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We have previously reported that nanoparticles (NPs) loaded with IL-2 and TGF-β and targeted to T cells induced polyclonal T regulatory cells (Tregs) that protected mice from graft-versus-host disease (GvHD). Here, we evaluated whether administration of these NPs during alloantigen immunization could prevent allograft rejection by converting immunogenic responses to tolerogenic ones. Using C57BL/6 mice and BALB/c mice as either donors or recipients of allogeneic splenocytes, we found that treatment with the tolerogenic NPs in both strains of mice resulted in a marked inhibition of mixed lymphocyte reaction (MLR) to donor cell alloantigen but not to third-party control mouse cells after transfer of the allogeneic cells. The decreased alloreactivity associated with a four- to fivefold increase in the number of CD4 + and CD8 + T regulatory cells (Tregs) and the acquisition of a tolerogenic phenotype by recipient dendritic cells (DCs) in NP-treated mice. As allogeneic cells persisted in NP-treated mice, these findings suggest that tolerogenic NPs can induce alloantigen-specific Tregs and tolerogenic DCs promoting tolerogenic responses to alloantigen. By inhibiting reactivity to allotransplant, this approach could help reduce the need for immune suppression for the maintenance of allografts., Competing Interests: DH is co-founder of General Nanotherapeutics LLC and has a financial interest in the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Horwitz, Wang, Kim, Kang, Brion, Bickerton and La Cava.)

Competing Interests: DAH is the CEO of and holds stock in General Nanotherapeutics, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

We recently reported that poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with interleukin (IL)-2 and targeted to T cells inhibited the development of lupus-like disease in BDF1 mice by inducing functional T regulatory cells (Tregs). Here we show that the protection from disease and the extended survival of BDF1 mice provided by IL-2-loaded NPs targeted to T cells is not only due to an induction of Tregs but also contributed by an inhibition of T follicular helper (T FH ) cells. These results identify a dual protective activity of IL-2 in the control of lupus autoimmunity, namely the inhibition of effector T FH cells, in addition to the previously known induction of Tregs. This newly recognized activity of IL-2 delivered by NPs can help better explain the beneficial effects of low-dose IL-2 immunotherapy in systemic lupus erythematosus (SLE), and might be considered as a new strategy to slow disease progression and improve outcomes in lupus patients., Competing Interests: Conflict of Interest Antonio La Cava is a Co-Editor-in-Chief of the journal. The article was subject to the journal's standard procedures, with rigorous peer-review handled independently by unrelated reviewers. David Horwitz is the founder of General Nanotherapeutics, LLC and has a financial interest in the Company. The research was conducted in the absence of any commercial or financial aspects that could be construed as a potential conflict of interest., (© 2021 Concetta Ferretti et al., published by Sciendo.)

Autoimmune diseases are disorders of immune regulation where the mechanisms responsible for self-tolerance break down and pathologic T cells overcome the protective effects of T regulatory cells (Tregs) that normally control them. The result can be the initiation of chronic inflammatory diseases. Systemic lupus erythematosus (SLE) and other autoimmune diseases are generally treated with pharmacologic or biological agents that have broad suppressive effects. These agents can halt disease progression, yet rarely cure while carrying serious adverse side effects. Recently, nanoparticles have been engineered to correct homeostatic regulatory defects and regenerate therapeutic antigen-specific Tregs. Some approaches have used nanoparticles targeted to antigen presenting cells to switch their support from pathogenic T cells to protective Tregs. Others have used nanoparticles targeted directly to T cells for the induction and expansion of CD4+ and CD8+ Tregs. Some of these T cell targeted nanoparticles have been formulated to act as tolerogenic artificial antigen presenting cells. This article discusses the properties of these various nanoparticle formulations and the strategies to use them in the treatment of autoimmune diseases. The restoration and maintenance of Treg predominance over effector cells should promote long-term autoimmune disease remission and ultimately prevent them in susceptible individuals., Competing Interests: DAH is the Founder of General Nanotherapeutics, LLC and has a financial interest in the Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Horwitz, Bickerton and La Cava.)

Artificial antigen-presenting cells (aAPCs) are synthetic versions of naturally occurring antigen-presenting cells (APCs) that, similar to natural APCs, promote efficient T effector cell responses in vitro . This report describes a method to produce acellular tolerogenic aAPCs made of biodegradable poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) and encapsulating IL-2 and TGF-β for a paracrine release to T cells. We document that these aAPCs can induce both human CD4 + and CD8 + T cells to become FoxP3 + T regulatory cells (Tregs). The aAPC NP-expanded human Tregs are functional in vitro and can modulate systemic autoimmunity in vivo in humanized NSG mice. These findings establish a proof-of-concept to use PLGA NPs as aAPCs for the induction of human Tregs in vitro and in vivo , highlighting the immunotherapeutic potential of this targeted approach to repair IL-2 and/or TGF-β defects documented in certain autoimmune diseases such as systemic lupus erythematosus., Competing Interests: DH owns stocks and stock options at General Nanotherapeutics and Toralgen, Inc. DH has filed a US patent application (63118863) on the methods described in this manuscript to produce tolerogenic NPs for the treatment of immune-mediated diseases. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Giang, Horwitz, Bickerton and La Cava.)

We recently reported that the treatment with nanoparticles (NPs) loaded with tolerogenic cytokines suppressed the manifestations of lupus-like disease induced by the transfer of donor CD4 + T cells from DBA/2 mice into (C57BL/6 × DBA/2)F 1  (BDF1) mice. Although the protective effects were ascribed to the induction of adaptive CD4 + and CD8 + T regulatory cells, the results suggested that another population of immune cells could be involved. Here we report that NK cells critically contribute to the protection from lupus-like disease conferred by NPs to BDF1 mice, and that this effect is TGF-β-dependent., Competing Interests: DH is an officer of General Nanotherapeutics LLC and owns stock. DH and TF own stocks and stock options at Toralgen, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Horwitz, Liu, Bickerton, Castaldo, Matarese, Fahmy and La Cava.)

Objective: To develop a nanoparticle (NP) platform that can expand both CD4+ and CD8+ Treg cells in vivo for the suppression of autoimmune responses in systemic lupus erythematosus (SLE)., Methods: Poly(lactic-co-glycolic acid) (PLGA) NPs encapsulating interleukin-2 (IL-2) and transforming growth factor β (TGFβ) were coated with anti-CD2/CD4 antibodies and administered to mice with lupus-like disease induced by the transfer of DBA/2 T cells into (C57BL/6 × DBA/2)F 1 (BDF1) mice. The peripheral frequency of Treg cells was monitored ex vivo by flow cytometry. Disease progression was assessed by measuring serum anti-double-stranded DNA antibody levels by enzyme-linked immunosorbent assay. Kidney disease was defined as the presence of proteinuria or renal histopathologic features., Results: Anti-CD2/CD4 antibody-coated, but not noncoated, NPs encapsulating IL-2 and TGFβ induced CD4+ and CD8+ FoxP3+ Treg cells in vitro. The optimal dosing regimen of NPs for expansion of CD4+ and CD8+ Treg cells was determined in in vivo studies in mice without lupus and then tested in BDF1 mice with lupus. The administration of anti-CD2/CD4 antibody-coated NPs encapsulating IL-2 and TGFβ resulted in the expansion of CD4+ and CD8+ Treg cells, a marked suppression of anti-DNA antibody production, and reduced renal disease., Conclusion: This study shows for the first time that T cell-targeted PLGA NPs encapsulating IL-2 and TGFβ can expand both CD4+ and CD8+ Treg cells in vivo and suppress murine lupus. This approach, which enables the expansion of Treg cells in vivo and inhibits pathogenic immune responses in SLE, could represent a potential new therapeutic modality in autoimmune conditions characterized by impaired Treg cell function associated with IL-2 deficiency., (© 2018, American College of Rheumatology.)

The cytokine milieu is critical for orchestration of lineage development towards effector T cell (Teff) or regulatory T cell (Treg) subsets implicated in the progression of cancer and autoimmune disease. Importantly, the fitness and survival of the Treg subset is dependent on the cytokines Interleukin-2 (IL-2) and transforming growth factor beta (TGF-β). The production of these cytokines is impaired in autoimmunity increasing the probability of Treg conversion to aggressive effector cells in a proinflammatory microenvironment. Therapy using soluble TGF-β and IL-2 administration is hindered by the cytokines' toxic pleiotropic effects and hence bioavailability to CD4(+) T cell targets. Thus, there is a clear need for a strategy that rectifies the cytokine milieu in autoimmunity and inflammation leading to enhanced Treg stability, frequency and number. Here we show that inert biodegradable nanoparticles (NP) loaded with TGF-β and IL-2 and targeted to CD4(+) cells can induce CD4(+) Tregs in-vitro and expand their number in-vivo. The stability of induced Tregs with cytokine-loaded NP was enhanced leading to retention of their suppressive phenotype even in the presence of proinflammatory cytokines. Our results highlight the importance of a nanocarrier-based approach for stabilizing and expanding Tregs essential for cell-immunotherapy of inflammation and autoimmune disease., (Copyright © 2015. Published by Elsevier Ltd.)

Recent studies have demonstrated that thymus-derived naturally occurring CD4(+)Foxp3(+) regulatory T cells (Tregs) in human and mouse may be unstable and dysfunctional in the presence of proinflammatory cytokines. All-trans RA (atRA), the active derivative of vitamin A, has been shown to regulate Treg and T effector cell differentiation. We hypothesize atRA stabilizes human natural Tregs (nTregs) under inflammatory conditions. atRA prevents human nTregs from converting to Th1 and/or Th17 cells and sustains their Foxp3 expression and suppressive function in vitro or in vivo following encounters with IL-1 and IL-6. Interestingly, adoptive transfer of human nTregs pretreated with atRA significantly enhanced their suppressive effects on xenograft-vs.-host diseases (xGVHDs), and atRA- but not rapamycin-pretreated nTregs sustained the functional activity against xGVHD after stimulation with IL-1/IL-6. atRA suppresses IL-1 receptor (IL-1R) up-regulation, accelerates IL-6R down-regulation, and diminishes their signaling events as well as prevents the up-regulation of STIP1 homology and U-Box containing protein 1 on Foxp3(+) cells following IL-1/IL-6 stimulation. atRA also increases histone acetylation on Foxp3 gene promoter and CpG demethylation in the region of Foxp3 locus (i.e., Treg-specific demethylated region). These results strongly implicate that nTregs primed with atRA may represent a novel treatment strategy to control established chronic immune-mediated autoimmune and inflammatory diseases.

TGF-β and Foxp3 expressions are crucial for the induction and functional activity of CD4(+)Foxp3(+) regulatory T (iTreg) cells. Here, we demonstrate that although TGF-β-primed CD8(+) cells display much lower Foxp3 expression, their suppressive capacity is equivalent to that of CD4(+) iTreg cells, and both Foxp3(-) and Foxp3(+) CD8+ subsets have suppressive activities in vitro and in vivo. CD8(+)Foxp3(-) iTreg cells produce little IFN-γ but almost no IL-2, and display a typical anergic phenotype. Among phenotypic markers expressed in CD8(+)Foxp3(-) cells, we identify CD103 expression particularly crucial for the generation and function of this subset. Moreover, IL-10 and TGF-β signals rather than cytotoxicity mediate the suppressive effect of this novel Treg population. Therefore, TGF-β can induce both CD8(+)Foxp3(-) and CD8(+)Foxp3(+) iTreg subsets, which may represent the unique immunoregulatory means to treat autoimmune and inflammatory diseases.

Interplay between Foxp3(+) regulatory T cells (Treg) and dendritic cells (DCs) maintains immunologic tolerance, but the effects of each cell on the other are not well understood. We report that polyclonal CD4(+)Foxp3(+) Treg cells induced ex vivo with transforming growth factor beta (TGFβ) (iTreg) suppress a lupus-like chronic graft-versus-host disease by preventing the expansion of immunogenic DCs and inducing protective DCs that generate additional recipient CD4(+)Foxp3(+) cells. The protective effects of the transferred iTreg cells required both interleukin (IL)-10 and TGFβ, but the tolerogenic effects of the iTreg on DCs, and the immunosuppressive effects of these DCs were exclusively TGFβ-dependent. The iTreg were unable to tolerize Tgfbr2-deficient DCs. These results support the essential role of DCs in 'infectious tolerance' and emphasize the central role of TGFβ in protective iTreg/DC interactions in vivo.

Objective: Transferred CD4+CD25+FoxP3+ Treg cells can prevent autoimmune disease, but generally fail to ameliorate established disease. This study was undertaken to compare the effects of antigen-specific Treg cells induced with interleukin-2 (IL-2) and transforming growth factor β (TGFβ) ex vivo (induced Treg [iTreg] cells) to the effects of equivalent expanded thymus-derived natural Treg (nTreg) cells on established collagen-induced arthritis (CIA)., Methods: CIA was induced in DBA/1 mice by immunization with type II collagen (CII), and before or shortly after immunization, mice were treated with iTreg or nTreg cells that were generated or expanded in vitro. Clinical scores were determined. Inflammatory responses were determined by measuring the levels of anti-CII antibody in the serum and examining the histologic features of the mouse joints. The Th1/Th17-mediated autoreactive response was evaluated by determining the cytokine profile of the draining lymph node (LN) cells of the mice by flow cytometry., Results: Following transfer, nTreg cells exhibited decreased FoxP3 and Bcl-2 expression and decreased suppressive activity, and many converted to Th17 cells. In contrast, transferred iTreg cells were more numerous, retained FoxP3 expression and their suppressive activity in the presence of IL-6, and were resistant to Th17 conversion. Notably, 10 days after the transfer of donor iTreg cells, predominance was shifted from Th17 cells to Treg cells in the draining LNs of recipient mice., Conclusion: These findings provide evidence that transferred TGFβ-induced iTreg cells are more stable and functional than nTreg cells in mice with established autoimmunity. Moreover, iTreg cells can have tolerogenic effects even in the presence of ongoing inflammation. The therapeutic potential of human iTreg cells in subjects with chronic, immune-mediated inflammatory diseases should be investigated., (Copyright © 2012 by the American College of Rheumatology.)

Objectives: The objective was to determine if emergency department (ED) patients with diabetes mellitus (DM) who have primary care providers (PCPs) have better control of their DM than patients with no PCPs., Methods: This was a prospective, cross-sectional, observation study at a large, adult, urban, academic ED with 85,000 annual visits. ED patients with a history of DM were eligible. Patients with severe systemic disease, diabetic ketoacidosis (DKA), sepsis, active steroid use, pregnancy, or cognitive impairment were excluded. Consenting patients had hemoglobin A1c (HgbA1c) analysis and completed a questionnaire regarding demographics, lifestyle, medication usage, educational level attained, and health care access, including whether or not they had PCPs. HgbA1c levels were compared between subjects with and without PCPs using medians with interquartile ranges (IQRs). A continuous plot was developed to demonstrate the proportion of patients without PCPs (PCP-) compared to those with PCPs (PCP+) at every level of %HgbA1c across the entire measured range. Multivariate logistic regression analysis was used to determine which clinical and demographic factors obtained from the questionnaire were associated with improved glycemic control (increased relative risk [RR] of having a %HgbA1c < 8%)., Results: A total of 284 patients were screened; 227 were enrolled, had HgbA1c analysis performed, and had complete PCP, race, and sex information. Complete demographic data (insurance status, employment status, etc.) were available on 209 subjects. Sixty-four of the 227 patients (28.2%) denied having PCPs. Median HgbA1c was 7.7% (IQR = 6.5% to 9.68%) in PCP+ versus 8.9% (IQR = 6.8% to 11.3%) in PCP- patients (p = 0.01). Ninety-one of 163 (55.8%) PCP+ subjects had a median HgbA1c < 8% versus 25 of 64 (39.1%) in the PCP- group (p = 0.02). After adjusting for multiple clinical and demographic variables, having a PCP remained significantly associated with a median HgbA1c value less than 8% (RR = 1.43; p = 0.04)., Conclusions: Diabetes control was significantly better in patients with PCPs, even after adjusting for a number of potentially confounding social and demographic factors., (© 2012 by the Society for Academic Emergency Medicine.)

Background: Protective CD4+CD25+ regulatory T cells bearing the Forkhead Foxp3 transcription factor can now be divided into three subsets: Endogenous thymus-derived cells, those induced in the periphery, and another subset induced ex-vivo with pharmacological amounts of IL-2 and TGF-β. Unfortunately, endogenous CD4+CD25+ regulatory T cells are unstable and can be converted to effector cells by pro-inflammatory cytokines. Although protective Foxp3+CD4+CD25+ cells resistant to proinflammatory cytokines have been generated in mice, in humans this result has been elusive. Our objective, therefore, was to induce human naïve CD4+ cells to become stable, functional CD25+ Foxp3+ regulatory cells that were also resistant to the inhibitory effects of proinflammatory cytokines., Methodology/principal Findings: The addition of the vitamin A metabolite, all-trans retinoic acid (atRA) to human naïve CD4+ cells suboptimally activated with IL-2 and TGF-β enhanced and stabilized FOXP3 expression, and accelerated their maturation to protective regulatory T cells. AtRA, by itself, accelerated conversion of naïve to mature cells but did not induce FOXP3 or suppressive activity. The combination of atRA and TGF-β enabled CD4+CD45RA+ cells to express a phenotype and trafficking receptors similar to natural Tregs. AtRA/TGF-β-induced CD4+ regs were anergic and low producers of IL-2. They had potent in vitro suppressive activity and protected immunodeficient mice from a human-anti-mouse GVHD as well as expanded endogenous Tregs. However, treatment of endogenous Tregs with IL-1β and IL-6 decreased FOXP3 expression and diminished their protective effects in vivo while atRA-induced iTregs were resistant to these inhibitory effects., Conclusions/significance: We have developed a methodology that induces human CD4(+) cells to rapidly become stable, fully functional suppressor cells that are also resistant to proinflammatory cytokines. This methodology offers a practical novel strategy to treat human autoimmune diseases and prevent allograft rejection without the use of agents that kill cells or interfere with signaling pathways.

Regulatory/suppressor T cells (Tregs) maintain immunologic homeo-stasis and prevent autoimmunity. In this article, past studies and recent studies of Tregs in mouse models for lupus and of human systemic lupus erythematosus are reviewed concentrating on CD4+CD25+Foxp3+ Tregs. These cells consist of thymus-derived, natural Tregs and peripherally induced Tregs that are similar phenotypically and functionally. These Tregs are decreased in young lupus-prone mice, but are present in normal numbers in mice with established disease. In humans, most workers report CD4+Tregs are decreased in subjects with active systemic lupus erythematosus, but the cells increase with treatment and clinical improvement. The role of immunogenic and tolerogenic dendritic cells in controlling Tregs is discussed, along with new strategies to normalize Treg function in systemic lupus erythematosus.

To determine how transforming growth factor-beta (TGF-beta) controls T cell function, Marie et al. (2006) and Li et al. (2006a) created mice with T cells lacking TGF-beta signaling receptors. Both report that TGF-beta signaling by T cells is absolutely essential for tolerance and homeostasis.

Certain CD4+CD25+ T cells can induce and maintain T-cell non-responsiveness to donor alloantigens and have therapeutic potential in solid organ transplantation. Peripheral CD4+CD25- cells alloactivated with IL-2 and transforming growth factor beta (TGF-beta) ex vivo express the transcription factor FoxP3, and become potent antigen-specific CD4+CD25- suppressor cells. Here we report that the transfer of TGF-beta-induced regulatory CD4+ and CD8+ T cells (Tregs) co-incident with transplantation of a histoincompatible heart resulted in extended allograft survival. To account for this result, we injected non-transplanted mice with a single dose of CD4+ and CD8+ Tregs and transferred donor cells every 2 weeks to mimic the continuous stimulation of a transplant. We observed increased splenic CD4+CD25+ cells that were of recipient origin. These cells rendered the animals non-responsive to donor alloantigens by an antigen-specific and cytokine-dependent mechanism of action. Both the increased number of CD4+CD25+ cells and their tolerogenic effect were dependent on continued donor antigen boosting. Thus, Tregs generated ex vivo can act like a vaccine that generates host suppressor cells with the potential to protect MHC-mismatched organ grafts from rejection.

Recently, considerable attention has been focused on thymus-derived CD4+ regulatory T cells that constitutively express CD25 and have a contact-dependent, cytokine-independent mechanism in vitro. However, peripheral CD4+ and CD8+ T cells can also be induced to become regulatory T cells. Here we review our studies using the combination of IL-2 and transforming growth factor beta (TGF-beta) to generate regulatory T cell subsets ex vivo, and the work of others using IL-10 to induce suppressive activity. Under certain conditions, the autocrine effects of TGF-beta and IL-10 induce peripheral T cells to produce immunosuppressive levels of each of these cytokines. This effect of TGF-beta is IL-2 dependent. Under other conditions IL-2 and TGF-beta can induce CD4+ cells to develop potent contact-dependent, cytokine-independent regulatory activity. At present, there is considerable confusion concerning the mechanism of action of CD4+ CD25+ cells because cytokine-producing regulatory T cells generated in the periphery can express CD25 and other markers displayed by naturally occurring, thymus-derived regulatory T cells. We, therefore, propose a nomenclature that identifies thymus-derived and peripheral regulatory cells, and that also differentiates T regulatory cells from T helper cells. Because T regulatory cells broadly control T helper cell reactivity, the mechanisms that control regulatory cell function are also reviewed. Finally, the potential use of regulatory T cells generated ex vivo as an adoptive immunotherapy for certain autoimmune diseases, to prevent organ graft rejection, or to prevent pathologic host responses to infectious agents is discussed.

Natural killer (NK) cells are a third lymphocyte population especially important in innate immunity. NK cells may also have an important role in the regulation of acquired immunity. These lymphocytes spontaneously produce large amounts of both active and latent transforming growth factor-beta (TGF-beta). NK-cell-derived TGF-beta1 enabled activated CD8(+) T cells to inhibit antibody production by blocking the induction of this response. Production of lymphocyte-derived TGF-beta is decreased in systemic lupus erythematosus. Insufficient levels of this cytokine in SLE and other autoimmune diseases may contribute to defective T regulatory cell function characteristic of this and other autoimmune diseases. NK cells are found in mucosal tissues and the TGF-beta spontaneously released by these cells could contribute to the usual tolerogenic response of T cells to antigens presented at these sites. Thus, in addition to its well known immunosuppressive effects, TGF-beta could have an equally important role in the generation of regulatory T cells.

Objective: To determine the profile of Th1-type and Th2-type cytokines produced by mononuclear cells from patients with recent-onset systemic lupus erythematosus (SLE), prior to the initiation of treatment with corticosteroids., Methods: Using sensitive radioimmunoassays, interleukin-4 (IL-4), IL-10, IL-12 p40, tumor necrosis factor alpha (TNF alpha), interferon-gamma (IFN gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) released into the culture supernatants of various unstimulated and stimulated blood mononuclear cell populations from 10 SLE patients was assessed in comparison with 10 matched healthy controls studied in parallel., Results: In early SLE, monocyte-enriched cells constitutively produced increased amounts of IL-10 and decreased amounts of IL-12 following stimulation. Lymphocyte-enriched cells in SLE produced decreased amounts of IFN gamma and TNF alpha following stimulation. In "rested" cells, these defects were accentuated and a defect in IL-12 production was suggested. Depletion studies suggested that CD8+ cells were a major source of TNF alpha and IFN gamma in controls, but not in SLE patients. Increased IL-4 production or abnormalities in GM-CSF production were not observed., Conclusion: This study suggests that even early in the course of SLE, monocyte production of IL-10 is increased and that of IL-12 is decreased. Decreased production of Th1-type cytokines in SLE may be secondary to this imbalance between IL-10 and IL-12. A contributory role of dysfunctional CD8+ cells is suggested.

Objective: To determine whether genes participating in programmed cell death, including bcl-2, IL-10, Fas-L, and CTLA-4, may contribute to the genetic predisposition to systemic lupus erythematosus (SLE)., Methods: First, intragenic markers for the bcl-2, IL-10, Fas-L, and CTLA-4 genes were characterized and their extent of polymorphism in normal populations was determined. The allelic distribution of these gene markers in a large Mexican American SLE cohort of 158 patients and 223 ethnically matched controls was determined using fluorescent-labeled primers and semiautomated genotyping., Results: The bcl-2, Fas-L, and IL-10 loci showed significantly different allelic distribution in SLE patients compared with controls, indicating an association between these genes and SLE. No association was found between SLE and the CTLA-4 gene. Further analysis revealed a synergistic effect between susceptibility alleles of the bcl-2 and IL-10 genes in determining disease susceptibility. Alone, the presence of each of these alleles was associated with a moderate increase in SLE risk, while the occurrence of these alleles together increased the odds of developing SLE by more than 40-fold., Conclusion: The results suggest that individuals carrying specific genotypes of both bcl-2 and IL-10 are at significant risk of developing SLE.

Objective: To assess the ability of T cells from patients with systemic lupus erythematosus (SLE) to respond to a mitogenic combination of anti-CD2 monoclonal antibodies (MAb), and to learn the molecular basis of the documented defect., Methods: Peripheral blood mononuclear cell (PBMC) populations from individuals with SLE and paired controls were stimulated in vitro with anti-CD2, and the proliferative response was compared with that evoked by stimulation with phytohemagglutinin (PHA) and anti-CD3. Surface markers on lymphocyte populations were assessed by flow cytometry after staining with specific MAb., Results: The proliferative response to anti-CD2 was decreased to a greater extent than was the response to anti-CD3 or PHA in SLE patients. This defect was found in approximately one-half of the patients examined, was not associated with disease activity, and was maintained upon repeated testing. Since either monocytes or resting B cells can serve as accessory cells for T cells following activation by anti-CD2, we examined the T cell response after depletion of adherent cells. In approximately two-thirds of the individuals with a decreased response, depletion of monocytes or substitution of monocytes with allogeneic, resting B cells from normal donors corrected the defect. The addition to PBMC of anti-CD28, but not of a neutralizing antibody to interleukin-10, largely reversed the anti-CD2 proliferative defect. Significantly fewer CD8+ T cells expressed CD28 in SLE, and this defect was also documented, to a lesser extent, in CD4+ cells., Conclusion: This study provides evidence that some functional T cell defects in SLE may be due, at least in part, to decreased CD28-mediated costimulatory activity following the interaction of T cells with conventional accessory cells.

Objective: To determine whether there is impaired generation and cytolytic function of CD56+ T cells and non-T cells in human systemic lupus erythematosus (SLE)., Methods: Peripheral blood mononuclear cells (PBMC) were obtained from 73 patients with SLE, 39 normal controls, and 9 pairs of monozygotic (MZ) twins discordant for SLE. PBMC were stimulated with anti-CD3 monoclonal antibody, maintained in interleukin-2, and assayed for percentages of total CD56+ cells and CD56+ T cells by flow cytometry, and for cytolytic activity against 51Cr-labeled Daudi target cells., Results: Despite normal total cell expansion, the percentages of recovered CD56+ T cells and total CD56+ cells were 1.6-fold and 1.8-fold lower, respectively, in patients with SLE compared with normal controls (P = 0.011 and P < 0.001, respectively). Cytolytic activities of isolated total CD56+ cells and CD56+ T cells and were also reduced in patients with SLE compared with normal controls (P = 0.033). These defects associated with SLE were independent of disease activity and immunosuppressive medications, and they reflected impaired maturation of cytolytic effector cells rather than a deficiency in precursor cell number. In MZ twins discordant for SLE, recovered percentages of CD56+ cells and cytolytic responses were very low in 4 of 8 and 6 of 9 co-twins with SLE, respectively. Cellmixing experiments with the PBMC of the MZ twins demonstrated that the E+ cell fractions (containing all T cells and CD56+ non-T cells) from the co-twins with SLE had decreased ability to generate cytolytic activity compared with the corresponding E+ cell fractions from the healthy co-twins. However, recovered percentages of CD56+ cells and non-T cells and cytolytic responses were also depressed in 4 of 8 and 4 of 9 healthy co-twins, respectively., Conclusion: Impaired CD56+ T cell and non-T cell responses are a feature of SLE and may antedate the onset of clinical disease.

CD8+ T cells have suppressor effector functions, but the mechanisms involved in the generation of this activity are poorly understood. We report that natural killer (NK) cells have an important role in the acquisition of this function. CD8+ cells induce NK cells to produce transforming growth factor-beta (TGF-beta) which, in turn, stimulates CD8+ T cells to become suppressors of antibody production. Using a monocyte-dependent and -independent method to induce antibody production, we first observed that the addition of NK cells to CD8+ cells was required for optimal suppression. Next, we determined that the interaction of CD8+ T cells with NK cells resulted in a striking increase NK cell TGF-beta mRNA and its production. This cytokine appeared to be involved in the induction of T suppressor cell activity since: (a) anti-TGF-beta 1 completely abrogated the suppression of immunoglobulin G synthesis; (b) TGF-beta 1 could substitute for NK cells in inducing CD8+ T cells to develop suppressor activity; and (c) a short exposure of T cells to TGF-beta 1 in the absence of B cells was sufficient for the generation of suppressor activity by CD8+ T cells. Interferon gamma did not have this property. These studies provide strong evidence that in addition to its suppressive properties, TGF-beta is involved in the generation of CD8+ T suppressor effector cells. Because NK cell function is decreased in many autoimmune diseases, these cells may fail to interact properly with these individuals' CD8+ cells in generating suppressors of aggressive anti-self responses.

Objective: To study the effects of N-chlorotaurine on collagenase activity, as a model of the effects of neutrophil activation in inflammatory arthritis., Methods: Collagen degradation by collagenase was measured by the release of acid-soluble counts from 3H-collagen., Results: N-chlorotaurine inhibited the degradation of collagen by bacterial collagenase. This result is explained by a direct inhibition/inactivation of collagenase, since N-chlorotaurine had no effect on the proteolytic susceptibility of collagen itself. The effect appears to be specific to N-chlorotaurine since N-chloroalanine, N-chloroleucine, and HOCl/OCl- failed to inhibit collagenase; in fact, N-chloroalanine and N-chloroleucine actually increased the proteolytic susceptibility of collagen., Conclusion: N-chlorotaurine may minimize damage to cartilaginous joint structures in inflammatory arthritis by inhibiting/inactivating collagenase.

The cellular requirements for B cell hyperactivity in systemic lupus erythematosus (SLE) were studied. Removal of either CD8+ or CD4+ lymphocyte markedly decreased the spontaneous in vitro production of polyclonal IgG and of antigen-specific (anti-double-stranded DNA and antinucleoprotein) antibodies by SLE peripheral blood mononuclear cells (PBMC). The CD8+ lymphocytes that sustained IgG production were CD3+, HLA-DR+, and their activity was abrogated by preincubation with anti-HLA-DR monoclonal antibody. When both CD4+ and CD8+ cells were removed, the readdition of either subset partially restored polyclonal IgG production, but both cell subsets were required to reconstitute autoantibody production. Purified SLE B cell cultures, which generated only 15% of the IgG produced by unseparated PBMC, were fully reconstituted only by mixtures of CD4+ with CD8+ cells, and with CD8-, CD4-, CD16+ cells. At least part of the support for spontaneous IgG production can be attributed to endogenous interleukin-2 and interleukin-6.

Methodological problems which affect the assessment of humoral effects on mitogenic reactivity include: (1) the source and concentration of serum used to support cell cultures; (2) the day to-day variability of inhibitory effects and (3) the specific activity of [3H]thymidine added to the culture. These problems were alleviated by addition of half concentration (7-5%) of pooled normal human serum to all cultures, the intoruction of anti-lymphocyte serum as a suitable internal control for monitoring the suppressability of lymphocytes and a reduction of specific activity of the [3H]thymidine to 1-3 C2/mM. Inhibitory factors were loosely bound to the lymphocyte surface and eluted off after incubation at 37 degrees C for 1 hr. Cells from twenty-five subjects and paired controls were cultured simultaneously in medium containing either 15% normal human serum (NHS) or 7-5% patient and 7-5% NHS. The cells were stimulated with various dilutions of phytohaemagglutinin, Con A or pokeweed mitogen. Lupus serums suppressed the reactivity of autologous lymphocytes to PHA and pokeweed mitogen. Serums from subjects with RA and scleroderma did not significantly inhibit blastogenesis of autologous lymphocytes. One-half of the lupus serums significantly inhibited the reactivity of homologous lymphocytes to two of three mitogens. Only one of eight scleroderma serums and none of twelve RA serums and none of twelve RA serums had this effect. All patients serums were examined for antilymphocyte antibodies by microcytotoxicity and immunofluorescent techniques. These antibodies were usually found in SLE, and were often observed in subjects with rheumatoid arthritis but not scleroderma. A firm relationship between serum suppressors of lymphocyte blastogenesis and anti-lymphocyte antibodies was not found.

In the absence of antigenic or mitogenic stimulation, certain peripheral blood lymphocytes exhibit proliferative and lymphokine-activated killer (LAK) cell activities when cultured with recombinant IL-2. Both activities were found to be an exclusive property of lymphocytes expressing type 3 complement receptors (CR3) identified by anti-CD11 monoclonal antibodies. CD11+ lymphocytes were then fractionated into three subsets by two-color flow cytometry. These included CD16+ cells, which display distinctive Fc receptors for IgG (CD16). Using anti-CD5, the CD11+ CD16- lymphocytes were separated into non-T cell and T cell subsets. The two non-T cell subsets (CD11+ CD16+ and CD11+ CD16- CD5-), but not the T cell subset (CD11+ CD16- CD5+), could proliferate in response to IL-2. Both CD11+ non-T cell subsets, but not the CD11+ T cell subset, had the capacity to mediate natural killer cell activity. However, all three CD11+ lymphocyte subsets were capable of generating LAK activity. These findings are consistent with the concept that two signals are required to stimulate T cells to proliferate. However, at least a small subset of blood T cells can be activated by IL-2 to become LAK cells.

The mitogenic reactivity of lymphocytes from subjects with systemic lupus erythematosus, rheumatoid arthritis and scleroderma was studied. Cultures containing either unseparated or separated lymphocytes were stimulated with phytohaemagglutinin, Con A and pokeweed mitogen after inhibitory serum factors were eluted from the cell surface. Incorporation of [3H]thymidine in patient cultures was compared to that of normal controls. Greatly decreased reactivity was found in SLE to all three mitogens. Significantly decreased values to some mitogens was also observed in rheumatoid arthritis and scleroderma, but the defect was less severe. Cultures of study subjects contained significantly fewer small lymphocytes than normal controls and this finding explained at least in part the decreased mitogenic reactivity.

Lymphocytes displaying iC3b (Type 3) complement receptors (CR3) were quantified by flow cytometry in patients with systemic lupus erythematosus. The percentages and absolute numbers were compared to age and sex matched controls. Total CR3+ lymphocytes identified by the monoclonal antibodies OKM1 or Leu 15 were significantly decreased in patients with symptomatic arthritis, serositis or vasculitis and those with lupus nephritis, whereas values for CR3+ lymphocytes in patients with inactive disease were similar to normal donors. The phenotype of CR3+ lymphocytes was markedly different in patients with active SLE. In normals granular lymphocytes bearing Fc receptors for IgG (L cells) comprised two-thirds of CR3+ lymphocytes. However, in SLE this subset was reduced to 20% and there was a corresponding increase in CR3+ lymphocytes co-expressing the T3 marker. Percentages of CR3 T4+ but not CR3+ T8+ lymphocytes were significantly increased in SLE. Although patients with active disease were lymphopenic, absolute numbers of CR3+ lymphocytes co-expressing T cell markers were similar to normal controls. Since L cells are non-specific suppressors of Ig production, the reduction of this subset along with the increase in CR3 T4+ cells could contribute to unregulated antibody production characteristic of SLE.

Interleukin-2 (IL-2) production in vitro is depressed in systemic lupus erythematosus (SLE) patients. It is not known whether this abnormality is caused by a defect in the producer lymphocytes or by excessive suppression. We report that removal of OKT8 (Leu 2a)+ cells increased the IL-2 production by in vitro-stimulated lymphocytes to normal or above normal levels in 19 of 21 SLE patients. This increase was more apparent in those patients with clinically inactive disease and/or receiving less than 7.5 mg of prednisone. Removal of OKT8+ cells from normals did not significantly increase IL-2 activity. SLE, but not normal, OKT8+ cells decreased IL-2 production when added back to autologous OKT8-depleted cells. In some experiments, OKT8+ cells from normal donors also suppressed IL-2 production in SLE. This result suggests that the defect in IL-2 production is complex and may involve multiple cell interactions. Three lines of evidence suggest that the SLE OKT8+ cells actively inhibit the production of IL-2 rather than passively absorb this lymphokine: (a) only 3.2% of SLE lymphocytes expressed IL-2 receptors as detected with anti-Tac; (b) freshly prepared SLE lymphocytes did not absorb IL-2; and (c) cell-free supernatants from SLE OKT8+ cells inhibited IL-2 production, but not IL-2 activity. Double-labeling studies by flow cytometry revealed that 19.3% of SLE OKT8+ cells were also Ia-positive, and approximately 33% co-expressed the natural killer cell marker, HNK-1 (Leu 7). Removal of Leu 7+ cells also significantly elevated IL-2 production in SLE. These studies suggest that one or more circulating mononuclear cell subsets in SLE patients can suppress IL-2 production and that one subset may possibly belong to a non-T, non-B "third mononuclear population."

Lymphocytes with Fc receptors (FcR) for IgG active in natural cytotoxicity and antibody-dependent cellular cytotoxicity were separated into sheep erythrocyte rosetting (E+) and nonrosetting (E-) fractions, and examined for reactivity with the OK panel of hybridoma-produced monoclonal antibodies. Few cells in either the E+ FcR+ or the E- FcR+ fraction reacted with seven antibodies used to define T cells in various stages of differentiation (OK3, OKT4, OKT5, OKT6, OKT8, OKT9, OKT10). Neither fraction expressed an Ia-like antigen (detected by OKI1), but both were highly reactive with OKM1, an antibody that reacts with monocytes and granulocytes. Incubation of these cytotoxic effector cells with OKM1 plus complement abolished all cytotoxic reactivity, but incubation with a pan-T cell antibody (OKT3) plus complement had no significant effect. These cells were not monocyte precursors, because they could not be induced in vitro to develop macrophage characteristics. The data indicate that most cytotoxic effector cells in natural cytotoxicity and antibody-dependent cellular cytotoxicity are not in the T cell lineage, but have a myeloid origin.

The cytotoxic activities of human blood mononuclear cells against certain established cell lines were evaluated prospectively in 17 patients with rheumatoid arthritis before and after treatment with low (150 mg per week) and moderate doses (300 mg per week) of levamisole. Spontaneous or "natural" killer activity (NK) and antibody-dependent cellular cytotoxicity (ADCC) of plastic adherent cells ("monocytes") and lymphocytes were studied. We report a selective cytotoxic defect in monocyte NK and a correlation of this defect with severely active disease. The patients with the most severe defect responded to low-dose levamisole, but others with normal values did nor respond as well to treatment. This cytotoxic defect may be an important pathogenetic factor in rheumatoid arthritis and this new assay may be helpful in selecting candidates who are most likely to respond to levamisole.

Two separate lymphocyte populations, each bearing easily detectable surface immunoglobulin, have been detected in human peripheral blood. The first, B cells, has surface determinants that are stable at 37degreeC, but are removed by pronase and regenerate in culture. The cells are nylon adherent and have a receptor for C3, and studies with unit gravity sedimentation indicate they are mostly small lymphocytes. B cells comprise 9.5% of the total lymphocytes, with the normal range from 3-16%. As many or more lymphocytes lack membrane-incorporated Ig determinants but have an Fc receptor that binds IgG1 and IgG3 in normal serum maximally at 4degreeC. This receptor for cytophilic IgG is removed by pronase but not by trypsin. The second population has been named L lymphocytes because of membrane-labile IgG determinants. L cells do not adhere to nylon, do not form rosettes with sheep erythrocytes sensitized with antibody and mouse complement, and are larger than small lymphocytes. These lymphocytes with cold-reactive Fc receptors for serum IgG do not form E-rosettes or respond to phytohemaggutinin. Since L cells do not have surface markers of T and B lymphocytes, it is likely that they comprise a separate population.

Levels of interleukin-2 receptors (IL-2R), as measured by a double-antibody "sandwich" enzyme-linked immunosorbent assay technique, were markedly elevated in the serum of patients with systemic lupus erythematosus, rheumatoid arthritis, and bacterial endocarditis, but not in patients with acute gout. Serum levels of IL-2R correlated strongly with clinical and laboratory indicators of disease activity in patients with lupus and in those with rheumatoid arthritis. This relationship was confirmed by sequential determinations in individual patients. Serum IL-2R values correlated with disease activity better than did the Westergren erythrocyte sedimentation rate. Our findings indicate that serum levels of IL-2R may serve as a reliable serologic indicator of disease activity in inflammatory diseases characterized by immune system activation.

Human blood lymphocytes that express Type 3 complement receptors (CR3) can be divided into a major subset with high density Fc receptors for IgG (FcR) identified with the monoclonal antibody Leu 11 and two minor subsets which display either CD8 (Leu 2) or CD4 (Leu 3) markers. We isolated CR3+ lymphocyte subsets and examined them for regulatory effects on pokeweed mitogen (PWM) stimulated cells. The FCR CR3+ cell suppressed PWM-induced proliferation and Ig production. Pretreatment of these lymphocytes with immune complexes was required to suppress proliferation, but not IgG production. The CR3+ Leu 2+ FCR- subset also had suppressive activity, but this effect was not observed unless the CR3+ Leu 3+ enriched subset was removed. In fact, the CR3+ Leu 3+ enriched subset enhanced IgG synthesis. Brief exposure of CR3+ lymphocytes to recombinant interleukin 2, recombinant alpha-interferon, but not gamma-interferon, markedly enhanced the inhibitory effect. Time course studies and a comparison of inhibition of Ig synthesis with natural killer cell activity suggested that CR3+ lymphocytes act shortly after lymphocytes are exposed to PWM and that Ig production was regulated by suppression rather than cytotoxicity. These CR3+ lymphocyte subsets may have broad antigen non-specific effects on immunoglobulin synthesis.

A method is described which demonstrates that human adherent mononuclear cells have a strong capacity to kill human nucleated target cells. Cytotoxicity was measured by the release of 51chromium from two established cell lines. Freshly prepared monolayers killed both non-sensitized and anti-body coated K562 and CLA-4 target cells. These spontaneous events decreased after short term culture. A second peak of cytotoxic activity was induced by activating the effector cells with endotoxin-treated serum or phytohaemagglutinin. Cytotoxicity was inhibited by silica particles. Studies with alpha-naphthyl esterase showed that 95% of freshly prepared cells and 99% of monolayers cultured for 68 h were monocytes. These studies suggest that adherent monocytes are the predominant cytotoxic effector cell although a contributing effect by the small numbers of contaminating lymphocytes can not be excluded. Evaluation of monocyte cytotoxicity should have a useful role in clinical investigation.

Histochemical staining for alpha-naphthyl (non-specific) esterase has been employed to define subpopulations of human peripheral blood lymphocytes and monocytes. To define optimal conditions for staining, various fixatives, incubation times and cell preparations were compared. The great majority of human blood lymphocytes were found to have discrete granules of reaction product. More than 80% of T lymphocytes separated by E rosetting are esterase-positive whereas non-T lymphocytes are esterase-negative. Lymphocytes transformed by polyclonal mitogens lose their esterase-staining granules, which suggests that immature T cells are esterase-negative. Most blood monocytes show a diffuse cytoplasmic esterase reaction product and are phagocytic. However, about 20% of diffusely stained cells are not phagocytic. When leucocytes are cultured for 24 to 48 hr, the total number of esterase-positive cells increases and the great majority are phagocytic. This is interpreted as maturation of precursors into mature esterase-positive phagocytic monocytes. When cultured for longer periods, some lose phagocytic capacity and acquire the characteristics of secretory cells. Esterase-staining of lymph node sections allowed the identification of T- and B-dependent areas as well as macrophages related to sinuses. The esterase-staining technique could play a useful role in clinical and experimental immunology.

Lymphocytes from patients with primary immunodeficiency were tested histochemically for ecto 5'-nucleotidase (5'N) and alpha-naphthyl (non-specific) esterase. More than half the patients with 'common variable' hypogammaglobulinaemia, all those with X-linked hypogammaglobulinaemia and some of those with selective IgA deficiency had a very low percentage of lymphocytes staining for 5'N as compared to controls. A lack of B cells probably explains the finding in X-linked hypogammaglobulinaemia, but does not fully explain the results in the other groups. Most patients with 'common variable' hypogammaglobulinaemia had a very low percentage of lymphocytes with granular staining for alpha-naphthyl (non-specific) esterase in contrast to normal numbers in those with X-linked hypogammaglobulinaemia and most of those with selective IgA deficiency. Granules containing non-specific esterase are characteristically found in 'mature' T lymphocytes. The enzyme abnormalities in the T and B cells of 'common variable' hypogammaglobulinaemic patients could be explained by 'immature' cell types.

The contribution of immune regulation to the etiology of systemic lupus erythematosus (SLE) is poorly understood. Using the monoclonal antibodies OKT4 and OKT8, we quantitated, by flow cytometry, T inducer/helper and T cytotoxic/suppressor cells in patients with SLE. Serologically active patients, who had clinical manifestations such as arthritis or rash and were not receiving prednisone, were characteristically lymphopenic due to a marked reduction in OKT4+ cells. Prednisone therapy produced the same phenomenon. Untreated patients, who were serologically inactive, demonstrated no abnormalities. These studies have thus revealed two presumably independent factors that can produce similar immunoregulatory aberrations.

IL-2 production by peripheral blood mononuclear cells (PBM) is decreased in patients with systemic lupus erythematosus (SLE). This defect can be reversed by the removal of CD8+ lymphocytes. The purpose of these studies was to determine whether the CD8+ IL-2 suppressor cells comprise a specific subset or whether all CD8+ cells have this activity. Lymphocyte subsets were identified and separated by two-colour flow cytometry prior to a 48 h mitogen stimulation. The CD8+ cells that suppressed IL-2 production co-expressed HLA DR and were radiosensitive. Other markers co-expressed by CD8+ cells which are found on suppressor cells such as Leu 15 (CD11), Leu 11 (CD16), and Leu 7 were also found on the CD8+ IL-2 suppressor cell population in SLE. In healthy subjects, removal of CD16+, but not of CD8+ cells markedly elevated the production of IL-2. The CD8- CD16+ non-T cell subset suppressed IL-2 production by normal and SLE PBM in autologous and allogeneic combinations. This subset may be a human equivalent of the murine natural suppressor cells. These results demonstrate that the cells that suppress IL-2 production in SLE are heterogeneous, and suggest that they belong to more than one lineage.

Human blood lymphocytes with high affinity Fc receptors for IgG will bind small aggregates of this immunoglobulin at 4 degrees C. These cells have been named L lymphocytes because of membrane-labile IgG determinants. L cells possess a profile of surface markers and functional characteristics which differ from T and B cells. Immunofluorescence methods have been employed to quantify L lymphocytes in subjects with connective tissue diseases and certain infections, and these values have been compared with those for T and B cells. The mean values of L lymphocytes in groups of patients with systemic lupus erythematosus, rheumatoid arthritis and scleroderma ranged between 14% and 18%; values similar to normals. Groups with acute pneumonia and tuberculosis, however, had significantly increased percentages of L lymphocytes. The absolute number of L cells was decreased in subjects with connective tissue diseases, as was the number of T and B cells. L lymphocytes in those with infections were not significantly decreased. Only L lymphocytes were depleted by immobilized antigen--antibody complexes, another characteristic which distinguishes them from T and B cells.

The origin and function of the increased of "atypical lymphocytes" which appear in the blood of patients with many inflammatory diseases is not known. Leukocyte suspensions from eight patients with systemic lupus erythematosus (SLE), five patients with other rheumatic diseases, and five patients with infectious diseases were pulse-labeled with tritiated thymidine (Tdr-(3)H) and sampled after 5 and 72 hr in vitro. Radioautographs indicated that 35% of the total large, nonphagocytic mononuclear leukocytes incorporated Tdr-(3)H during the initial 5 hr of culture. Tdr-(3)H-labeled large phagocytic or glass-adherent cells were observed only infrequently. After 72 hr one-third of the original number of Tdr-(3)H-labeled cells from patients with SLE developed the morphology of macrophages and the capacity to phagocytose latex particles. Similar findings were observed in patients with other rheumatic diseases and bacterial infections. In contrast, the thymidine-labeled cells from patients with infectious hepatitis and infectious mononucleosis were poorly viable in culture and rarely became macrophages. Tdr-(3)H-labeled small lymphocytes were uncommon. The present experiments suggest that in patients with certain inflammatory diseases large, proliferating "lymphocytelike" cells are very immature monocyte precursors which appear in response to tissue injury. These DNA-synthesizing cells together with mature monocytes may serve as the circulating source of macrophages.

Tumor immunity in patients with primary intracranial tumors was assessed in relation to the general status of host immunocompetence. Lymphocyte sensitization to tumor-specific membrane antigens was demonstrated by the proliferative response of lymphocytes in the presence of autochthonous tumor cells. Paradoxically, one-half of the patients could not be sensitized to a primary antigen, dinitrochlorobenzene; existing delayed hypersensitivity was also depressed, as measured by skin tests and lymphocyte transformation in response to common antigens. A heat-stable factor in patients' sera blocked cell-mediated tumor immunity. In addition, these "enhancing" sera consistently suppressed the blastogenic response of autologous and homologous lymphocytes to phytohemagglutinin and to membrane antigens on allogeneic cells in the one-way mixed lymphocyte culture. When patients' leukocytes were washed and autologous plasma replaced with normal plasma, reactivity in the mixed lymphocyte culture increased to normal values. In vitro immunosuppressive activity in patients' plasma or sera correlated with depressed delayed hypersensitivity. After removal of the tumor, suppressor activity disappeared. IgG fractions of patient sera contained strong immunosuppressive activity. These data suggest that the suppressor factor may be an isoantibody elicited by the tumor that also binds to receptors on the lymphocyte membrane. In addition to specifically blocking cell-mediated tumor immunity, enhancing sera may broadly depress host immunocompetence.