Cells Expressing Tumor Necrosis Factor Research Articles

Identifying genetic variants that influence the abundance of cell states in single-cell data.

Disease risk alleles influence the composition of cells present in the body, but modeling genetic effects on the cell states revealed by single-cell profiling is difficult because variant-associated states may reflect diverse combinations of the profiled cell features that are challenging to predefine. We introduce Genotype-Neighborhood Associations (GeNA), a statistical tool to identify cell-state abundance quantitative trait loci (csaQTLs) in high-dimensional single-cell datasets. Instead of testing associations to predefined cell states, GeNA flexibly identifies the cell states whose abundance is most associated with genetic variants. In a genome-wide survey of single-cell RNA sequencing peripheral blood profiling from 969 individuals, GeNA identifies five independent loci associated with shifts in the relative abundance of immune cell states. For example, rs3003-T (P = 1.96 × 10-11) associates with increased abundance of natural killer cells expressing tumor necrosis factor response programs. This csaQTL colocalizes with increased risk for psoriasis, an autoimmune disease that responds to anti-tumor necrosis factor treatments. Flexibly characterizing csaQTLs for granular cell states may help illuminate how genetic background alters cellular composition to confer disease risk.

Circulating Regulatory T Cells Expressing Tumor Necrosis Factor Receptor Type 2 Contribute to Sepsis-Induced Immunosuppression in Patients During Septic Shock.

Septic shock remains a major cause of death that can be complicated by long-term impairment in immune function. Among regulatory T (Treg) cells, the tumor necrosis factor receptor 2 positive (TNFR2pos) Treg-cell subset endorses significant immunosuppressive functions in human tumors and a sepsis mouse model but has not been investigated during septic shock in humans. We prospectively enrolled patients with septic shock hospitalized in intensive care units (ICU). We performed immunophenotyping and functional tests of CD4+ T cells, Treg cells, and TNFR2pos Treg cells on blood samples collected 1, 4, and 7 days after admission to ICU. We investigated 10 patients with septic shock compared to 10 healthy controls. Although the proportions of circulating Treg cells and TNFR2pos Treg-cell subsets were not increased, their CTLA4 expression and suppressive functions in vitro were increased at 4 days of septic shock. Peripheral blood mononuclear cells from healthy donors cultured with serum from septic shock patients had increased CTLA4 expression in TNFR2pos Treg cells compared to TNFR2neg Treg cells. In patients with septic shock, CTLA4 expression and suppressive function were increased in circulating TNFR2pos Treg cells. We identify TNFR2pos Treg cells as a potential attractive target for therapeutic intervention.

Regulatory T Cells Expressing Tumor Necrosis Factor Receptor Type 2 Play a Major Role in CD4+ T-Cell Impairment During Sepsis.

Sepsis causes inflammation-induced immunosuppression with lymphopenia and alterations of CD4+ T-cell functions that renders the host prone to secondary infections. Whether and how regulatory T cells (Treg) are involved in this postseptic immunosuppression is unknown. We observed in vivo that early activation of Treg during Staphylococcus aureus sepsis induces CD4+ T-cell impairment and increases susceptibility to secondary pneumonia. The tumor necrosis factor receptor 2 positive (TNFR2pos) Treg subset endorsed the majority of effector immunosuppressive functions, and TNRF2 was particularly associated with activation of genes involved in cell cycle and replication in Treg, probably explaining their maintenance. Blocking or deleting TNFR2 during sepsis decreased the susceptibility to secondary infection. In humans, our data paralleled those in mice; the expression of CTLA-4 was dramatically increased in TNFR2pos Treg after culture in vitro with S. aureus. Our findings describe in vivo mechanisms underlying sepsis-induced immunosuppression and identify TNFR2pos Treg as targets for therapeutic intervention.

Involvement of Tumor Necrosis Factor Receptor Type II in FoxP3 Stability and as a Marker of Treg Cells Specifically Expanded by Anti-Tumor Necrosis Factor Treatments in Rheumatoid Arthritis.

To study the involvement of Treg cells expressing tumor necrosis factor receptor type II (TNFRII) in exerting control of inflammation in experimental models and in the response to anti-TNF treatments in patients with rheumatoid arthritis (RA) or spondyloarthritis (SpA). The role of TNFRII in Treg cells was explored using a multilevel translational approach. Treg cell stability was evaluated by analyzing the methylation status of the Foxp3 locus using bisulfite sequencing. Two models of inflammation (imiquimod-induced skin inflammation and delayed-type hypersensitivity arthritis [DTHA]) were induced in TNFRII-/- mice, with or without transfer of purified CD4+CD25+ cells from wild-type (WT) mice. In patients with RA and those with SpA, the evolution of the TNFRII+ Treg cell population before and after targeted treatment was monitored. Foxp3 gene methylation in Treg cells was greater in TNFRII-/- mice than in WT mice (50% versus 36.7%). In cultured Treg cells, TNF enhanced the expression, maintenance, and proliferation of Foxp3 through TNFRII signaling. Imiquimod-induced skin inflammation and DTHA were aggravated in TNFRII-/- mice (P < 0.05 for mice with skin inflammation and P < 0.0001 for mice with ankle swelling during DTHA compared to WT mice). Adoptive transfer of WT mouse Treg cells into TNFRII-/- mice prevented aggravation of arthritis. In patients with RA receiving anti-TNF treatments, but not those receiving tocilizumab, the frequency of TNFRII+ Treg cells was increased at 3 months of treatment compared to baseline (mean ± SEM 65.2 ± 3.1% versus 49.1 ± 5.5%; P < 0.01). In contrast, in anti-TNF-treated patients with SpA, the frequency of TNFRII+ Treg cells was not modified. TNFRII expression identifies a subset of Treg cells that are characterized by stable expression of Foxp3 via gene hypomethylation, and adoptive transfer of TNFRII-expressing Treg cells ameliorates inflammation in experimental models. Expansion and activation of TNFRII+ Treg cells may be one of the mechanisms by which anti-TNF agents control inflammation in RA, but not in SpA.

Successful activation of rat T lymphocytes by sperm specific antigens in vitro.

Autoimmune orchitis is a condition related to cellular immunity. A disease model involving transfer of T lymphocytes activated by known antigens would be useful for defining pathogenical molecules. Since no method for activating rat T cells using specific antigens is available, we started the study to develop the method. T cells were collected from draining lymph nodes of immunized rats, then co-cultured with syngeneic splenocytes as antigen-presenting cells (APC) in antigen-supplemented medium (= stimulation). The cells were then incubated in medium without antigens and APC (= resting). Repetitive stimulation and resting increased the number of the T cells more than 100-fold. The antigen-specific activation was demonstrated by cell proliferation assay and ELISA assay for interferon gamma. Flow cytometry revealed that > 95% of the cells expressed tumor necrosis factor alpha, a cytokine responsible for autoimmune orchitis. The present method will provide a new procedure to evaluate antigenicity of sperm molecules.

Aberrant frequency of TNFR2+ Treg and related cytokines in patients with CIN and cervical cancer.

Regulatory T (Treg) cells expressing tumor necrosis factor receptor 2 (TNFR2) are highly suppressive and are associated with immune homeostasis in various diseases. However, the role of TNFR2+Treg subset and relevant cytokines in the development of cervical cancer (CC) remained unclear. In this study, 72 patients with CC, 30 patients with cervical intraepithelial neoplasia (CIN) and 30 healthy volunteers were enrolled. The level of circulating TNFR2+Tregs was investigated through flow cytometry. The plasma concentrations of soluble TNFR1 (s-TNFR1) and soluble TNFR2 (s-TNFR2) were determined by enzyme-linked immunosorbent assay. In addition, the mRNA expression levels of TNF-α, TNFR1, TNFR2, and Foxp3 were measured using real-time polymerase chain reaction. Results showed that both peripheral and tumor infiltrating TNFR2+Tregs significantly increased in patients with CIN and CC and levels of circulating s-TNFR1 and s-TNFR2 increased in patients with CC. Moreover, the percentage of peripheral TNFR2+Tregs was inversely correlated with the clinical stages of CC. Furthermore, the mRNA expression levels of TNF-α, TNFR2, and Foxp3 increased in patients with CIN and CC. Overall, these results indicate that TNFR2+Tregs and relevant cytokines contribute to CC development and are promising targets in future immunotherapeutic approaches.

Interleukin-10- and transforming growth factor β-independent regulation of CD8⁺ T cells expressing type 1 and type 2 cytokines in human lymphatic filariasis.

Lymphatic filariasis is known to be associated with diminished CD4⁺ Th1 and elevated CD4⁺ Th2 responses to parasite-specific antigens. The roles of cytokine-expressing CD8⁺ T cells in immune responses to filarial infections are not well defined. To study the roles of CD8⁺ T cells expressing type 1, type 2, and type 17 cytokines in filarial infections, we examined the frequencies of these cells in clinically asymptomatic, patently infected (INF) individuals, directly ex vivo and in response to parasite or nonparasite antigens; these frequencies were compared with the results for individuals with filarial lymphedema (i.e., clinical pathology [CP]) and those without active infection or pathology (i.e., endemic normal [EN]). INF individuals exhibited significant decreases in the frequencies of CD8⁺ T cells expressing tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin-22 (IL-22) at baseline and/or in response to filarial antigens, compared with CP and EN individuals. In contrast, the same individuals exhibited significant increases in the frequencies of CD8⁺ T cells expressing IL-4, IL-5, IL-9, IL-13, and IL-21, compared with CP and/or EN individuals. Curative treatment resulted in significantly increased frequencies of CD8⁺ T cells expressing IL-2 and significantly decreased frequencies of CD8⁺ T cells expressing type 2 cytokines. Finally, the regulation of these responses appears to be independent of IL-10 and transforming growth factor β (TGF-β), since blockade of IL-10 or TGF-β signaling did not significantly alter the frequencies of type 1 or type 2 cytokine-expressing CD8⁺ T cells. Our findings suggest that alterations in the frequencies of cytokine-expressing CD8⁺ T cells are characteristic features of lymphatic filarial infections.

Stimulation of Matrix Metalloproteinases by Tumor Necrosis Factor–α in Human Pulp Cell Cultures

IntroductionThe purpose of this study was to investigate whether in vitro stimulation of pulp cells leads to increased secretion of matrix metalloproteinases (MMPs) and, if so, to identify which MMPs are affected. MethodsCells cultured from dental pulp were stimulated with tumor necrosis factor–α (TNF-α) (10 ng/mL) for 24 hours, and lysates were analyzed with an antibody array (Bio-Rad Laboratories, Hercules, CA). The mRNA and protein levels of MMP-3, -10, and -13 were measured by real-time polymerase chain reaction (real-time PCR), enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and zymography. In addition, tumor necrosis factor receptors in the pulp cells were assayed by flow cytometry. The ELISA and real-time PCR results were analyzed by paired t tests. ResultsThe expression of MMP-3, -10, and -13 was up-regulated in the pulp cells after 24 hours of stimulation with TNF-α (10 ng/mL) as seen in the antibody array, real-time PCR, and ELISA results, but MMP-10 was not detected by Western blotting or casein zymography. Flow cytometry analysis showed that the majority of the pulp cells expressed tumor necrosis factor receptor 1. ConclusionsIn regions of inflammation, TNF-α may initiate the degradation of dental connective tissue by activating MMP-3 and MMP-13. These proteins may play an important pathologic role in the inflammation of dental pulp.

Concomitant apoptosis and regeneration of liver cells as a mechanism of liver-tumor promotion by β-naphthoflavone involving TNFα-signaling due to oxidative cellular stress in rats

β-Naphthoflavone (BNF) is a strong inducer of cytochrome P450 1A enzymes, and exerts liver tumor-promoting activity through enhancement of oxidative stress responses in rats. This study investigated the role of the tissue environment surrounding hepatocellular preneoplastic lesions in the early tumor-promotion stage by BNF, using enzymatically modified isoquercitrin (EMIQ) as an anti-oxidative chemopreventive agent. Male F344 rats were fed a diet containing BNF (0.5%) for 6 weeks, with or without EMIQ (0.2%) in the drinking water, 2 weeks after initiation with N-diethylnitrosamine, and were subjected to two-thirds partial hepatectomy 1 week after starting BNF-promotion. BNF-treatment increased concentrations of liver thiobarbituric acid-reactive substances, single liver cells expressing glutathione S-transferase placental form or heme oxygenase (HO)-1, and concomitant apoptosis and proliferation of liver cells. Transcript upregulation of anti-oxidative enzymes ( Aldh1a1 and Nqo1), cell cycle-related molecules ( Cdc20 and Cdkn2b) and inflammation-related molecules including proinflammatory cytokines ( Ccl2, Col1a1, Il6, Nos2 and Serpine1) was also evident. Furthermore, BNF increased HO-1-expressing Kupffer cells and liver cells expressing tumor necrosis factor receptor 1 (TNFR1) and the TNFR1-associated death domain. Most of these BNF-induced fluctuations disappeared or were suppressed by EMIQ in conjunction with suppression of tumor-promotion. Tnf transcript levels with BNF were also suppressed by EMIQ. These results suggest that BNF-induced oxidative stress causes single liver cell toxicity, allowing subsequent concomitant apoptosis and regeneration involving inflammatory responses including TNFα-signaling, contributing to tumor promotion. Kupffer cells may act to protect against inflammatory stimuli induced as a result of oxidative cellular stress by BNF, causing proinflammatory cytokine level fluctuations.

Natural Killer Cells Are Polarized Toward Cytotoxicity in Chronic Hepatitis C in an Interferon-Alfa–Dependent Manner

Patients with chronic hepatitis C virus (HCV) infection display great variability in disease activity and progression. Although virus-specific adaptive immune responses have been characterized extensively and found to be impaired in chronic hepatitis C, the role of innate immune responses in disease activity and progression of chronic hepatitis C is not well understood. We studied 42 HCV-infected patients and 12 healthy uninfected controls. We found an increased frequency of natural killer (NK) cells expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), NKp44, NKG2C, and CD122 in chronic hepatitis C as compared with healthy controls (P < .05 for all markers) and stronger activation of NK cells in the liver than in the blood (P < .05). This NK cell phenotype was associated with polarization of NK cell function toward CD107a expression as a marker of degranulation, but with not increased interferon (IFN)-gamma production of CD56(dim) NK cells. The polarized NK cell phenotype correlated with alanine aminotransferase levels (r(2) = 0.312, P = .03). To investigate whether in vivo exposure of NK cells to HCV-induced type I IFN was causing this NK cell phenotype, peripheral blood mononuclear cells from 10 healthy controls and 8 HCV-infected patients were stimulated in the presence of IFN-alfa, which resulted in increased NK cell expression of TRAIL and CD107a (P < .001), but not IFN-gamma. Collectively, these results describe a polarized NK cell phenotype induced by chronic exposure to HCV-induced IFN-alfa. This phenotype may contribute to liver injury through TRAIL expression and cytotoxicity, whereas the lacking increase in IFN-gamma production may facilitate the inability to clear HCV.

Lower motor neuron loss in multiple sclerosis and experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is considered a chronic inflammatory and demyelinating disease of the central nervous system. Evidence that axonal and neuronal pathology contributes to the disease is accumulating, however, the distribution of neuronal injury as well as the underlying mechanisms have not yet been fully clarified. Here, we investigated the role of neuronal cell loss in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). We performed electrophysiological investigations in MS patients, including assessment of compound muscle action potentials and motor unit numbers and quantified neuronal cell loss in human MS samples and different EAE models by high-precision stereology. Both electrophysiological and morphological analyses indicated a massive loss of lower motor neurons in MS patients. We regularly found dying spinal motor neurons surrounded by CD3+ (CD4+ as well as CD8+) T cells expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We observed a similar degree of damage and immune attack in different variants of EAE; the lower motor neurons were preserved in adoptive transfer EAE induced with TRAIL-deficient T lymphocytes. Our study indicates that damage to lower motor neurons and TRAIL-mediated inflammatory neurodegeneration in the spinal cord contribute to MS pathology.

An increased proportion of inflammatory cells express tumor necrosis factor alpha in idiopathic achalasia of the esophagus

Achalasia is a motility disorder characterized by the absence of coordinated peristalsis and incomplete relaxation of the lower esophageal sphincter. The etiology remains unclear although dense inflammatory infiltrates within the myenteric plexus have been described. The nature of these infiltrating cells is unknown. The aim of this study was to evaluate the expression of proinflammatory cytokines - namely, tumor necrosis factor alpha and interleukin-2 - in the distal esophageal muscle in patients with achalasia. Lower esophageal sphincter muscle from eight patients undergoing myotomy or esophagectomy for achalasia of the esophagus were obtained at the time of surgery. Control specimens consisted of similar muscle taken from eight patients undergoing operation for cancer or Barrett's esophagus. The expression of tumor necrosis factor alpha and interleukin-2 were assessed by immunohistochemistry. The total number of inflammatory cells within the myenteric plexus were counted in five high power fields. The percentage of infiltrating cells expressing tumor necrosis factor alpha or interleukin-2 was calculated. Clinical data including demographics, preoperative lower esophageal sphincter pressure, duration of symptoms, and dysphagia score (1 = no dysphagia to 5 = dysphagia to saliva) were obtained through electronic medical records. Statistical comparisons between the groups were made using the unpaired t-test, Fisher's exact test, or Mann-Whitney U test, with a two-tailed P-value less than 0.05 being considered significant. The total number of inflammatory cells was found to be similar between the groups. A significantly higher proportion of inflammatory cells expressed tumor necrosis factor alpha in achalasia as compared with controls (22 vs. 11%; P= 0.02). A similar percentage of infiltrating cells expressed interleukin-2 (40 vs. 41%; P= 0.87). Age, gender, preoperative lower esophageal sphincter pressure, or dysphagia score were not correlated to expression of these cytokines. There was, however, a significant inverse correlation between duration of symptoms and the proportion of inflammatory cells expressing tumor necrosis factor alpha in achalasia (P= 0.007). In conclusion, a higher proportion of infiltrating inflammatory cells expressed tumor necrosis factor alpha in achalasia. Furthermore, this proportion appears to be highest early in the disease process. Further studies are required to more clearly delineate the role of tumor necrosis factor alpha in the pathogenesis of this idiopathic disease.

Simultaneous Induction of Apoptotic and Survival Signaling Pathways in Macrophage-Like THP-1 Cells by Shiga Toxin 1

Shiga toxins have been shown to induce apoptosis in many cell types. However, Shiga toxin 1 (Stx1) induced only limited apoptosis of macrophage-like THP-1 cells in vitro. The mechanisms regulating macrophage death or survival following toxin challenge are unknown. Differentiated THP-1 cells expressed tumor necrosis factor receptors and membrane-associated tumor necrosis factor alpha (TNF-alpha) and produced soluble TNF-alpha after exposure to Stx1. However, the cells were refractory to apoptosis induced by TNF-alpha, although the cytokine modestly increased apoptosis in the presence of Stx1. Despite the partial resistance of macrophage-like THP-1 cells to Stx1-mediated killing, treatment of these cells with Stx1 activated a broad array of caspases, disrupted the mitochondrial membrane potential (DeltaPsi(m)), and released cytochrome c into the cytoplasm. The DeltaPsi(m) values were greatest in cells that had detached from plastic surfaces. Specific caspase inhibitors revealed that caspase-3, caspase-6, caspase-8, and caspase-9 were primarily involved in apoptosis induction. The antiapoptotic factors involved in macrophage survival following toxin challenge include inhibitors of apoptosis proteins and X-linked inhibitor of apoptosis protein. NF-kappaB and JNK mitogen-activated protein kinases (MAPKs) appeared to activate survival pathways, while p38 MAPK was involved in proapoptotic signaling. The JNK and p38 MAPKs were shown to be upstream signaling pathways which may regulate caspase activation. Finally, the protein synthesis inhibitors Stx1 and anisomycin triggered limited apoptosis and prolonged JNK and p38 MAPK activation, while macrophage-like cells treated with cycloheximide remained viable and showed transient activation of MAPKs. Collectively, these data suggest that Stx1 activates both apoptotic and cell survival signaling pathways in macrophage-like THP-1 cells.

A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles

Recently activated, but not resting, CD4(+) T cells express CD154, providing costimulatory signals to B cells and antigen-presenting cells (APCs). Therefore, de novo CD154 expression after stimulation identifies antigen-specific CD4(+) T cells. Previous assays were limited by the transient nature of surface CD154 expression; we overcame this by including fluorescently conjugated CD154-specific antibody during stimulation. Our assay is fully compatible with intracellular cytokine staining, and can be used for stimulations as long as 24 h. Notably, it is nonlethal, providing a means to purify viable antigen-specific CD4(+) T cells for further analysis. Using this assay, we found that stimulated cells expressing tumor necrosis factor (TNF)-alpha, interleukin (IL)-2 or interferon (IFN)-gamma were predominantly CD154(+). Furthermore, some cells expressing none of these cytokines also expressed CD154, suggesting that CD154 marks cells with other effector functions. For vaccine- or pathogen-specific responses, we found substantial heterogeneity in expression of CD154 and cytokines, suggesting previously unrecognized diversity in abilities of responding cells to stimulate APCs through CD40.

Histologic and Molecular Correlates of Fatal Measles Infection in Children

The purpose of this study is to document three cases of fatal measles infection in children who ranged in age from 1 to 6 years old. In each case, there was a rapidly progressive illness marked by severe respiratory and central nervous system disease; in two cases, tonsillar herniation occurred. The lung tissues showed marked interstitial pneumonitis with diffuse endothelial cell and pneumocyte degeneration; occasional multinucleated giant cells were observed. Brain sections showed a paucicellular inflammatory infiltrate with diffuse neuronal damage. Measles nucleoprotein and measles RNA were detected in each case by immunohistochemistry and reverse transcriptase (RT) in situ PCR, respectively. In the lung tissues, the viral protein and RNA localized primarily to pneumocytes and macrophages; infected endothelial cells were also evident. In the brain sections, the virus-infected cells cytologically had the appearance of neurons and microglial cells. The viral load, defined by the percentage of cells infected in a given field, was very high in the lung, spleen, and brain. Viral infection was associated with a marked increase in the number of cells expressing tumor necrosis factor alpha and concomitant reduction in the cells expressing suppressors of cytokine signaling (SOCS). It is concluded that measles infection should be in the differential diagnosis of a rapidly progressive illness in young children in the United States and that the pathogenesis is based, in part, on massive viral infection with up-regulation of cytokine expression that likely reflects, in part, down-regulation of inhibitors of cytokine mRNA receptor synthesis.

Molecular detection of rabies encephalitis and correlation with cytokine expression

The purposes of this study were to elucidate the role of cytokine upregulation in the pathogenesis of rabies encephalitis and to compare the detection of Negri bodies with that of rabies protein by immunohistochemistry and rabies RNA by reverse transcriptase (RT) in situ PCR for its diagnosis. Negri bodies were evident in 4/7 of the documented rabies cases; viral protein and viral RNA were detected in each case. The average number of rabies-infected cells, determined by counting 150 neurons in serial sections in areas where viral protein was evident, with the three different detection methods was: Negri bodies (<1/150), immunohistochemistry (4/150), and RT in situ PCR (49/150). No rabies protein or RNA was detected in four control brain tissues that were read with the rabies cases in a blinded fashion. The ratio of cells expressing tumor necrosis factor α (TNFα) or inducible nitric oxide synthetase (iNOS) to 1 SSI-1/SOCS-1 (suppressors of cytokine signaling) expression, which is a novel class of negative feedback regulators of cytokine receptor signaling, was markedly increased only in the areas where many viral infected cells were present. Colabeling experiments showed that most of the cells expressing iNOS or TNFα were not virally infected, but rather adjacent to rabies-infected neurons. We conclude that RT in situ PCR for rabies virus is the most accurate test for the determination of viral load in rabies encephalitis. Further, the disease is characterized by massive viral infection of neurons in a markedly focal distribution in conjunction with a concomitant upregulation of cytokine expression in adjacent, noninfected cells that may be due, in part, to SOCS downregulation.

V gamma 9V delta 2 T cells in human legionellosis.

In humans, expansion of circulating Vgamma9Vdelta2 T cells seems to be a pathophysiological denominator shared by protozoan and intracellular bacterial diseases. The assumption was tested here on legionellosis, a condition conforming to the category but not yet described with respect to gammadelta T cells. Levels of Vgamma9Vdelta2 T cells in peripheral blood were measured at various intervals in 14 subjects undergoing a Pontiac fever-like disease, shown by serological investigation to be caused by Legionella micdadei. In samples obtained 4 to 6 days after the onset of the disease, the mean percentage (+/- the standard deviation) of Vgamma9Vdelta2+ T cells among CD3+ cells was 1.0% +/- 0.5%, compared to 5.0% +/- 3.9% in healthy control subjects (P < 0.001). Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset, mean peak levels were as high as approximately equal to 15%. During the next 6 months, values slowly declined, although without reaching the normal range. Percentages of gammadelta+ T cells expressing tumor necrosis factor alpha or gamma interferon in response to phorbol myristate acetate were assayed in vitro. At 14 to 16 days after the onset of disease, the expression of both cytokines was increased (P < 0.01), whereas at 5 to 7 weeks, the expression of tumor necrosis factor alpha was decreased (P < 0.05), possibly reflecting modulation of an inflammatory response. In conclusion, Pontiac fever was found to be associated with a pronounced and long-lasting expansion of Vgamma9Vdelta2 T cells, implying that the subset may also be pathophysiologically important in a mild and transient form of intracellular bacterial diseases. Surprisingly, the expansion was preceded by a depletion of circulatory Vgamma9Vdelta2 T cells. Possibly, Vgamma9Vdelta2 T cells are initially recruited to a site of infection before they expand in response to antigen and occur in high numbers in blood.

T-cell subsets mediate graft-versus-myeloid leukemia responses via different cytotoxic mechanisms

Analysis of the cytotoxic effector mechanisms by which T-cell subsets mediate graft-versus-leukemia (GVL) activity is complicated by systems that use unfractionated T cells and leukemias that express alloantigens in addition to tumor-specific antigens. In this study, we used MMB1.10, a myeloid leukemia of C57Bl/6 (B6) origin, to examine the cytolytic pathways employed by syngeneic GVL-mediating, and therefore tumor antigen-specific, T-cell subsets. Wright-Giemsa staining and flow cytometric analysis indicated that MMB1.10 cells exhibited the morphology and markers most consistent with a monocytic-myeloid origin. Although reverse transcription-polymerase chain reaction analysis revealed that MMB1.10 cells expressed tumor necrosis factor (TNF) receptor types I and H, in vitro assays suggested that these cells were resistant to TNF-alpha-mediated cytotoxicity. For study of in vivo GVL responses, mice were challenged with MMBl.10 cells, lethally irradiated, and administered anti-Thy-1-treated (T-cell-depleted) bone marrow (ATBM) either alone or in combination with T-cell subsets from MMB1.10-presensitized mice. In regard to CD4+ donor T cells, 4 x 10(6) MMB1.10-presensitized wild-type (wt) cells exhibited increased GVL responses and survival values relative to tumor-challenged recipients of ATBM only. CD4 T cells from either perforin-deficient (pfp0) or Fas ligand (FasL)-deficient (gld) mice exhibited a lower level of GVL activity but did not produce any long-term survivors. Recipients of 5 x 10(6) wt B6 CD8+ T cells had significantly improved survival relative to tumor-challenged mice that received ATBM only. The same dose of gld CD8+ T cells exhibited a reduced but significant level of GVL activity, whereas cells from mice that were perforin-deficient or cytotoxicity doubly deficient (cdd) (ie, lacking perforin and FasL) exhibited no discernable GVL activity. Doubling the gld CD8+ T-cell dose to 10(7) cells resulted in further improved survival of recipients. We conclude that GVL effects mediated by CD4+ T cells can depend on either perforin- or FasL-mediated mechanisms, whereas the CD8+ T-cell subset is heavily dependent on perforin-mediated cytotoxicity. Biol Blood Marrow Transplant 2000;6(3):231-40.

Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes.

Progressive destruction of the insulin-producing beta cells in nonobese diabetic mice is observed after infiltration of the pancreas with lymphocytes [Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K. & Tochino, Y. (1980) Exp. Anim. (Tokyo) 29, 1-13]. We show that the genes for tumor necrosis factor alpha and granzyme A, a serine protease associated with cytoplasmic granules of cytotoxic cells, are expressed during the development of spontaneous diabetes mellitus in the nonobese diabetic mouse. Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration. Tumor necrosis factor alpha expression is exclusively observed in the intra-islet infiltrate, predominantly in lymphocytes adjacent to insulin-producing beta cells, the targets of the autoimmune destruction, implying that tumor necrosis factor alpha expression is induced locally--i.e., in the islet. A considerable portion of cells expressing tumor necrosis factor alpha appear to be CD4+ T cells. This T-cell subset was previously shown to be necessary for development of the disease. Thus, these findings may be important for understanding the pathogenesis of autoimmune diabetes mellitus and potentially also for that of other T-cell-mediated autoimmune diseases.