Migration and differentiation of bone marrow lymphocytes: development of surface immunoglobulin, Fc receptor, complement receptor, and functional responsiveness as studied with anti-allotype serum

Immunofluorescent studies using fluorescein isothiocyanate-conjugated mouse anti-allotype antibody were carried out to study the migration pattern and the development of surface Ig (SIg), Fc receptor for IgG (FcR gamma), and complement receptor (CR) or mouse bone marrow lymphocytes following intravenous injection into congenic mice. After transfer of bone marrow cells from CSW mice into untreated congenic CWB mice, the absolute number of donor-type SIg-bearing (SIg+) cells and the proportion of either FcR gamma- or CR-bearing (FcR gamma+ or CR+) cells in donor-type SIg+ cells were evaluated in the recipient spleen and the results were compared with those obtained after the transfer of CSW spleen cells. After injection of donor bone marrow cells, detectable donor-type SIg+ cells, although few initially, increased from day 1 to Day 2 and reached a plateau thereafter. The proportion of FcR gamma+ cells in donor-type SIg+ cells, although very low in the donor marrow inoculum, increased progressively after 1 day to reach a maximum at Day 5 (90%). On the other hand, following the transfer of spleen cells, the proportion of FcR gamma+ cells remained at high levels (90%) for 5 days after transfer. Likewise, the proportion of CR+ cells in donor-type SIg+ cells was very low (less than 1%) in the original donor bone marrow cells but high (60%) in the donor spleen cells. However, in transferring bone marrow cells this proportion also increased in the recipient spleen to reach a maximum (49%) at Day 5 although it was lower compared to the percentage of FcR gamma+ cells in donor SIg+ cells. Furthermore, the ability of functional responsiveness to antigen was also examined in the same system by detecting plaque-forming cells (PFC) from donor origin. In transferring donor bone marrow cells into recipient, the participation of donor cells in the PFC response was very low when the recipients were primed with sheep red blood cells at Day 3 after transfer. However, when the recipients were primed at Days 7 to 21 after transfer, increasing numbers of the donor marrow-derived cells were involved in the PFC response. Thus, the present study demonstrates that the bone marrow-derived lymphocytes, albeit lacking both distinctive surface receptors (IgM, FcR gamma, CR) and the functional responsiveness to antigen, continue their development along the B-cell lineage after migrating into the spleen, as evidenced by the surface receptor expression and participation in the antibody response.     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

In vitro and in vivo activation of murine gamma/delta T cells induces the expression of IgA, IgM, and IgG Fc receptors.

The present studies examined resting and activated murine gamma/delta T lymphocytes, in vitro and in vivo, for surface expression of FcR. Polyclonal gamma/delta TCR+ lymphocytes selectively grown from the spleen and intestine of normal mice did not express FcR when the cells were in a resting state, but when cells were activated with anti-CD3 antibody virtually all of the splenic gamma/delta lymphocytes and a large subpopulation of the intestinal gamma/delta lymphocytes expressed IgA and IgM FcR. This was confirmed by using transgenic mice. Resting gamma/delta TCR+ lymphocytes from the spleen, thymus, lymph node, and blood of gamma/delta TCR transgenic mice did not express FcR for any of the five major classes of Ig H chains. Activation of the gamma/delta TCR+ cells via the CD3/TCR complex induced high levels of IgM and IgA FcR and low levels of IgG FcR. Finally, in hepatic granulomas of schistosome-infected mice, activated gamma/delta TCR+ cells are present and express high levels of IgA and IgM FcR and low levels of IgG FcR. These investigations establish that transition of gamma/delta TCR+ lymphocytes from a resting to an activated state (triggered via the T3Ti TCR complex) is accompanied by the induction of surface membrane receptors specific for Ig H chain isotypes. The activation-linked expression of FcR on gamma/delta TCR+ lymphocytes provides potential mechanisms for coupling the functional activities of gamma/delta T lymphocytes with immune mechanisms that involve Ig molecules, such as antibody-dependent cellular cytotoxicity.     

The distribution of rapidly and slowly renewed T, B, and "null" lymphocytes in mouse bone marrow, thymus, lymph nodes, and spleen.

Combined radioautography and immunofluorescence were employed to discern the proportions of rapidly renewed (RR) and slowly renewed (SR) T, B, and null cells in mouse lymph nodes (LN), spleen (Spl), thymus (Thy), and the lymphocyte-rich fraction of bone marrow (BML). Thy and BML were found to consist predominantly of cells with a rapid turnover rate (95.4 and 76.9%, respectively) in accord with their roles as primary lymphoid organs. In contrast, the secondary lymphoid organs were primarily composed of SR cells (LN, 73.4%; Spl, 67.8%) and contained a more even admixture of the six lymphocyte subsets. Most cells in Thy were RR T cells (93.3%), whereas the predominant lymphocyte subpopulation in both LN and Spl consisted of SR T cells (56 and 50%, respectively), with RR T cells being much less frequent (14.6% LN; 6.8% Spl). BML contained both RR and SR cells which stained with the T-cell reagent (7.8 and 7.1%). These percentages are probably overestimates, however, since this reagent stained some nonlymphoid cells in BML. RR B cells were most plentiful in BML (31.4%) and Spl (23.3%), less common in LN (11.2%), and rare in Thy (0.4%). SR B cells were equally numerous in Spl (16%), LN (15.4%), and BML (15.3%). RR null cells were the most prevalent cell type in BML (37.7%), but were infrequent in other tissues (1.6% Thy, 2.1% LN, 2.1% Spl). SR null cells were rare in all tissues (0% Thy, 2% LN, 2.1% Spl, 0.7% BML). These experiments represent the first comprehensive investigation of the composition of the lymphomyeloid organs in terms of RR and SR T, B, and null cells. They conclusively demonstrate RR and SR lymphocytes in all three functional categories (T, B, and null) and show characteristic tissue distributions of the six lymphocyte subsets.     

Regulation of lymphocyte production in the bone marrow. I. Turnover of small lymphocytes in mice depleted of B lymphocytes by treatment with anti-IgM antibodies

To examine the concept that the genesis of lymphocytes in the bone marrow may be regulated by homeostatic feedback signals from peripheral B lymphocytes or their products, lymphocyte production was measured in mice selectively depleted of B lymphocytes by repeated administration of anti-IgM antibodies from birth. The turnover of small lymphocytes was quantitated radioautographically after DNA labeling by continuous infusion of 3H-thymidine. In the femoral marrow of anti-IgM-treated mice, the number of small lymphocytes was reduced and their turnover time was shorter than in control mice, presumably reflecting the premature elimination from the marrow of maturing cells about to express surface IgM. The absolute number of small lymphocytes being produced per femur in unit time, however, was identical in anti-IgM-treated and control mice. Lymphocyte production in the thymus was also unaffected by anti-IgM suppression whereas in the spleen the turnover of small lymphocytes was reduced due to the lack of young immigrant B lymphocytes from the bone marrow. The results demonstrate that the normal large-scale production of lymphocytes in mouse bone marrow is independent of the magnitude of the peripheral pool of B lymphocytes or the level of circulating immunoglobulins, suggesting the process is not subject to feedback control. Some implications for the genesis and diversity of primary B lymphocytes are discussed.     

Fc receptors for IgA on human B, and human non-B, non-T lymphocytes.

Recently, receptors for IgA were demonstrated on subpopulations of human T lymphocytes. In this report, TNP-modified ox erythrocytes coated with the IgA myeloma MOPC-315 were used to detect IgA receptor-bearing lymphocytes within the human non T cell lymphocyte population. A mean of 5.3% (range 2.9 to 12.4%) of E-rosette negative human lymphocytes bound IgA-coated indicator cells. Blocking studies with soluble IgA, IgG, and IgM demonstrated that the IgA receptors on the non-T cell populations were separate and distinct from the Fc-receptors for IgG and IgM. Fractionation of the non-T lymphocytes on anti-human (Fab)2 columns into sIg+ and sIg- populations or by rosetting with EAC to provide CRL+ and CRL- populations demonstrated that Fc-IgA receptors were present on a subpopulation of sIg+, CRL+ lymphocytes, and also on sIg- (non-T, non-B) lymphocytes.     

Ontogeny of the antibody-forming cell line in mice. IV. Appearance of cells bearing Fc receptors, complement receptors, and surface immunoglobulin.

The ontogeny of Ig, FcR, and CR-bearing cells in liver and spleen has been followed by using rosetting procedures. These studies demonstrated a sequential appearance of surface receptors during development. Two types of Ig+ cells could be distinguished according to their rosette morphology and adherence to carbonyl iron: 1) an adherent cell which bound few erythrocytes was found predominantly in fetal liver from 13 days gestation and 2) a nonadherent cell which bound larger numbers of erythrocytes appeared in small numbers in fetal liver from day-16 gestation but represented the major Ig+ cell type after birth. Changes in the proportions of receptor-bearing populations occurred at two particular periods during ontogeny. The first was at birth, where an increase in the proportion of FcR+ cells occurred and the proportion of type 2 Ig+ cells rose rapidly. This probably represented the first appearance of FcR+ B lymphocytes even though cells bearing FcR were detected in fetal liver of all ages (days 12 to 18). The second period was around 10 days after birth when the proportion of Ig+ cells again increased concomitant with the appearance of CR+ nonadherent cells.     

Functional significance of the Fc receptor on mixed lymphocyte culture-activated T blasts.

Fc receptor (FcR)-carrying blast cells were separated from nonFcR blast cells after priming in primary mixed lymphocyte culture (MLC) by erythrocyte-antibody rosetting and by 1-g velocity sedimentation. Both types of blast cells were cytotoxic to relevant allogeneic target cells in vitro. The FcR-positive and FcR-negative blasts were then plated on a feeder layer syngeneic to the primary MLC responder cells. After feeder layer culture both types of cells reverted into secondary small lymphocytes. When restimulated with the original stimulator cells, both types of secondary lymphocytes produced the relevant secondary cell-mediated lysis responses. Thus no functionally dissimilar subclasses of secondary T lymphocytes can be distinguished in the MLC-stimulated T-cell population on the basis of the FcR.     

Regulation of bone marrow lymphocyte production: III. Increased production of B and non-B lymphocytes after administering systemic antigens

To examine the influence of exogenous stimuli on the genesis of lymphocytes in mouse bone marrow, the production rate and subsets of marrow lymphocytes were examined after a systemic injection of sheep red blood cells (SRBC). Radioautographic analysis after either pulse labeling or infusion of [³H]thymidine revealed a pronounced increase in the number of newly formed small lymphocytes appearing in the marrow, maximal 4–5 days after SRBC injection and dose related. The resulting expansion of the marrow lymphocyte population included both immature B cells and null cells, as shown by cell surface and cytoplasmic markers. Similar stimulation of marrow lymphocyte production followed an injection of either bovine serum albumin or mineral oil. No comparable stimulation occurred in either the thymus or the spleen. The results demonstrate that antigens and nonspecific irritants can exert a central effect in the bone marrow, producing a surge in the production of both primary B and non-B lymphocytes. The possible role of external stimulants in determining the normal rate of bone marrow lymphocyte production is discussed.     

Cytotoxicity from cultured cells: analysis of precursors involved in generation of human cells mediating natural and antibody-dependent cell-mediated cytotoxicity.

Natural cell-mediated cytotoxicity of human peripheral blood lymphocytes natural killer (NK) against K-562 and antibody-dependent cellular cytotoxicity (ADCC) against Chang cells, as measured in a 4-hr ⁵¹Cr release assay, were both completely removed by depletion of Fc receptor-positive (FcR+) cells. After in vitro culture for 7 days, however, NK- and ADCC-like activities spontaneously regenerated. The nature of precursor cells was studied by examination of lymphocyte subpopulations required for generation of this cytotoxicity. After depletion of FcR+ cells from PBL, the following subpopulations were prepared: sheep erythrocyte rosette-forming cells (E+), surface membrane immunoglobulin-positive cells (SmIg+), and null cells (lacking E+, SmIg+, or FcR+ markers). Separate cell types or mixtures were cultured in vitro in medium containing 10% fetal calf serum for 7 days and then tested for NK and ADCC. Whereas unseparated FcR-depleted cells developed substantial cytotoxic activity, each of the subpopulations cultured alone was negative or had low activity. Addition of SmIg+ cells to other cell types had no effect; however, mixture of 80% E+ and 20% null cells resulted in optimal NK and ADCC. It is not presently clear which population the precursors were in. However, the requirement for proliferation by the null cell population but not by the E+ cells (as indicated by sensitivity to radiation and mitomycin C) suggested that the precursors for NK cells may be null cells.     

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.     

Fc receptor heterogeneity: immunofluorescent studies of B, T, and "third population" lymphocytes in human blood with rabbit IgG b4/anti-b4 complexes.

IgG Fc receptors on human peripheral blood lymphocytes (PBL) were characterized by immunofluorescence studies with defined rabbit IgG b4 allotype/anti-allotype complexes. Three discrete types of Fc receptor-bearing cells, totaling approximately 33% of PBL, were identified. Fc receptors of the three types differed in their sensitivity to trypsin and in either absolute or localized density (topography) as determined by variable requirements for anti-IgC cross-linking in order to visualize bound complexes microscopically. The question of additional heterogeneity related to differences in individual Fc receptor affinity for complexed IgG was not approached in this study. Ten to 15% of PBL had pronase-sensitive, trypsin-resistant Fc receptors readily detected by direct immunofluorescence by using large fluorescein-conjugated complexes prepared near equivalence. Double label and lymphocyte fractionation experiments established this population to be largely distinct from suface IgM+ B cells and T cells, and identical to EA Ripley rosette-forming cells. Approximately 50% of surface IgM+ B cells and approximately 10% of T cells had lower density Fc receptors identified by indirect immunofluorescence with small complexes prepared in antigen excess or by cross-linking fluorescein-conjugated complexes with anti-rabbit IgG anti-serum. An additional approximately 15% peripheral T and B cells had very low density Fc receptors detectable by complexing the IgG on the cell surface by sequential incubations of cells with b4 IgG and anti-b4. Fc receptors on B and T cells were sensitive to both pronase and trypsin digestion. The heterogeneity of IgG Fc receptors on different lymphocyte subpopulations as defined by these these experiments may be of relevance for further analysis of normal and abnormal immune function.     

Activation of lymphocytes alters Fc receptor-beta 2-microglobulin interrelationship on the lymphocyte surface

The effect of anti-beta 2-microglobulin (anti-B2Mi) on the expression of Fc receptors (FcR) of human lymphocytes was compared on resting and activated cells. Previously we reported that anti-B2Mi induces a "co-shedding" of FcR with the beta 2-microglobulin (B2Mi)-anti-B2Mi complexes when used under the conditions where the redistribution of membrane molecules is allowed (Sármay et al., Cell. Immunol. 56, 452, 1980; Sármay et al. Immunology 36, 339, 1979). Furthermore our group also described two types of FcR-bearing cells, one which shed their FcR during a temperature shift from 4 to 37 degrees C (FcRI+ cells) and the other which has an immobile type FcR under the same circumstances (FcRI+ cells) (Sándor et al., Immunology 38, 553, 1979; Sármay et al., Immunology 34, 315, 1978). In this work we have characterized the FcR released from the membrane as a consequence of anti-B2Mi treatment. We have found that they are the mobile, FcRI type. It was proved that the shedding of this FcRI is a consequence of the anti-B2MI-induced transformation of FcRII into the FcRI form on the membrane of the antibody-treated lymphocytes. On the activated T cells, however, anti-B2Mi is incapable of inducing the same phenomenon in the early phase of activation. In contrast, FcR expression is blocked by anti-B2Mi treatment similarly to that on resting lymphocytes, on the surface of activated B cells, or on activated T cells in the later phases of activation.     

Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.     

A phenotypic and functional analysis of long-lived B and T lymphocytes

The lymphocyte composition of spleen, lymph nodes, bone marrow, and thymus of mice submitted to hydroxyurea treatments for four consecutive days was studied. The treatment selects for small lymphocyte populations that represent between 4 and 20% of control numbers in the various organs. Spleen and bone marrow contain the same B cell population with a low IgM, high IgD, low I-E phenotype, which respond to LPS at control clonal frequencies. The T cell compartment is equally depleted, and the lymphocytes remaining contain frequencies of clonable cells in response to mitogens and IL-2 that are comparable to those detected in normal spleen cells. Overall, the results suggest that only a minor fraction of all lymphocytes in a normal young adult mouse have life spans longer than 4 days.     

Characterization of small lymphocytes in the bone marrow of mice after prolonged treatment with anti-IgM antibodies.

In mice treated with anti-IgM antibodies from birth, small lymphocytes in the bone marrow and spleen have been characterized by their incidence, surface markers, and turnover. Essentially complete elimination of IgM-bearing small lymphocytes followed injections of anti-IgM but this treatment did not deplete the IgM-negative small lymphocytes in the marrow. These IgM-negative cells also lacked other markers of B lymphocytes, such as receptors for Fc and complement; they failed to bind anti-mouse T-lymphocyte serum and they formed a rapidly renewing population within the marrow. This population may represent cells whose normal differentiation has been aborted by the anti-IgM antibodies or, alternatively, they may be “null” cells, distinct from B lymphocytes.     

Differentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling

DNA labeling by ³H-thymidine in vitro and antiglobulin-¹³¹I binding in vitro were used to determine the development and turnover of immunoglobulin-bearing lymphocytes in mouse bone marrow.Bone marrow cells from CBA mice previously injected repeatedly with ³H-thymidine for 1–84 hr were exposed to ¹³¹I-labeled rabbit-antimouse globulin for 30 min at 0 °C, and examined radioautographically. The antiglobulin-binding cells in bone marrow were predominantly (97–98%) nondividing small lymphocytes. Some plasmacytoid and monocytoid cells, but not the proliferating large lymphoid cells, also bound antiglobulin. The ³H-thymidine labeling index of the small lymphocyte population showed a rapid exponential increase (50% in 32 hr). The first small lymphocytes to show ³H-thymidine labeling were those lacking antiglobulin-binding capacity, reaching approximately 90% ³H-thymidine labeling after 2 days. Small lymphocytes which bound antiglobulin-¹³¹I at a concentration of 1.0 μg/ml became labeled with ³H-thymidine only after a lag of approximately 1.5 days. More avid antiglobulinbinding cells were delayed a further 12 hr in ³H-thymidine labeling. During in vitro culture the proportion of antiglobulin-binding small lymphocytes increased progressively in bone marrow but decreased in spleen cell suspensions.The results demonstrate a continuous, rapid renewal of immunoglobulin-bearing small lymphocytes in adult mouse bone marrow. Surface immunoglobulin molecules are not detectable when marrow small lymphocytes are first formed, but they appear and increase progressively in density as the cells mature.     

Differentiation of lymphocytes in mouse bone marrow. I. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues

The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding.Cell suspensions from CBA mice were exposed to ¹²⁵I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin-¹²⁵I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin-¹²⁵I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin-¹²⁵I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti-θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice.The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ-bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.     

Mobility and density of AgB, "Ia", and Fc receptors on the surface of lymphocytes from young and old rats.

Analysis of spleen cell populations from old Lewis rats (greater than 24 months) and from young Lewis rats (3 to 4 months) in a fluorescence-activated cell sorter indicated that with aging there is a loss of brightly stained Ia and Fc receptor- (FcR) positive cells. The density of AgB, Ia, and FcR was diminished on the surface of cells from old rats. The rate of capping of all three membrane proteins was slower on cells from old rats. Colchicine treatment allows capping of AgB with a single ligand only in young rats. Fluorescence photobleach recovery experiments (FPR) show that the fluidity of the lymphocyte membrane from old rats is diminished and the lateral diffusion of AgB is decreased. The colchicine and FPR experiments suggest that the changes in capping in old rats are due to, in part, alterations in membrane fluidity and cytoskeletal function.     

Regulation of bone marrow lymphocyte production: IV. Cells mediating the stimulation of marrow lymphocyte production by sheep red blood cells: Studies in anti-IgM-suppressed mice,athymic mice,and silica-treated mice

Some cellular requirements have been examined for the stimulation of lymphocyte production in mouse bone marrow by injected sheep red blood cells (SRBC). The increased genesis of marrow lymphocytes after a single dose of SRBC assayed radioautographically after [³H]thymidine labeling was unimpaired in the marrow of mice treated with anti-IgM antibodies from birth to eliminate B lymphocytes, and in congenitally athymic mice lacking T lymphocytes. However, pretreatment of mice with silica to depress macrophage function completely abolished the SRBC effect both on the total lymphocyte production and on the number of B and null small lymphocytes in the marrow. Comparative studies were performed on the thymus and spleen. The results demonstrate that the stimulation of marrow lymphocyte production by SRBC is mediated by a silica-sensitive mechanism, does not require B or T lymphocytes, and is independent of the humoral immune response. Thus, extrinsic agents may amplify the production of primary B cells and other lymphocytes in the bone marrow by an antigen-nonspecific mechanism, putatively mediated by macrophages.