Sequential appearance of T-cell receptor gamma delta- and alpha beta-bearing intestinal intra-epithelial lymphocytes in mice after irradiation.

We have previously reported that T-cell receptor (TcR) gamma delta-bearing T cells precede TcR alpha beta-bearing T cells in appearance in the thymus after whole-body irradiation. In the present study, the kinetics of appearance of intestinal intra-epithelial lymphocytes (IEL) was examined in mice after whole-body irradiation with a lethal dose of 9.5 Gy or with a sublethal dose of 6 Gy. The number of CD3+ IEL decreased to the lowest value 4 days after irradiation with 9.5 Gy, and thereafter increased to half as many as the normal level by day 7. Thy-1+TcR alpha beta- IEL and Thy-TcR alpha beta- IEL recovered considerably by day 7 after the irradiation, whereas Thy-1+TcR alpha beta+ IEL and Thy-1+TcR alpha beta+ IEL hardly recovered at this stage. All mice died within 12 days after irradiation with a lethal dose of 9.5 Gy. On the other hand, when irradiation dose was decreased to 6 Gy, all mice survived beyond 40 days after irradiation. The number of CD3+ IEL recovered to the normal level by 10 days after irradiation with 6 Gy. Consistently with the results in mice irradiated with a lethal dose, the first cells to increase in IEL of mice irradiated with a sublethal dose were TcR gamma delta+ IEL expressing Thy-1 antigen. The number of Thy-1+TcR gamma delta+ IEL increased to approximately two-fold as many as that in normal mice by day 10, while TcR alpha beta+ IEL began to increase in number from day 20 after irradiation and recovered to the normal level by day 40 after irradiation. Thus, sequential appearance of TcR gamma delta+ and TcR alpha beta+ IEL was evident after irradiation, similar to that seen in the thymus after irradiation. The IEL on day 10 after a sublethal irradiation, which is composed mainly of Thy-1+TcR gamma delta+ IEL, exhibited a strong cytolytic activity against P815 in the presence of anti-CD3 mAb, suggesting that the early appearing Thy-1+TcR gamma delta+ IEL may play important roles in epithelial immunity at an early stage after irradiation.     

Augmentation of natural killer cytotoxicity by alpha or gamma natural and recombinant interferons and interferon inducers. Effect of monocytes

We investigated the effect of human peripheral blood monocytes on the augmentation of natural killer cytotoxicity by alpha or gamma natural and recombinant interferons (IFN) and certain interferon inducers. We observed that: (1) in the majority of the donors examined (75%) human peripheral blood monocytes do not affect natural killer cytotoxicity, determined by a 4-hour chromium-51 release assay, against target cells from hemopoietic human tumor cell lines. (2) Monocytes are not required and do not affect the augmentation of natural killer cytotoxicity by Escherichia coli-derived IFN-gamma, natural human IFN-gamma, E. Coli-derived IFN-alpha 2 or natural human IFN-alpha. E. Coli-derived IFN-gamma and natural human IFN-gamma have been reported to activate monocyte cytotoxicity determined in 72-hour assay. (3) Monocytes are not required for the augmentation of natural killer cytotoxicity against target cells from hemopoietic tumor cell lines by polyinosinic acid-polycytidylic acid or staphylococcal enterotoxin A.     

An increase in number of T-cell receptor gamma/delta-bearing T cells in athymic nude mice treated with complete Freund's adjuvants.

It has been reported previously that environmental antigens such as intestinal microflora play an important role in an age-associated increase in number of T-cell receptor gamma/delta-bearing T cells. To extend the scope of this finding, this study examined the influences of local inflammation on the accumulation and/or proliferation of gamma/delta T cells in athymic nude mice injected subcutaneously with complete Freund's adjuvants (CFA). The inguinal lymph nodes (ILN) in nude mice injected with CFA in their hind footpads 7 days previously contained an increased number of CD3+CD4-CD8- cells. Increased levels of proliferative responses against syngeneic stimulator cells were noted in the LN cells of CFA-injected nude mice in association with increases in expression of gamma- and delta-chain gene messages. Furthermore, the LN cells showed an early proliferative response to purified protein derivative (PPD) from Mycobacterium bovis. These results suggest that local inflammation by CFA may induce functional gamma/delta T cells in nude mice, which may proliferate rapidly at the inflamed sites and represent a first line of defence in such animals against the invasion of various pathogens.     

A monoclonal antibody to gamma interferon blocks augmentation of natural killer cell activity induced during systemic cryptococcosis.

These studies demonstrate that the cytotoxic activity of splenic natural killer (NK) cells is augmented in both nu/nu and nu/+ mice during systemic cryptococcosis. Both the kinetics and the regulation of NK cell activity differed in Cryptococcus neoformans-infected nu/nu and nu/+ mice. Greater augmentation was observed following challenge with 10(5) cells than with smaller inocula, and augmented NK cell activity was not always associated with enhanced control of systemic cryptococcosis. Infection with a nonencapsulated strain of C. neoformans induced an early but transient increase in splenic NK cell activity in nu/nu and nu/+ mice. Injection of capsular polysaccharide induced a transient augmentation of splenic NK cell activity in nu/+ mice but caused a persistent increase in splenic NK cell activity in nu/nu mice. In vivo treatment with monoclonal antibody to gamma interferon abrogated the augmentation of splenic NK cell activity induced during cryptococcal infections in both nu/nu and nu/+ mice and enhanced the susceptibility of nu/+ mice to C. neoformans to a greater extent than it did that of nu/nu mice. These results suggest that gamma interferon is an important mediator of resistance to C. neoformans.     

Increase of T-cell receptor gamma/delta-bearing T cells in cord blood of newborn babies obtained by in vitro stimulation with mycobacterial cord factor.

Cord blood T lymphocytes proliferated in vitro in response to mycobacterial organisms but did not proliferate in the presence of tuberculin purified protein derivative. Components recognized by cord blood T cells were resistant to protease digestion. In contrast, T lymphocytes derived from tuberculin-positive adult peripheral blood proliferated when stimulated by the protease-sensitive component of mycobacterial organisms or purified protein derivative, confirming that adult T cells respond to protein components whereas cord blood T cells respond to the nonpeptide component of mycobacteria. In vitro culture of cord blood lymphocytes stimulated by either mycobacterial lysates or the lipid fraction showed increases in the numbers of T-cell receptor (TcR) gamma/delta T lymphocytes with no changes in the numbers of TcR alpha/beta T lymphocytes in contrast to the in vitro cultures of adult blood lymphocytes stimulated with mycobacterial ligands in which no increase of TcR gamma/delta cells was observed. Interleukin-2 receptor (CD25) and Ia antigen (HLA-DR) analyses evidenced the activation of a large proportion of cord blood gamma/delta T cells which had increased after stimulation with mycobacteria in vitro. Further characterization of mycobacterial ligand suggested that the lipid fraction of mycobacterial lysate or trehalose dimycolate-cord factor was the most plausible cause for T-cell proliferation in cord blood. These results suggest that when the gamma/delta T cells in a newborn infant not yet sensitized to any pathogenic organisms are confronted by a mycobacterium, they respond nonspecifically to the mycobacterial organism or its lipid component (cord factor). gamma/delta T cells may therefore play a distinct role in forming the first line of the host defense system against certain microorganisms.     

Analysis of the role of natural killer cells in Listeria monocytogenes infection: relation between natural killer cells and T-cell receptor gamma delta T cells in the host defence mechanism at the early stage of infection.

We have reported that T cells bearing T-cell receptors (TcR) of gamma delta type (gamma delta T cells) appear in the peritoneal cavity in a relatively early stage of primary intraperitoneal (i.p.) Listeria monocytogenes infection, and play a significant role against the infection. To elucidate the protective role of natural killer cells which also appear in the early stage of L. monocytogenes infection, mice were treated with anti-NK1.1 monoclonal antibody (mAb) to deplete NK cells before the infection. They exhibited accelerated clearance of L. monocytogenes, accompanied by enhanced induction of gamma delta T cells in the peritoneal cavity compared with non-treated mice. When the mice were depleted of gamma delta T cells by in vivo administration of anti-TcR gamma delta mAb, the bacterial burdens of organs from infected mice were not affected by NK cell depletion. These results suggest that, although NK cells increase significantly during the early stage of L. monocytogenes infection, they do not take part in the early host resistance against i.p. L. monocytogenes infection. It is also suggested that increased gamma delta T cells in the peritoneal cavity of NK cell-depleted mice can be one of the factors responsible for the enhanced clearance of L. monocytogenes in the early stage of infection.     

The immunobiology of natural killer cells and bone marrow allograft rejection.

Natural killer (NK) cells mediate the acute rejection of bone marrow cell (BMC) allografts, but not solid tissue grafts, in lethally irradiated mice. However, the mechanisms underlying this capability for rejecting BMC remain unclear. NK cells express (1) inhibitory receptors specific for major histocompatibility complex (MHC) class I molecules and (2) activating receptors with diverse specificities. Inhibitory NK receptors confer to NK cells the ability to discriminate between MHC class I-positive and -negative target cells and are therefore involved in the control of NK cell tolerance to self, as well as in the elimination of cells that have downregulation of MHC class I molecules. Preclinical studies in mice have provided good evidence that subsets of NK cells that bear different combinations of both inhibitory and activating Ly49 receptors can interact with each other and target specific BMC rejection, as well as NK cell responses toward tumor cells. Recent clinical studies have also shown that the use of killer cell immunoglobulin-like receptor ligand incompatibility in patients with leukemia who received hematopoietic stem cell transplants correlated not only with the elimination of graft rejection, but also with eradication of tumor and prevention of graft-versus-host disease; this offers a significant advantage for survival. In this review, we attempt to bring together literature regarding the biology of NK cells and discuss the current issues in bone marrow transplantation and the potential clinical role of NK cell alloreactivity in the efficacy of this procedure for immunotherapy of cancer and infectious states.     

Recombinant interferon alpha 2 stimulation of target-binding by natural killer cells

Summary Even though the enhancement of the lyitc capacity and the kinetics of lysis of natural killer cells (NK) by interferon has been well documented, an increase of the target-effector cell binding percentage is still disputed. We, therefore, modified the Grimm-Bonavida single-cell assay so that 400 to 600 cells per individual determination could be reliably evaluated. Using this assay, which makes possible separate determination of effector-target cell binding and target lysis, we demonstrated that, in addition to lytic capacity, target-effector cell binding is also increased by preincubating NK with 100 to 1,000 IU interferon alpha 2 per 10⁶ cells. Our data indicate that interferon alpha 2 induces pre-NK cells to bind target cells and that it activates these pre-NK cells to kill the targets.Abbreviations NK Natural killer cells - LCMV Lymphocytic choriomeningitis virus - IFN Interferon - FCS Fetal calf serum - RPMI 1640 Culture medium Dedicated to Prof. H.D. Waller on the occasion of his 60^th birthday     

Impairment of natural killer cell activity in Indian kala-azar: restoration of activity by interleukin 2 but not by alpha or gamma interferon.

Indian kala-azar patients have normal numbers of peripheral blood NK cells but impaired functional activity due to decreased binding and lysis of target cells. This impairment of NK activity could not be corrected by exogenous recombinant human alpha or gamma interferon. However, recombinant human interleukin 2 was able to restore this activity by augmenting conjugate formation and lysis of target cells.     

Characterization of Fc epsilon RI gamma in human natural killer cells.

We report the characterization of the Fc epsilon RI gamma chain which associates with the transmembrane form of CD16 to form the low affinity receptor for IgG (Fc gamma RIII) expressed on human natural killer (NK) cells. cDNA cloning and sequence analysis of Fc epsilon RI gamma from a polyclonal CD3-CD16+ NK line established that this molecule is identical to Fc epsilon RI gamma previously identified in human basophils as part of a high affinity receptor for IgE. Polymerase chain reaction analysis of Fc epsilon RI gamma gene expression in a series of CD3+CD16- and CD3-CD16+ NK clones reveals that Fc epsilon RI gamma is not directly linked to NK activity since clones of the CD3+CD16- phenotype lack Fc epsilon RI gamma RNA but nevertheless mediate cytotoxicity. Taken together, these results demonstrate that the Fc epsilon RI gamma molecule is expressed in various types within the hematopoietic system as part of multimeric surface receptors involved in different biological functions.     

Stimulation and expansion of a human T-cell subpopulation by a monoclonal antibody to T-cell receptor molecule

A murine monoclonal antibody (MAb) was obtained that showed unique specificity for the immunizing T-cell line HPB-ALL. This antibody, C37 (an IgG1,K) also reacted with a small (2-5%) population of normal peripheral blood T (PBL-T) cells. These C37-positive (C37+) cells were found in both the T4/Leu3+ and T8/Leu2+ subsets. Like OKT3 antibody, C37 induced T-cell mitogenesis with a peak proliferative response at day 3. In long-term cultures containing irradiated autologous feeder cells and IL-2, C37 antibody caused the selective expansion of C37+ T cells. On HPB-ALL cells C37 induced comodulation of the T3 molecule. C37 precipitated a disulfide-linked dimer characteristic of the T-cell antigen receptor consisting of an alpha-subunit (45-48 kD) and a beta-subunit (38-42 kD) from both C37+ T-cell blasts of a normal individual and HPB-ALL cells that were surface radioiodinated. However, the precipitated molecule isolated from C37 antibody-activated T-cell blasts exhibited a different pI from that isolated from HPB-ALL cells. Our studies indicate that C37 recognizes an epitope on the T-cell receptor molecule that is shared by a subpopulation of human T cells, which raises the possibility that multiple variable-region associated and/or framework-like determinants of the T-cell antigen receptor can be defined serologically and used in functional and molecular studies of T-cell subsets.     

Analysis of the cytolytic activity mediated by natural killer cells from acquired immunodeficiency syndrome patients in response to phytohemagglutinin or anti-CD16 monoclonal antibody

The aim of this study was to assess the cytolytic potential of natural killer (NK) cells from human immunodeficiency virus type 1 (HIV-1)-infected patients, at different stages of the disease. Twenty HIV-1 seronegative donors as well as sixty HIV-1 seropositive patients were studied. Phytohemagglutinin and/or the anti-CD16 monoclonal antibody Kd1 were used to redirect the peripheral blood lymphocyte lysis of these patients to the ⁵¹Cr-labeled Fcγ receptor-positive P815 murine mastocytoma target cell line. In parallel, NK cytotoxicity to tumor targets was investigated. Seronegative as well as HIV-1 Center for Disease Control (CDC) stage II patients showed maintained cytolytic activity. The cytolytic potential declined with disease progression, starting with CDC IVC2 patients, and was strongly diminished in acquired immunodeficiency syndrome stage patients. This defect was accompanied by decreased cytolytic activity to tumor targets and was not corrected by the in vitro addition of interleukin-2. The number of cells bearing a mature NK phenotype was normal in all the study groups. Our data suggest that the impaired NK cytotoxicity to tumor targets described during the progression of HIV-1 disease may be related to the progressive loss of function of surface receptors involved in NK cell triggering.     

Presence of restricted killer immunoglobulin-like receptor repertoire and monoclonal T-cell receptor gamma rearrangement as evidence of mixed NK/T-cell differentiation in a subset of sinonasal lymphomas

Most sinonasal lymphomas have a restricted killer immunoglobulin-like receptor (KIR) repertoire without a monoclonal T-cell receptor-gamma (TCR-gamma) rearrangement, implying an NK lineage. However, the lineage assignment of sinonasal lymphoma with a monoclonal TCR-gamma rearrangement is unclear because of its mixed NK/T phenotype. The possibility of a mixed NK/T lineage arises with the discovery of T cells with NK features, such as KIR(+) T cells or Valpha24(+) NKT cells. The former might transform into a T-cell lymphoma with both a monoclonal TCR-gamma rearrangement and a restricted KIR repertoire; the latter might give rise to a T-cell lymphoma with a monoclonal Valpha24 rearrangement and possibly a restricted KIR repertoire. To identify such mixed-lineage lymphomas, we undertook a survey of 15 consecutive sinonasal lymphomas and found six with both a restricted KIR repertoire and a monoclonal TCR-gamma rearrangement, consistent with KIR(+) T-cell lymphomas. Among these six cases, four female CD56(-)/CD44(-)/CD8(-)/CD45RO(+)/CD45RA(-) cases constituted a distinct group with a better prognosis than the rest of the male cases of sinonasal lymphomas. None of the six cases had a monoclonal Valpha24 repertoire, thus excluding a derivation from NKT cells. The predominance of KIR(+) T cells that normally function in chronic viral infections over Valpha24(+) NKT cells that typically recognize glycolipid antigens is consistent with the known association of Epstein-Barr virus infection with sinonasal lymphoma. The demonstration of mixed lineage in a mature lymphoid neoplasm is unusual and echoes the World Health Organization classification that placed NK-cell and T-cell lymphomas in a mixed group.     

Inhibition of mouse natural killer activity by cyclosporin A.

Cyclosporin A(CSA), administered in vivo or in vitro, inhibited the spontaneous cytotoxicity of C57BL/6 and NZB/WF1 mouse spleen cells against YAC and K562 target cells in a 4 hr 51Cr release assay. Inhibition of natural killing (NK) by CSA occurred rapidly, was dose-dependent, and did not require the presence of T cells, B cells or macrophages. CSA depressed NK activity by direct interaction with NK cells. There was no evidence that inhibition of NK by CSA was mediated through suppressor cells.     

Phenotype of rat natural killer cells defined by monoclonal antibodies marking rat lymphocyte subsets.

The fluorescence-activated cell sorter and a rosette-depletion technique were used to separate rat spleen lymphocytes and BCG-activated lymphocytes into subpopulations with and without the antigens defined by W3/13, W3/23 and OX8 monoclonal antibodies. The resultant populations were then tested for natural killer (NK) activity in a quantifiable 6 hr 51Cr release assay. The data establish that rat natural killer cells are heterogeneous with respect to their surface antigen expression and that subpopulations of rat NK cells express the OX8 and W3/13 defined antigens. However, rat NK cells do not express the antigen defined by W3/24 monoclonal antibody.     

Differential staining of human alpha beta and gamma delta T cells by the fluorescein conjugate of an anti-CD3 monoclonal antibody.

The enumeration of total T cells, an important function of the clinical immunology laboratory, utilizes antibodies to CD3, the macromolecular complex associated with the antigen-specific receptors of T cells. We compared the ability of some commonly employed commercial anti-CD3 reagents to stain human peripheral blood lymphocytes. Surprisingly, the fluorescein isothiocyanate (FITC) conjugate of Coulter clone T3 (FITC-T3) stained most T cells brightly, but selectively stained gamma delta T cells very dimly or not at all. In contrast, the other anti-CD3 reagents studied (FITC-Leu 4, PE-T3, PE-Leu 4, and indirectly labelled T3 and Leu 4) stained all T cells equivalently. Dual-colour flow cytometric analysis with FITC-T3 and PE-Leu 4 readily demonstrated a FITC-T3-/PE-Leu 4+ population of T cells. This unique population stained dimly or not at all with a combination of anti-CD4 and anti-CD8 monoclonal antibodies and positively with the pan-gamma delta T cell antibody TCR delta 1. Moreover, an excellent correlation was found between the number of FITC-T3-/PE-Leu 4+ cells and the number of TCR delta 1+ cells in 32 normal individuals. Thus, the FITC-T3-/PE-Leu 4+ phenotype accurately marks all gamma delta T cells. In contrast to FITC-T3, both PE-conjugated and unconjugated T3 stained gamma delta T cells brightly. Therefore, T3 binds to an epitope present on all T cells, but fluoresceinylation specifically attenuates this antibody's ability to bind to gamma delta T cells. These findings indicate that the use of FITC-T3 can result in a significant and variable underestimation of peripheral blood T cell number and demonstrate further that the CD3 complexes of human alpha beta and gamma delta T cells are significantly different.     

Growth of murine natural killer cells from bone marrow in vitro: role of TNF alpha and IFN gamma.

It has been previously shown that natural killer (NK) cell growth can be induced by interleukin-2 (IL-2) in bone marrow (BM) cultures and that other cytokines (CKs), including IL-1 alpha, act synergistically with IL-2. However, as the effect of IL-2 and IL-1 alpha could be due to direct stimulation of NK progenitor cell growth, as well as to the induction of other factors, we analysed the role of the endogenous production of CKs in BM cultures. Results show that mRNAs specific for tumour necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) are detectable within hours in BM cultures supplemented with IL-2 and IL-1 alpha, and that the amount is higher when both IL-2 and IL-1 alpha are present. Antibodies directed against TNF alpha and IFN gamma abrogate the NK cell development, indicating that these CKs play an essential role. The antibodies, however, had no effect on mature NK cells. Furthermore, pretreatment of BM cells with TNF alpha or IFN gamma before culturing with IL-2, enhances IL-2 responsiveness and NK cell growth. These results suggest that induction of cytokines production may be important for growth of NK cells from BM precursors and that the synergistic effect of IL-1 alpha could be due, at least in part, to increased TNF alpha and IFN gamma production.     

Subunit composition of the pre-T-cell receptor complex analysed by monoclonal antibody against the pre-T-cell receptor alpha chain.

The pre-T-cell receptor (TCR) complex, which consists of a heterodimer of the TCR beta-chain and the pre-TCR alpha-chain, is known to regulate early thymocyte development. The pre-TCR complex contains CD3 subunits as a signal-transducing molecule, but the exact subunit composition of the fully assembled pre-TCR complex remains to be elucidated. In particular, the association of the CD3 zeta-chain with the pre-TCR is controversial. In the present study, we have generated a monoclonal antibody against the cytoplasmic portion of the pre-TCR alpha-chain, and analysed a subunit composition of the pre-TCR complex. We demonstrated that the CD3 zeta-chain is physically associated with the pre-TCR in immature T cells. Thus, the result strongly supports the previous findings that CD3 zeta contributes to signalling mediated through the pre-TCR complex.     

Homogenous expression of killer cell immunoglobulin-like receptors (KIR) on polyclonal natural killer cells detected by a monoclonal antibody to KIR2D

The activity of human natural killer (NK) cells is in part regulated by the expression of killer cell immunoglobulin (Ig)-like receptors (KIR) that recognize major histocompatibility complex (MHC) class I and can inhibit NK cell cytotoxicity. A monoclonal anti-KIR antibody was established and designated Lig1. Lig1 was shown to be specific for KIR in cell-surface staining and to react with all KIR2D, except KIR2DL4 which lacks a D1 domain, but not with KIR3D molecules in an enzyme-linked immunoadsorbent assay (ELISA) and Western blotting. Unlike other anti-KIR antibodies, Lig1 did not inhibit binding of KIR-Ig-fusion proteins to MHC-class I expressing cells nor did it interfere with KIR-mediated inhibition of NK cell cytotoxicity in a functional assay. Lig1 reacted with all NK cells in polyclonal NK populations from different donors, demonstrating that all NK cells express at least one KIR2D receptor.