Activation of intra-epithelial lymphocytes; their morphology,marker expression and ultimate fate

Intraepithelial lymphocytes (IELs) have been considered to play a key role in the defense system of the small intestine. Its mechanism has not been made sufficiently clear. Studies on IELs have been extremely limited to functions of αβ T-cell receptor (αβTCR) IELs (αβ-IELs). Since, in the mouse duodenum and jejunum, γδ-IELs consist 75 % of IELs, it thus would be inappropriate to argue the mechanism without extensive discussions over the functions of γδ-IELs. In previous studies, we found that the anti-CD3 monoclonal antibody (mAb) injection induced DNA fragmentation in intestinal epithelial cells (IECs) and DNA repair immediately after, that these responses were reproduced by anti-γδTCR mAb not by anti-αβTCR mAb and that the DNA fragmentation was induced by Granzyme B secreted by IELs, totally independent of Perforin. To further explore the functions of IELs in situ, we undertook experiments exclusively focused on IELs, on their changes and ultimate fate after the stimulation in mouse in vivo system. The current study demonstrated that the injected anti-CD3 mAb bound to CD3 on IELs, that the mAb activated γδ-IELs, leading to their degranulation, that changes occurred irreversibly in IELs and finally that activated IELs died in situ. γδ-IELs could be considered to respond to various stimulations most likely without the need of accessory cells (“always ready for rapid response”), to die in situ (“disposable”) and thus to respond to the stimulation only once (“a one-shot responder”). These characteristics of γδ-IELs are important to further elucidate the functions of γδ-IELs in the intestinal defense system.     

Maximum immunobioactivity of murine small intestinal intraepithelial lymphocytes resides in a subpopulation of CD43+ T cells

CD43 has been linked to many function-associated T cell activities. Using mAbs that recognize two different CD43 determinants, we show that, although mouse small intestinal intraepithelial lymphocytes (IELs) expressed the CD43 core molecule reactive with mAb R2/60, only about one-half of the total IELs-including some but not all of the TCRalphabeta and TCRgammadelta cells-expressed the CD43 S7(-) reactive determinant. CD43 S7(+) IELs secreted more IL-2, IL-4, IL-10, IL-17, and IFN-gamma following anti-CD3 stimulation, and were >4-fold more cytotoxic in fresh isolates and >16-fold more cytotoxic after anti-CD3 stimulation, than S7(-) IELs. S7(+) but not S7(-) IELs from the ileum of IL-10(-/-) mice spontaneously produced IFN-gamma. In vivo BrdU uptake by IELs in non-Ag-primed mice was greatest in the S7(+) population, indicating that significantly more S7(+) IELs than S7(-) IELs undergo cell expansion under normal homeostatic conditions. DNA microarray analyses showed that S7(+) IELs expressed higher levels of genes associated with activated T cells, whereas S7(-) IELs expressed genes used in the regulation of NK cells. These findings define two functionally distinct populations of IELs based on CD43 expression independent of TCR class, and they identify a subset of IELs that may serve as a target to better control intestinal inflammation.     

Intestinal IgA responses to Giardia muris in mice depleted of helper T lymphocytes and in immunocompetent mice

Immunocompetent mice infected with Giardia muris generate an intestinal antibody response to this parasite and clear G. muris infection. Previous work has shown that G. muris infection is prolonged in mice that have been depleted of helper (CD4+) T lymphocytes by treatment with a monoclonal antibody (mAb) directed against the murine CD4 antigen. The aim of the present study was to compare the intestinal anti-Giardia antibody response in immunocompetent mice and in mice depleted of helper T (Th) lymphocytes by treatment with anti-CD4 mAb. Immunocompetent mice generated an IgA response to G. muris, as judged by the presence of IgA on Giardia trophozoites harvested from the intestine of these animals more than 10 days after the start of the infection. The anti-Giardia IgA response was impaired in mice depleted of Th lymphocytes, as judged by virtual absence of immunofluorescent staining of trophozoites from these animals for surface-bound IgA. Clearance of G. muris infection was impaired by treatment of mice with anti-CD4 mAb. The results suggest that Th (CD4+) lymphocytes are important for the generation of a local IgA response against G. muris trophozoites in the mouse intestine and that IgA anti-trophozoite antibody may contribute to the clearance of G. muris from the intestine of immunocompetent mice.     

Evidence of synergy between Thy-1 and CD3/TCR complex in signal delivery to murine thymocytes for cell death.

The potential role of Thy-1 in CD3/TCR complex-mediated signal delivery to murine thymocytes was studied. Ag-mimicking cross-linked anti-CD3 mAb stimulated suspension of thymocytes from adult (6 to 8 wk old) mice for a brisk free cytoplasmic calcium ion ([Ca2+]i) rise, low level of inositol phosphate production, and marginal increase in tyrosine-specific phosphorylation of 110/120-kDa and 40-kDa cellular proteins. Weak but sustained [Ca2+]i rise, low inositol phosphate production, and weak protein tyrosine phosphorylation were also induced by the cross-linked anti-Thy-1 mAb that mimicked the putative natural ligand. The signal delivered via either of these two pathways was however insufficient for definitively promoting cell death and DNA fragmentation in the adult thymocytes. Here we demonstrated that anti-Thy-1 mAb synergized with anti-CD3 mAb for inducing a long-lasting prominent [Ca2+]i rise, definite inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakiphosphate production, and extensive tyrosine-specific phosphorylation of 110/120-, 92-, 75-, and 40-kDa proteins, which resulted in marked promotion of cell death and DNA fragmentation in the adult thymocytes. This unique anti-Thy-1 antibody activity was confirmed to be directed to glycosylphosphatidylinositol-anchored Thy-1, and was distinguished from the known anti-L3T4 activity that augmented the CD3-mediated signal transduction in a different manner. The synergistic actions of anti-CD3 and anti-Thy-1 mAb obligatorily required the cross-linking of the two mAb together. The anti-CD3 and anti-Thy-1 mAb cross-linked together acted on immature thymocytes from newborn (less than 24 h after birth) mice for rather more extensive promotion of protein tyrosine phosphorylation and cell death. In addition, they affected peripheral T lymphocytes for accelerating protein tyrosine phosphorylation but not cell death. These results suggest a novel function of glycosylphosphatidylinositol-anchored Thy-1 as a possible unique intrathymic intensifier of the CD3/TCR complex-delivered signal for negative thymocyte selection.     

Upregulation of ICOS on CD43+ CD4+ murine small intestinal intraepithelial lymphocytes during acute reovirus infection

Murine intestinal intraepithelial lymphocytes (IELs) can be classified according to expression of a CD43 glycoform recognized by the S7 monoclonal antibody. In this study, we examined the response of S7+ and S7- IELs in mice during acute reovirus serotype 3 (Dearing strain) infection, which was confirmed by virus-specific real-time PCR. In vivo proliferation increased significantly for both S7- and S7+ IELs on day 4 post-infection as determined by BrdU incorporation; however, expression of the inducible costimulatory (ICOS) molecule, which peaked on day 7 post-infection, was upregulated on S7+ CD4+ T cells, most of which were CD4+8- IELs. In vitro ICOS stimulation by syngeneic peritoneal macrophages induced IFN-gamma secretion from IELs from day 7 infected mice, and was suppressed by treatment with anti-ICOS mAb. Additionally, IFN-gamma mRNA increased in CD4+ IELs on day 6 post-infection. These findings indicate that S7- and S7+ IELs are differentially mobilized during the immune response to reovirus infection; that the regulated expression of ICOS is associated with S7+ IELs; and that stimulation of IELs through ICOS enhances IFN-gamma synthesis during infection.     

Rotavirus-specific cytotoxic T lymphocytes appear at the intestinal mucosal surface after rotavirus infection.

The gastrointestinal tract is constantly exposed to a variety of potentially invasive bacteria and viruses. The first line of defense of the host against these pathogens is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IELs), mucus, and secretory immunoglobulins. Little is known about the function, memory, or trafficking of IELs after intestinal infection. We found that IELs obtained 6 days after oral inoculation of mice with the intestinal pathogen rotavirus (simian strain RRV) lysed rotavirus-infected target cells; cytotoxic T lymphocytes (CTLs) were responsible for rotavirus-specific cytotoxic activity. Rotavirus-specific cytotoxic activity by IELs was (i) eliminated by treatment with Thy 1.2-specific immunoglobulin M plus complement, (ii) restricted by proteins encoded at the major histocompatibility complex, and (iii) absent in mock-infected animals. Oral inoculation of mice with RRV also induced rotavirus-specific CTLs in splenic and intestinal lymphocytes (mesenteric lymph nodes, Peyer's patch). Parenteral inoculation induced rotavirus-specific CTLs in splenic, intestinal (IELs, mesenteric lymph nodes, Peyer's patch), and nonintestinal lymphocytes (inguinal nodes). Therefore, presentation of rotavirus to the intestinal mucosal surface was not necessary to induce IELs with virus-specific cytotoxic activity. At 4 weeks after oral or parenteral inoculation of mice with RRV, rotavirus-specific CTL precursors appeared among splenic, Peyer's patch, inguinal, and mesenteric node lymphocytes, but not among IELs. IELs with rotavirus-specific cytotoxic activity may be generated from precursors at a site other than the intestinal mucosal surface. Part of the response of the host to enteric infection may include surveillance and lysis of virus-infected villus epithelial cells by IELs.     

DNA repair after DNA fragmentation in mouse small intestinal epithelial cells

In our earlier work, we found that, in mice, i.p. injection of anti-CD3 monoclonal antibody activated intraepithelial lymphocytes (iIEL), leading to DNA fragmentation in villous epithelial cells of the duodenum and jejunum within 30 min. By 2 h after injection, nearly half of the enterocytes had detached from the villi, and DNA fragmentation could barely be detected in the remaining villous epithelium. We hypothesized that DNA had been repaired in enterocytes in which DNA fragmentation had previously been induced. In this study, enterocytes became negative for TUNEL staining at 60 min after anti-CD3 treatment, prior to detachment. The remaining villous epithelial cells, after DNA fragmentation and detachment, were found to be positive for 5-bromo-2-deoxyuridine labeling. To confirm whether fragmented DNA had been repaired in situ, we investigated the appearance and/or mobilization of DNA-repair-related proteins. Focus formation, a typical staining pattern of repair-related proteins including phosphorylated H2AX, phospo-ATM substrate, and Nbs1, was observed 30 min after anti-CD3 injection, with the kinetics virtually identical to that of DNA fragmentation. The co-localization of γ-H2AX and phospo-ATM substrate was also confirmed. The disappearance of a positive reaction for TUNEL staining in previously fragmented DNA, the appearance of representative DNA-repair-related proteins, the coincidence of the kinetics of DNA fragmentation and this appearance of DNA-repair-related proteins, and the co-localization of two of the repair-related proteins strongly indicated that enterocyte DNA could be repaired after it had been fragmented in vivo. Thus, DNA fragmentation per se may not necessarily be an immediate sign of cell death. This work was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (16590132 to T.M., 16390045 to T.I., and 20590181 to M.O.).     

Apoptotic death concurrent with CD3 stimulation in primary human CD8+ T lymphocytes: a role for endogenous granzyme B

We exposed primary CD8(+) T cells to soluble CD3 mAb plus IL-2 and limited numbers of monocytes (3%). These cells were activated but concurrently subjected to ongoing apoptosis ( approximately 25% were apoptotic from day 2 of culture). However, their costimulated CD4(+) counterparts were much less prone to apoptosis. The apoptotic signaling pathway bypassed Fas and TNFRs, and required the activity of cathepsin C, a protease which performs the proteolytic maturation of granzyme (Gr) A and GrB proenzymes within the cytolytic granules. Silencing the GrB gene by RNA interference in activated CD8(+) T cells prevented the activation of procaspase-3 and Bid, and indicated that GrB was the upstream death mediator. A GrB-specific mAb immunoprecipitated a approximately 70-kDa molecular complex from cytolytic extracts of activated CD8(+) (but not resting) T cells, that was specifically recognized by a nucleocytoplasmic protease inhibitor 9 (PI-9) specific mAb. This complex was also detected after reciprocal immunoprecipitation of PI-9. It coexisted in the cytosol with the 32-kDa form of GrB. As neither were detected in the cytosol of CD4(+) bystander T cells (which poorly synthesized GrB), and as silencing the perforin (Pf) gene had no effect in our system, endogenous GrB was likely implicated. Immunoprecipitation experiments failed to reveal Pf in the cytosol of CD8(+) T cells, and only a tiny efflux of granular GrA was detected by ELISA. We propose that some GrB is released from cytolytic granules to the cytosol of CD8(+) T lymphocytes upon CD3/TCR stimulation and escapes PI-9, thereby mediating apoptotic cell death.     

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-CD3, anti-TCR, or anti-TCR 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-TCR mAb, but not by anti-TCR 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- 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, TCR⁺ IEL, when treated with anti-CD3 or anti-TCR 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 TCR⁺ IELs in the ileum.K. Yaguchi and S. Kayaba contributed equally to this workThis work was in part supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (07407066, 10470002, and 13670002 to T.I., and 10770001 to H.S.), and by The Funds for Comprehensive Research on Long Term Chronic Diseases from the Ministry of Health and Welfare of Japan (to T.I.)     

Specific T-cell factor production and lymphocytes in the direct surroundings of a subcutaneous allogeneic tumor.

Antigen-specific T-cell factors (TCF) play a role in the initiation of cellular immune responses. In allogeneic mouse-tumor models lymphocytes from the direct tumor surroundings of both euthymic and nude mice produce TCF. These lymphocytes produce TCF when collected already 1 day after subcutaneous (sc) injection of tumor cells. In contrast to euthymic mice, draining lymph nodes and spleen of nude mice did not contain TCF-producing lymphocytes at any stage after sc tumor cell injection. In sensitized euthymic mice TCF production by lymphocytes is significantly higher in the direct tumor surroundings than in draining lymph nodes or spleen. At 2 and 5 days after tumor cell injection, the mononuclear cell infiltrate of the tissue surrounding the tumor in euthymic mice showed low expression of Thy 1, CD3, TCR alpha beta, TCR gamma delta, CD4, CD8, and asialo GM1, whereas several lymphocytes and mast cells were positive for monoclonal antibody (mAb) 14-30 (directed against TCF). In both euthymic and nude mice, sc injected tumor cells showed apoptosis. In conclusion, the direct tumor surroundings are the first (and, for nude mice, the only) site of TCF production, sc injection of tumor cells attracts mAb 14-30-positive lymphocytes and renders mast cells positive for mAb 14-30.     

IL-10 modulates intestinal damage and epithelial cell apoptosis in T cell-mediated enteropathy

In vivo T cell activation by anti-CD3 monoclonal antibody (mAb) results in intestinal damage characterized by loss of villi and epithelial cell apoptosis. The role of the increased interleukin (IL)-10 released during this process is not clear. We assessed the effects of IL-10 on T cell-induced mucosal damage in vivo using IL-10-deficient C57BL/6 [IL-10 knockout (KO)] mice. IL-10 KO and wild-type C57BL/6 mice were injected with anti-CD3 mAb and observed for diarrhea. Changes in serum cytokine levels were measured by ELISA. Histological changes and epithelial cell apoptosis were analyzed on hematoxylin- and eosin-stained tissue sections. Fas expression on intestinal epithelial cells was assessed by flow cytometry analysis of freshly isolated intestinal epithelial cells. Anti-CD3-treated IL-10 KO mice developed more severe diarrhea, a greater loss of intestinal villi, and an increase in the numbers of apoptotic cells in the crypt epithelium. This difference in IL-10 KO mice was associated with an increase in serum tumor necrosis factor-alpha and interferon-gamma levels and with an increase in Fas expression on fresh, isolated, small intestinal epithelial cells. In addition, the enhanced intestinal tissue damage induced by anti-CD3 in IL-10 KO mice was significantly diminished by treatment with recombinant murine IL-10. Therefore, the lack of IL-10 allowed for an increased T cell-induced intestinal tissue damage, and this was associated with an increase in T cell cytokine release and an increase in epithelial cell Fas expression.     

Memory and distribution of virus-specific cytotoxic T lymphocytes (CTLs) and CTL precursors after rotavirus infection.

The gastrointestinal tract is constantly exposed to a variety of potentially invasive bacteria, viruses, and parasites. The first line of defense against these pathogens is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IELs), mucus, and secretory immunoglobulins. In addition, the intestine is a rich source of lymphocytes located within Peyer's patches and the lamina propria. Little is known about the function, memory, trafficking, or origin of intestinal T lymphocytes after intestinal infection. We studied the murine cytotoxic T-lymphocyte (CTL) response to the intestinal pathogen rotavirus (simian strain RRV). Adult mice were inoculated orally or via the hind footpad with RRV; virus-specific cytotoxic activities in intestinal and nonintestinal lymphocyte populations were determined by 51Cr release assays. In addition, virus-specific CTL precursor (CTLp) frequencies were determined by limiting-dilution analysis. IELs containing rotavirus-specific cytotoxic activity were detected after oral but not footpad inoculation and expressed alpha/beta but not gamma/delta cell surface protein; virus-specific CTLs did not appear to arise from CTLp among IELs. In addition, the site at which RRV was presented to the immune system determined the site at which RRV-specific CTLp first appeared. Frequencies of rotavirus-specific CTLp detected in Peyer's patches were 25- to 30-fold greater after oral than after footpad inoculation. However, regardless of the route of inoculation, rotavirus-specific CTLp were distributed throughout the lymphoid system 21 days after infection. Implications of these findings for vaccine design are discussed.     

Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28

Antagonist anti-CD28 antibodies prevent T-cell costimulation and are functionally different from CTLA4Ig since they cannot block CTLA-4 and PDL-1 co-inhibitory signals. They demonstrated preclinical efficacy in suppressing effector T cells while enhancing immunoregulatory mechanisms. Because a severe cytokine release syndrome was observed during the Phase 1 study with the superagonist anti-CD28 TGN1412, development of other anti-CD28 antibodies requires careful preclinical evaluation to exclude any potential immunotoxicity side-effects. The failure to identify immunological toxicity of TGN1412 using macaques led us to investigate more relevant preclinical models.

We report here that contrary to macaques, and like in man, all baboon CD4-positive T lymphocytes express CD28 in their effector memory cells compartment, a lymphocyte subtype that is the most prone to releasing cytokines after reactivation. Baboon lymphocytes are able to release pro-inflammatory cytokines in vitro in response to agonist or superagonist anti-CD28 antibodies. Furthermore, we compared the reactivity of human and baboon lymphocytes after transfer into non obese diabetic/severe combined immunodeficiency (NOD/SCID) interleukin-2rγ knockout mice and confirmed that both cell types could release inflammatory cytokines in situ after injection of agonistic anti-CD28 antibodies. In contrast, FR104, a monovalent antagonistic anti-CD28 antibody, did not elicit T cell activation in these assays, even in the presence of anti-drug antibodies. Infusion to baboons also resulted in an absence of cytokine release.

In conclusion, the baboon represents a suitable species for preclinical immunotoxicity evaluation of anti-CD28 antibodies because their effector memory T cells do express CD28 and because cytokine release can be assessed in vitro and trans vivo.     

Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28

Antagonist anti-CD28 antibodies prevent T-cell costimulation and are functionally different from CTLA4Ig since they cannot block CTLA-4 and PDL-1 co-inhibitory signals. They demonstrated preclinical efficacy in suppressing effector T cells while enhancing immunoregulatory mechanisms. Because a severe cytokine release syndrome was observed during the Phase 1 study with the superagonist anti-CD28 TGN1412, development of other anti-CD28 antibodies requires careful preclinical evaluation to exclude any potential immunotoxicity side-effects. The failure to identify immunological toxicity of TGN1412 using macaques led us to investigate more relevant preclinical models. We report here that contrary to macaques, and like in man, all baboon CD4-positive T lymphocytes express CD28 in their effector memory cells compartment, a lymphocyte subtype that is the most prone to releasing cytokines after reactivation. Baboon lymphocytes are able to release pro-inflammatory cytokines in vitro in response to agonist or superagonist anti-CD28 antibodies. Furthermore, we compared the reactivity of human and baboon lymphocytes after transfer into non obese diabetic/severe combined immunodeficiency (NOD/SCID) interleukin-2rγ knockout mice and confirmed that both cell types could release inflammatory cytokines in situ after injection of agonistic anti-CD28 antibodies. In contrast, FR104, a monovalent antagonistic anti-CD28 antibody, did not elicit T cell activation in these assays, even in the presence of anti-drug antibodies. Infusion to baboons also resulted in an absence of cytokine release. In conclusion, the baboon represents a suitable species for preclinical immunotoxicity evaluation of anti-CD28 antibodies because their effector memory T cells do express CD28 and because cytokine release can be assessed in vitro and trans vivo.     

Multiple levels of activation of murine CD8(+) intraepithelial lymphocytes defined by OX40 (CD134) expression: effects on cell-mediated cytotoxicity, IFN-gamma, and IL-10 regulation.

The involvement of OX40 (CD134) in the activation of CD8(+) intestinal intraepithelial lymphocytes (IELs) has been studied using freshly isolated IELs and in vitro CD3-stimulated IELs. Although freshly isolated CD8(+) IELs exhibited properties of activated T cells (CD69 expression and ex vivo cytotoxicity), virtually all CD8(+) IELs from normal mice were devoid of other activation-associated properties, including a lack of expression of OX40 and the ligand for OX40 (OX40L) and an absence of intracellular IFN-gamma staining. However, OX40 and OX40L expression were rapidly up-regulated on CD8 IELs following CD3 stimulation, indicating that both markers on IELs reflect activation-dependent events. Unlike IELs, activated lymph node T cells did not express OX40L, thus indicating that OX40-OX40L communication in the intestinal epithelium is part of a novel CD8 network. Functionally, OX40 expression was exclusively associated with IELs with active intracellular IFN-gamma synthesis and markedly enhanced cell-mediated cytotoxicity. However, OX40 costimulation during CD3-mediated activation significantly suppressed IL-10 synthesis by IELs, whereas blockade of OX40-OX40L by anti-OX40L mAb markedly increased IL-10 production. These findings indicate that: 1) resident CD69(+)OX40(-) IELs constitute a population of partially activated T cells poised for rapid delivery of effector activity, 2) OX40 and OX40L expression defines IELs that have undergone recent immune activation, 3) OX40(+) IELs are significantly more efficient CTL than are OX40(-) IELs, and 4) the local OX40/OX40L system plays a critical role in regulating the magnitude of cytokine responses in the gut epithelium.     

Lack of DNA degradation in target cells lysed by granules derived from cytolytic T lymphocytes

It has been shown previously that fragmentation of target cell DNA is an early event in lysis mediated by cytolytic T lymphocytes (CTL). In this study, we have investigated whether CTL-derived granules that exhibit lytic activity also induce DNA fragmentation in murine target cells. Cytolytic granules isolated from three different alloreactive CTL clones were tested for the induction of DNA fragmentation in P815 and EL4 target cells, by using a Triton X-100-facilitated, radiolabeled DNA release assay. In contrast to the CTL clones from which they were derived, the cytolytic granules did not induce DNA fragmentation. Agarose gel electrophoretic analysis of DNA confirmed the lack of discrete DNA fragments in target cells lysed by CTL-derived granules. Possible explanations for the difference in the ability of CTL and CTL-derived granules to trigger DNA fragmentation are discussed.     

Recovery from chronic rotavirus infection in mice with severe combined immunodeficiency: virus clearance mediated by adoptive transfer of immune CD8+ T lymphocytes.

Severe combined immunodeficient (SCID) mice lack both functional T and B cells. These mice develop chronic rotavirus infection following an oral inoculation with the epizootic diarrhea of infant mice (EDIM) rotavirus. Reconstitution of rotavirus-infected SCID mice with T lymphocytes from immunocompetent mice allows an evaluation of a role of T-cell-mediated immunity in clearing chronic rotavirus infection. Complete rotavirus clearance was demonstrated in C.B-17/scid mice 7 to 9 days after the transfer of immune CD8+ splenic T lymphocytes from histocompatible BALB/c mice previously immunized intraperitoneally with the EDIM-w strain of murine rotavirus. The virus clearance mediated by T-cell transfer was restricted to H-2d-bearing T cells and occurred in the absence of rotavirus-specific antibody as determined by enzyme-linked immunosorbent assay, neutralization, immunohistochemistry, and radioimmunoprecipitation. Temporary clearance of rotavirus was observed after the transfer of immune CD8+ T cells isolated from the intestinal mucosa (intraepithelial lymphocytes [IELs]) or the spleens of BALB/c mice previously infected with EDIM by the oral route. Chronic virus shedding was transiently eliminated 7 to 11 days after spleen cell transfer and 11 to 12 days after IEL transfer. However, recurrence of rotavirus infection was detected 1 to 8 days later in all but one SCID recipient receiving cells from orally immunized donors. The viral clearance was mediated by IELs that were both Thy1+ and CD8+. These data demonstrated that the clearance of chronic rotavirus infection in SCID mice can be mediated by immune CD8+ T lymphocytes and that this clearance can occur in the absence of virus-specific antibodies.     

Natural killer (NK) cell-mediated cytolysis of Plasmodium falciparum-infected human red blood cells in vitro

The ability of human NK cells to inhibit the growth in vitro of the asexual blood stages of Plasmodium falciparum was tested. Purified NK cells from donors with no prior exposure to malaria significantly inhibited parasite growth after 48 hours of co-culture in the presence of human immune serum. This inhibition was completely abrogated by pre-treatment of the NK cells with an anti-CD95 (anti-Fas) monoclonal antibody and human Fas-Fc soluble protein. The level of growth inhibition was also substantially reduced by pre-treatment with an anti-CD56 antibody. These two antibodies caused reductions, to varying levels, of the amounts of NK cell-derived granzyme B (GrB) and pro-inflammatory cytokines, but only the anti-CD95 antibody affected the production of soluble Fas ligand (sFasL). Direct destruction of parasite-infected red cells by NK cells, in the absence of serum, was also observed in a standard 51Cr cytotoxicity test, during which N-carboxybenzoxy-L-lysine thiobenzil ester (BLT esterase) activity, which catalyzes serine protease granule release, was detected. The results obtained are indicative of a novel mechanism of NK cell-mediated cytotoxic activity against Plasmodium falciparum-infected red cells, which is mediated in part by both Fas and by GrB.     

Temperature requirements and kinetics of T cell signaling. Velocity of triggering correlates with functional effects of anti-CD3 mAb

Antibody reactive with the CD3 complex on the surface of T lymphocytes can either: inhibit CTL lysis of target cells expressing Ag; or redirect CTL to lyse target cells expressing FcR in the absence of Ag expression. To investigate these phenomena we examined the effect of anti-CD3 mAb on two indicators of CTL activation, the release of esterase and target cell lysis. Esterase release by long term allo-reactive human CTL in response to target cells (JY or HLA transfected K562 cells) was found to be Ag specific and correlate with target cell lysis. Addition of anti-CD3 to either JY targets or K562 cells expressing FcR resulted in a high level of esterase release. Triggering of esterase release was found with both soluble intact and Fab fragment of anti-CD3 in the absence of cells expressing measurable FcR. This apparent FcR-independent triggering of esterase release occurred at 37 degrees C but not at 24 degrees C. In contrast esterase activity was released from CTL at both 24 and 37 degrees C in response to intact target cells, JY or K562 cells plus intact anti-CD3 mAb. Addition of anti-CD3 mAb, at a level capable of blocking target cell lysis by greater than 50%, resulted in an initial velocity of esterase release almost twice that found in response to JY target cells. With a low level of anti-CD3 mAb, able to block JY lysis by approximately 10%, the initial rate of esterase release was much slower than that found in response to target cells. In contrast when FcR+ cells, K562, were added along with a low level of anti-CD3 the initial velocity of esterase release was about twofold more than the velocity of esterase release triggered by soluble anti-CD3 alone. These results indicate that soluble antibody can trigger long term active CTL and the velocity of this triggering correlates with anti-CD3-mediated inhibition as well as redirected lysis.