Injection Of anti-CD3 mAb Research Articles

The Cytosolic Microbial Receptor Nod2 Regulates Small Intestinal Crypt Damage and Epithelial Regeneration following T Cell-Induced Enteropathy.

Loss of function in the NOD2 gene is associated with a higher risk of developing Crohn's disease (CD). CD is characterized by activation of T cells and activated T cells are involved in mucosal inflammation and mucosal damage. We found that acute T cell activation with anti-CD3 mAb induced stronger small intestinal mucosal damage in NOD2(-/-) mice compared with wild-type mice. This enhanced mucosal damage was characterized by loss of crypt architecture, increased epithelial cell apoptosis, delayed epithelial regeneration and an accumulation of inflammatory cytokines and Th17 cells in the small intestine. Partial microbiota depletion with antibiotics did not decrease mucosal damage 1 d after anti-CD3 mAb injection, but it significantly reduced crypt damage and inflammatory cytokine secretion in NOD2(-/-) mice 3 d after anti-CD3 mAb injection, indicating that microbial sensing by Nod2 was important to control mucosal damage and epithelial regeneration after anti-CD3 mAb injection. To determine which cells play a key role in microbial sensing and regulation of mucosal damage, we engineered mice carrying a cell-specific deletion of Nod2 in villin and Lyz2-expressing cells. T cell activation did not worsen crypt damage in mice carrying either cell-specific deletion of Nod2 compared with wild-type mice. However, increased numbers of apoptotic epithelial cells and higher expression of TNF-α and IL-22 were observed in mice carrying a deletion of Nod2 in Lyz2-expressing cells. Taken together, our results demonstrate that microbial sensing by Nod2 is an important mechanism to regulate small intestinal mucosal damage following acute T cell activation.

Gamma/delta intraepithelial lymphocytes in the mouse small intestine.

Although many studies of intraepithelial lymphocytes (IELs) have been reported, most of them have focused on αβ-IELs; little attention has been paid to γδ-IELs. The function of γδ-IELs remains largely unclear. In this article, we briefly review a number of reports on γδ-IELs, especially those in the small intestine, along with our recent studies. We found that γδ-IELs are the most abundant (comprising >70% of the) IELs in the duodenum and the jejunum, implying that it is absolutely necessary to investigate the function(s) of γδ-IELs when attempting to delineate the in vivo defense system of the small intestine. Intraperitoneal injection of anti-CD3 mAb stimulated the γδ-IELs and caused rapid degranulation of them. Granzyme B released from their granules induced DNA fragmentation of duodenal and jejunal epithelial cells (paracrine) and of the IELs themselves (autocrine). However, perforin (Pfn) was not detected, and DNA fragmentation was induced even in Pfn-knockout mice; our system was therefore found to present a novel type of in vivo Pfn-independent DNA fragmentation. We can therefore consider γδ-IELs to be a novel type of large granular lymphocyte without Pfn. Fragmented DNA was repaired in the cells, indicating that DNA fragmentation alone cannot be regarded as an unambiguous marker of cell death or apoptosis. Finally, since the response was so rapid and achieved without the need for accessory cells, it seems that γδ-IELs respond readily to various stimuli, are activated only once, and die 2-3days after activation in situ without leaving their site. Taken together, these results suggest that γδ-IELs are not involved in the recognition of specific antigen(s) and are not involved in the resulting specific killing or exclusion of the relevant antigen(s).

Granzyme B-dependent and perforin-independent DNA fragmentation in intestinal epithelial cells induced by anti-CD3 mAb-activated intra-epithelial lymphocytes

We previously found that an i.p. injection of anti-CD3 monoclonal antibody (mAb) into mice caused DNA fragmentation in the intestinal villous epithelial cells (IVECs) of the duodenum and the jejunum. In this study, in order to elucidate the mechanism of DNA fragmentation in IVECs, we searched for the inducer(s) of DNA fragmentation by using immunohistochemistry. The release of cytoplasmic granules from intraepithelial lymphocytes (IELs) and the formation of large gaps between IELs and IVECs were observed electron microscopically after antibody administration. The presence and distribution pattern of Granzyme B (GrB), a serine protease in cytolytic granules present in cytotoxic T lymphocytes and natural killer cells and considered to be the responsible molecule for DNA fragmentation in target cells, was examined in detail in intestinal villi by immunohistology. GrB was detected in cytoplasmic granules in nearly all IELs. The time-kinetics of granule release from IELs after mAb injection coincided not only with that of the extracellular diffusion of GrB, but also with that of DNA fragmentation in IVECs. On the other hand, perforin (Pfn), assumed to cooperate with GrB in DNA fragmentation, could not be detected in IELs, and its release was not confirmed after the anti-CD3 mAb injection. Anti-CD3 mAb injection also induced DNA fragmentation in IVECs in Pfn-knockout mice. These results support the notion that DNA fragmentation in IVECs by the stimulated IELs in the present study is induced by a mechanism involving GrB, but independent of Pfn.

“Default” Generation of Neonatal Regulatory T Cells

CD4(+)Foxp3(+) regulatory T (Treg) cells were shown to control all aspects of immune responses. How these Treg cells develop is not fully defined, especially in neonates during development of the immune system. We studied the induction of Treg cells from neonatal T cells with various TCR stimulatory conditions, because TCR stimulation is required for Treg cell generation. Independent of the types of TCR stimulus and without the addition of exogenous TGF-beta, up to 70% of neonatal CD4(+)Foxp3(-) T cells became CD4(+)Foxp3(+) Treg cells, whereas generally <10% of adult CD4(+)Foxp3(-) T cells became CD4(+)Foxp3(+) Treg cells under the same conditions. These neonatal Treg cells exert suppressive function and display relatively stable Foxp3 expression. Importantly, this ability of Treg cell generation gradually diminishes within 2 wk of birth. Consistent with in vitro findings, the in vivo i.p. injection of anti-CD3 mAb to stimulate T cells also resulted in a >3-fold increase in Treg cells in neonates but not in adults. Furthermore, neonatal or adult Foxp3(-) T cells were adoptively transferred into Rag1(-/-) mice. Twelve days later, the frequency of CD4(+)Foxp3(+) T cells converted from neonatal cells was 6-fold higher than that converted from adult cells. Taken together, neonatal CD4(+) T cells have an intrinsic "default" mechanism to become Treg cells in response to TCR stimulations. This finding provides intriguing implications about neonatal immunity, Treg cell generation, and tolerance establishment early in life.

ANTI‐CD3 therapy expands the numbers of CD4+ and CD8+ treg cells and induces sustained amelioration of collagen‐induced arthritis

To assess the therapeutic potential of anti-CD3 monoclonal antibodies (mAb) for rheumatoid arthritis, using collagen-induced arthritis as an animal model. Arthritis was induced in DBA/1 mice by immunization with type II collagen. After disease onset, a single injection of anti-CD3 mAb (20 microg/mouse) was administered, and arthritis severity was monitored over a 10-day period. Anti-CD3 mAb treatment resulted in a sustained reduction in disease activity, which was associated with an increase in the proportion of naturally occurring CD4+CD25+FoxP3+ regulatory T (Treg) cells and the generation of a population of CD8+CD25+FoxP3+ Treg cells. Anti-CD3 mAb treatment did not alter the capacity of CD4+ Treg cells to suppress effector T cell proliferation and interferon-gamma (IFNgamma) production in vitro. However, CD4+ Treg cells from both anti-CD3 mAb-treated and control mice were unable to suppress interleukin-17 (IL-17) production. In contrast, CD8+ Treg cells induced by anti-CD3 therapy suppressed IL-17 production as well as CD4+ T cell proliferation and IFNgamma production. These results show that anti-CD3 mAb treatment has important therapeutic potential for rheumatoid arthritis and has the capacity to generate antiarthritic CD8+ Treg cells and expand the relative numbers of CD4+ Treg cells.

Histone Deacetylase Inhibitor SAHA Reduces Cytokine Storm and Facilitates Induction of Chimerism When Combined with Anti-CD3 mAb in Conditioning of Recipients.

Donor T cell-mediated graft versus leukemia (GVL) and graft versus autoimmunity (GVA) plays a critical role in the treatment of hematological malignancies and refractory autoimmune diseases using allogeneic hematopoietic cell transplantation (HCT). However, graft versus host disease (GVHD) remains a major obstacle in classical HCT, where recipients are usually conditioned with total body irradiation or high dose chemotherapy. We have recently reported a radiation-free and GVHD preventive anti-CD3-conditioning regimen, which allows donor CD8+ T cells to facilitate engraftment and mediate GVL without causing GVHD (PNAS, 2007 and J. I. 2007). In order to promote the clinical application of the anti-CD3-conditioning regimen, we need to overcome the cytokine storm that causes flu-like syndrome in patients after anti-CD3 mAb injection and reduce the required donor bone marrow cell dose. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, was reported by others to induce apoptosis of human T cell lymphoma cell lines and to reduce serum levels of pro-inflammatory cytokines in HCT recipients. Therefore, we tested whether conditioning with a combination of anti-CD3 and SAHA can reduce the cytokine storm caused by anti-CD3 and reduce the resistance of engraftment mediated by residual host T cells. Accordingly, titrated dose of SAHA (0.125–8 μM) were added to cultures of BALB/c spleen cells stimulated with plate-bound anti-CD3. We found that SAHA augmented apoptosis of anti-CD3 activated T cells in a dose-dependent manner. Although low-dose SAHA (0.125–0.25 μM) did not augment apoptosis, it rendered the residual live T cells partially unresponsive. At the same time, SAHA significantly reduced the pro-inflammatory cytokines (IL-2, IFN-γ, TNF-α, and IL-6) in the culture supernatants. Similarly, in vivo treatment with anti-CD3 and low-dose SAHA (40 μg/g) led to significant reduction of serum levels of those cytokines and partial unresponsiveness of the residual host T cells. Finally, conditioning with combined anti-CD3 and SAHA (40 μg/g) induced complete chimerism without GVHD in 12/12 of old BALB/c as well as in 8/8 of old autoimmune lupus NZB/NZW F1 recipients after two injections of BM cells (2 ×106/g) and CD4+ T-depleted spleen cells (4 ×106/g), although conditioning with anti-CD3 alone did not induce any chimerism (0/8). The chimeric NZB/NZW F1 recipients showed complete reversal of autoimmune glomerulonephritis and proteinuria. These results indicate that SAHA can not only reduce cytokine storm but also facilitate engraftment when combined with anti-CD3 for conditioning of recipients. This radiation free and GVHD preventive conditioning regimen may provide a novel approach for clinical HCT.

Acute inflammation alters bicarbonate transport in mouse ileum

T-cell mediated acute inflammation of the ileum may occur during Crohn's disease exacerbations. During ileal inflammation, absorption of nutrients and electrolytes by villus cells is decreased with a concomitant increase in crypt and/or villus fluid secretion. These alterations lead to fluid accumulation and the subsequent diarrhoea. Net intestinal fluid secretion consists of HCO3--rich plasma-like fluid. However, the regulation and mechanisms of HCO3- secretion in normal and acutely inflamed ileum are not clearly understood. To study this phenomenon, anti-CD3 monoclonal antibody (mAb)- induced in vivo ileal inflammatory mouse models was used for in vitro functional studies with Ussing chamber and pH stat techniques. Three hours after anti-CD3 mAb injection, ileal mucosa stripped of muscular and serosal layers showed a significant increase in short circuit current (Isc) (0.58+/-0.07 microEq h(-1) cm2 versus 1.63+/-0.14 microEq h(-1) cm2). The cAMP-stimulated Isc component was sensitive to glibenclamide but not to DIDS, suggesting that a cystic fibrosis transmembrane conductance regulator (Cftr)-mediated anion conductance was responsible. Basal Cl--dependent HCO3- secretion, measured using a pH stat technique, was decreased significantly in anti-CD3-injected mice, with a simultaneous increase in Cl--independent HCO3- secretion that was also inhibited by glibenclamide. Experiments using Cftr-/- mice showed neither an increase in Isc nor an increase in HCO3- secretion, confirming the role for Cftr protein in stimulating anion secretion following anti-CD3 treatment. Western blot analysis indicated that Cftr protein levels were unaltered by anti-CD3 treatment, at least acutely. Finally, an immunoassay for cAMP showed significant increases in intracellular cAMP in villus cells, but not in crypt cells. These studies therefore suggest a shift from a predominantly electroneutral Cl-HCO3- exchange in normal mice, to a predominantly electrogenic anion secretion including HCO3- that occurs via functional Cftr during anti-CD3-mediated acute inflammation.

AsialoGM1+CD8+ central memory-type T cells in unimmunized mice as novel immunomodulator of IFN- -dependent type 1 immunity

In unimmunized specific pathogen-free mice, there are unique memory-type CD8(+) T cell populations expressing asialoGM1 (ASGM1). These cells were classified into central memory-type T cells (T(CMT)) judging from their expression profile of CD44, IL-2Rbeta, CD62L and CCR7 cell-surface molecules. Among CD44(high)CD8(+) so-called memory CD8(+) T cell population, ASGM1(+)CD44(high)CD8(+) T(CMT), but not ASGM1(-)CD44(high)CD8(+) memory T cells, produced IFN-gamma by stimulation with anti-CD3 mAb. The physiological significance of ASGM1(+)CD8(+) T(CMT) as early source of IFN-gamma was also demonstrated in vivo. Namely, intravenous injection of anti-CD3 mAb (2 microg) resulted in early activation of IFN-gamma-producing ASGM1(+)CD8(+) T(CMT) cells as well as NKT and NK cells. Unexpectedly, however, few IFN-gamma-producing CD4(+) T cells were detected until 4 h after anti-CD3 mAb administration. Thus, ASGM1(+)CD8(+) T(CMT) were demonstrated to be early IFN-gamma producer, which may be crucial for T(h)1-dependent cellular immunity. Indeed, co-culture of naive CD4(+) T cells with ASGM1(+)CD8(+) T(CMT) but not ASGM1(-)CD8(+) T cells caused a great acceleration of IFN-gamma-producing T(h)1 cells in vitro. Finally, we found that T(h)1-prone C57BL/6 mice possessed higher percentage (10%) of ASGM1(+)CD8(+) T(CMT) in CD8(+) T cells compared with that (3%) of T(h)2-prone BALB/c mice. Moreover, ASGM1(+)CD8(+) T(CMT) derived from C57BL/6 mice produced higher levels of IFN-gamma compared with those from BALB/c mice. Thus, ASGM1(+)CD8(+) T(CMT), whose differentiation in vivo is genetically controlled, appear to play a critical role in the control of type 1 immunity, which is essential for therapy of tumors and infectious diseases.

DNA fragmentation and detachment of enterocytes induced by anti-CD3 mAb-activated intraepithelial lymphocytes.

To elucidate the role of intraepithelial lymphocytes (IEL) and enterocytes in the defense mechanism of the small intestine, we designed experiments to stimulate the IEL by anti-CD3epsilon, anti-TCRalphabeta, or anti-TCRgammadelta monoclonal antibodies (mAbs), and to examine the subsequent changes to the enterocytes. The enterocytes of the duodenum and jejunum, but not of the ileum, showed massive DNA fragmentation 30 min after intraperitoneal injection of anti-CD3 mAb. These responses were also induced by anti-TCRgammadelta mAb, but not by anti-TCRalphabeta mAb, and were completely inhibited by cyclosporin A. Nearly half of the enterocytes of the villi in the duodenum and jejunum were exfoliated into the lumen 4 h after the injection of the mAb. Administration of anti-CD3 mAb also induced DNA fragmentation in Fas-deficient MRL/lpr mice, indicating that the Fas-Fas ligand system was not involved in these events. The anti-CD3 mAb treatment also induced massive DNA fragmentation in the intestinal epithelium of the duodenum and jejunum in TNF-receptor-1-deficient mice, whereas TNF-alpha induced only the detachment of intestinal epithelium of wild-type mice, implying the dissociation of two independent factors and/or mechanisms for DNA fragmentation and the subsequent epithelial cell detachment in the murine duodenum and jejunum. The mAb failed to exfoliate the epithelium in TNF-R1-deficient mice. Thus, TCRgammadelta(+) IEL, when treated with anti-CD3 or anti-TCRgammadelta mAbs, induced rapid DNA fragmentation and subsequent detachment of the duodenal and jejunal epithelia, but not in the ileum ("the silent ileum"), partly because of the paucity of TCRgammadelta(+) IELs in the ileum.

Nitric oxide targets bronchiolar epithelial cells in murine cytomegalovirus-associated disease in lungs that are free of the virus.

At 4 weeks after intraperitoneal (i.p.) injection of 0.2LD50 (50% lethal dose) of murine cytomegalovirus (MCMV) in adult BALB/c mice, productive virus and the viral DNA were detected only in the salivary glands, but not in the lungs. A single i.p. injection of anti-CD3 mAb to these mice provoked pulmonary lesions, which included a thickening of the interstitium and peribronchiolar areas, accompanied with a cellular infiltration in those areas. As a result, half of the mice died. In a histochemical analysis with anti-nitrotyrosine polyclonal Ab, bronchiolar epithelial cells were stained with this Ab, thus demonstrating that peroxynitrite, which was biochemically derived from nitric oxide (NO), injured those cells. Similarly, when T cells of iNOS+/+ mice, which had been infected with MCMV 4 weeks before, were activated by a single injection of anti-CD3 mAb, 37.5% of the mice died. Nitrotyrosine was also detected in the bronchiolar epithelial cells in these mice. In contrast, none of MCMV-infected iNOS-/- mice died after the anti-CD3 injection. No pathological changes were noted in the histological findings of the lungs of those mice. An intranasal injection of bacterial superantigen, staphylococcal enterotoxin B (SEB), demonstrated the same histopathological changes in the lungs and mortality in BALB/c mice as those in mice i.p. injected with anti-CD3. Therefore, T-cell responsiveness to stimulation with anti-CD3 or a superantigen was presumably modified by MCMV infection. MCMV-associated pneumonitis in the present study was thus mediated not by a direct viral attack but by iNOS-derived NO, which can be induced by the cytokines from the T cells. In addition, it was demonstrated that the NO produced by the cytokine-mediated pathway targeted bronchiolar epithelial cells.

Down-regulation of interleukin-2 and interferon-gamma and maintenance of interleukin-4 and interleukin-10 production after administration of an anti-CD3 monoclonal antibody in mice.

Activating anti-CD3 monoclonal antibodies (mAbs), such as OKT3, are potent immunosuppressive agents that are widely used in clinical transplantation. We investigated whether the in vivo induction of T cell unresponsiveness contributes to the immunosuppressive properties of the anti-mouse-CD3 mAb 145-2C11. After a single in vivo administration of 145-2C11 residual T cells were restimulated in vivo and in vitro to assess cytokine production. Mice were also transplanted with allogeneic skin 9 days after 145-2C11 administration to investigate whether the immunosuppressive properties of the antibody persist after the reexpression of the T cell receptor. Pretreatment with anti-CD3 mAbs caused a profound deficit in both interleukin- (IL) 2 and interferon- (IFN) y secretion upon restimulation in vivo, whereas IL-4 was only partially inhibited and IL-10 production was significantly increased. Purified T cells obtained from mice injected with anti-CD3 mAb also displayed deficient IL-2 and IFN-gamma production together with persisting IL-4 and IL-10 secretion. 145-2C11 had immunosuppressive properties that per sisted after the reexpression of the T cell receptor because mice transplanted with allogeneic skin 9 days after a single anti-CD3 mAb injection still had significantly prolonged graft survival (14.1+/-0.6 days vs. 10.7+/-0.4 days in controls, P<0.02). Blocking IL-4 and IL-10 by neutralizing mAbs further prolonged skin graft survival in mice injected with 145-2C11 (18.3+/-0.7 vs. 14.8+/-0.6 days, P<0.02). The in vivo administration of the 145-2C11 anti-CD3 mAb results in the selective inhibition of Thl-type cytokine secretion upon restimulation, which correlates with a state of immunosuppression. The persistent production of Th2-type cytokines does not contribute to the anti-CD3 mAb-mediated prolonged survival of skin allografts in our experimental model.

β-Selection of immature thymocytes is less dependent on CD45 tyrosinephosphatase

Tyrosine kinase p56 lck plays a pivotal role in β-selection from CD4 −8 − (DN) to CD4 +8 + (DP) developing pathway, but it is unclear how CD45 transmembrane tyrosinephosphatase is involved in this process although CD45 activates p56 lck by dephosphorylating its tyrosine-505. To analyze this issue, we produced double mutant mice of T-cell receptor transgenic mice (TCR-Tg) or RAG-2 knock out mice backcrossed with either p56 lck or CD45 knock out mice. In TCR-Tg, CD25 +DN thymocytes almost disappeared and CD25 −44 −DN cells of further developing stage increased, implying that all DN thymocytes can undergo β-selection due to the expression of functionally rearranged TCR- β on CD25 +DN thymocytes. However, CD25 + thymocytes increased in DN stage when TCR-Tg were backcrossed with p56 lck deficient mice but not with CD45 deficient mice. Similarly, DP thymocyte induction with CD25 + cell reduction in RAG-2 knock out mice by injection of anti-CD3 mAb was inhibited in p56 lck deficient but not in CD45 deficient mice. This suggests that CD45 is dispensable for β-selection though p56 lck is required.

IL-4-producing NK T cells are biased towards IFN-γ production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the microenvironment, NK T lymphocytes can preferentially produce either IL-4 or IFN-gamma. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4-CD8-TCRalphabeta+ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-gamma in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-gamma is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-gamma production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-gamma secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant upregulation of the capacity of NK T cells to produce IFN-gamma after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.

MHC class I-selected CD4-CD8-TCR-alpha beta+ T cells are a potential source of IL-4 during primary immune response.

Differentiation of naive CD4+ lymphocytes into either Th1 or Th2 cells is influenced by the cytokine present during initial Ag priming. IL-4 is the critical element in the induction of Th2 response; however, its origin during a primary immune response is not well defined. In the present study, we characterized a novel potential source of IL-4, the class I-selected CD4-CD8-TCR-alpha beta+ T cells. In a first set of experiments, we demonstrated that CD4-CD8-TCR-alpha beta+ thymocytes produce a large amount of IL-4 after in vitro anti-CD3 stimulation. This phenomenon was not observed in class I-deficient mice, demonstrating that among these cells, the class I-selected subset was predominantly responsible for IL-4 production. Further studies focused on the in vivo IL-4-producing capacity of peripheral CD4-CD8-TCR-alpha beta+ T cells. To this end, a single injection of anti-CD3 mAb, which promptly induces IL-4 mRNA expression, was used. Peripheral CD4-CD8-TCR-alpha beta+ T cells express high levels of IL-4 mRNA in response to in vivo anti-CD3 challenge. Furthermore, analysis performed in mice lacking MHC class I or class II molecules demonstrates that both the class I-selected subset of CD4-CD8-TCR+ and CD4+ peripheral T lymphocytes are the major IL-4 producers after in vivo anti-CD3 stimulation. These findings suggest that class I-selected CD4-CD8-TCR-alpha beta+ and CD4+ T cell populations are important sources of IL-4 probably implicated in the development of specific Th2 immune responses.