Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.     

Expression of Leu-19 (NKH-1) antigen on IL 2-dependent cytotoxic and non-cytotoxic T cell lines

The Leu-19 (NKH-1) antigen is expressed on human peripheral blood NK cells and a subset of peripheral blood cytotoxic T lymphocytes that kill "NK-sensitive" tumor cell targets without major histocompatibility complex restriction. In the present study, we demonstrate that the Leu-19 (NKH-1) antigen is also expressed on most interleukin 2 (IL 2) dependent T cell lines and clones that have been maintained in long term culture. The Leu-19 (NKH-1) antigen expressed on an antigen-specific, class I directed cytotoxic T lymphocyte cell line was an approximately 200,000 to 220,000 dalton protein, similar to Leu-19 (NKH-1) protein expressed on natural killer cells and KG1a, an immature stem cell leukemia cell line. Furthermore, Leu-19 (NKH-1) was expressed on both CD4+ and CD8+ IL 2 dependent T cell clones, and was present on both cytotoxic and non-cytotoxic T cell clones. Thus expression of Leu-19 (NKH-1) antigen on cultured cell lines does not directly correlate with cytotoxic function, antigenic specificity, or cell lineage.     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

Generation of lymphokine-activated killer cell activity from human thymocytes

We have generated lymphokine-activated killer (LAK) cells from human thymocytes in order to assess the relationship between LAK cells and T cells. Fresh thymocytes lack natural cytotoxic activity, and cytotoxicity cannot be stimulated by short term (1 hr) incubation with interferon or recombinant interleukin 2 (rIL-2). In addition, thymocytes are phenotypically devoid of cells bearing the natural killer (NK)-associated markers cluster designation (CD) 16 and NKH-1. After culture for 5 to 8 days with rIL-2, thymocytes display high levels of cytotoxic activity against both NK-sensitive and NK-resistant targets. Thymocytes require slightly more IL-2 than do peripheral blood lymphocytes to generate LAK activity. We have examined the phenotype of the thymocyte LAK precursor and effector cells. Thymocyte LAK precursors are of low to medium density, CD1-negative, and predominantly CD3-negative. Although CD3-positive cells proliferate in response to rIL-2, they are low in cytolytic capabilities. The effector cells, like the LAK precursors, are low to medium density lymphocytes. The cytotoxic cells are predominantly CD3-negative, and cytotoxic activity cannot be blocked with the use of anti-CD3 monoclonal antibodies. The effector cells also lack most NK-associated markers (HNK-1, and the CD16 markers Leu-11b and B73.1) but possess the NK-associated marker NKH-1 (N901). The responsive cell appears to be at a very early stage of thymic development, and it does not appear to either require or express the CD3-T cell receptor complex.     

Leu-Leu-OMe sensitivity of human activated killer cells: delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets

Sensitivity to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) was used to characterize the phenotype of human activated killer cells. Natural killer cells (NK) and the precursors of both the alloantigen-specific cytotoxic T lymphocytes (CTL) and the NK-like activated killer cells generated after stimulation with allogeneic cells were deleted from human peripheral blood lymphocytes by preincubation with Leu-Leu-OMe. It was noted, however, that cytotoxic lymphocytes could be generated from Leu-Leu-OMe-treated lymphocyte precursors after 2 to 6 days of culture with the nonspecific mitogen, phytohemagglutinin (PHA). The characteristics of these killer cells indicated that they were a unique population that could be distinguished from other cytotoxic cells. Killing by these cells exhibited slow kinetics in that 18 hr cytotoxicity assays were required to detect full cytotoxic potential. When 18 hr assays were used, PHA-stimulated cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes were able to kill both NK-sensitive K562 cells and the relatively NK-resistant renal cell carcinoma cell line, Cur. These cytotoxic lymphocytes were HNK-1, Leu-11b (CD16), and OKM1 (CR3)-negative at both the precursor and effector stage of activation. Furthermore, these cells were derived from a CD3-positive precursor. Finally, killing by activated effectors was inhibited by OKT3. Unlike activation of Leu-Leu-OMe-sensitive large granular lymphocytes, generation of these cytotoxic T cells was totally prevented by treatment with mitomycin c before stimulation. Thus, a unique class of tumoricidal T cells can be characterized by resistance of lymphocyte precursors to a concentration of Leu-Leu-OMe, which has been shown to ablate NK, mixed lymphocyte culture-activated NK-like cytotoxic precursors, and the precursors of alloantigen-specific CTL.     

Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen

Monoclonal antibodies against the CD3 antigen and certain lectins can induce interleukin 2 dependent antigen-specific T cell clones to mediate non-antigen specific cytotoxicity. On the basis of this observation, we predicted that it may be possible to identify cytotoxic T lymphocytes (CTL) in peripheral blood without knowing the antigen specificity of these in vivo primed CTL. By using this strategy, peripheral blood lymphocytes were separated into low and high-density fractions on Percoll gradients and were tested for cytotoxic activity in the presence or absence of concanavalin A (Con A) or anti-Leu-4 antibody. Lectin-dependent cellular cytotoxicity (LDCC) and anti-CD3 induced cytotoxicity against both natural killer (NK)-insensitive and NK-sensitive targets were exclusively mediated by low-density CD3+ T lymphocytes. Additional studies indicated that low-density CD3+ T lymphocytes co-expressing Leu-7 antigen preferentially mediated this activity, although in some individuals, significant activity was also observed in the low-density T cells lacking Leu-7. In contrast, high-density CD3+ T lymphocytes and CD16+ (Leu-11+) NK cells (both Leu-7 and Leu-7+) did not mediate nonantigen-specific cytotoxicity under these conditions. The finding that NK cell-mediated cytotoxicity was unaffected by these lectins refutes the hypothesis that lectin-dependent cellular cytotoxicity is simply a result of effector and target agglutination. T cell-mediated cytotoxicity was both lectin and antibody specific. Phytohemagglutinin, Con A, and pokeweed mitogen induced cytolytic activity in the Leu-7+ T cells, whereas wheat germ agglutinin did not. Of the antibodies against T cell-associated differentiation antigens (anti-Leu-2,3,4, and 5), only anti-Leu-4 induced cytotoxicity. This anti-CD3-induced cytotoxicity was essentially completely inhibited by the presence of anti-LFA-1 or anti-CD2 monoclonal antibodies, implicating these molecules in the triggering process. A proportion of the CD3+, Leu-7+ CTL expressed HLA-DR antigens, indicating possible in vivo activation. Because previous clinical studies have indicated that lymphocytes with this phenotype may be elevated in clinical situations associated with immunosuppression and chronic viral infection, this unique subset of CD3+ T lymphocytes may represent a population of in vivo primed CTL possibly against viral antigens.     

The immunocompetence of murine stromal cell-associated thymocytes

Thymocyte subpopulations that associate in vivo with distinct nonlymphoid cells of the thymus have been isolated, and their immunocompetence was analyzed. Previous studies have indicated that greater than 95% of such cells bear a surface antigen phenotype representative of immature thymocytes, and are among the earliest thymic compartments repopulated by bone marrow-derived cells after lethal and sub-lethal irradiation. Stromal cell-associated thymocytes may be activated in vivo because they proliferate well in vitro with no additional stimulus, and show little increase in proliferation with the addition of T cell mitogens or allogeneic spleen cells. Stromal cell-associated T cells contain cytotoxic T lymphocyte (CTL) precursors that are indistinguishable from mature peripheral T cells by the parameters of self tolerance, alloreactivity, H-2 restriction, and stringency of self H-2 preference. CTL precursor frequencies and the cytotoxic activity of cells further separated on the basis of high levels of Thy-1 expression argue against the possibility that stromal cell-associated CTL activity is due solely to contaminating mature lymphocytes. Our data suggest that stromal cell-associated thymocytes represent an intermediate subpopulation of thymocytes that is functionally mature and that expresses an immature surface phenotype. Furthermore, the imposition of self tolerance and MHC restriction specificity appears to be tightly associated with the acquisition of immunocompetence in these thymocyte subpopulations.     

Induction of natural killer effectors from human thymus with recombinant IL-2

The NKH1 Ag is expressed on all cells in human peripheral blood capable of mediating spontaneous non-MHC restricted cytolytic function (i.e., natural killing). The majority of NK cells do not express CD3 Ag and do not express TCR gene products. However, approximately 20 to 25% of NKH1+ cells coexpress CD3 and TCR proteins. Both NKH1+CD3+ and NKH1+CD3- effectors can proliferate in response to IL-2 which also results in enhancement of cytolytic function. In the present studies, we examined thymocytes after incubation with rIL-2 for the presence of NKH1+ cells and for the development of non-MHC restricted cytolytic function. NKH1+ cells and NK activity could not be detected in fresh thymus. After culture with rIL-2 only, NK activity appeared in 3 days, reached a maximum after 7 days, and was effective against a panel of NK-sensitive targets. NK activity was correlated with the expression of NKH1 on the surface of in vitro proliferating thymocytes and immunofluorescent cell sorting demonstrated that almost all cytolytic activity was mediated by NKH1+ cells. As expected given the thymic origin of these cells, the majority of NKH1+ cells in culture expressed CD3. However, all cultures contained NKH1+CD3- effector cells which represent 15 to 40% of the NKH1+ population. As in peripheral blood, both NKH1+CD3- and NKH1+CD3+ exhibited non-MHC-restricted cytotoxicity, but only CD3+ effectors could be inhibited by anti-T3 mAb. These findings demonstrate that rIL-2 alone can induce subpopulations of thymocytes to proliferate, to express the NKH1 marker and become NK active in vitro. Furthermore, they suggest that the thymus which plays a role in the differentiation of NKH1+CD3+ NK effectors may also play a role in the differentiation or maturation of NKH1+CD3- NK effectors.     

Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. II. A novel CTL-produced cytotoxin that is antigenically distinct from tumor necrosis factor and alpha-lymphotoxin

We have cloned lines of IL 2-dependent human T cells derived from alloantigen, soluble antigen (tetanus toxoid), mitogen, or IL 2-stimulated peripheral blood lymphocytes and characterized their surface marker expression and cytolytic activity. The surface phenotype and cytolytic function was compared with the ability of these T cell clones to release cytotoxic lymphokines in response to mitogenic lectins. The cytotoxins released by these CTL clones were detected on the murine L929 target cells in a 16-hr assay. All of the T cell clones, whether stimulated by HLA alloantigens, tetanus toxoid, or mitogens, exhibited killer cell activity and the capacity to secrete a soluble cytotoxin(s). Specific polyclonal antisera to recombinant human tumor necrosis factor (rTNF) and human alpha-lymphotoxin (alpha LT) were unable to neutralize the cytotoxic activity released by most of these CTL clones. These results indicate that human CTL produce a novel antigenic form(s) of cytotoxin that we have termed CTL-toxin. Supernatants from several CTL clones yielded a cytotoxic activity that was partially neutralized (10 to 40%) by saturating levels of anti-TNF (but not anti-alpha LT) indicating that human CTL may be capable of producing a TNF-like molecule. Only two out of 60 CTL clones studied thus far produced a cytotoxic activity that was partially neutralized by anti-alpha LT (20 to 40%). Collectively, these results suggest that although both the CD4 and the CD8 subpopulations of human cytotoxic T cells may be capable of releasing several types of cytotoxins in response to mitogenic signals, the predominant cytotoxin is distinct from alpha LT and TNF.     

The stem cell antigens Sca-1 and Sca-2 subdivide thymic and peripheral T lymphocytes into unique subsets

Stem cell Ag 1 and 2 (Sca-1 and Sca-2), so named due to their expression by mouse bone marrow stem cells, were evaluated for expression by populations of cells within the thymus. Immunohistochemical analysis demonstrated that Sca-1 was expressed by cells in the thymic medulla and by some subcapsular blast cells, as well as by the thymic blood vessels and capsule. Sca-2 expression, which was limited to the thymic cortex, could be associated with large cycling thymic blast cells. Both Sca-1 and Sca-2 were expressed on a sub-population of CD4-CD8- thymocytes, and this subpopulation was entirely contained within the Ly-1lo progenitor fraction of cells. Sca-1 expression by a phenotypically mature subset of CD4+CD8- thymocytes was also noted. Conversely, Sca-2 expression was observed on a phenotypically immature or nonmature subpopulation of CD4-CD8- thymocytes. MEL-14, an antibody that defines functional expression of a lymphocyte homing molecule, identified a small population of thymocytes that contained all four major thymic subsets. Sca-2 split the MEL-14hi thymocyte subset into two Sca-2+ non-mature/immature phenotype fractions and two Sca-2- mature phenotype fractions. In peripheral lymphoid organs, Sca-1 identified a sub-population of mature T lymphocytes that is predominantly CD4+CD8-, in agreement with the thymic distribution of Sca-1. Peripheral T cells of the CD4-CD8+ phenotype were predominantly Sca-1-. In contrast, Sca-2 did not appear to stain peripheral T lymphocytes, but recognized only a subset of B lymphocytes which could be localized by immunohistochemistry to germinal centers. Thus, expression of Sca-1 is observed throughout T cell ontogeny, whereas Sca-2 is expressed by some subsets of thymocytes, including at least one half of thymic blasts, but not by mature peripheral T lymphocytes.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. I. Requirement of both killer-helper factor(s) and interleukin 2 for CTL generation from a subpopulation of thymocytes

The requirement for signals in the induction of cytotoxic T lymphocytes (CTL) from thymocyte precursors has been investigated. Either unfractionated or peanut agglutinin-binding (PNA+) C3H/He thymocytes were stimulated with mitomycin C(MMC)-treated, 2,4,6-trinitrophenyl(TNP)-modified syngeneic spleen cells in the presence of a variety of lymphokine preparations. Cellfree supernatant (CFS) from purified protein derivatives(PPD-CFS) stimulated Mycobacterium tuberculosis (Tbc)-primed cells, or partially purified interleukin 2 (IL 2) mediated strong cytotoxic responses in unfractionated thymocytes, whereas only PPD-CFS at final concentrations beyond 30% was active for CTL generation in PNA+ thymocytes. Neither IL 2 at concentrations of below 60 U/ml nor a low concentration of PPD-CFS (at final below 10%) had such a capacity. The addition of monoclonal anti-IL 2 receptor antibody completely blocked CTL generation induced by PPD-CFS in PNA+ thymocytes. In contrast, anti-immune interferon (IFN-gamma) antibody showed a marginal effect. PPD-CFS (10%) and IL 2 (10 U/ml) could synergistically trigger PNA+ thymocytes to induce CTL generation. These results suggested that both IL 2 and "helper" factors other than IL 2 are required for CTL generation from PNA+ thymocytes. We refer to these kinds of helper factors as killer helper factors (KHF). Partially purified IL 2-free KHF show two peaks of activities at apparent m.w. 14,000 to 34,000 and 44,000 to 90,000, and are heterogeneous with respect to isoelectric point, which is between 4.5 and 5.1. Cultures that received TNP-modified syngeneic cells and KHF on day 0 and IL 2 on day 2 generated potent CTL responses, whereas the addition of IL 2 on day 0 followed by the addition of KHF on day 2 to the culture was ineffective, suggesting that KHF is required in the early phase of the culture to achieve optimal CTL responses.     

Phenotypic and functional heterogeneity of thymic and splenic lymphokine activated killer cells relative to ornithine sensitivity

We have previously reported the selective inhibition of cytotoxic T lymphocytes (CTL) by 10 mM ornithine (ORN) relative to natural killer (NK) cell-derived lymphokine activated killer cells (LAK). To determine if this were due to differences in the progenitor cells or the type of stimulus, we used cortisone-resistant thymocytes (CRT) as a source of mature T cells for induction of LAK and CTL, and compared the results with spleen. Thymic and splenic CTL precursors (CTLp) from C57B1/6 (B6) mice were CD8+, ASGM1-, ORN sensitive. Splenic LAK precursors (LAKp) were CD8-, ASGM1+, ORN resistant when assayed against both YAC-1 and P815 tumor targets. In contrast, CRT-derived LAKp were CD8-, ASGM1+, ORN resistant against YAC-1, whereas LAKp against P815 were CD8+, ASGM1+, ORN sensitive. ORN sensitivity was also observed among CTL and LAK in DBA/2 mice and was associated with CD8+ phenotype. Therefore, our initial observation of differential ORN sensitivity in CTL vs LAK was a function of the progenitor cells; furthermore, CD8+ cytolytic cells are ORN sensitive whether activated by antigen (CTL) or IL-2 (T-LAK).     

New approaches to eliciting protective immunity through T cell repertoire manipulation: the concept of thymic vaccination

Conventional vaccines afford protection against infectious diseases by expanding existing pathogen-specific peripheral lymphocytes, both CD8 cytotoxic effector (CTL) and CD4 helper T cells. The latter induce B cell maturation and antibody production. As a consequence, lymphocytes within the memory pool are poised to rapidly proliferate at the time of a subsequent infection. The "thymic vaccination" concept offers a novel way to alter the primary T cell repertoire through exposure of thymocytes to altered peptide ligands (APL) with reduced T cell receptor (TCR) affinity relative to cognate antigens recognized by those same TCRs. Thymocyte maturation (i.e. positive selection) is enhanced by low affinity interaction between a TCR and an MHC-bound peptide in the thymus and subsequent emigration of mature cells into the peripheral T lymphocyte pool follows. In principal, such variants of antigens derived from infectious agents could be utilized for peptide-driven maturation of thymocytes bearing pathogen-specific TCRs. To test this idea, APLs of gp33-41, a Db-restricted peptide derived from the lymphocytic choriomeningitis virus (LCMV) glycoprotein, and of VSV8, a Kb-restricted peptide from the vesicular stomatitis virus (VSV) nucleoprotein, have been designed and their influence on thymic maturation of specific TCR-bearing transgenic thymocytes examined in vivo using irradiation chimeras. Injection of APL resulted in positive selection of CD8 T cells expressing the relevant viral specificity and in the export of those virus-specific CTL to lymph nodes without inducing T cell proliferation. Thus, exogenous APL administration offers the potential of expanding repertoires in vivo in a manner useful to the organism. To efficiently peripheralize antigen-specific T cells, concomitant enhancement of mechanisms promoting thymocyte migration appears to be required. This commentary describes the rationale for thymic vaccination and addresses the potential prophylactic and therapeutic applications of this approach for treatment of infectious diseases and cancer. Thymic vaccination-induced peptide-specific T cells might generate effective immune protection against disease-causing agents, including those for which no effective natural protection exists.     

Human T cell clones use a CD3-associated surface antigen recognition structure to exhibit both NK-like and allogeneic cytotoxic reactivity

In the present study, we developed human non-MHC-restricted CTL clones from human peripheral blood mononuclear cells activated in vitro with recombinant IL 2 and subsequently expended with PHA. The CD3/Ti+ clones were selected for their ability to exhibit non-MHC-restricted CTL reactivity by killing various tumor cell lines in culture, including the line K562 which does not express MHC antigens. We report that, at least for some of the NK-like T cell clones, it is possible to establish an allo-CTL activity, and that the CD3-associated surface antigen recognition structure might be involved in both reactivities.     

Lymphocytes infiltrating human ovarian tumors. I. Role of Leu-19 (NKH1)-positive recombinant IL-2-activated cultures of lymphocytes infiltrating human ovarian tumors

Tumor-infiltrating lymphocytes (TIL) were obtained from human ovarian tumors, expanded in the presence of IL-2 in culture and studied for cytotoxicity against fresh autologous and allogeneic ovarian carcinoma (CA) targets. TIL from ovarian tumors grew well in long term cultures, achieving from 8- to 682-fold expansion. TIL cultured with IL-2 were cytotoxic against both autologous and allogeneic fresh ovarian CA targets, and no specificity for autologous tumor could be demonstrated in any of the cultures. In all fresh TIL preparations, CD3+ lymphocytes were the major cell type and contained a high proportion (up to 51%) of activated (IL-2R+) cells as determined by two-color flow cytometry. Sorting of bulk TIL cultures followed by cytotoxicity assays identified the Leu-19+ cells, both CD3+ and CD3-, as effectors of cytotoxicity against autologous and allogeneic tumor cell targets. Cold target inhibition assays showed that allogeneic targets (both ovarian CA and a sarcoma) competed effectively with autologous ovarian CA targets for Leu-19+ effectors in TIL cultures. mAb to Leu-19 or Leu-2a did not block lysis of autologous targets by sorted effectors. OKT3 antibody augmented lysis of autologous targets by CD3+Leu-19- effectors only. These results show that non-MHC-restricted Leu-19+ effectors in cultures of TIL with 1000 U/ml of rIL-2 mediate lysis of autologous and allogeneic tumor cells. The CD3+Leu-19- cells, the main population in these cultures, do not mediate tumor lysis. To determine the phenotype of antitumor effectors in IL-2 cultures of TIL, cell sorting followed by functional assays are necessary.     

Colony formation by subpopulations of human T lymphocytes. VI. Further studies on colony phenotype, function, and cloning efficiency

Phytohemagglutinin (PHA)-induced colony formation in semisolid agar medium by human peripheral blood T lymphocytes showed an increasing cloning efficiency with decreasing numbers of cultured cells. Ninety percent of CD4+ cells (inducer/helper phenotype) and 20% of CD8+ cells (cytotoxic/suppressor phenotype) formed colonies when cultured at 10-200 cells/ml culture in the presence of sheep red blood cells (SRBC) and a source of interleukin-2 (IL-2). Probably all T-colony-forming cells, but none of the subsequent colony cells, expressed the Leu-8 antigen. The cloning efficiencies of FACS-sorted cells expressing the natural killer antigenic phenotypes Leu-7+ and CD16+ were found to be less than 1%. The costimulatory effect of red blood cells for colony formation was specific for SRBC and not observed in the presence of red cells obtained from seven other species including man. All T-lymphocyte colonies obtained from unseparated peripheral blood mononuclear cells expressed the CD25 antigen (IL-2 receptor) and colonies were always composed of either CD4+ or CD8+ cells. None of the colony cells expressed the Leu-8 or the CD16 antigens. By their specific morphology in agar culture the majority of colonies composed of CD4+ cells were easily recognized, but but approximately one-third of the CD4+ colonies could not be distinguished from colonies composed of CD8+ cells. On expansion of individual colonies in liquid subculture in the presence of interleukin-2, approximately 15% of the colonies developed natural killer (NK)-like cytotoxic activity, being capable of direct killing of K562 tumor cells. It is concluded that the present method for growing human T colonies exhibits the same cloning efficiency as the most efficient liquid culture systems. Individual T colonies are composed exclusively of T inducer/helper or T cytotoxic/suppressor cells, they are never of mixed phenotype, and they do not contain cells of natural killer phenotype. Regulatory mechanisms influencing colony formation are operating between and within the various subsets of T lymphocytes.     

Developmental regulation of the intrathymic T cell precursor population

The maturation potential of CD4-8- thymocytes purified from mice of different developmental ages was examined in vivo after intrathymic injection. As previously reported, 14-day fetal CD4-8- thymocytes produced fewer CD4+ than CD8+ progeny in peripheral lymphoid tissues, resulting in a CD4+:CD8+ ratio of less than or equal to 1.0. In contrast, adult CD4-8- thymocytes generated CD4+ or CD8+ peripheral progeny in the proportions found in the normal adult animal (CD4+:CD8+ = 2 to 3). Here we have shown that CD4-8- precursor cells from the 17-day fetal thymus also produced peripheral lymphocytes with low CD4+:CD8+ ratios. Precursors from full term fetuses produced slightly higher CD4+:CD8+ ratios (1.1-1.6) and precursors from animals three to 4 days post-birth achieved CD4+:CD8+ ratios intermediate between those produced by fetal and adult CD4-8- thymocytes. Parallel changes in the production of alpha beta TCR+ peripheral progeny were observed. Fetal CD4-8- thymocytes generated fewer alpha beta TCR+ progeny than did adult CD4-8- thymocytes. However, peripheral lymphocytes arising from either fetal or adult thymic precursors showed similar proportions of gamma delta TCR+ cells. The same pattern of progeny was observed when fetal CD4-8- thymocytes matured in an adult or in a fetal thymic stromal environment. In contrast to fetal thymic precursors, fetal liver T cell precursors resembled adult CD4-8- thymocytes by all parameters measured. These results suggest that fetal thymic precursors are intrinsically different from both adult CD4-8- thymocytes and fetal liver T cell precursors. Moreover, they lead to the hypothesis that the composition of the peripheral T cell compartment is developmentally regulated by the types of precursors found in the thymus. A model is proposed in which migration of adult-like precursors from the fetal liver to the thymus approximately at birth triggers a transition from the fetal to the adult stages of intrathymic T cell differentiation.     

Thymic alterations in EphA4-deficient mice

In the present work, we have demonstrated in vivo an altered maturation of the thymic epithelium that results in defective T cell development which increases with age, in the thymus of Eph A4-deficient mice. The deficient thymi are hypocellular and show decreased proportions of double-positive (CD4+CD8+) cells which reach minimal numbers in 4-wk-old thymi. The EphA4 (-/-) phenotype correlates with an early block of T cell precursor differentiation that results in accumulation of CD44-CD25+ triple-negative cells and, sometimes, of CD44+CD25- triple-negative thymocytes as well as with increased numbers of apoptotic cells and an important reduction in the numbers of cycling thymocytes. Various approaches support a key role of the thymic epithelial cells in the observed phenotype. Thymic cytoarchitecture undergoes profound changes earlier than those found in the thymocyte maturation. Thymic cortex is extremely reduced and consists of densely packed thymic epithelial cells. Presumably the lack of forward Eph A4 signaling in the Eph A4 -/- epithelial cells affects their development and finally results in altered T cell development.     

Human thymomas: evidence of immunohistologically defined normal and abnormal microenvironmental differentiation

Fifteen human thymomas were analyzed by immunoperoxidase studies on frozen and paraffin-embedded tissue sections in an attempt to identify the existence of immunologically defined microenvironments. All nine lymphocyte predominant thymomas contained a predominance of lymphocytes bearing the phenotype of cortical thymocytes and dendritic Class II major histocompatibility complex antigen-positive epithelial cells, thus defining cortical-like microenvironments. Medullary-like foci were also seen in all of these cases. Minor phenotypic abnormalities in Leu-2 and -3 antigen expression were seen in three cases. In contrast, the two epithelial predominant thymomas and four mixed thymomas all exhibited features of aberrant microenvironmental differentiation, with only two cases showing demarcation into cortical and medullary foci. A lack of Class II major histocompatibility complex antigens was associated with a decrease in the lymphoid populations and an increase in Leu-1 antigen expression by T cells of otherwise normal cortical phenotype when lymphocytes were present. In contrast, lack of Class I antigen on epithelial cells was not associated with any abnormality in lymphocyte phenotype or microenvironmental organization. We document for the first time abnormal microenvironments in thymomas that may offer insights into understanding normal thymic differentiation.