Brain-associated-theta antiserum. Differential effects on lymphocyte subpopulations

Subpopulations of lymphocytes were delineated, both in vitro and in vivo, using brain associated-θ antisera. Lymphocytes were depleted from the cortical areas of the thymus and the T-dependent regions of the spleen. No depletion occurred in the medullary area of the thymus or in the deep cortical areas of the lymph node. These histological findings correlated well with the effects of brain associated-θ antisera using an in vitro cytotoxicity assay. Greater concentrations of antisera were required to cause cell death of lymphocytes from lymph node than lymphocytes from either spleen or thymus. These data would suggest that the θ antigen may be either lost or modulated during the process of lymphocyte differentiation.     

Specificity of Anti-θ Sera in Cytotoxicity and Functional Tests on T Lymphocytes

THE theta (θ) alloantigen is a marker for thymus-dependent (T) lymphocytes in mice^1–4 and anti-θ serum has been used to study the distribution, differentiation and function of T lymphocytes⁵. We report here studies which indicate that anti-θ sera raised in conventional inbred mice are usually contaminated with a number of antibodies directed against surface antigenic determinants other than θ, at least one of which is present on thymus-independent B lymphocytes. These contaminating antibodies have been demonstrated by cytotoxicity tests with rabbit complement and by rosette-forming cell (r.f.c.) inhibition studies. In the presence of guinea-pig complement, however, most anti-θ sera can be used selectively to kill and/or inhibit T cells.     

The differentiation of T lymphocytes. I. Proliferation kinetics and interrelationships of subpopulations of mouse thymus cells

Differential quantitative cytotoxic assays were used to distinguish the major high θ population of mouse thymus from the minor low θ subpopulation. The low θ cells were isolated in good yield by killing all high θ cells with controlled anti-θ and complement treatment, followed by a damaged cell removal step. A third population of labile cells, subject to rapid death in culture, was distinguished as a variable component within the high θ category. The corticosteroid sensitivity and anatomical location of these subpopulations was briefly considered. Large and medium sized dividing lymphocytes were studied by pulse labeling with tritiated thymidine, followed by radio-autography of separated subpopulations. Both the high θ and low θ categories included large dividing cells. Kinetic studies under conditions of continuous labeling were used to explore precursor-product relationships among the small thymocytes. Both low θ and high θ small lymphocytes showed continuous and close to linear accumulation of labeled cells, with no evidence for a marked lag in labeling. The turnover time of high θ small lymphocytes was three–four times that of low θ elements. The results suggest largely independent pathways are involved in the development of the two antigenically defined subpopulations. They do not support a direct transfer of “immature” high θ, TL positive small thymocytes in mature, active, low θ,TL negative cells. Some alternative models of T cell development are discussed.     

Immunologic properties of mouse thymus cells: membrane antigen patterns associated with various cell subpopulations

The membrane antigen components of mouse thymus cells and fractions derived from BSA density gradient centrifugation were assayed by quantitative cytotoxicity tests. Two subpopulations were identified on the basis of average density and antigen patterns. The major subpopulation consisted of small lymphoid cells and comprised 80%–90% of all cells, was of high relative density and rich in θ, TL, G_IX, Ly-A, Ly-B, and Ly-C, but contained little or no H-2. The minor subpopulation was chiefly large lymphoid cells, comprised 10%–15% of cells, was of low relative density, was relatively rich in H-2 but low in θ and Ly antigens, and contained no detectable TL or G_IX. This minor subpopulation was identical in density and antigen patterns to those cells remaining in the thymus after short-term cortisone treatment or whole-body irradiation. It could also be reproduced by treating whole thymus with anti-TL or anti-θ sera. The antigenic attributes of this minor subpopulation differed from those of spleen lymphocytes only with respect to average density.     

Modulation of antibody synthesis by cholera exotoxin: Influence on helper thymocytes

This report demonstrates that a major biological influence of cholera exotoxin (CT) on antibody formation to sheep erythrocytes is mediated by the toxin's influence on the helper function of thymic lymphocytes. Hemolytic plaque-forming cell (PFC) responses for lethally X-irradiated mice repopulated with isologous thymus-marrow cell suspensions were stimulated three- to fivefold when 1.0 μg CT was administered at cell transfer. For mice given marrow cells only, CT did not elevate the PFC response; however, CT stimulated as many PFC in spleens of mice given marrow and 1 × 10⁷ thymus cells as mice given marrow and 4 × 10⁷ thymus cells. In other transfer experiments, depressed PFC responses were observed when irradiated mice were given marrow and thymus cells from donor mice inoculated with CT 24 hr prior to cell preparation and infusion, or given marrow cells from normal mice and thymus cells from CT-treated mice. In contrast, mice given marrow from CT-treated mice and thymus cells from normal mice responded as well as mice repopulated with normal thymus-marrow cell suspensions.     

Immune responses in vitro. VII. Loss of susceptibility of functional theta-bearing cells to cytotoxic action of anti-theta serum and complement in vitro

θ-Bearing lymphocytes become progressively less susceptible to cytolytic effects of specific anti-θ serum and complement with incubation in tissue culture. Thymus-derived cell function, in terms of “helper cell” capacity for antibody responses and DNA synthetic responses to phytomitogens, however, remains intact. The data indicate that the loss of susceptibility to cytoloytic effects of anti-θ serum and complement is due to a decrease in the amount of θ antigen on these cells. This phenomenon hampers attempts to determine the length of time that collaborative or “helper” influences of thymusderived cells are required for development of optimal antibody responses in vitro.     

Properties of Educated T Cells for Rosette Formation and Cooperation with T Cells

IT has been shown that the humoral antibody response in mice to many antigens requires cooperation between thymus derived lymphocytes (T cells) and bone marrow derived lymphocytes (B cells)^1,2. The B cells are the direct precursors of antibody secreting cells and, although T cells react specifically with antigen, their role is unknown^2–6.     

Cooperative monomer binding by polynucleotides. Effect of multiple-loop configurations on formation of triple-stranded complexes

Simulated binding curves for the reaction 2 polymer + monomer = triple-stranded complex are presented, in which loop formation and sliding degeneracy of the polymer adsorption surface are considered. Exact calculations for a polymer chain length N of 11 units suggest that configurations of two or more loops have negligible effect on the isotherm when SW > 1, where S and W are exponential weighting factors for monomer–monomer S and polymer–polymer W nearest neighbor interactions. There is a pronounced effect, however, when SW ? 1. Limiting expressions (N ? 1, but finite) for the single-loop configurations suggest these configurations are negligible at any degree of saturation θ if θ (1 ? θ)^2–k N^3–k ? SW, where k is defined by the weighting factor (j + 1)^?k for a ring of j units. This expression suggests that single monomer-stack configurations are the only significant contributors to the grand partition function at the midpoint of the isotherm if N^3–k ? SW. Furthermore, single-loop configurations are negligible below θ = 0.5 but become dominant above the isotherm midpoint when SW ～ 1 (random binding) if 2 < k < 3. For k > 3 and N → ∞, loop configurations have no effect in any region of the random binding isotherm usually analyzed experimentally (θ < 0.95). Equivalence of matrix and sequence generating methods is also demonstrated.     

Evidence for Subpopulation of Mature Lymphocytes within Mouse Thymus

THERE is increasing evidence that thymus cells migrate from the thymus to the peripheral lymphoid tissues where they make up most, if not all, of the thymus-dependent population of lymphocytes^1–3. The term “thymus-derived” is thus appropriately applied to this population. Yet most thymocytes are different from peripheral lymphocytes, both in immunological competence and in surface antigenic characteristics. For example, thymocytes have more theta (θ)⁴ and less H2 antigen⁵ than do peripheral lymphocytes and in TL-positive strains of mice only thymocytes normally express the TL antigen⁶. Recently, Lance et al.⁷ found that injected thymocytes which had migrated to lymph nodes and spleen were progressively less susceptible to anti-TL and more susceptible to anti-H2 serum over the first 24 h. I report here experiments in which thymus cells injected intravenously into irradiated syngeneic mice and harvested as early as 3 h later from the peripheral lymphoid tissues can be shown to have the surface antigenic properties of peripheral thymus-derived lymphocytes rather than thymocytes. A second experiment demonstrates that at least part of the differentiation from thymocyte to thymus-derived lymphocyte seems to occur within the thymus.     

Activation of T and B thymus cells to recognize histocompatibility antigens

Lethally irradiated (A × CBA) F₁ or (A × C57BL/6) F₁ mice were injected with 10⁷ A strain thymus cells in attempts to activate donor cells to recognize CBA or C57BL/6 histocompatibility antigens, respectively. Activation could be revealed by injecting activated thymus cells (day 5 irradiated F₁ hybrid spleen cells) into corresponding unirradiated F₁ hybrid hosts. The alloantibody titers formed by these cells and the antirecognition structure (anti-RS) antibody titers induced by them were similar to those observed after injection of normal parental strain spleen cells, indicating that thymus cells had become endowed with recognition structures (RS). Alloantibodies, but no anti-RS antibodies, were present in the serum of F₁ mice given activated thymus cells treated with anti-θ and complement. It, therefore, appeared that activated thymus cells contained sufficient B cells differentiated into antibody-forming cells to give a measurable alloantibody response. On the other hand, receptors responsible for anti-RS antibody induction presumably were located on T cells. Specificity and restriction of antigenic recognition were revealed by negative results obtained when activated thymus cells were injected into F₁ hosts not containing the antigens against which activation had been directed.     

Study of the immune responses to nucleated cells. I. In vitro functional evaluation of the immune effectors responsible for complement-dependent cellular cytotoxicity

A modification of the ⁵¹Cr cytotoxic test has made it possible to assess under the same conditions not only cytotoxic serum antibodies, and cell-mediated immunity, but also cells releasing cytotoxic antibody. The measurement of these antibody-releasing cells was carried out with nucleated target cells, both normal and leukemic, across θ or H-2 antigenic differences. This test was found to be specific. The release of ⁵¹Cr from the labeled target cells was proportional to the ratio of immune cells to target cells, and for a given ratio to the incubation time, 60 min usually being the optimum time at ratios of 50–100 to 1. The test was not affected by treatment of the effector cells with an anti-θ serum; however, pretreatment of these cells with an anti-IgM serum, even without complement, inhibited the test with cells taken during primary responses. Both cytotoxic IgM and IgG antibodies were detected by the assay directly without the addition of enhancing serum; discrimination between these two γ-globulins can be made by suppressing the cytotoxicity due to either Ig class consequent to the addition of the appropriate specific anti-globulin serum during the incubation.     

ANALYSIS OF TIME DEPENDENT CHANGES OF LYMPHOPOIETIC ACTIVITY IN ORGAN CULTURES OF EMBRYONIC CHICKEN THYMUS

The time dependent development of lymphocytes in organ cultures of the thymus obtained from 10-day-old chick embryos was characterized by an initial phase of exponential increase of the number of lymphocytes per thymus followed by a plateau phase with no further increase in cell number. The proportion of cells in DNA synthesis dropped rapidly during the first 10 days of culture. Simultaneously the lymphocytes turned progressively smaller, as evidenced by both cell diameter and dry mass and constituted a homogeneous population of small cells at the end of the culture period. Thymic anlagen partially depleted of lymphoid precursor cells by a short hot pulse with ³H-TdR showed a prolonged exponential phase and reached normal plateau cell numbers 2–4 days later than usual. Furthermore, at least in the first part of the plateau phase, a reduction in the number of lymphoid cells per thymus resulted in a recovery in terms of the cell number which was associated with increased DNA synthesis. These results are compatible with the regulation of thymic lymphopoiesis in organ culture through a mechanism operating via cell density.     

Nonspecific suppressor elements in murine allogeneic radiation chimeras.

Spleen cells from long-term mouse allogeneic radiation chimeras were tested for their ability to modulate the graft-versus-host (GVH) or plaque-forming cell (PFC) response of normal lymphocytes transplanted in lethally X-irradiated recipients. In vivo GVH proliferation of normal lymphocytes (syngeneic to donor cells of the chimera) against antigens of host-type in which the chimeric state had been established was reduced by chimera cells. Inhibition varied, some chimeras suppressing GVH more than others and a few not suppressing at all. The suppressive effect was abrogated if the chimera cells were treated with anti-θ; treatment with anti-IgM did not eliminate this activity. When mixtures of normal donor lymphocytes and chimera cells were given to irradiated recipients genetically different from host or donor, reduction of donor cell GVH also occurred. Further, chimera cells reduced the GVH activity of normal host cells in irradiated recipients differing from the host at one H-2 locus and from the donor at minor histocompatibility loci. The modulating effect of spleen cells from chimeras on the PFC response by normal lymphocytes also varied. Six chimeras induced a 25 to 90% suppression, two enhanced the response, and one showed no effect. Where suppression occurred, treatment of chimera cells with anti-θ most often, but not always, restored PFC production. Our results show that the suppressive action of splenic lymphoid cells by chimeras is highly nonspecific and variable in expression. We suggest that tolerance in chimeras may be mediated by nonspecific suppressor elements leading to unresponsiveness to a variety of antigens including SRBC.     

Development of T lymphocytes in Xenopus laevis: appearance of the antigen recognized by an anti-thymocyte mouse monoclonal antibody

Development of T lymphocytes in Xenopus laevis was studied using a mouse monoclonal antibody (mAb), XT-1, that was produced against surface determinants on thymocytes of J strain frog. Ontogenic studies, employing immunofluorescence, showed that cells positive for the determinant recognized by XT-1 mAb (XT-1⁺ cells) were first detected in the thymus of J strain Xenopus by Nieuwkoop and Faber stage 48 (7 days postfertilization) and then in the spleen, liver and kidney by stage 52 (20 days postfertilization). Percentages of XT-1⁺ cells in the thymus increased rapidly by stage 49 (10 days postfertilization) and reached adult levels by stage 52, and those in the spleen, liver, and kidney reached adult levels by stage 56 (40 days postfertilization). Electron microscopic immunohistochemistry revealed that most XT-1⁺ cells in thymuses from stage 56 larvae were typical small lymphocytes (4–7 μm in diameter). In contrast, many XT-1⁺ cells in larval thymuses at stage 49 are large (8–10 μm in diameter) lymphoblastoid cells. Thymectomy at stage 46 (5 days postfertilization) depleted XT-1⁺ cells in larval and adult lymphoid organs to background levels. These results suggest that the XT-1⁺ cells are differentiated from the lymphoid precursor cells in the thymus before the appearance of small lymphocytes and migrate into peripheral lymphoid organs. The cell surface determinant recognized by the XT-1 mAb may provide an important marker for the differentiation of T lymphocytes in Xenopus.     

Biological characterization of T and B lymphocytes separated from normal mouse spleen by a physical adherence column method

The capacity of spleen cell populations enriched for T and B lymphocytes by a physical adherence column method to respond in vitro to phytomitogens and allogeneic lymphocytes was determined. Column filtrate cells (T lymphocytes) responded well to phytohaemagglutinin- and mitomycin-C-treated allogeneic spleen cells, but poorly to pokeweed mitogen. Adherent cell populations from the column (B and some T lymphocytes) responded well to pokeweed mitogen, but poorly to phytohaemagglutinin- and mitomycin-C-treated allogeneic cells.Purified peripheral T lymphocytes prepared from normal mouse spleen by the column method reconstituted the depleted in vitro antibody response to the thymic-dependent SRBC antigen of all B lymphocyte sources tested, namely, spleen cells from congenitally athymic mice, neonatally thymectomized mice, and adult thymectomized mice which had been reconstituted with bone marrow, and a lymphocyte population prepared by incubating spleen cells with anti-θ serum and complement. When transferred with sheep erythrocytes to congenitally athymic mice, purified peripheral T cells restored the in vivo IgM and IgG responses of these animals. These results confirm that the column filtrate is a thymus derived subpopulation of cells capable of cell-mediated immunity and cooperation with B lymphocytes in humoral immunity both in vitro and in vivo.     

A Stochastic Model for Surname Evolution

Surnames are inherited in much the same way as biological traits like alleles of one locus. Assuming the heritability of surnames, a simple stochastic model for X, the total number of occurrences of a surname, the Consul distribution defined by the probability mass function: for x = 1, 2, 3,… and zero otherwise and where either (i) m is a positive integer when 0 ≤ θ ≤ 1 such that θ ≦ mθ ≦ 1, or (ii) m≤0, θ ≤0 such that mθ 1, can be arrived at by considering the branching process mechanism. Some applications of the model to real data are also considered.     

Kinetics of B cell memory development during a thymus "independent" immune response

Some parameters of the development of immunological memory to B. abortus (BA) and sheep erythrocytes (SE) in the mouse have been compared. The thymus-independence of the BA response allowed evaluation of B-cell memory in vivo and in adoptive immune responses. A reduced responsiveness to BA was seen during the first few days after the primary injection, whereas enhanced ability to give responses to SE (thymus dependent) occurred at that time.The ability of primed spleen cells to transfer 19S and 7S memory responses to SE developed in parallel. In contrast, the earliest appearance of 19S memory to BA on Days 5–7 after priming was not yet accompanied by memory for the 7S response, but by Day 10 both 19S and 7S memory were present. At 1–2 months after priming, 100-fold fewer cells than needed for transfer of the primary response still transferred excellent 19S and 7S memory responses to BA. Anti-θ treatment of long-term memory 19S and 7S spleen cells did not affect their ability to respond to challenge even with limiting BA doses. It is suggested, however, that the T-independency of the response to BA applies only to the specific induction by antigen of preexisting B cells into antibody secreting cells, whereas optimal B cell memory formation to any antigen may be a separate T-dependent function.Serial spleen cell transfers into lethally irradiated recipients at 1–2 week intervals with antigen challenge at each transfer, appeared to interfere with the development of memory to BA, particularly for the 7S response. No such effect was seen on the responses to SE.     

Genomic Structure and Precise Mapping of a Thymic Regulatory Region on Mouse Chromosome 17 Revealed by a c-mycTransgene Insertion

In one transgenic strain harboring a human c-mycproto-oncogene construct, the transgene was actively and exclusively expressed in the thymus, where it contributed to the development of lymphoma that corresponded to CD4⁺CD8⁺cells. Here, we have pursued the analysis of transgene expression in healthy transgenic mice and show that transgene activation occurs in the thymus 3 days before birth, at a time when CD4⁺CD8⁺lymphocytes emerge. In the adult, its expression is restricted to the CD4⁺CD8⁺cells. The region flanking the transgene insertion site was isolated and made it possible to map the preintegration locus, hereafter calledTsil(for thymus-specific integration locus) on chromosome 17 betweenD17Rp11eandRas12-3.A YAC that contains bothTsiland thePim2locus, previously shown to be involved in progression of T-cell lymphoma, was isolated. Analysis ofTsiloffers a unique opportunity to identify a regulatory region or a gene that might play an important role in T-cell maturation.