-dependent, for viability of cells exposed to antibody alone is unaltered. When excess isoantibody and optimum concentrations of complement are used, killing begins as soon as these reagents are mixed with the cells, and no further killing occurs after 5 to 15 minutes at 37°C. About 80 per cent of cells are killed with an isoantiserum containing antibody to two isoantigenic components of the H-2d complex. That the cytotoxic action is mediated through the H-2 isoantigen is shown by (a) isoantiserum containing only anti H-2d antibody produces maximal cell killing, and (b) isoantiserum from which anti H-2d antibody has been removed by absorption loses all cytotoxic activity. Variant cells resistant to the cytotoxic action of anti H-2d isoantibody were isolated from lymphoma cell populations surviving multiple exposures to isoantibody and complement. These variants can be distinguished morphologically from the isoantibody-sensitive parent cell line. Although variants are resistant to anti H-2d isoantibody, these cells possess H-2d isoantigen but in a lower concentration than found in cells of the parent line. The basis for resistance to cytotoxic isoantibody is discussed.
PMCID: PMC2137607  PMID: 14018309

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