Limiting Dilution Culture System Research Articles

The B cell repertoire of patients with rheumatoid arthritis. Frequencies and specificities of peripheral blood B cells reacting with human IgG, human collagens, a mycobacterial heat shock protein and other antigens.

Using a potent in vitro limiting dilution culture system, we have activated human peripheral blood B cells to proliferate and to differentiate into antibody-secreting cells (ASC). Under these conditions 25-100% of B cells are clonally expanded and produce IgM, IgG or IgA. Culture supernatants were tested for antibodies binding to human IgG-Fc fragments (RF), the 65-kD heat shock protein of Mycobacterium bovis (hsp60), human collagens type I, II, IV, V, transferrin, lactoferrin, albumins, and gelatine. All blood samples contained precursors of ASC (p-ASC) able to produce IgM binding to these antigens in frequencies above 0.03% of B cells. Most interestingly, a significant difference exists between rheumatoid arthritis (RA) patients and controls, concerning the relative frequencies of p-ASC able to produce monospecific or multireactive RF. Whereas most p-ASC(RF) in RA patients are monospecific (mean ratio 3.7), most p-ASC(RF) in healthy control persons are cross-reactive with at least one of five other antigens tested (mean ratio 0.2). The data suggest a disease-specific expansion of p-ASC committed to the production of monospecific rheumatoid factors.

Phase I/II feasibility study evaluating the generation of leukemia-reactive cytotoxic T lymphocyte lines for treatment of patients with relapsed leukemia after allogeneic stem cell transplantation

Graft-versus-host-disease may be avoided and the likelihood of a graft-versus-leukemia reaction increased by infusion of in vitro generated, leukemia-reactive, cytotoxic T lymphocyte (CTL) lines as treatment for patients with relapsed leukemia after allogeneic stem cell transplantation, instead of donor lymphocyte infusion. The aim of this study phase I/II study was to assess the feasibility of large-scale in vitro generation of leukemia-reactive CTL for clinical use. Using a modified limiting dilution culture system donor T cells were stimulated with HLA-identical leukemic antigen presenting cells. Feasibility experiments demonstrated that in 16 of 27 donor-recipient pairs tested a CTL line could be generated. Twelve of these 16 patients developed a relapse and for 11 of these 12 patients a CTL line was generated under Good Manufacturing Practice conditions. The CTL lines showed moderate to high cytotoxic activity against original recipient leukemic cells in vitro. Eight patients with a relapse received from one to seven CTL lines. One patient entered a complete remission after CTL infusion only, one entered a complete remission after combined CTL infusion and donor lymphocyte infusion, two patients had temporarily stable disease, and in four patients no response was observed. Although the current procedure to generate these CTL lines is feasible, the strategy is logistically complex and time-consuming, and needs further improvement. Key words: cellular immunotherapy, CTL, leukemia, allogeneic stem cell transplantation.

Dissection of Systems, Cell Populations and Molecules

This paper summarizes studies on antibody formation in the bone marrow and the suppressive effects of intravenous immunization with allogeneic blood cells on T-cell function in mice. The latter studies were extended by employing the limiting dilution culture system developed in Ivan Lefkovits' laboratory and implemented in collaboration with Lucien Aarden. Thereby, the functional data were complemented with frequencies of alloantigen-activated helper (Th) and suppressor T cells after intravenous alloimmunization. These results led the Rotterdam group to studies on the prevention of rejection of the foetal 'allograft'. Th cells are central in foetal allograft rejection and pregnancy success. Characteristic for human pregnancy is the production of the glycoprotein chorionic gonadotropin (hCG) hormone. The in vivo liberated peptide fragments originating from nicking of the sequence MTRVLQGVLPALPQ in the beta-chain of hCG were considered for their immunoregulating capacity related to pregnancy success. These peptides - prepared synthetically - (MTR, MTRV, LQG, LQGV, VLPALP and others) indeed showed a remarkable spectrum of biological effects (e.g. modulation of angiogenesis, inhibition of septic shock syndrome, prevention of diabetes and reduction of ischaemia-reperfusion damage). The paper interprets and generalizes these findings and projects them into various research directions, especially towards the proteomics framework studies built up in Ivan Lefkovits' laboratory in the nineties. During the time period, when Ivan spent a mini-sabbatical in Rotterdam (months after closing down the BII) more detailed discussions were intiated. This paper is meant to keep the discussions between the involved research groups going on.

Lack of specific antibody response in common variable immunodeficiency (CVID) associated with failure in production of antigen-specific memory T cells. MRC Immunodeficiency Group.

Several T cell defects have been described in the antibody deficiency disease, CVID, but there have been few data on the generation of responses of specific T cell populations to primary neoantigens. We have now used immunization with the neoantigens, keyhole limpet haemocyanin (KLH) and DNP-Ficoll, to evaluate immune responses in CVID patients and normal donors. B and T cell responses were examined 2 and 4 weeks post-immunization. Sera were examined for IgM and IgG anti-KLH responses by ELISA and for anti-DNP-Ficoll activity by haemagglutination. The frequency of KLH-responsive T cells was measured by DNA synthesis in a limiting dilution culture system. Low density cells enriched for dendritic cells were pulsed with KLH and cultured with different numbers of autologous T cells. T cells from normal donors and from patients showed a low frequency of antigen-specific precursor T cells (< or = 1:200,000). After KLH immunization the frequency increased in normal donors (1:60,000 and 1:30,000 at 2 and 4 weeks, respectively), while in CVID patients it did not change from the pre-immunization level. The defect may extend to a dysfunction of antigen-specific cells, rather than being solely due to the reduced numbers of cells, since mean responses of 'positive' wells were also reduced. The serum-specific antibody response paralleled the T cell data, in that all normal donors but none of the CVID patients generated IgG KLH-specific antibodies. CVID patients did produce IgM antibodies against the T-independent DNP-Ficoll, but at a lower level than normal controls. These data show that both T and B cells from CVID patients have defective responses to specific antigen, implicating both lineages in the antibody deficiency.

Interleukin-12 up-regulates the induction of an antigen-specific antibody response in cultures of human lymphocytes.

The influence of interleukin-12 (IL-12) on the induction of a specific antibody response to the T-dependent antigen sheep erythrocytes (SRBC) in cultures of human blood lymphocytes was investigated. The response, evaluated as number of antigen-induced antibody-producing cells, was greatly increased in the presence of IL-12. When a two-stage limiting dilution culture system was used, the plot of the number of seeded cells versus the logarithm of the fraction of negative cultures deviated from linearity in antigen- and IL-12-stimulated cultures. However, linearity was reached when IL-2 was added in the second stage. Under these latter conditions, since single-hit criteria were fulfilled, it was possible to estimate the frequency of SRBC-specific B cell precursors able to respond to the antigen and to show that such frequency was increased upon addition of IL-12. Thus, the enhancing effect of IL-12 may be based on an increased frequency of responding precursor cells. The results here presented demonstrate, to our knowledge for the first time, a definite role of IL-12 in the induction of a specific antibody response in human cells. Further, they stress the importance for such studies of appropriate in vitro systems. Finally, they show that the induction of primary immune responses in cultures of human peripheral blood lymphocytes mostly depends on the proper cytokine balance at different time points.

A new limiting dilution culture system for the detection of T cell subsets in T cell-depleted marrow grafts.

T cell depletion (TCD) has been achieved using techniques that cause the inactivation, lysis, or physical removal of T cells from the donor marrow. The clinical results of TCD reflect, in part, the degree of TCD achieved and the subsets that are removed. To better evaluate TCD using the monoclonal antibody (mAb) T10B9, we have performed a series of flow cytometry and mAb blocking studies and have developed a new limiting dilution assay (LDA) that allows the detection of T cell subsets that survive treatment. T cell growth was stimulated with PHA, rIL-2, and irradiated feeder PBMC in a total well volume of 20 microliters. Growth was scored by microscopic examination on days 14-16 of incubation. Immunomagnetic beads coated with mAb were added to the growing wells and incubated, then the plates were fixed to a template of samarium cobalt magnets before washing away nonadherent cells. Wells in which > 50 cells bound > or = 2 beads were scored as positive. Flow cytometry indicated that T10B9 recognized all T cells, but complement-mediated lysis spared a significant proportion of the TCR gamma delta + subset. The epitope recognized by T10B9 on TCR gamma delta + cells appears to be differentially expressed compared with TCR alpha beta + T cells based on antibody blocking studies. In contrast to antibodies to CD3 epsilon, T10B9 binds less well to TCR gamma delta + cells, possibly resulting in incomplete complement-mediated lysis of this subset. The relative sparing of TCR gamma delta + cells was found in marrow and peripheral blood. Subset LDA confirmed that the TCR gamma delta + cells detected by flow cytometry were capable of growth and further showed that OKT3 did not spare TCR gamma delta + cells. The subset LDA should prove useful in helping to assess the role of T cell subsets in clinical events post-TCD bone marrow transplantation.

Optimising a limiting dilution culture system for quantitating frequencies of alloreactive cytotoxic T lymphocyte precursors

We have developed a limiting dilution assay for estimating the frequencies of allo-MHC reactive cytotoxic T lymphocyte precursors (CTL-p) in peripheral blood. The culture conditions were optimised in order to give maximum sensitivity while at the same time maintaining a high degree of specificity. While the addition of filler cells, interleukin-1 and interleukin-4 were unnecessary, the addition of interleukin-2 (IL-2) was crucial for optimal expansion of the CTL-ps. However, excess exogenous IL-2 was found to give rise to nonspecific cytotoxicity, and 5 U/ml final concentration of recombinant IL-2 added on Days 3 and 6 was optimal. The establishment of baseline control cultures for determining positivity and negativity of the test cultures is discussed. Using this modified assay system, we demonstrated that the culture conditions fulfil “single hit” criteria and that the assay is highly specific.

Clonal repertoire analysis of murine B cells specific for repeat sequence antigens of Plasmodium falciparum

Clonal analysis of the murine B-cell repertoire has been used to investigate the possible role of tandem repeat sequence epitopes of Plasmodium falciparum in immune evasion. A limiting dilution culture system was used whereby murine spleen cells were stimulated with the B-cell mitogen lipopolysaccharide (LPS) in the presence of 3T3 fibroblast filler cells. One in three B cells were shown to produce clones secreting immunoglobulin measurable by an ELISA. The frequency of antibody forming cell precursors (AFCp) specific for the 3' repeat epitopes of the ring injected erythrocyte surface antigen (RESA) was estimated in non-primed mice and found to be low. However, an accurate frequency determination was not possible using this method since the detection of the few positive cultures was found to depend on the presence of more than one AFCp or its products. Limiting dilution analysis was used to assess the frequency and repertoire of splenic AFCp at various times after immunization with a synthetic peptide of the RESA 3' repeat epitope (8 x 4-mer), presented in various ways. There was no marked increase in LPS-responsive AFCp specific for this antigen at the level of either IgM or IgG secretion. This was in marked contrast to the antibody response in vivo, where moderate IgG antibody titres, normally indicative of a secondary response, were seen in the serum of the same mice used for AFCp assay. This discrepancy between serum titre and AFCp frequency following immunization was not apparent with a non-malarial antigen, keyhole limpet haemocyanin (KLH). It was concluded that the LPS-stimulated limiting dilution culture system was not registering RESA-specific memory AFCp. These results raise the possibility that the malarial antigens are deficient in memory B-cell generation, or that secondary responses to these determinants may arise from a distinct B-cell progenitor which is non-responsive to LPS in vitro.

Antigen-presenting T cells. II. Clonal responses of alloreactive and virus-specific self-restricted human cytotoxic T cell responses stimulated by T lymphoblasts.

The capacity of various stimulator cell types to present alloantigens or viral antigens to resting human CD8+ cytotoxic lymphocyte precursors (CLP) was analyzed in a limiting dilution culture system. Cell sorter-separated T lymphoblasts of both CD4+ and CD8+ phenotypes but not resting T cells were found to efficiently stimulate the clonal development of allogeneic CD8+ CLP. Thus, 5000 CD4+ T lymphoblasts activated as many (one out of 200 to one out of 300) allogeneic CLP as 50,000 peripheral blood mononuclear stimulator cells. This potent stimulator activity was found in CD4+ and CD8+ T lymphoblasts activated by mitogen, anti-T3 monoclonal antibody, or mixed leukocyte reactions. Cytotoxic T cells generated in this system were highly specific for HLA class I antigens. Furthermore, T lymphoblasts infected with mumps virus efficiently induced development of autologous CLP into CTL clones that were virus specific and self-HLA restricted, as shown by split-well analysis. The possible in vivo significance of antigen-presenting T lymphoblasts is discussed.

Comparative analysis of infection and transformation of lymphocytes from African buffalo and Boran cattle with Theileria parva subsp. parva and T. parva subsp. lawrencei

This study compared infection and transformation of peripheral blood mononuclear cells (PBM) of Boran cattle and African buffalo in vitro to determine whether differences occurred which could account for the greater susceptibility of Boran cattle to infection with Theileria parva subsp. parva and T. parva subsp. lawrencei. PBM from buffalo and cattle had a similar percentage of cells which bound T. parva subsp. parva sporozoites (24 to 34%) and in which schizonts developed during the first week after infection (18 to 23%). Using a limiting dilution culture system, it was established, however, that a significantly higher proportion of cattle PBM transformed into continuously replicating cell lines after infection with T. parva subsp. parva than did buffalo PBM. The evidence suggests that the low capacity of T. parva subsp. parva to establish infections in buffalo compared with cattle is related to the lower frequency of buffalo cells which undergo transformation. With T. parva subsp. lawrencei, however, the frequency of transformation of buffalo PBM was higher than that for cattle PBM. The frequency of cells transformed by T. parva subsp. lawrencei, therefore, cannot account for the greater resistance of buffalo to infections with T. parva subsp. lawrencei. Buffalo must have other mechanisms, either innate or acquired, which control infection with T. parva subsp. lawrencei more efficiently than in cattle.

Heterogeneity of EBV-transformable human B lymphocyte populations.

Although most human B cells express receptors for Epstein Barr virus (EBV), few (usually less than 1%) are readily transformed into B lymphoblastoid cell lines after exposure to EBV. Transformable cells previously have been found to be mostly resting B lymphocytes. We recently developed a limiting dilution culture system which permits the growth of EBV-transformed B lymphocytes with high efficiency. Because in this system up to over 30% of peripheral blood- or tonsil-derived B cells respond to EBV, we re-examined the properties of EBV-transformable cells. Frequencies of transformable lymphocytes were determined by Poisson analysis. EBV-susceptible B cells committed to IgM, IgG, or IgA secretion were found to occur in the range of 3 to 27, 0.1 to 6, and 0.1 to 5 per 100 B cells, respectively. Under our culture conditions, a major proportion of the IgM-committed cells derived from large lymphocytes which appeared to have entered the cell cycle. This population contains most of the EBV-responsive cells detected and, therefore, most of the additional cells responding in our culture system. In contrast, precursors of IgG- or IgA-producing lymphoblast lines were small cells with DNA contents typical for the G0/G1 phases of the cell cycle. Anti-immunoglobulin antibodies were used in panning experiments to separate B cell subpopulations which expressed different immunoglobulin isotypes on their surface. In limiting dilution cultures of these purified B lymphocytes subsets, it was found that virtually all precursors of IgM-producing cell lines expressed surface IgM (sIgM) before their infection and transformation by EBV. The "cloning efficiency" of positively selected, large sIgM+ cells approached 100%. In contrast, sIgG or sIgA were found only on cells committed to the production of IgG or IgA, respectively. The expression of sIgD was examined by using sequential panning procedures. Virtually all IgM-committed lymphocytes and a subset of cells committed to IgA secretion were found among the sIgD+ cells. The majority of cells committed to IgA production and all IgG-committed cells were found in the sIgD- B cell population. Our findings indicate that the EBV-susceptible B cell subset of normal lymphocytes is heterogeneous with respect to cell size, cell cycle, sIg determinants, and efficiency of transformation. On the basis of our findings and previous reports, we propose a model in which transformability is a B cell-inherent property. Factors unrelated to the virus but present in our culture system appear responsible for the enhanced vulnerability to viral transformation in some cells which entered into the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal development of functional T cell subsets in the mouse: a frequency analysis of mitogen reactive precursors of proliferating, of cytotoxic and of IL 2 producing T cells

In order to study the postnatal development of functional T cell subsets in the mouse, amitogen-driven limiting dilution culture system was used for a precursor frequency analysis of proliferating, of cytolytic and of IL 2-producing T cells, respectively, present in spleen and thymus of mice from neonatal to adult age. In adult mice, the majority (up to 100%) of splenic T cells was capable to respond to Concanavalin A. In contrast, an up to tenfold lower frequency of mitogen reactive precursors was found within positively selected Thy-1 + spleen cells of neonatal mice. Within this fraction of Con A reactive neonatal T cells, there was an apparent imbalance in the CTL p/PTL p ratio within the first to second week after birth. Accordingly, significant numbers of immunocompetent precursors of HTL p were detected in the spleen shortly after birth, while the vast majority of CTL p developed later on. This differential development of CTL p and PTL p was not seen with thymocytes of the same mice, where from the age of two to three days on up to the adult age the frequency of both CTL pand PTL p remained largely unchanged. Analysis of the Lyt-antigen expression, in addition, revealed phenotypical differences between neonatal and adult Thy-1 + spleen cells, such thatLyt-2 antigen density but not the proportion of Lyt-2 positive T cells, was considerably lower in newborn mice. An age related increase in both Lyt-2 antigen density and CTL p frequency was parallelled by the rapid increase in the total number of splenic T cells during the second and third postnatal week, reaching 60–70% of adult values. At this time, a normalisation of the CTL p/PTL p ratio at approximately 0.4 had occurred.

Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet.

The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.

Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and “spontaneously” occurring “background” plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of “spontaneously” occurring (“background”) IgM-, IgG-, and IgA-secreting cells as well as the “background” IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the “background” IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by “background” immunoglobulin-secreting cells.

SIgD-negative B cells from neonatal mice do not respond to the thymus-independent antigen TNP-BA in limiting dilution cultures.

The majority of B lymphocytes in adult mice express both IgM and IgD on their surface (sIgM and sIgD). A small percentage of sIgM+ splenic B cells lack (or express very low levels of) sIgD. These cells have been termed "mu-predominant" (mu p) B cells. In neonatal mice (5 to 12 days of age), mu p B cells account for more than 50% of the sIg+ cells. There is conflicting evidence concerning the immunocompetency of mup cells in vitro. To study this question further, splenocytes from neonatal BALB/c mice were depleted of sIgD+ B cells by a panning procedure. A portion of the nonadherent (mu p) cell population was analyzed for residual sIgD+ cells by using indirect immunofluorescence in conjunction with the fluorescence-activated cell sorter (FACS). Such cells were then tested for their responsiveness to the thymus-independent (TI) antigen, trinitrophenyl Brucella abortus (TNP-BA), by using a limiting dilution culture system. Results indicate that the depletion of sIgD+ B cells and the decrease in the precursor frequency of splenocytes responding to TNP-BA are very similar, suggesting that virtually all of the responding B cells bear sIgD.

Primary in vitro generation of cytotoxic cells specific for human minor histocompatibility antigens between HLA-identical siblings.

A limiting dilution culture system was developed for the primary in vitro detection of human minor histocompatibility antigens by cytotoxic T lymphocytes (CTL). CTL were generated in primary in vitro culture between two HLA-identical sibling pairs and propagated as stable CTL lines. Population and family studies indicate that these CTL lines recognize minor histocompatibility antigens in an HLA-restricted manner. The antigen recognized by one CTL line is detected on six (out of 37) HLA-B7-positive donors but not on 32 HLA-B7-negative donors. The cytotoxicity of this CTL line is mediated by T3+, T8+ effector cells. The antigen detected by this CTL population is different from all known human minor histocompatibility antigens. The data of this study, like those in the mouse system, suggest that a suppressor cell is diluted out in a limiting dilution culture, which allows the activation of the CTL precursors.

T-T cell interactions during in vitro cytotoxic T cell responses. VI. The role of T cell-derived colony-stimulating factor in helper T cell activation.

A limiting dilution culture system has been used to analyze the role of T cell-derived colony stimulating factor (CSF) during the activation of IL 2-producing helper T lymphocytes (HTL). EL4 thymoma-derived supernatant (EL4-SN) increased about 4-fold the frequency of HTL precursors responding to metabolically active allogeneic stimulator cells. Upon biochemical separation this biological activity within the EL4-SN segregated from interleukin 2 (IL 2) and gamma interferon but was associated with or identical to CSF. Further, a comparable rise in HTL precursor frequencies was observed when semipurified interleukin 1 (IL 1) derived from the P388 D1 cell line was added to limiting dilution cultures. In contrast to IL 1, semipurified CSF failed to facilitate the activation of HTL precursors when heat-treated allogeneic spleen cells were used as stimulator cells. Because EL4-derived CSF was found to induce IL 1 production by macrophages we conclude that T cell-derived CSF amplifies, via stimulation of antigen-presenting cells the number of inducible HTL precursors. Therefore, the CSF/IL 1-dependent increase in HTL precursor frequencies reported here may reflect the differential activation threshold of HTL-precursors, most of which will not be activated by antigen per se but only in presence of additional cytokines.

Quantitative representation of all T cells committed to develop into cytotoxic effector cells and/or interleukin 2 activity-producing helper cells within murine T lymphocyte subsets.

A limiting dilution culture system based on stimulation with concanavalin A (Con A) has been used to study the quantitative distribution of helper and of cytotoxic precursor cells in Lyt-2-defined subpopulations of murine T cells. Virtually all of the selected Lyt-2+ and Lyt-2-T cells grow and expand to large clonal colonies within an 8-9-day culture period. Our data show that upon stimulation with Con A, 90% of the Lyt-2-T cells were capable to produce interleukin 2 (IL 2) activity. In addition, IL 2 activity is produced by 8-10% of Lyt-2+ T cells. However, at the clonal level, the average of the IL2 activity produced by Lyt-2+ T cells is about 8-fold less as compared to Lyt-2-T cells. Precursors of cytotoxic T cells were almost exclusively found in the Lyt-2+ population, of which about 70% displayed lytic activity in a lectin-dependent cytolysis test. For the vast majority of clones analyzed the capacity to produce IL 2 activity and the capacity to express lytic activity, was found to be mutually exclusive. A minority of clones (less than 3%) was found to simultaneously produce IL 2 activity and to express cytotoxicity. These latter cells are therefore considered as bifunctional T cells.

Differentiation from precursors in athymic nude mouse bone marrow of unusual spontaneously cytolytic cells showing anti-self-H-2 specificity and bearing T cell markers.

We describe an in vitro limiting dilution culture system that supports growth and differentiation of nylon wool nonadherent bone marrow cells from athymic nude mice. Cells were seeded at low cell numbers (5-120 cells per 20-microliter microculture well) in the absence of added filler or feeder cells but in the presence of conditioned medium. Microwells positive for growth appeared to contain a single clone of cells that adhered together to form a tight cluster referred to here as a colony. A fraction of colonies contained cells that expressed an unusual spontaneous cytolytic activity. They lysed syngeneic or semisyngeneic Con A blast or tumor cell targets but seldom lysed H-2-incompatible Con A blast or tumor target cells, even in a lectin-facilitated assay. A large fraction of colonies contained lymphoid cells that expressed the T cell markers Thy-1 and Lyt-1. Colonies expressing spontaneous cytolytic activity and also containing cells with Thy-1+ and/or Lyt-1+ markers could be grown from nylon wool nonadherent nude marrow cells depleted rigorously by cell sorting of cells expressing either of these markers. Expression of Thy-1 and spontaneous cytolytic activity in a particular colony was significantly correlated. Short-term lines established from cytolytic colonies with T cell markers maintained both characteristics. The cytolytic effector cells observed in these cultures may represent an early stage in the development of the T cell repertoire.

Frequency analysis of functional immunoglobulin C and V gene expression in murine B cells at various ages.

The frequencies of lipopolysaccharide- (LPS) reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of mice at different ages. A limiting dilution culture system was employed that allows the growth and development of every LPS-reactive B cell into a clone an IgM-secreting cells that are capable of switching to other Ig heavy chain isotypes (C gene expression). The secretion of IgM and IgG1 was assessed in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V gene expression) were detected with the use of plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The frequencies of LPS-reactive B cells in the spleen and BM of C3H/Tif, C57BL/Ka, BALB/c, and CBA/Rij mice appeared to be similar in 6- to 12- and 100-wk-old animals, as was the switch frequency to IgG1 secretion in three strains tested. Moreover, no age-related changes were observed in the frequencies of antigen-specific B cells within the pool of LPS-reactive B cells in the spleen and BM of C57BL/Ka mice. The frequencies ranged from 1 in 10 to 1 in 20 for NIP4- and NNP2-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 4000 for SRBC, HRBC, and GRBC. The specificity repertoire of the "spontaneously" occurring ("background") IgM-secreting cells in the spleen and BM, on the other hand, did differ between young and old C57BL/Ka mice. During aging the frequencies of the tested specificities decreased in the spleen but increased in the BM. Our data indicate that in unintentionally immunized mice the clonal selection of B lineage cells by antigen takes place at the level of the mature, antigen-reactive B cell.