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INTRODUCTION: Critically ill patients are at high risk of nosocomial infections, with between 20% and 40% of patients admitted to the intensive care unit (ICU) acquiring infections. These infections result in increased antibiotic use, and are associated with morbidity and mortality. Although critical illness is classically associated with hyperinflammation, the high rates of nosocomial infection argue for an importance of effect of impaired immunity. Our group recently demonstrated that a combination of 3 measures of immune cell function (namely neutrophil CD88, monocyte HLA-DR and % regulatory T cells) identified a patient population with a 2.4-5-fold greater risk for susceptibility to nosocomial infections.METHODS AND ANALYSIS: This is a prospective, observational study to determine whether previously identified markers of susceptibility to nosocomial infection can be validated in a multicentre population, as well as testing several novel markers which may improve the risk of nosocomial infection prediction. Blood samples from critically ill patients (those admitted to the ICU for at least 48 hours and requiring mechanical ventilation alone or support of 2 or more organ systems) are taken and undergo whole blood staining for a range of immune cell surface markers. These samples undergo analysis on a standardised flow cytometry platform. Patients are followed up to determine whether they develop nosocomial infection. Infections need to meet strict prespecified criteria based on international guidelines; where these criteria are not met, an adjudication panel of experienced intensivists is asked to rule on the presence of infection. Secondary outcomes will be death from severe infection (sepsis) and change in organ failure.ETHICS AND DISSEMINATION: Ethical approval including the involvement of adults lacking capacity has been obtained from respective English and Scottish Ethics Committees. Results will be disseminated through presentations at scientific meetings and publications in peer-reviewed journals.TRIAL REGISTRATION NUMBER: NCT02186522; Pre-results.

Natural Regulatory T cells (Tregs) are defined by stable expression of the cell surface proteins CD4 and CD25, low surface expression of CD127 and expression of the transcription factor FOXP3. The contribution of Treg to the prevention of autoimmunity and the maintenance of immune homoestasis is the subject of ongoing interest, as alterations in Treg numbers and function are implicated in a wide range of diseases. The in vitro benchmark for determining Treg function is suppression of proliferation of unmatched effector T cells in a mixed lymphocyte reaction (MLR) over a 3-6-day time period. As an alternative to this assay, we show that a 7-h CD154 expression assay is rapid, simple and provides a reliable readout of suppressor function. Using multiple Treg-like cell types including natural (n)Treg, inducible (i)Treg and Treg cell lines, we show that suppression of CD154 expression is a surrogate for suppression of proliferation. We propose this as a suitable alternative to the MLR assay, as it is rapid and may be more amenable to high-throughput screening, analysing large cohorts of clinical samples or assaying transiently suppressive populations.

Advancements in cell analysis capabilities have significantly expanded our understanding of the complexity of immunological systems. As the need for deeper analysis has increased, many laboratories have employed a set of lineage markers to define a cell population, followed by the addition of unique markers specific to their biological question. Utilizing regulatory T cells (Treg) as a model population, we describe the design of an 8-color backbone panel on a 12-Color BD FACSLyric™ flow cytometer that can be supplemented with 4 drop-in markers (colors) to characterize two critical facets of Treg biology: activation and homing. Intelligent panel design enabled identification of human naïve and activated Treg cells utilizing established Treg markers (CD3, CD4, CD25, CD127, FoxP3, CD45RA). CD15s and CD161 were included in the 8-color backbone panel to identify functionally suppressive effector and/or pro-inflammatory cytokine secreting Tregs. Importantly, addition of a 4-color drop-in activation panel (PI16, CD147, CD39 and HLA-DR) or a 4-color drop-in homing panel (CCR4, CCR6, CXCR3 and CD31) did not impact resolution of the backbone Treg population(s), while providing new and interesting insights into Treg biology. We highlight the utility of a modular panel design approach that provides an efficient, scalable, and standardized solution for complex analysis of essentially any cell population of interest.

The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice. Restimulation of Tregs after 8–12 days of culture with CD28 costimulation in the presence of rapamycin resulted in >1000-fold expansion of Tregs in

A spontaneously arising murine plasmacytoma, HPC-202, derived from a BALB/c.H-2 b congenic mouse that lacks any detectable H-2 determinants on its cell surface is described. However, the expression of H-2 determinants is inducible by interferon-γ. The H-2 negative cell surface phenotype permits the HPC-202 tumor to escape H-2 allospecific cytotoxic cell lysis but not NK cell lysis, as well as to grow, to varying degrees, in some H-2 incompatible hosts. In those strains which exhibit a resistance to HPC-202 growth, resistance does not map to a single gene within the major histocompatibility complex of the mouse. Resistance is also radiosensitive and is therefore presumably due to a rapidly dividing cell population. The utility of this tumor as a model system to study both the non-H-2-restricted natural resistance to tumor growth, and the mechanism by which H-2 genes are regulated by cells is discussed.

Introduction: CD23 is a 45-kDa transmembrane glycoprotein corresponding to the low-affinity receptor for the immunoglobulin E (IgE). There are two isoforms - CD23a expressed on B-cells, and CD23b primarily expressed on monocytes, T-cells and platelets. The CD23 protein can be cleaved and released into the serum as a soluble form of CD23 (sCD23). The sCD23 is a 25kDa fragment that can be found in serum, plasma and urine in patients with chronic lymphocytic leukemia. Objective: To evaluate a novel and robust method of sCD23 detection using CBA detection, and correlate with known markers of prognostication and clinical outcome. Methods: At Barts & The London School of Medicine, historical serum samples have been taken at diagnosis from patients with CLL between 1984 to 2012 with corresponding clinical data. Serum analysis was obtained from 60 healthy controls and assessed for sCD23 in ng/ml (France and Canada). sCD23 levels (ng/ml) were evaluated against known prognostic markers and clinical features, including: ZAP-70 (>20%), CD38 (>20%), B2M, cytogenetics (17p-/11q- vs 12+/13q-/normal), LDH, splenomegaly, hepatomegaly, and lymphadenopathy. 107 samples from 86 patients underwent retrospective assessment of sCD23 from stored serum. The sCD23 cytometric bead array (CBA) flow cytometric assay (BD Biosciences) captures the soluble analyte with beads of known size and fluorescence.1 Capture beads are identified and sCD23 quantification (in ng/ml) is performed based on PE fluorescence excitation. Results were acquired using FACSDiva (BD Biosciences) and analysed using FCAParray. Statistical analysis was performed and significance was set at Results: sCD23 levels from the 86 patients ranged from 1.92 to 2441 ng/ml with a median value of 234.5 ng/ml. The sCD23 levels from the healthy control group ranged from 0.43 to 5.16 ng/ml with a median value of 1.56 ng/ml (figure 1). Figure 1: sCD23 levels between healthy controls and patients Figure 1:. sCD23 levels between healthy controls and patients Within the CLL patient cohort, elevated sCD23 significantly correlated with 6 out of the 8 prognostic & clinical indicators (Table 1). Table 1: Median sCD23 (ng/ml) for CLL patients by prognostic risk stratification. Indicator Median sCD23 (ng/ml) Level of significance (MWU-test) ZAP-70 positive (n=19) 1,096.0 P In addition, there was a strong correlation with response to therapy. Those patients who never required therapy (n=21) had significantly lower levels of sCD23 (mean 93.36ng/ml) over those who achieved a partial remission (n=21, mean 650.2 ng/ml p Figure 2: Mean sCD23 level (ng/ml) at diagnosis against response to therapy when required. Figure 2:. Mean sCD23 level (ng/ml) at diagnosis against response to therapy when required. Two patients had sequential serum samples taken throughout therapy. The patient with elevated sCD23 at diagnosis remained elevated through each treatment cycle to only achieve partial response, whilst the patient with low sCD23 at diagnosis responded well to therapy and achieved a complete remission (p=0.01, Figure 3). Figure 3: sCD23 following therapy. sCD23 non-responder did not respond to FC or FCR therapy . The responder received Chlorambucil+R then FCR. Figure 3:. sCD23 following therapy. sCD23 non-responder did not respond to FC or FCR therapy . The responder received Chlorambucil+R then FCR. Conclusion: We have evaluated in a pilot study the sCD23 CBA assay, which is an easy and reproducible assay. Elevated levels of sCD23 at diagnosis correlated with “physical” prognosis, as well as known biological markers of prognostication. Those patients with elevated levels of sCD23 at diagnosis correlated with response to therapy regardless of therapy given, and there is evidence that patients who do not respond to therapy retain their elevated levels of sCD23 during therapy. This simple assay could provide the basis for individualised risk stratification and prediction of response to therapy based on an individual analyte. Ref: 1 Grelier A et al. Cytometry B. 2014 Mar;86(2):91-7. Disclosures Farren: BD Biosciences: Research Funding. Nauwelaers:BD Biosciences: Employment. Keenan:BD Bioscience: Employment. Warner:BD Biosciences: Employment. Agrawal:BD Biosciences: Research Funding.

Introduction Over the past decade, treatments for patients with chronic lymphocytic leukemia (CLL) have produced complete remissions (CR) without evidence for minimal residual disease (MRD), particularly for younger and/or fitter patients. In this setting, achieving an MRD-negative CR has prognostic implications, yielding longer progression free survival (PFS) and overall survival (OS) than for patients who achieve a CR with persistent MRD. Complicating efforts to incorporate testing for MRD in clinical practice has been the lack of a defined consensus on the methods of MRD detection. In this study, we report on a novel combination of mAbs for MRD detection by flow cytometry based on two antigens: the NK-cell receptor and tumor specific antigen, CD160; and the tumor associated antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR-1). Objective To compare a novel single-tube, tumor-specific (CD160+ROR1) targeted approach to MRD detection against the previously published CD160 flow cytometric assay (CD160FCA) (Farren et al, 2011) and the new, single-tube 8-color ERIC assay. Methods Between October 2012 and July 2013, prospective assessment of MRD was performed on peripheral blood in 56 patients (86 samples). We developed a flow cytometric assay using mAbs specific for CD160 or ROR1 (Fukuda et al, 2008). For this we used the following mAb from BD Biosciences: CD2 FITC, CD5 Pe-Cy7, CD19 PerCP5.5, CD45V500, CD160PE, ROR-1 AF647 (“ROR-160FCA”) and a sequential gating strategy. This was compared with CD160FCA and the 8-color ERIC consortium protocol (unpublished). Light chain analysis (LCR) was performed in all cases and reported where detectable. A proof of concept spiking experiment simulating MRD was prepared by mixing CLL and normal peripheral blood leukocytes in a serial dilution to a level of 10-5(n=3). Statistical analysis was performed using Spearman Rank correlation coefficients, Mann-Whitney t-test, and Bland-Altman method comparison. Significance was set at Results To establish the proof of principle, MRD levels ranging from 0.001% to 100% (Neat CLL) were prepared by serial dilutions, in which MRD levels could be established by the ROR-160FCA to 10-5 (n=3). Assessment of the observed incidence against expected incidence of CLL MRD demonstrated a highly significant correlation (R2=0.96, p=0.01). In the study, the range of detectable disease went from 54 samples had levels of disease Conclusion Monitoring minimal residual disease in CLL is a key focus for clinical trials, as MRD is an important prognostic marker in CLL in terms of PFS and OS. Here we provide a single tube assay, ROR-160FCA, which is unique by targeting two antigens restricted to malignant B-cells, CD160 and ROR1. ROR-160FCA is equivalent in MRD detection compared to both CD160FCA and the current ERIC assay under development. The two tumor-specific antigens give ROR-160FCA the potential for improved sensitivity, particularly where limited sample is available. Furthermore, it only requires a simple sequential gating strategy, is rapid, and appears more cost effective than other Methods. References Farren TW, Giustiniani J, Liu FT et al. Blood. 2011;118 (8):2174-2183. Fukuda T, Chen L, Endo T et al. Proc Natl Acad Sci U S A. 2008;105 (8):3047-3052. Disclosures: Farren: BD Biosciences: Research Funding. Warner:BD Biosciences: Employment, Research Funding. Agrawal:BD Biosciences: Research Funding.

The binding of murine IgG1, IgG2a, IgG2b, IgG3 protein molecules to Staphylococcal A protein is presented. Differential elution by sodium thiocyanate gradients is described. Proteins of the IgG2 class (either a or b subclass) require between 1.5 and 2.0 M for complete elution, whereas, IgG1 proteins are fully eluted with only 0.5 M NaSCN. These differential elution patterns can be utilized to distinguish between, or prepare proteins of different Ig classes. It is proposed that this quantitative rather than qualitative distinction between Ig subclass binding to Staph A, indicates the existence of multiple binding sites per molecule, with different markers of such sites being present on the heavy chain of the different Ig classes.

Normal C3H bone marrow cells were grown 7 days in medium containing L cell-derived colony stimulating factor-1 (CSF-1). During the first 4 days of culture, erythroid and granulocytic cells decreased while macrophages increased exponentially with a doubling time of about 31 hr. Only 0.3% of all cells in the initial bone marrow suspension formed discrete colonies of mononuclear phagocytes, but by day 6 60% of the nonadherent cells were capable of forming macrophage colonies, representing a 200-fold enrichment of the original progenitor population. Using flow cytometry, mononuclear phagocytes obtained after 4 days of culture were separated into two distinct phenotypes based on their autofluorescence. Nonadherent cells were a discrete population of small cells exhibiting low autofluorescence, and the adherent cells were a broad heterogenous population of large cells exhibiting high autofluorescence. A panel of currently available rat monoclonal antibodies (MABs) against murine hematopoietic cells were used to determine whether unique subsets of macrophages could be resolved. The MABs RA 31B6 and H-11 stained virtually all the nonadherent cells but not adherent cells. The MABs E-2 and 11–4.1 (anti-H-2Kk) stained almost all the adherent cells and demonstrated no significant staining of nonadherent cells. Nearly all the nonadherent and adherent cells were stained by the MABs DNL 4.4 and MAC-1. Additionally, the data suggest that the epitopes for MAC-2 and MAC-3 and γ2a Fc receptors develop late in nonadherent progenitor cells as they mature into adherent macrophages.

Simultaneous dual immunofluorescence and flow cytometry was used to study sixteen lymphocyte phenotypes in 209 men including: healthy homosexuals, lymphadenopathy patients (LAN), and AIDS patients. Significant differences between the distribution of lymphocytes in healthy homosexuals and healthy heterosexuals were decreased percentages of helper/inducer T cells (Leu 3), increased cytotoxic/suppressor T cells (Leu 2), and consequently a decreased Leu 3/Leu 2 ratio. The increased Leu 2 cells were identified as functionally cytotoxic subset Leu 2+ 15- phenotype rather than suppressor cells which are Leu 15+. Leu 2 and Leu 3 bearing cells exhibited an excess of membrane-bound immunoglobulins which were easily elutable at 37 degrees C. An increased percentage of an HLA-DR framework determinant bearing T cells were also detected. Within the NK cell family, Leu 7 cells were moderately increased and the functionally unidentified Leu 2+ 7+ population was strikingly elevated. LAN or AIDS patients were compared to healthy homosexual controls. Lower percentages of Leu 3 cells and higher percentages of Leu 2 cells were evident in LAN patients. These subsets were similar in LAN and AIDS patients. The increase in Leu 2+ cells was due to the Leu 2+ 15- cytotoxic subset. Fewer T cells had immunoglobulin in LAN and AIDS. A definite increase in Leu 2+ DR+ cells but not Leu 3+ DR+ cells occurred in AIDS compared to LAN or healthy controls. NK cell changes already present in healthy homosexuals persisted in LAN and AIDS patients. No differences in the distribution of B cells was detected in any intergroup comparisons. Changes in monocytes or pan-T cells were relatively insensitive measures of immunologic alterations among any of the groups. These results indicate many of the changes in lymphocyte subsets seen in AIDS and LAN subjects are already present in a carefully screened population of healthy homosexuals in San Francisco. Many of the changes in Leu 2 and NK family of cells suggest a possible adaptive response to viral or neoplastic challenge. Whether these interesting phenotypic alterations relate to functional changes in response to such challenge of the identified subsets waits further investigation.

Allotypic analysis of IgG 2 anti-red cell autoantibodies in NZB hybrid mice was performed by the Coombs (antiglobulin) test using alloantisera to mouse immunoglobulins. The results in 34 of 35 mice showed that only one parental allele was expressed in the autoantibody population of any given mouse. Either allele could be expressed and no preference for either parental type was evident. These results are held to favor the concept of a monoclonal or restricted origin of the autoantibody producing cell population, and do not indicate a role for allotype linked immune response gene control in autoantibody production.

Monoclonal antibody (mAb) anti-Leu M5 reacts with a two-chain molecule composed of a 150-kDa alpha subunit noncovalently associated with a 95-kDa beta subunit and probably is specific for an epitope on the 150-kDa alpha chain. This p150/95 antigen is the third member of a family of polypeptides sharing a common 95-kDa beta chain, which includes the lymphocyte function-associated antigen LFA-1 (p177/95) and complement receptor CR3 (Mo1/MAC-1/OKM1; p165/95) antigens. Sequential immunoprecipitation with anti-p95 beta chain mAb specifically removed the antigens detected by anti-LFA-1, anti-CR3 and anti-Leu M5 mAb. Certain patients with recurrent bacterial infections are genetically deficient in expression of the LFA-1 and Mo1 antigens, and have impaired granulocyte function. Granulocytes from a patient with this disease also failed to react with anti-Leu M5. Stimulation of normal granulocytes with f-Met-Leu-Phe, C5a-desArg, or calcium ionophore resulted in increased expression of Mo1 and Leu M5 antigens on the cell surface, but did not significantly increase expression of LFA-1 antigen. In functional assays, anti-Leu M5 did not inhibit T cell-mediated or natural killer cell-mediated cytotoxicity. In addition, anti-Leu M5 neither inhibited the binding of complement-coated particles to CR1 or CR3 nor did it affect the binding of EC3dg to neutrophils (CR4). These studies clearly indicate that the p150/95 antigen recognized by the anti-Leu M5 antibody is a structurally distinct member of the LFA-1/CR3 family.

We have previously reported that the addition of monocytes results in enhanced modulation of the T65 antigen when normal or leukemic lymphoid cells were cultured in vitro with the T101 monoclonal antibody. In the present investigation, we extend these findings to demonstrate that monocyte-enhanced modulation is a phenomenon that occurs with a variety of T and B lymphoid antigens identified by murine monoclonal antibodies. Two patterns of monocyte-enhanced modulation were observed: (1) augmentation by monocytes of existing antigen modulation by the T101 and anti-Leu-4 antibodies, and (2) induction by monocytes of previously unrecognized modulation with the anti-Leu-2 and anti-Leu-9 antibodies. Enhancement of modulation by monocytes was also detected with antibodies to surface IgM and HLA-DR antigens. Antigen modulation on lymphoid cell lines appeared to be more variable than on fresh cells, with or without monocytes. Monocyte-enhanced antigen modulation was not demonstrated with two monoclonal antibodies against solid tumors. Monocyte-enhanced modulation was shown to be dependent upon the Fc portion of the antibody, but independent of proteolytic or oxidative compounds released by monocytes. These findings indicate that the results obtained during in vitro studies of antigen modulation may vary with the source of cells and the extent to which monocytic cells are present. In addition, these findings suggest an enhanced role for Fc receptor-bearing cells of monocytic origin in antigen modulation following in vivo administration of monoclonal antibodies.

Since preliminary evidence indicated that a gene locus associated with the chocolate coat color had an inhibitory effect on the spontaneous development of autoimmune hemolytic anemia–which in turn was associated with a dominant gene–we accordingly initiated the development of a congenic line of NZB mice carrying the chocolate coat color locus (and other genes closely linked to this locus). After eight backcross generations to NZB, an inbred line of mice carrying the chocolate coat color gene was established and then studied for incidence of anti-RBC autoantibodies. The congenic line NZB.ch shows a considerably lower incidence of such autoantibodies, particularly in male mice. Although further analysis is still required, we propose that the genetic control of autoimmunity is based on the resultant interaction of several gene loci: 1) a single dominant locus (A) that predisposes to an autoimmune state rather than the maintenance of (self) tolerance, 2) a modifying locus (M) linked to ch., subject to gene dosage, which particularly in the recessive state can inhibit the effect of the A locus, and 3) a cluster of specific immune response genes that are associated with specific autoantibody responses, e.g. antinucleic acid antibodies, and are probably H2 linked. Our current analysis does not suggest that the A or M loci are linked to the MHC. It is also proposed that the wild-type allele of the M locus is carried by normal mouse strains, that NZB mice have a completely inactive allele at this locus, and that NZC mice have a third allele detectable only in the homozygous state.

Many recent studies have demonstrated that cytotoxic T lymphocytes (CL) activated to various antigens other than those of the H-2 complex, will lyse target cells only when H-2 compatibility exists between the CL and target cell. From these observations, it has been inferred that T lymphocytes might only be capable of responding to H-2 antigen or antigens that become associated with H-2 region gene products. Our results suggest that this is not the case, and that in some situations, cytotoxic T lymphocytes can specifically lyse target cells of different H-2 types. Two in vitro systems are described where primary induction of cytotoxic T lymphocytes to oncofetal and plasmacytoma antigens results in CL capable of lysing suitable targets bearing these antigens, of either syngeneic or allogeneic derivation. Thus it is proposed that although interaction antigens involving H-2 components may preferentially activate T lymphocytes, this does not imply a restriction on the recognition potential of T lymphocytes.

We have observed that a subset of E+ cells bind human monomeric IgG (FcR-IgG). In the present study, we have separated the E+, FcR-IgG+ cells by flow cytometry and tested their NK activity against the tumor KS62 using the chromium-release assay. Virtually all the NK activity residing within the E+ subset was mediated by E+, FcR-IgG+ cells. These findings are discussed in relation to the cellular lineage of the human NK cell.

A new antigenic specificity, designated Lym 7.3, defines a third allele of the Ly 7 locus. The monoclonal antibody recognizing this determinant was produced by a hybridoma derived from immunization of B10.D2 Ign mice against a (BALB/c X NZB) F1 murine B lymphoma cell line (WEHI-5). Previously, we reported that another antibody (BD5-334.5) from this fusion reacted with an alloantigen similar or identical to the Ly 7.2 antigen originally described by McKenzie and coworkers. The Lym 7.3 specificity is qualitatively and quantitatively identical to the Lym 7.2 determinant in tissue distribution. Both antibodies react with a majority of splenocytes, peripheral blood lymphocytes, lymph node cells, and mitogen activated blasts from positive strains, but do not react with kidney, liver, or brain tissues by absorption analysis. B lymphocytes expressed significantly higher cell surface density of Lym 7.2 and Lym 7.3 than T lymphocytes. However, these antibodies clearly could be distinguished on the basis of inbred and congenic strain distribution. Three distinct phenotypes were presented: (1) Lym 7.2+ Lym 7.3+; (2) Lym 7.2+ Lym 7.3-; and (3) Lym 7.2- Lym 7.3-. Genetic studies suggested both specificities were encoded by a single locus.

Various murine lymphoid tumor cell lines were examined for their alterations in intracellular levels of cyclic AMP following exposure to several pharmacologic agents. The agents tested in the present study included 3-isobutyl-1-methylxanthine (MIX), isoproterenol (ISO), prostaglandin E2 (PGE), histamine, and cholera toxin (CT). B cell tumors were generally found to be less responsive to beta-adrenergic and PGE-induced increases in cyclic AMP than were T cell tumors. An exception to this general finding was the murine B lymphoma cell line WEHI-231, which demonstrated marked sensitivity to ISO and PGE. Macrophage tumors were generally found to be even less responsive to PGE than were the B cell tumors, again with some notable exceptions (P388.D1). Cholera toxin produced differential effects in B, T, and macrophage tumors, both in terms of the absolute magnitude of the cyclic AMP response and the kinetics of this response. A T lymphoma cell line (EL-4) was identified that seems to be totally unresponsive to cell surface receptor-mediated increases in intracellular levels of cyclic AMP. The results of this study are discussed in terms of the usefulness of murine lymphoid tumors for the study of pharmacologic modulation of the immune response by cyclic AMP-elevating agents. These results demonstrate the high degree of cellular heterogeneity that can exist within normal lymphoid populations of cells, and suggest that cloned cell lines may be useful in the biochemical characterization of subsets of lymphoid cells.

Radiation injury in defined populations of B cells was investigated utilizing an allotype congenic transfer system. The amount of donor immunoglobulin present in the recipient's serum was found to be directly proportional to the number of viable cells injected, and on this basis approximate D37 values for the inoculated B cells were determined in several experiments and found to fall in the 70 to 145 rad range depending upon the specific experimental conditions. Since the sensitivity of T cells to radiation-induced interphase death can be modified by prior exposure to select mitogens and antigens, an attempt was made to demonstrate a similar phenomenon with respect to B cells. The results indicate that the radiosensitivity of B cells can be slightly influenced by prior incubation with mitogens and only equivocally changed by activation with antigen.

Murine B cell lymphomas and myelomas were examined for the expression of a determinant previously found exclusively on normal pluripotent stem cells colony-forming unit-spleen (CFU-s). This determinant(s), which is defined by a rabbit antimouse brain antiserum (R alpha MB), is present on the tumor stem cell population of some but not all B cell neoplasms examined. The determinant is not detected on tumor cells of the macrophage or T cell lineage. Absorption of the activity in R alpha MB with myeloma cells, concomitantly removed reactivity with the normal stem cell, CFU-s, and the myeloma stem cell, plasmacytoma CFU-s. Sorting analysis further showed that the antigen was diminished within a positive tumor population as cells acquired the capacity to secrete immunoglobulin. These studies suggest that this normal stem cell-associated antigen may also be an early differentiation antigen for the B cell lineage, and is expressed on some stem cells of B cell tumors.

A microculture system for the in vitro induction of tumour-specific immunity with syngeneic spleen cells and plasma-cell tumours is described. The system generates cytotoxic lymphocytes assessed by their ability to lyse 51Cr-labelled target cells in a micrototoxicity assay. Both the inductive and effector phases are subject to many variables in materials and methodology and the optimal conditions are defined in this study. Of particular importance are the responder- to stimulator-cell ratios, the presence of 2-mercaptoethanol, the day of harvest, the method of target-cell 51Cr labelling, and preincubation of the cytotoxic lymphocytes prior to assay.

In semisolid agar cultures containing mercaptoethanol, cells from the spleen, lymph nodes, marrow, peritoneal cavity, thoracic duct, and blood of normal mice generated clusters and colonies of up to 3,000 cells. Colony numbers and growth were markedly enhanced by the addition of sheep red cells. The frequency of colony-forming cells in the spleen or lymph nodes was 0.5-2.0%, and cluster forming cells were approximately five times more numerous. The mononuclear cells comprising these colonies had the electronmicroscopic morphology of immature lymphoid and plasma cells. The majority of the cells possessed Fc receptors, 61-69% reacted with anti-mu-serum and 4-11% with anti-gamma2-serum. Analysis of single cells from individual colonies indicated a higher frequency of the cells with membrane immunoglobulin and a clonal pattern of anti-mu or anti-gamma-reactivity. The clonal nature of colonies was supported by an analysis of NIP-binding cells in colonies grown from CBA spleen cells enriched for NIP-binding cells. Mass-harvested colony cells synthesized immunoglobulin in short-term liquid cultures. It is concluded that the colonies are clones of functionally active B-lymphoid cells.

In this report we demonstrate that when the murine macrophage tumor cell line P- 388D1 is incubated for 48-72 h with either concanavalin A-stimulated rat spleen cell supernatant or cloned murine immune interferon (IFN-gamma), the cultured cells release a cell-free factor activity that in turn induces the cell surface expression of Ia antigen on the murine monocyte cell line WEHI-3. This IFN-gamma-stimulated, Ia-inducing activity cannot be blocked with an anti-IFN-gamma heteroantiserum that does block the induction of Ia expression on WEHI-3 by both cloned murine IFN-gamma and rat Con A supernatant. The Ia-inducing factor ( IaIF ) generated from P- 388D1 after stimulation by IFN-gamma does not demonstrate any antiviral activity. The P- 388D1 -derived IaIF is not shed plasma membrane Ia glycoprotein molecules, as demonstrated by the inability of the active component to bind specifically to an anti-I-Ad affinity column or to a protein A column after the active supernatant is first treated with an excess of anti-I-E/Cd,k monoclonal antibody.

The binding of Staph A protein to murine immunoglobulins has previously been thought to be restricted to the IgG2 and IgG3 classes. In this study five IgM proteins were assessed for binding, and of these one showed marked Staph A binding. Pepsin digestion of this IgM molecule produced several different sized fragments, and the binding studies with these fragments indicated that the binding site is in the CH2 domain.

Chicken embryos injected with pooled spleen cells from outbred embryos are tolerant to histocompatability antigens of the donor population. Tolerance can be shown by the failure of peripheral blood cells (PBC) from tolerant birds to produce graft versus host lesions on the chorioallantoic membrane of outbred chicken embryos. It is shown that PBC from tolerant chickens can reduce by up to 85% the GVH reactivity of PBC from normal chickens. These results are interpreted to indicate that the PBC of birds tolerant to allogeneic cells contain active suppressor cells that can reduce the activity of normal cells in their response to allogeneic stimulation. The implications of these results are discussed in relation to the pathogenesis of Marek's disease—a common herpes virus-induced lymphoid neoplasm of chickens. It is suggested that resistance to neoplasia in this disease occurs because of the establishment of a population of active suppressor cells that prevents a neoplastic lymphoid proliferation.

The presence of oncofetal antigens (OFA) on a wide variety of murine tumor cells was demonstrated to a totally in vitro system of cellular immunity. Nonimmune spleen lymphocytes were cocultivated with irradiated syngeneic fetal liver cells and, at various times after initiation of culture, were tested for the presence of cytotoxic lymphocytes (CL) by 51Cr-release assay with labeled tumor target cells. Significant cytotoxic activity was regularly detected after such culture, whereas only minor levels appeared in control cultures of spleen lymphocytes with irradiated syngeneic spleen cells. Specificity of the reaction was assessed by inhibition tests in which nonlabeled cells were admixed to the CL and 51Cr-labeled tumor targets. Fetal liver cells gave significant inhibition; however, no inhibition was found with adult spleen cells. Various tumor types gave inhibition, and fibrosarcomas were more effective than plasmacytomas or lymphomas. The results suggested that all tumor types tested possess such OFA, as well as their unique or virus-associated, tumor-associated transplantation antigens, and that the in vitro system permits a more active response to the tumor-associated OFA than that observed in in vivo studies.

A tumour-associated antigen has been identified which is expressed on both BALB/c plasmacytomas and T lymphomas, but which does not induce a detectable cell-mediated immune response in the strain of origin of the tumours. The (BALB/c x C57BL) F1 hybrid, however, makes a readily detectable response both in vivo and in vitro in vitro experiments with various BALB/c H-2 F1 cogenic mice indicated that the genetic control of the response was not H-2 linked, and was associated with other C57BL genes.

In previous studies with in vitro activated cytotoxic T lymphocytes, we have demonstrated the presence of oncofetal antigens (OFA) on a range of murine tumor cells. The present studies with the same tumor lines attempt to determine whether these antigens are also capable of activating lymphocyte responses in vivo. Several experimental designs were followed, each being performed many times (1). Preimmunization of mice with irradiated fetal liver cells and followed by challenge with viable tumor cells did not consistently produce a state of tumor resistance. However, injection of live fetal cells frequently led to enhanced tumor growth (2). The growth of tumors in multiparous mice can be inhibited, in contrast to controls, but this effect was relatively short lived after parturition (3). Preimmunization with fetal cells in vivo did not result in augmented secondary cytotoxic T cell responses in vitro (4). Immunization of mice with irradiated tumor cells frequently led to a state of resistance to the clonal growth of hemopoietic fetal cells, although again the level of resistance was usually relatively weak. From the overall results, we conclude that OFA are relatively poor immunogens on tumor cells or fetal cells in vivo, and in contrast to in vitro responses, do not act as potent tumor transplantation antigens.

Monoclonal antibodies directed against rat kappa light chains have been generated by immunizing SJL/J mice with soluble rat immunoglobulin, followed by fusion of immune spleen cells with the P3-X63-Ag8 myeloma cell line. Monoclonal antibodies from three of these hybridoma cell lines, MAR 18.5, 80.2, and 103.6, have been extensively characterized. MAR 18.5, 80.2, and 103.6 antibodies are of the gamma 2a kappa isotype, and bind strongly to protein A, allowing easy purification. Monoclonal antibody from clone 18.5 binds equally well to Ig of both RI-1a and RI-1b allotypes, whereas 80.2 and 103.6 antibodies selectively bind to RI-1b. These monoclonal antibodies can be FITC conjugated for use as a second antibody in indirect immunofluorescence assays, or radiolabeled for use in radio immunoassays requiring a specific antirat kappa antibody. The antiallotype specific monoclonal antibodies also may be of use in the study of rat immunoglobulin genetics.

Although the Lyt-1 antigen has previously been considered a T cell-specific marker, recent evidence suggests that a population of Thy-1-, Lyt-1+ cells exists in normal lymphoid tissues. In this study, we have observed that the WEHI-55, WEHI-259, and CH5 B cell lymphomas express high levels of the Lyt-1 antigen, as detected by monoclonal antibodies using the fluorescence activated cell sorter. Three other B cell lymphomas of the 11 examined also gave weak but detectable reactions with the anti Lyt-1 monoclonal antibody. Except for the expression of the Lyt-1 antigen, these lymphomas are typical of cells in the B cell lineage with respect to surface phenotype. The Lyt-1 glycoprotein immunoprecipitated from metabolically labeled WEHI-55 cells is similar in structure to the Lyt-1 glycoprotein on thymocytes. These findings are similar to recent reports that B-type human lymphocytic leukemia cells express the putative human homologue of Lyt-1, the Leu-1 antigen.

Tumor-associated antigens (TAA) were demonstrated on plasmacytomas derived from BALB/c, NZB, and C3H mouse strains, by in vivo and in vitro techniques. By immunizing the appropriate F1 hybrid mice with these tumors, it was possible to show that all the plasmacytomas expressed cross-reactive tumor-associated transplantation antigens. When cytotoxic lymphocytes (CL) were induced in vitro by the coculturing of syngeneic or F1 hybrid spleen cells with irradiated plasmacytoma cells, "shared" and "unique" plasmacytoma TAA were demonstrable. This was accomplished by inducing CL in vitro against one syngeneic plasmacytoma and assaying for lytic activity on a range of 51Cr-labeled BALB/c, NZB and C3H plasmacytoma cells in vitro. In a second in vitro assay, unlabeled plasmacytoma cells were tested for their ability to inhibit the lysis of a particular 51Cr-labeled plasmacytoma, with the use of CL induced in vitro against it. The possibility that these TAA were "self" antigens was excluded by demonstrating in the inhibition assay that they were not present on T lymphomas and spleen cells of the same strain, and that CL "autosensitized" in vitro could not significantly lyse 51Cr-labeled plasmacytoma cells in vitro. From both in vivo and in vitro studies of immunity to these tumors, it was concluded that any one plasmacytoma line possesses multiple TAA of both shared and unique types.

Binding of antibody to the surface of B lymphocytes was shown to involve the Fc piece of the immunoglobulin molecule. This property was not shared equally by all immunoglobulin classes as revealed by direct binding and inhibition studies. Total IgG globulin was found to label cells more heavily than IgM, and IgG1-containing fractions more heavily than IgG2 fractions lacking IgG1. The ability of various purified myeloma proteins to inhibit attachment of antibody to B cells was examined. Pretreatment of B cells with excess IgG2a, IgA, or light chain proteins failed to inhibit, whereas IgG1 proteins and to a lesser extent Ig2b and IgM proteins at the same concentrations did so. At lower protein concentrations, IgG1 myeloma protein alone retained the capacity to inhibit binding. The conclusion was reached that the receptor on B cells for antibody has a marked predilection for the IgG1 class. Although IgM and IgG2b antibody may bind, they do so with lower avidity and probably in insignificant amounts if IgG1 antibody is present in excess. No evidence was found to implicate complement in the binding process. For example, heat-inactivated sera at high dilution retained the ability to label B cells, while the use of purified low molecular weight anticomplementary factor, a potent inhibitor of C'3, did not interfere with the formation of the bond between antibody and cell surface. The failure of anti-mouse immunoglobulin F(ab)'2 fragments to prevent access of antibody to B cells implied that the antibody-binding receptor and antigen-binding (immunoglobulin) receptor are discrete entities on the B cell membrane. Despite this, a marked similarity between their surface distribution was observed on electron microscopy. The antibody-binding receptor was shown to be a marker for mature B cells. It did not appear to be present on hematopoietic precursor stem cells and was lost during differentiation to antibody-forming cells.

SummaryThe NZC strain of mouse is known to have a defect involving the transplantation of hematopoietic stem cells. Attempts to test the immune system of this strain show a normal antibody response in vivo. However, in adoptive transfer systems, NZC cells were shown to be minimally responsive on transfer, although (Balb X NZB) F1 cells could respond in the NZC environment. In vitro studies of the response of NZC cells to SRBC, phytohemagglutinin and pokeweed all show reduced responses. Evidence is presented which suggests that there is a defect in the number of progenitor cells for the immune system, possibly involving only one component cell population.We are grateful to Dr. Malcolm Moore for valuable discussions in the course of this work, and to Mrs. D. Barr, and Misses G. Cousins and P. Bell for competent technical assistance.

Nineteen different radiation-induced thymomas originating in BALB/c, NZB and NZW mouse strains were tested for Ig synthesis by short-term culture with 14C-amino acids followed by radioimmunoelectrophoresis. Twelve were negative, six produced light (L) chains and one synthesized a protein similar to IgM. The IgM producer, WEHI-22, and one of the L chain producers, WEHI-105, were established as cloned cultured cell lines and shown to possess the lymphoid cell characteristics of susceptibility to inhibition by low concentrations of thymidine and of cortisol. Both were also shown to bear the θ antigen, a thymus-derived (T) lymphocyte marker. Cells of the WEHI-22 line also possessed surface immunoglobulin readily detectable by binding of labeled anti-Ig antibody and were able to bind mouse antibody-coated sheep erythrocytes forming rosettes, while WEHI-105 did neither. The specificity of the Ig receptor mediating rosette formation appeared from myeloma protein competition experiments to be very similar to that of the receptor described for non-thymus-derived (B) cells. It was considered that WEHI-22 cells either possess a mixture of T and B cell characteristics or are neoplastic variants of T cells possessing surface IgM with antiglobulin activity.

Addition of 125I-labeled anti-immunoglobulin protein to cell suspensions from mouse thymus, spleen and thoracic duct lymph (TDL) resulted in labeling of surface immunoglobulins of lymphoid-like cells. Near-optimal conditions for cell labeling were established with varying anti-immunoglobulin concentrations and times of exposure. On the average 20.1% to 36.3% of TDL and spleen cells labeled under such conditions, whereas thymus cells labeled less heavily (0.3% to 3.7%) with identical materials. Normal rabbit sera (NRS) or anti-immunoglobulin absorbed with purified immunoglobulins produced essentially no labeling. The total heavy chain labeling on TDL and spleen cells exceeded light chain labeling (64.6% vs 31.8% and 79.8% vs 33.0%, respectively). This might suggest that labeled cells had multiple surface heavy chain determinants or that the anti-heavy chain antibodies cross-reacted with an unidentified immunoglobulin-like surface antigen. There was increased labeling with TDL suspensions from neonatally thymectomized mice (65% with anti-light chain). This was consistent with the hypothesis that primarily non-thymus-derived cells were labeled by these techniques.

Rabbit antisera against intact sheep erythrocytes (SRBC) were assayed by hemolysis and PCA. In one serum, antibodies mediating both PCA and hemolysis were present, whereas another serum contained mainly hemolytic antibodies. The PCA reaction could be developed by intravenous challenge with either fresh SRBC or their lysate, but not with stroma or formalin-fixed SRBC. Both stroma and formalin-fixed cells could, however, bind the hemolytic antibodies, which the lysate failed to do. The results indicate that one of the anti-SRBC serum contained antibodies against stromal antigens, which could be detected only by direct red cell binding assays (hemolysis or hemagglutination), and antibodies against a soluble internal antigen, possibly hemoglobin, which could be detected only by PCA reactions.

Summary Rabbits were immunized with either normal mouse immunoglobulins or isolated mouse myeloma proteins from BALB/c or (BALB/c × NZB)F1 plasma cell tumors. The antisera were tested for antibodies directed to allotypic antigenic specificities by the method of inhibition of precipitation of I125 labeled antigens. Three γG2a and one γG2b allotypic specificities were detected with one or another of these rabbit antisera. These specificities each corresponded in mouse strain distribution with one of the allotypic specificities previously defined through the use of mouse isoantisera. The possible nature of the allotypic sites versus the class specific antigen sites was briefly discussed and the potential usefulness of these heterologous antisera in allotypic systems noted.

Experiments were designed to test the possibility that thymus-derived (T) cells cooperate with nonthymus derived (B) cells in antibody responses by acting as passive carriers of antigen. Thoracic duct lymphocytes (TDL) from fowl γG-tolerant mice were incubated in vitro with fowl anti-mouse lymphocyte globulin (FALG), which was shown not to be immunosuppressive in mice. On transfer into adult thymectomized, irradiated, and marrow protected (TxBM) hosts together with a control antigen, horse RBC, a response to horse RBC but not to fowl γG was obtained. By contrast, TxBM recipients of nontolerant, FALG-coated TDL responded to both antigens and the antibody-forming cells were shown to be derived from the host, not from the injected TDL. These findings suggested that, under the conditions of the experiment, triggering of unprimed B cells in the spleens of TxBM hosts was not achieved with antigen-coated tolerant lymphocytes. Another model utilized the ability of B cells to bind antibody-antigen complexes. Spleen cells from TxBM mice, incubated in vitro with anti-fowl γG-fowl γG·NIP, were injected with or without normal TDL (a source of T cells) into irradiated hosts. Only mice given both cell types could produce an anti-NIP antibody response. In a further experiment, spleen cells from HGG·NIP-primed mice were injected together with NIP-coated B cells (prepared as above) into irradiated hosts. A substantial anti-NIP antibody response occurred. If, however, the T cells in the spleens of HGG·NIP-primed mice were eliminated by treatment with anti-θ serum and complement, the NIP response was abolished. It was concluded that antigen-coated B cells could not substitute for T cells either in the primary or secondary response. Treatment of T cells from unprimed or primed mice with mitomycin C impaired their capacity to collaborate with B cells on transfer into irradiated hosts. Taken together these findings suggest that before collaboration can take place T cells must be activated by antigen to differentiate and in so doing may produce some factor essential for triggering of B cells.

Progeny mice were confronted with maternal γ-globulin of a different allotype by either back-cross mating, intercross mating, or by foster nursing. In all cases, many mice subsequently produced alloantibodies directed against the incompatible maternal type of IgG2a-globulin. In one series of experiments, immunologic tolerance to the maternally derived γ-globulin was demonstrated to exist in the period before formation of spontaneous antibody. The state of tolerance was then lost, unless maintenance injections of foreign γ-globulin were given. These studies demonstrate in a natural situation that maternally derived foreign proteins can first induce a state of immunological tolerance which is followed, after disappearance of the antigen, by a state of immunity. As such, this parallels the experimental induction of tolerance to foreign proteins by neonatal injections.

Bone marrow colony-derived mononuclear phagocytes are macrophages on the basis of morphology, glass adherence, the phagocytosis of a range of particles including erythrocytes, yeast, and bacteria, and the possession of receptors for IgG of predominantly IgG2a specificity.

Hormonally bursectomized (HBx) chickens were repeatedly immunized with Brucella antigen. The proportion of antibody-responding HBx chickens increased from 16% at 6-day primary, to 48% at 6-day secondary, and to 60% at 15- and 21 day-tertiary. Despite this continued immunization, however, 40% of the HBx chickens still remained completely unable to produce any detectable antibody response even with the use of an extremely sensitive agglutinin assay. Following a secondary immunization with HGG 64% of HBx chickens failed to produce any detectable antibody. Natural hemagglutinins to rabbit red blood cells were also absent from the sera of HBx chickens. Serum immunoglobulins were determined by immunodiffusion and immuno-electrophoresis using specific anti IgM and IgG sera. Serum IgG levels were quantitated with the use of an extremely sensitive assay employing the inhibition of precipitation of 125I-labeled IgG by specific antiserum. Approximately 50% of HBx chickens had less than 1% of the control IgG level. Some birds were completely agammaglobulinemic, having less than 1/10,000 the control amount of IgG. Several of the HBx chickens with about 1% of IgG had normal or elevated levels of IgM. Serum α2-macroglobulin levels were normal in HBx chickens. Several HBx chickens that survived until 8 months of life were rechallenged with various antigens. A total lack of antibody production to most antigens was still found, even though some of these birds had considerable amounts of IgG and/or IgM. In vitro cultures of spleen and appendix confirmed the serum results on immunoglobulins, in showing that lack of circulating IgG and IgM was associated with a failure of its synthesis. The results presented in this paper strongly deny claims that bursectomy cannot totally prevent the development of all antibody-forming capacity or immunoglobulin synthesis. They reinforce the concept of an absolute dissociation of immunologic responsiveness in chickens, in that the bursa is the sole site of control of the development of the antibody-forming system.

Mice and guinea pigs were immunized with dinitrophenyl (DNP) protein complexes in low doses combined with aluminium hydroxide gel adjuvant. This scheme led to the production of two different types of antibodies mediating passive cutaneous anaphylaxis (PCA) reactions in each species. Both species made a heat stable IgG1 antibody capable of giving PCA reactions with a short sensitization period, which was removed by treatment with specific anti-IgG1 serum, and migrated electrophoretically with the total IgG1 globulins of each species. The other antibody provoking PCA reactions in each species was detected with a longer sensitization period (2 days in mice and 4 days in guinea pigs). These antibodies did not show identical properties to each other. The mouse antibody was completely heat labile, was not removed by a specific anti-IgG1 serum, and migrated electrophoretically slightly more slowly than did IgG1. In distinction, the guinea pig antibody was only marginally affected by heating, was removed by pretreatment with a specific anti-IgG1, and had a faster electrophoretic mobility than IgG1 globulin. These results support a previous observation of Parish, that guinea pigs may possess two heat stable IgG1-type antibodies that can mediate PCA, possibly as well as having a true reaginic antibody.

Spleen cells, thoracic duct lymphocytes and adherent peritoneal exudate cells from mice immunized to syngeneic plasma cell tumors were capable of transferring specific protective immunity to these tumors. Pre-treatment of these cells with anti-Θ serum or anti-lymphocyte serum, but not with anti-ΘK serum, effectively abolished the ability of lymphoid cells to transfer this immune response. These studies demonstrate that T cells are essential in the adoptive transfer of immunity. Furthermore, immunization of athymic (nude) mice to plasma cell tumors was not achieved. Attempts to activate normal macrophages in vitro with sensitized lymphoid cells were unsuccessful. Admixture of immune lymphoid cells and target tumor cells to unrelated tumor cells did not inhibit the growth of the unrelated tumor cells. It is proposed that the immune response to syngeneic tumor associated antigens of plasma cell tumors not only involves essential sensitization of T lymphocytes, but that activated cytotoxic T lymphocytes are directly responsible for inhibiting target tumor growth.

Mouse IgA myeloma proteins produced by plasma cell tumors derived from BÀLB/c, NZB and (BALB/c × NZB)F1 hybrid mice were examined electrophoretically under dissociating conditions by using reduced and unreduced samples. In confirmation of previous studies by Abel and Grey (3) our electrophoretic patterns are consistent with the conclusion that no L-H disulfide bonds are present in BALB/c-derived IgA proteins. However, 3 out of 3 NZB derived IgA proteins appeared to possess L-H disulfide bonds, as did 5 of 10 NZB hybrid derived proteins. The presence of L-H disulfide bonds in the hybrid-derived proteins completely correlated with the presence of Ig-2e (NZB type) IgA allotypic antigens.

The present study was designed to devise and characterize an indirect or sandwich radioimmunolabeling technique for the study of lymphocyte surface receptors of immunoglobulin nature. Mouse lymphocytes from various sources were treated by the method of Shortman et al. to remove debris and damaged cells. This was an important preliminary step, as without it, little meaning could be attached to bulk scintillation counting of labeled cell suspensions, in view of the marked tendency of dead or damaged cells to adsorb protein nonspecifically. Next, cells were reacted at 0°C for 30 min with graded dilutions of unlabeled rabbit antisera against defined mouse Ig chains. After washing, the cells were reacted with a sheep anti-rabbit globulin reagent labeled with 125I, again at graded concentrations. After further washing, lymphocyte labeling was quantitated by both bulk scintillation counting and radioautography. Conditions were defined in which nonthymus-derived cells (B cells) but not thymus-derived cells (T cells) could be labeled. Most B cells displayed κ- and µ-chains on their surface, but some also displayed α- and γ2-chains, though in smaller amounts. When the concentration of both the first and the second reagents were raised considerably, conditions were defined under which virtually all T cells could be labeled by polyvalent antiglobulin sera, anti-κ sera, or, with more difficulty, by anti-µ sera. A large series of control experiments confirmed the serologic specificity of this labeling. It was shown that under equivalent conditions, B cells bind 100–400 times more antiglobulin than do T cells. The theoretical implications of the results are briefly discussed. It is argued that the sandwich approach offers certain technical advantages over direct labeling procedures for further analyses of T cell receptors and for studies of receptor metabolism.