The effect of levamisole on human lymphocyte mediator production in vitro

Levamisole has previously been demonstrated to increase delayed hypersensitivity reactions in anergic patients. In order to elucidate the mechanism by which levamisole stimulates the immune response in vivo, we have studied the effect of this substance on both human lymphocyte proliferation and lymphocyte-mediator production in vitro. Our results indicate that in vitro levamisole augments the production of soluble mediators by mitogen-stimulated lymphocytes, while having no effect on lymphocyte proliferation.     

Self DNA from Lymphocytes That Have Undergone Activation-Induced Cell Death Enhances Murine B Cell Proliferation and Antibody Production

Systemic lupus erythematosus (SLE) is characterized by prominent autoinflammatory tissue damage associated with impaired removal of dying cells and DNA. Self DNA-containing immune complexes are able to activate both innate and adaptive immune responses and play an important role in the maintenance and exacerbation of autoimmunity in SLE. In this study, we used DNA from lymphocytes that have undergone activation-induced cell death (ALD-DNA) and analyzed its role on the activation and differentiation of B cells from normal BALB/c mice as well as lupus-prone MRL^+/+ and MRL/lpr mice. We found that ALD-DNA directly increased the expression of costimulatory molecules and the survival of naïve B cells in vitro. Although ALD-DNA alone had little effect on the proliferation of naïve B cells, it enhanced LPS-activated B cell proliferation in vitro and in vivo. In addition, ALD-DNA increased plasma cell numbers and IgG production in LPS-stimulated cultures of naïve B cells, in part via enhancing IL-6 production. Importantly, B cells from lupus mice were hyperresponsive to ALD-DNA and/or LPS relative to normal control B cells in terminal plasma cell differentiation, as evidenced by increases in CD138⁺ cell numbers, IgM production, and mRNA levels of B lymphocyte-induced maturation protein-1 (Blimp-1) and the X-box binding protein 1 (XBP1). Furthermore, ALD-DNA enhanced CD40-activated naïve B cell proliferation. Collectively, these data indicate that self DNA can serve as a DAMP (damage-associated molecular pattern) that cooperates with signals from both innate and adaptive immunity to promote polyclonal B cell activation, a common characteristic of autoimmune diseases.     

Chemiluminescence is increased in a subgroup of PHA-stimulated lymphocytes from patients with systemic lupus erythematosus and inhibited by 5-lipoxygenase inhibitors.

Chemiluminescence (CL) was examined in phytohemagglutinin (PHA)-stimulated control and lupus lymphocytes because oxidative radicals have the chemical potential to generate DNA changes recently observed in lupus lymphocytes. Increased CL was found in 30 of 65 PHA-stimulated lupus lymphocyte samples by a luminol assay. CL did not correspond statistically to oxidative potential measured by a nitroblue tetrazolium assay. CL did not appear to be related to disease activity, organ involvement, or drug therapy. However, six of six males tested had positive CL activity. Cocultivation of CL-positive PHA-stimulated lupus lymphocytes with metabolic inhibitors of various oxidative enzymes revealed that 50 microM arachidonic acid dramatically inhibited the excess oxidation. A specific inhibitor of 5-lipoxygenase activity, 3 microM nordihydroguaiaretic acid, abolished excess CL activity. These studies suggest that chemiluminescence assays can be used to better understand the oxidative metabolism in lupus lymphocytes. The enzyme 5-lipoxygenase may be dysfunctional in a subgroup of lupus patients.     

Association between T lymphocyte sub-sets apoptosis and peripheral blood mononuclear cells oxidative stress in systemic lupus erythematosus

Abstract

Increased oxidative stress and lymphocyte apoptosis are a hallmark of the autoimmune disease systemic lupus erythematosus (SLE). However, the association between oxidative stress and T lymphocytes apoptosis has still to be elucidated in SLE. In order to appraise the interaction between oxidative stress and T lymphocyte apoptosis with the severity of disease, oxidative stress profile and T lymphocytes apoptosis were studied. Increased levels of ROS, MDA and CD4⁺ lymphocyte apoptosis were positively associated with disease activity while decreased levels of GSH and percentage expression of CD4⁺ lymphocyte were negatively associated with disease activity. The decrease in intracellular levels of GSH was negatively associated with T lymphocyte, CD4⁺ lymphocyte, CD8⁺ lymphocyte apoptosis and intracellular caspase-3 expression. The present study suggests that increased T lymphocyte sub-sets apoptosis may be mediated by decreased intracellular glutathione concentration and severity of disease might be enhanced together by over-production of ROS in SLE.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Vitamin B6 deficiency and the lymphoid system: I. Effects on cellular immunity and in vitro incorporation of 3H-uridine by small lymphocytes

Thoracic duct lymphocytes from vitamin B₆-deficient rats were found to have a reduced capacity to respond to foreign lymphoid cells in the mixed lymphocyte reaction (MLR), to produce normal lymphocyte transfer reactions, and to incorporate ³H-uridine in vitro. These findings indicate that specific nutritional deficiencies may impair cellular immunity and that this impairment can be monitored by the MLR. It is suggested that the reduction in MLR activity and in ³H-uridine uptake by TDL cells reflected either a shift in the proportions of T and B cells in the TDL and/or an impairment in the capacity of such cells to function in the MLR and in the in vitro test for ³H-uridine incorporation.     

Amelioration of lupus nephritis by serum amyloid P component gene therapy with distinct mechanisms varied from different stage of the disease

Background

Our previous study revealed that administration of syngeneic female BALB/c mice with excessive self activated lymphocyte-derived DNA (ALD-DNA) could induce systemic lupus erythematosus (SLE) disease, indicating that overload of self-DNA might exceed normal clearance ability and comprise the major source of autoantigens in lupus mice. Serum amyloid P component (SAP), an acute-phase serum protein with binding reactivity to DNA in mice, was proved to promote the clearance of free DNA and prevent mice against self-antigen induced autoimmune response. It is reasonable to hypothesize that SAP treatment might contribute to alleviation of SLE disease, whereas its role in ALD-DNA-induced lupus nephritis is not fully understood.

Methodology/Principal Findings

The ratios of SAP to DNA significantly decreased and were negatively correlated with the titers of anti-dsDNA antibodies in ALD-DNA-induced lupus mice, indicating SAP was relatively insufficient in lupus mice. Herein a pcDNA3-SAP plasmid (pSAP) was genetically constructed and intramuscularly injected into BALB/c mice. It was found that SAP protein purified from the serum of pSAP-treated mice bound efficiently to ALD-DNA and inhibited ALD-DNA-mediated innate immune response in vitro. Treatment of ALD-DNA-induced lupus mice with pSAP in the early stage of SLE disease with the onset of proteinuria reversed lupus nephritis via decreasing anti-dsDNA autoantibody production and immune complex (IC) deposition. Further administration of pSAP in the late stage of SLE disease that had established lupus nephritis alleviated proteinuria and ameliorated lupus nephritis. This therapeutic effect of SAP was not only attributable to the decreased levels of anti-dsDNA autoantibodies, but also associated with the decreased infiltration of lymphocytes and the reduced production of inflammatory markers.

Conclusion/Significance

These results suggest that SAP administration could effectively alleviated lupus nephritis via modulating anti-dsDNA antibody production and the inflammation followed IC deposition, and SAP-based intervening strategy may provide new approaches for treating SLE disease.     

The effects of freeze/thawing on the function and phenotype of CD4+ lymphocyte subsets in normal individuals and patients with systemic lupus erythematosus

Several studies report on lymphocyte phenotypic and functional abnormalities in Systemic Lupus Erythematosus (SLE). Freezing and thawing may alter functional and phenotypic properties of cells. We assessed the effect of the freezing/thawing process (F/T) on Th1 (CD3⁺CD4⁺CCR4⁻CXCR3⁺CCR5⁺), Th2 (CD3⁺CD4⁺CCR5⁻CXCR3⁻CCR4⁺), Th17 (CD3⁺CD4⁺CCR6⁺CD161⁺), and Treg (CD3⁺CD4⁺CD25^highCD127^-) cell cultures in healthy controls and SLE patients. F/T was associated with decreased frequency of Th2 and Th17 cells in cultures from SLE patients but not from controls. F/T was also associated with increased frequency of apoptotic cells, as measured by annexin V labeling, in all T cell subtypes analyzed, as well as increased cell proliferation, as measured by Ki-67 labeling, in all cells except Th1 from SLE patients. Thus, F/T can have differentiated effects on T lymphocyte subtypes from SLE patients and controls, and can have significant effects on cell death and proliferation. These findings should be carefully considered when designing and interpreting studies on functional and phenotypic aspects of T lymphocytes in SLE.     

Abnormal activation of the Akt-GSK3β signaling pathway in peripheral blood T cells from patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease accompanied by the activation and proliferation of T cells and B cells. In this study, we found that the distributions of lymphocytes obtained from patients with SLE or SLE with renal disease (RSLE) were reduced in the G₀/G₁ phase and were elevated in the S phase after phytohemagglutinin treatment. Increased expression of CDK2 and decreased expression of cyclin-dependent kinase inhibitors p27^Kip1 and p21^WAF1/CIP1 were observed in RSLE and SLE lymphocytes. The phosphorylation levels of Akt473 and GSK3β (ser9) were increased in lymphocytes from the patients. Moreover, inhibition of GSK3β with lithium chloride or SB216763 induced T cell proliferation, and the most significant effects were observed in RSLE lymphocytes. These results indicate that upregulation of CDKs and downregulation of p27^Kip1 and p21^WAF1/CIP1 increased the proliferation of T lymphocytes in SLE patients. Abnormal activation of the Akt–GSK3β signaling pathway increased the proliferation of lupus lymphocytes.     

Enhancing and suppressive effects of macrophages on T-lymphocyte stimulation in vitro.

The immunoregulatory effect of peritoneal and splenic macrophages on Con A-stimulated mouse splenic T lymphocytes was investigated in vitro using [¹²⁵I]UdR incorporation as a measure of lymphocyte proliferation. [¹²⁵I]UdR incorporation was enhanced by the addition of increasing numbers of splenic or low doses of peritoneal adherent cells to macrophagedepleted splenic lymphocytes. The addition of increasing numbers of peritoneal macrophages beyond 5–10%, however, proportionally suppressed T-cell proliferation. Activated splenic macrophages obtained from mice 6 days after infection with Listeria monocytogenes were suppressive, whereas macrophages obtained from immune donors 9–10 days after infection were not, so that a chronological association appeared to exist between macrophage activation and immunosuppression. The addition of 2-mercaptoethanol to the cell cultures increased [¹²⁵I]UdR incorporation without affecting the stimulatory and suppressive effects of splenic and peritoneal macrophages, respectively. Heat-killed and freeze-thawed macrophages lost their capacity to enhance or inhibit lymphocyte transformation. Macrophages treated with mitomycin C to inhibit DNA synthesis retained their regulatory functions. These studies suggest differential regulatory roles for spleen versus peritoneal macrophages on T-lymphocyte responses to Con A stimulation in vitro.     

Y27, a novel derivative of 4-hydroxyquinoline-3-formamide,prevents the development of murine systemic lupus erythematosus-like diseases in MRL/lpr autoimmune mice and BDF1 hybrid mice

Introduction

Naturally occurring CD4⁺CD25⁺ regulatory T (Treg) cells are central to the maintenance of peripheral tolerance. Impaired activity and/or a lower frequency of these cells lead to systemic lupus erythematosus (SLE). Manipulating the number or activity of Treg cells is to be a promising strategy in treating it and other autoimmune diseases. We have examined the effects of Y27, a novel derivative of 4-hydroxyquinoline-3-formamide, on SLE-like symptoms in MRL/lpr autoimmune mice and BDF1 hybrid mice. Whether the beneficial effect of Y27 involves modulation of CD4⁺CD25⁺ Treg cells has also been investigated.

Methods

Female MRL/lpr mice that spontaneously develop lupus were treated orally by gavage with Y27 for 10 weeks, starting at 10 weeks of age. BDF1 mice developed a chronic graft-versus-host disease (GVHD) by two weekly intravenous injections of parental female DBA/2 splenic lymphocytes, characterized by immunocomplex-mediated glomerulonephritis resembling SLE. Y27 was administered to chronic GVHD mice for 12 weeks. Nephritic symptoms were monitored and the percentage of CD4⁺CD25⁺FoxP3⁺ Treg peripheral blood leukocyte was detected with mouse regulatory T cell staining kit by flowcytometry. Purified CD4⁺CD25⁺ Tregs were assessed for immune suppressive activity using the mixed lymphocyte reaction.

Results

The life-span of MRL/lpr mice treated with Y27 for 10 weeks was significantly prolonged, proteinuria and renal lesion severity were ameliorated, and blood urea nitrogen, triglyceride and serum anti-double-stranded DNA antibodies were decreased. Similar results were found in chronic GVHD mice. Administration of Y27 had little impact on percentage of the peripheral blood lymphocyte CD4⁺CD25⁺Foxp3⁺ Treg cells in both groups of mice. In contrast, the suppressive capacity of CD4⁺CD25⁺ Treg cells in splenocytes was markedly augmented in Y27-treated mice ex vivo.

Conclusions

Experimental evidence of the protect effects of Y27 against autoimmune nephritis has been shown. The mechanism may involve enhancement of the suppressive capacity of CD4⁺CD25⁺ Treg cells.     

Syngeneic adipose-derived stem cells with short-term immunosuppression induce vascularized composite allotransplantation tolerance in rats

Background aimsA clinically applicable tolerance induction regimen that removes the requirement for lifelong immunosuppression would benefit recipients of vascularized composite allotransplantation (VCA). We characterized the immunomodulatory properties of syngeneic (derived from the recipient strain) adipocyte-derived stem cells (ADSCs) and investigated their potential to induce VCA tolerance in rats.MethodsADSCs were isolated from Lewis (LEW, RT1A^l) rats; their immunomodulatory properties were evaluated by means of mixed lymphocyte reactions in vitro and VCAs in vivo across a full major histocompatibility complex mismatch with the use of Brown-Norway (BN, RT1Aⁿ) donor rats. Two control and four experimental groups were designed to evaluate treatment effects of ADSCs and transient immunosuppressants (anti-lymphocyte globulin, cyclosporine) with or without low-dose (200 cGy) total body irradiation. Flow cytometry was performed to quantify levels of circulating CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs).ResultsCultured syngeneic ADSCs exhibited CD90.1⁺CD29⁺CD73⁺CD45⁻CD79a⁻CD11b/c⁻ phenotype and the plasticity to differentiate to adipocytes and osteocytes. ADSCs dramatically suppressed proliferation of LEW splenocytes against BN antigen and mitogen, respectively, in a dose-dependent fashion, culminating in abrogation of allo- and mitogen-stimulated proliferation at the highest concentration tested. Accordingly, one infusion of syngeneic ADSCs markedly prolonged VCA survival in LEW recipients treated with transient immunosuppression; of these, 66% developed tolerance. Total body irradiation provided no additional VCA survival benefit. An important role for Tregs in tolerance induction/maintenance was suggested in vivo and in vitro.ConclusionsTreatment comprising syngeneic ADSCs and transient immunosuppression (i) increased levels of circulating Tregs and (ii) induced tolerance in 66% of recipients of major histocompatibility complex–mismatched VCAs.     

Structure and functional alterations in lymphocytes induced by cryopreservation

Surface markers, Con A-induced capping, blastogenic transformation stimulated by PHA and allogeneic mononuclear cells, and natural killer activity of Ficoll — Hypaque-separated lymphocytes were studied before and after varying periods of cryopreservation. An increase was observed in the relative number of E rosetteforming cells and in the incorporation of [³H]thymidine into DNA, by the unstimulated cryopreserved cells after thawing. On the other hand, a substantial drop occurred in the Con A-induced capping and the natural killer activity of cryopreserved cells. The possible causes for the variation in the effects of cryopreservation on lymphocyte functions as reported by different investigators were discussed. It was concluded that until universally accepted, standardized procedures for the assessment of lymphocyte functions in vitro become available, each laboratory should establish the changes induced by cryopreservation in lymphocyte function with the methods employed locally to allow the observations made on cryopreserved lymphocytes to be meaningful.     

Characterization of warm-reactive IgG anti-lymphocyte antibodies in systemic lupus erythematosus. Relative specificity for mitogen-activated T cells and their soluble products

In addition to previously described cold-reactive IgM anti-lymphocyte antibodies maximally cytotoxic for resting cells at 15 degrees C, sera from patients with systemic lupus erythematosus (SLE) were found to contain a new type of antibody preferentially reactive at physiologic temperatures with mitogen-activated lymphocytes. This antibody lacked specificity for unstimulated lymphocytes, and was shown to be of the IgG class both by indirect immunofluorescence and in immunochemical experiments. Certain SLE sera also contained IgG antibodies with the capacity to develop plaques with mitogen-activated T lymphocyte preparations used in a reverse hemolytic plaque assay, indicating reactivity with products released by activated cells. The elimination of the ability of SLE sera to develop plaques after absorption with viable mitogen-stimulated lymphocytes, but not with resting cells, suggested that these antibodies were directed toward activation "neoantigen(s)" shed from the cell surface membrane. Surface membrane phenotype analyses performed by using a variety of monoclonal antibody reagents indicated that the plaque-forming cells (PFC) detected with SLE sera were activated T lymphocytes not restricted to single OKT4+, OKT8+, or Ia antigen+ subpopulations. Essentially all PFC expressed transferrin receptors. The present data raise the possibility that certain of the interesting effects of anti-lymphocyte antibodies on immunologic function in SLE may be mediated by interactions of these new type(s) of antibodies with activated lymphocytes or their products, rather than through blocking or depletion effects on resting precursor cells.     

Resveratrol Possesses Protective Effects in a Pristane-Induced Lupus Mouse Model

Background

Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease characterized by the production of autoantibodies. To date, no therapy has been found to satisfactorily treat SLE. SIRT1 deficiency results in the development of an autoimmune syndrome in mice, including a high titer of anti-nuclear antibody in serum, immunoglobulin deposition in the kidney, and immune complex glomerulonephritis. Resveratrol is an activator of SIRT1 and possesses anti-inflammation and immune-regulatory properties.

Objective

To evaluate the preventative effects of resveratrol on a pristane-induced lupus animal model and assess its putative immune modulation effects.

Methods

BALB/c mice received a single intraperitoneal injection of 0.5 ml of pristane on day 1 and then various doses of resveratrol were given to the mice daily starting on day 2 and continuing for seven months. The autoantibodies in serum and supernatants were measured. Single cells isolated from spleen, isolated CD4+ T cells, and CD19+ B cells were cultured with or without resveratrol in vitro and assessed by flow cytometry.

Results

Resveratrol attenuated proteinuria, immunoglobuin depositon in kidney, and glomerulonephritis as well as IgG1 and IgG2a in serum in pristane-induced lupus mice. Resveratrol also suppressed CD69 and CD71 expression on CD4+ T cells as well as CD4+ T cell proliferation, induced CD4+ T cell apoptosis, and decreased CD4 IFNγ⁺ Th1 cells and the ratio of Th1/Th2 cells in vitro. In vitro antibody production and proliferation of B cells were also inhibited.

Conclusion

Resveratrol possesses protective effects in pristane-induced lupus mice and may represent a novel approach for the management of SLE.     

Effects of some 1,4‐dihydropyridine Ca antagonists on the blast transformation of rat spleen lymphocytes

Ca antagonists of different classes (verapamil, nifedipine, nicardipine, diltiazem) in a concentration of 10⁻⁵ m and higher are known to suppress Ca²⁺ transport into the lymphocyte cytosol, changing a normal response of lymphocytes to mitogens and antigens and so inhibiting their proliferation, as well as IL‐2‐induced cell proliferation, and their receptor expression on the surface of lymphocytes without cell cytotoxicity. In the present work we studied the effect of some 1,4‐dihydropyridines (DHP) such as nimodipine, nicardipine, nifedipine, niludipine, cerebrocrast, etaftoron, as well as metabolites of cerebrocrast: compounds 7 and 8, (four of the last were synthesized in the Latvian Institute of Organic Synthesis) on rat spleen isolated lymphocyte activation and proliferation in vitro following stimulation with the mitogens concanavalin A (Con A) and recombinant interleukin‐2 (IL‐2), insulin and insulin antibodies. Based on the experimental results we conclude that in low concentrations (10⁻⁷ to 10⁻⁹ M ) the tested 1,4‐DHP Ca antagonists stimulated the process of rat spleen lymphocyte proliferation and DNA synthesis, especially cerebrocrast. It is proposed that these Ca antagonists, as well as causing a concentration decrease of Ca²⁺, also activated phosphodiesterase, which in its turn, suppressed cAMP accumulation in the lymphocytes and eventually increased Ca²⁺ ion transport in the cells. Cerebrocrast among all the studied DHP Ca antagonists was the most potent in studies of activation of the lymphocytes in the presence of Con A, IL‐2 and insulin, which indicates the number of suppressor and helper lymphocytes and formation of insulin and interleukin receptors on their membrane surface. The increase in the lymphocyte suppressive activity produced by this compound effect can prevent diabetes mellitus types I and II at the stages of pre‐diabetes, early and distant diabetes, from hyperexpression of insulin and its receptor antibodies. Copyright © 1999 John Wiley & Sons, Ltd.     

Thymus and Bursa Dependence of Lymphocyte Mitogenic Factor in the Chicken

AMONG the soluble products generated during activation of rodent or human lymphocytes¹, lymphocyte-stimulating (‘mitogenic’) factors are detected by their ability to accelerate DNA metabolism of freshly cultured allogeneic or syngeneic lymphocytes^2–8. If such lymphocyte activation products function as mediators of cellular immunity^1,9–11, their activities will be generated independently of the antibody-forming system. In the chicken, antigen-stimulation of sensitized lymphocytes in vitro involves both thymus (T-) and bursa-(B-)-dependent cells^12–14. If antigen-induced lymphocyte transformations are generally mediated by lymphocyte mitogenic factors, both T-cells and B-cells should produce lymphocyte mitogenic factors when stimulated by specific antigen. We have therefore determined whether antigen-stimulated chicken lymphocytes generate a lymphocyte mitogenic factor and whether this response is affected by neonatal thymectomy or bursectomy.     

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.