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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

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.

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.

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.

The 51Cr release cytotoxicity assay was used to evaluate whether self-reacting clones of T cells develop in vitro when normal spleen cells are cultured with irradiated syngeneic cells. This phenomenon has previously been described as autosensitization and has been thought to be due to reactivity against self-determinants. It was shown, however, in this present study that the autosensitization phenomenon differs qualitatively and quantitatively from specific immune induction by oncofetal antigens and tumor-associated antigens. A variety of tumor cell lines of different H-2 types are lysed by autosensitized cells, and the lysis appears to be nonspecific and not restricted to identity at the major histocompatibility complex (MHC). Induction of autosensitization follows the same kinetic pattern, and lysis is semilogarithmically related to effector cell to target ratio as in other systems. Analysis of the specificity of the phenomenon by competitive inhibition with tumor cells or congenic derived spleen cells did not, however, indicate any reactivity for self-MHC determinants, but rather showed that a wide range of culture tumor cells were all capable of inhibiting lysis. Since it was further shown that the level of autosensitization was markedly dependent on the batch of fetal calf serum, it is provisionally concluded that this phenomenon does not represent specific T cell activation to self-determinants, but rather reactivity against minor antigens in the system, possibly a cell-bound fetal calf serum component.

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.

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.

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.

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.

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.

An NZB-derived IgG2a myeloma protein with anti-dinitrophenol (DNP) activity was assayed for its ability to elicit complement fixation (CF) and passive cutaneous anaphylaxis (PCA) in guinea pigs after it had been combined with DNP antigens. The myeloma protein agglutinates difluorodinitrobenzene-coupled red cells and this agglutination can be inhibited by monovalent DNP amino acids, albeit with approximately 1000-fold more inhibitor than that needed to inhibit agglutination by a mouse anti-DNP serum. Despite the ability to agglutinate DNP red cells, no passive hemolysis occurred. The myeloma protein gave reverse PCA reactions in guinea pig skin with as little as 0.12 μg and challenge by anti-mouse globulin. However, even with 40 μg per skin site, no PCA reactions could be elicited on challenge with DNP conjugates. It is concluded that the complete biologically active sites on IgG2a molecules for CF and PCA reactions are created by the energy of binding of the molecule to its antigen. The myeloma anti-DNP protein does not create these sites after antigen binding because of its extremely low affinity.

Unprimed mouse spleen cells were incubated in vitro with various class specific rabbit anti-mouse immunoglobulin sera. Cells were washed and transferred into syngeneic or allotype congenic irradiated recipients. Syngeneic recipients were stimulated at the time of cell transfer with sheep erythrocytes (SRBC) and Brucella abortus. At 7 days their IgM response to SRBC and B. abortus was significantly depressed following pretreatment with anti-µ chain sera, but not with anti-γ chain sera. Congenic recipients were serially bled and levels of donor type IgG immunoglobulins were determined by allotype specific inhibition of precipitation assays. Marked suppression of the transfer of donor type IgG of all classes was obtained by pretreatment with anti-µ chain sera.

The thymus of several species was examined for antigen-binding lymphocytes (ABL), by using autoradiography and 125I-labeled antigens, flagellin from Salmonella adelaide and hemocyanin from Jasus lalandii. Counts of ABL in thymic cell suspensions, expressed per 104 lymphocytes, varied according to the species and antigen used. For flagellin the count of thymic ABL was considerably greater in the human fetus (182 ± 50) than the mouse fetus (2.9 ± 0.5), but for hemocyanin was greater in the mouse (36 ± 8.5) than the human (10 ± 2.5). In man and mouse counts of thymic ABL fell steadily with increasing age, from fetal to late adult life. The property of antigen binding could not be attributed to cytophilic antibody because this was not passively conferred on to lymphocytes in vitro or in vivo by antibody to flagellin in high titer. The in vivo experiment was on rats immunized with flagellin before pregnancy; antibody crossed the placenta yet failed to confer antigen-binding properties on lymphocytes of various fetal tissues. The binding of flagellin and hemocyanin to thymic lymphocytes in man and mouse was blocked by monospecific antisera to light chains and µ chains, even in high dilution. Most of the ABL in thymus cell suspensions would be expected to be thymus-derived (T) lymphocytes rather than immigrant B lymphocytes and the data were therefore interpreted as showing that such lymphocytes carry antigen-binding receptors of IgM character.

Surface receptors for IgG immunoglobulins have been demonstrated on a mouse mast cell tumor growing in tissue culture. Inhibition of rosette formation between mastocytoma cells and IgG-coated SRBC by purified mouse immunoglobulins indicated receptor specificity for all known IgG globulin classes but not for IgM, IgA or light chain immunoglobulins. Pepsin-digested IgG antibody-coated SRBC did not bind to the mast cell. Mast cell receptors appear to react with immunoglobulin-antigen complexes more strongly than with immunoglobulin alone. Receptor activity was unaffected by phospholipase D, neuraminidase or azide and was reduced by phospholipase C, iodoacetamide and fluorescein isothiocyanate. Actinomycin D had no effect for periods up to 9 hr. Treatment of mast cells with trypsin enhanced receptor activity but did not alter specificity. Our initial conclusion is that the mastocytoma IgG-receptor is a stable phospholipid or phospholipoprotein which is protease-resistant.

Summary Pretreatment of normal adult mouse spleen cells, or peritoneal exudate cells from mice sensitized to DNP~CSA, with anti-immunoglobulin light polypeptide chain sera considerably suppressed the ability of these cells to induce the graft v8 host reaction or the transfer of delayed hypersensitivity. Anti-heavy chain sera did not cause this suppression. These results indicate that the antigen recognition site on lymphocytes involves an immunoglobulin light chain which is either free or bound to a new heavy chain type.

Two groups of human IgM proteins are described. One group interacts with a heat-labile component of rabbit or human serum which has characteristics similar to C′1q. The second group fails to interact with this factor. This grouping is not related to κ or λ chains or to previously described differences in human IgM molecules. Such a division may provide a tool for the identification of µ chain genetic markers.

Summary Mouse B lymphocytes and cells of a plasma cell tumor bound immune complexes and IgG but not IgA or IgM immunoglobulins or light chains. Binding required an intact Fc piece but was independent of complement. T lymphocytes did not bind immune complexes. The presence of surface receptors for IgG immunoglobulin on B lymphocytes and a plasma cell variant are consistent with the concept of an ancestor-progeny relationship between these cell lines.

Two general observations form the basis for considering the possible autoimmune etiology for plasma cell tumors in man and mouse. 1) Antibody specificity of myeloma proteins. Although the true percentage of such proteins with autoantibody activity is not clear, it is certainly apparent that a significant proportion of myeloma and Waldenström macroglobulins show autoantibody specificity. This particularly includes several types of antiglobulin specificity such as a true anti-antibody activity (Warner, McKenzie and Fudenberg), and several anti-DNP myelomas with cross-reaction to γ globulin (a mouse IgG2a globulin and a human Waldenström macroglobulin). 2) Genetic susceptibility to plasma cell tumor induction. Mice of different inbred strains and hybrids were treated with three injections of mineral oil early in life. A high incidence of plasma cell tumors subsequently developed in BALB/c, NZB and (BALB/c × NZB) F1 hybrid mice.