Specific lysis of allogeneic cells after activation of CD3- lymphocytes in mixed lymphocyte culture

Human CD3- lymphocyte populations were obtained by treating peripheral blood lymphocytes with mAbs directed to CD3, CD4, and CD8 surface antigens. The resulting populations were cultured with irradiated allogeneic cells; at day 4, 100 U/ml IL-2 were added and cultures continued for an additional 10 d. The resulting populations were CD3-CD2+CD7+ and displayed cytolytic activity against PHA-induced blast cells bearing the stimulating alloantigens but not against autologous or unrelated allogeneic blast cells. When CD3- populations were cultured with irradiated autologous cells, no cytolytic activity could be detected either against autologous or allogeneic blast cells. On the other hand, K562 target cells were lysed by both MLC-derived CD3- cell populations regardless of the origin (autologous or allogeneic) of the stimulating cells. CD3- clones were further derived from MLC-stimulated CD3- populations. These clones displayed a cytolytic pattern similar to the original MLC populations as only specific PHA blasts could be lysed. These clones did not express detectable surface TCR-alpha/beta or -gamma/delta molecules and lacked productive mRNA for TCR alpha and beta chains, while small amounts of TCR-gamma mRNA were detectable in one of four clones tested. Also mRNA for CD3 gamma and delta chains were undetectable in all clones, however, CD3 epsilon mRNA was consistently present.     

A monoclonal antibody specific for a common determinant of the human T cell receptor gamma/delta directly activates CD3+WT31- lymphocytes to express their functional program(s)

In an attempt to select mAbs specific for human TCR-gamma/delta, a polyclonal CD3+ 4-8-WT31- (TCR-gamma/delta+) cell line (MV1) was used for mice immunization. An mAb, termed BB3, reacted with MV1 cells but not with a large panel of CD3+ WT31+ (TCR-alpha/beta+) cell populations or clones. In addition, BB3 mAb reacted with the majority of CD3+ WT31- clones derived from six different donors. Double-color fluorescence experiments and FACS analysis showed that BB3+ cells were restricted to the CD3+ fraction of peripheral blood lymphocytes; in addition, in several donors the percentages (0.5-8% of total PBL) of BB3+ cells paralleled those of CD3+ WT31- cells. Surface molecules recognized by BB3 were susceptible to antibody-induced modulation; in addition, cell treatment with either BB3 or anti-CD3 mAb caused the simultaneous downregulation of the two molecules. That BB3 molecules are physically linked to CD3 antigen was further supported by immunoprecipitation experiments. Thus, under conditions that preserve the TCR-CD3 association, both BB3 and anti-CD3 mAb precipitated from 125I-labeled MV1 cells the same set of molecules. These consisted in the 18-28-kD CD3 molecules and in three bands of approximately 44, 42, and 38 kD under reducing conditions. When cell lysis was performed in 1% NP-40, the molecules immunoprecipitated by BB3 mAb were represented by an 80-kD band under nonreducing conditions, which resolved, under reducing conditions, in the three 44-, 42-, and 38-kD bands. Similar disulphide-linked forms of the TCR molecules were revealed in all of the other eight CD3+ WT31- BB3+ clones analyzed. Analysis of TCR molecules by electrophoresis (NEPHGE) showed that BB3 or anti-CD3 precipitated a 44-kD molecule displaying a basic PI (approximately 7.5) and two more acidic proteins (PI approximately 6) with a mol mass of 42 and 38 kD. Studies aimed to define whether stimuli directly acting on TCR-gamma/delta could induce CD3+ WT31- cell activation revealed that (a) In the presence of PMA, soluble BB3 mAb induced IL-2 production by MV1 cell line and by three other CD3+ WT31- BB3+ clones analyzed. (b) BB3 mAb-producing hybridoma used as triggering target, was efficiently lysed by CD3+ WT31- BB3+ effector cells (but not by CD3+ WT31+ BB3- conventional CTL). (c) Soluble BB3 mAb induced CD3+ WT31- BB3+ effector cells to lyse the Fc receptor-positive P815 target cells. (d) BB3-TCR-gamma/delta interaction on CD3+ WT31- BB3+ cells induced a rapid increase of [Ca2+]i levels, similar to that observed in response to anti-CD3 mAbs.     

CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta

The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells.     

Two subsets of human T lymphocytes expressing gamma/delta antigen receptor are identifiable by monoclonal antibodies directed to two distinct molecular forms of the receptor

Two mAbs directed to the TCR-gamma/delta were analyzed for their pattern of reactivity with CD3+WT31- cell populations or clones. In normal individuals, the BB3 mAb reacted with approximately 2/3 of peripheral blood CD3+WT31- lymphocytes, whereas delta-TCS-1 stained approximately 1/3 of such cells. In addition, the sum of the percentages of BB3+ and delta-TCS-1+ cells approximated the percentages of peripheral blood CD3+WT31- lymphocytes in seven normal donors tested. Also, in peripheral blood-derived polyclonal CD3+WT31- populations, cultured in IL-2, cells reacting with one or another mAb accounted for the whole cell population. On the other hand, only delta-TCS-1-reactive cells, but not BB3+ cells, could be detected in unfractionated as well as in CD4-8-thymocyte populations. Analysis of peripheral blood-derived CD3+WT31- clones showed that 70% of 72 clones analyzed reacted with BB3 mAb, but not with delta-TCS-1 mAb. On the other hand, delta-TCS-1 mAb stained the remaining BB3- clones. Five clones expressing medium-low amounts of CD8 antigen were BB3- delta-TCS-1+. Both types of clones lysed the Fc gamma receptor-bearing P815 target cell in the presence of anti-CD3 mAb (but not of mAb directed against HLA-DR, CD7 molecules, or TCR-alpha/beta). In this cytolytic assay, BB3 mAb induced target cell lysis only by BB3+ clones, whereas delta-TCS-1 mAb was effective only with delta-TCS-1+ clones. The CD3-associated surface molecules expressed by BB3+ or delta-TCS-1+ clones were analyzed after cell surface iodination and immunoprecipitation with the corresponding anti-TCR mAb or with anti-CD3 mAb (in digitonin-containing buffer). In SDS-PAGE, molecules immunoprecipitated from 13 BB3+ clones displayed, under nonreducing conditions, a molecular weight of 80 kD (in some cases, a minor 38-kD band could be detected). Under reducing conditions, two major components of 44 and 41 kD (and a minor component of 38 kD) were detected. On the other hand, TCR molecules immunoprecipitated from 11 different delta-TCS-1+ clones appeared as a diffuse band of 41-44 kD, both under reducing and nonreducing conditions (under non-reducing condition, an additional 38-kD band was present). Therefore, BB3+ cells express a disulphide-linked form of TCR-gamma/delta whereas delta-TCS-1+ cells express a non-disulphide-linked form.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human peripheral blood lymphocytes bearing T cell receptor gamma/delta. Expression of CD8 differentiation antigen correlates with the expression of the 55-kD, C gamma 2-encoded gamma chain

We analyzed the CD3-associated molecules present on peripheral blood-derived TCR-gamma/delta+ clones that express CD8 surface antigens. Clones were derived by limiting dilution from CD3+WT31- FACS-purified populations derived from several donors. Eight of greater than 300 TCR-gamma/delta+ clones analyzed expressed CD8 and reacted with delta-TCS-1 mAb. Cell numbers suitable for more detailed analyses could be obtained from four clones, including one derived from thymus. Analysis of CD3-associated TCR molecules immunoprecipitated by anti-Leu-4 (anti-CD3) mAb under conditions that preserve the CD3/TCR association (1% digitonin) showed a predominant 55-60-kD molecule both under reducing and nonreducing conditions. On the other hand, the delta-TCS-1-reactive molecules immunoprecipitated from 25 CD3+ delta-TCS-1+ CD8- clones, in all instances, displayed a 40-44-kD mol mass. In two-dimensional PAGE, TCR-gamma molecules precipitated from delta-TCS-1+ CD8+ clones appeared more acidic than those of BB3+ or delta-TCS-1+ CD8+ clones. Southern analysis confirmed that this type of non-disulphide-linked TCR-gamma/delta is also coded for by the C gamma 2 gene segment.     

Characterization of CD3+, CD4-, CD8- clones expressing the putative T cell receptor gamma gene product. Analysis of the activation pathways leading to interleukin 2 production and triggering of the lytic machinery

Four clones were derived from human peripheral blood T lymphocytes from which CD4+ and CD8+ cells had been removed by treatment with specific mAbs and complement. All expressed the CD2+, 3+, 4-, 8-, T44- phenotype, and did not react with the WT31 mAb, which is specific for a framework determinant of the CD3-associated alpha/beta heterodimer which serves as receptor for antigen on most human T lymphocytes. Surface iodination followed by crosslinking with dithiobis-succinimidyl propionate (DSP) and immunoprecipitation with anti-CD3 mAbs indicated that, in all four clones, the CD3-associated molecules consisted of a major 45 kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNA for the alpha chain was missing; beta chain mRNA was present in a defective form (1 kb instead of 1.3 kb). These data support the concept that these clones may express, in association with CD3, the molecular product of the T cell receptor gamma genes instead of the typical alpha/beta heterodimer. CD3+, WT31- clones lysed the NK-sensitive K562 target cells and produced IL-2 upon stimulation with PHA. In addition, they released IL-2 after triggering with soluble anti-CD3 mAbs or with an appropriate combination of anti-CD2 mAbs (in the presence of adherent cells). When CD3+, WT31- clones were incubated with an anti-CD3 producing hybridoma as triggering target, the latter was efficiently lysed. Target cell lysis also occurred when a suitable combination of anti-CD2 mAbs-producing hybridomas was used. Therefore, CD3+, WT31- cells appear to use two pathways of cell activation that function also in conventional CD3+, WT31+ T cells, but they lack a third putative pathway initiated by T44 surface molecules.     

Autologous tumor-specific cytotoxic T lymphocytes in the infiltrate of human metastatic melanomas. Activation by interleukin 2 and autologous tumor cells, and involvement of the T cell receptor

TIL from metastatic melanoma proliferated by greater than 1,000-fold (840-3,675, mean 1,543) after 6 wk in culture of mixtures of TIL and tumor cells with rIL-2 alone. Cytolysis was restricted to autologous tumor cells. CD8+ T cells were the predominant population of TIL before and after expansion, and were primarily responsible for autologous tumor-specific CTL activity. No other rIL-2-activated lymphocytes from peripheral blood, lymph nodes with melanoma metastasis, or TIL from sarcoma or renal cell carcinoma had autologous tumor-specific CTL activity. There were few or no CD16+ NK cells in TIL from metastatic melanoma before or after incubation with rIL-2, respectively. However, TIL from sarcoma or renal cell carcinoma contained a substantial proportion of CD3-CD16+ NK cells, which increased in number in culture with rIL-2. Purified CD16+ NK cells as well as CD3+CD16- T cells from rIL-2-activated TIL of renal cell carcinoma displayed MHC-nonrestricted cytotoxicity. At the clonal level as determined by limiting dilution, 8 of 10 clones from melanoma TIL displayed cytotoxicity restricted to autologous tumor cells, while all 13 clones from renal cancer TIL equally lysed autologous and allogeneic tumor cells. Anti-T cell receptor (TCR)-alpha/beta(WT31) mAb as well as anti-CD3 mAb inhibited autologous melanoma cell-specific CTL activity mediated by rIL-2-activated TIL at the effector phase. These two mAbs also inhibited rIL-2-dependent proliferation of these TIL when added to the culture. Pretreatment of fresh melanoma cells with mAb to MHC antigens followed by washing inhibited specific CTL activity. These results suggest that both TCR-alpha/beta on effector TIL and MHC antigens on fresh tumor cells are involved in the specific immune-recognition. After reaching maximum propagation, TIL from metastatic melanoma responded poorly to rIL-2 alone. However, stimulation with fresh autologous melanoma cells restored both CTL activity and proliferation in response to rIL-2. The latter is associated with IL-2 receptor (Tac antigen) expression on the surface. These results indicate that TIL from metastatic melanomas may have unique characteristics different from lymphocytes obtained from the other sources, and may contain precursor CTL sensitized in vivo to autologous tumor cells, and thus can be propagated in larger numbers with rIL-2 alone while retaining autologous tumor-specific CTL activity.     

Lysis of tumor biopsy cells by autologous T lymphocytes activated in mixed cultures and propagated with T cell growth factor

Blood lymphocytes from tumor patients were cocultivated with allogeneic lymphocytes (MLC) or autologous tumor cells (ATS), and their cytotoxicity was characterized. The main objective of the study was the lysis of autologous tumor biopsy cells by such effectors. Lymphocytes of patients activated in MLC lysed allogeneic third-party cells and in some cases also lysed autologous tumor cells. Allogeneic but not autologous PHA blasts were also damaged by these effectors. The cytotoxic potential of MLC-activated lymphocytes from healthy donors was similar; allogeneic tumors and phytohemagglutinin (PHA) blasts but not autologous PHA blasts were lysed. The cytotoxicity of lymphocytes activated in ATS were specific for the stimulator because they acted only on the autologous tumor cells. Allogeneic tumors and autologous and allogeneic PHA blasts were not lysed. The pattern of cytotoxicity with regard to this target panel was maintained when the MLC or ATS cultures were further propagated with TCGF. Results obtained in cold target competition assays suggested (a) activated lymphocyte lyse the third party tumor targets because of alloantigen recognition; (b) in MLC several different sets of alloreactive cytotoxic lymphocytes are present simultaneously; and (c) the alloreactive cells are different than those that act on the autologous tumor cells. Thus, the lysis of allogeneic tumor cells by lymphocytes of the patient is not due to recognition of cross-reacting tumor-related antigens, and the autotumor cytotoxicity of the patients' MLC-activated lymphocytes if performed by specifically reacting cells.     

Selective activation of gamma/delta + T cell clones by single anti-CD2 antibodies

The CD2 antigen is the target for an "alternative" T cell activation pathway. Numerous studies have demonstrated that pairs of monoclonal antibodies (mAbs) directed toward two different epitopes are required for activation of T cell receptor (TCR)-alpha/beta + T cells via CD2. We have now explored the activation of human TCR-gamma/delta + T cell clones by a panel of anti-CD2 mAbs directed against the sheep erythrocyte-binding (T11.1) epitope of CD2. Seven of seven gamma/delta + clones expressing different molecular forms of the TCR-gamma/delta responded to stimulation by a single anti-CD2 mAb (OKT11, 9E8, BW0110, M-T910) with IL-2 secretion and/or proliferation. Immobilization of anti-CD2 mAbs in microculture plates was essential for activation of gamma/delta + clones, which occurred in the absence of feeder cells. In addition to interleukin 2 (IL-2) production and proliferation, anti-CD2 mAbs also triggered cytotoxic effector activity in gamma/delta + clones as measured against FcR+ P815 target cells. In contrast to gamma/delta + clones (but in line with established data), none of five CD4+ or CD8+ TCR-alpha/beta + clones were activated by any of the tested individual anti-CD2 mAbs. Taken together, our results reveal a striking difference between cloned gamma/delta + and alpha/beta + T cells in that gamma/delta + T cells are selectively activated by a single anti-CD2 (T11.1) mAb, without need for the simultaneous signal of a second anti-CD2 mAb directed against another (T11.2 or T11.3) CD2 epitope.     

Evidence of a natural killer (NK) cell repertoire for (allo) antigen recognition: definition of five distinct NK-determined allospecificities in humans

Previous studies indicated that CD3-CD16+ natural killer (NK) cells are capable of specific alloantigen recognition. Thus, alloreactive NK clones lysed normal allogeneic target cells (phytohemagglutinin [PHA] blasts) bearing the stimulating alloantigen but did not lyse autologous cells or the majority of unrelated allogeneic cells. In this study we investigated whether NK cells isolated from single individuals could exhibit different allospecificities. To this end, we derived large numbers of CD3-CD16+ clones (in the presence of PHA) from fresh CD3- peripheral blood lymphocytes. Cloning efficiencies ranged between 5 and 10%. The resulting CD3-CD16+ clones were tested for their reactivity against a panel of allogeneic PHA blasts (derived from six donors). In a given individual (A), four distinct groups of clones could be identified according to their pattern of reactivity (over 400 clones have been analyzed). Clones that could be assigned to one or another group of specificity represented 36% of all clones derived from this donor. The remaining clones did not display cytolytic activity against any of the allogeneic target cells used in the panel. None of the clones lysed autologous (A) PHA blasts, yet, these cells were lysed by the representative clones G10 and H12 specific for donor A. Clones displaying a cytolytic pattern of reactivity identical to that defined for donor A were present in other individuals studied, however not all groups of allospecific clones were necessarily represented in different individuals. Allospecific clones belonging to the various groups were homogeneous in the expression of EB6/GL183-triggering surface molecules, and could thus be assigned to one or another of the previously defined subsets of NK cells. Genetic analysis of the new NK-defined alloantigens was performed in representative families. The corresponding characters were found to segregate independently and, at least for three of them, an autosomic recessive type of inheritance could be demonstrated. Moreover, the comparative analysis of the segregation of the major histocompatibility complex haplotypes and the recessive or dominant alleles of the genes governing the five specificities analyzed indicated that there is no independent sampling between the two genetic traits, thus suggesting that the genes regulating the NK-defined specificities are carried by chromosome 6. Finally, some donors expressed more than one specificity, thus providing evidence for an NK-defined complex haplotype.     

Specific recognition of human CD3-CD16+ natural killer cells requires the expression of an autosomic recessive gene on target cells

We analyzed the recently defined ability of CD3-CD16+ cells to specifically recognize and lyse normal allogeneic target cells (PHA-induced blasts). The susceptibility to lysis by a given alloreactive natural killer (NK) clone ("1 anti-A") was expressed by PHA blasts derived from 9 of 38 random donors analyzed. In all instances, the specific lysis of "susceptible" target cells was greater than 35% while that of "nonsusceptible" targets was less than 6% at an E/T cell ratio of 5:1. In addition to 1 anti-A, A anti-1 specific CD3-CD16+ clones could also be isolated from the reverse MLC combination. The relationship existing between lysis of normal allogeneic cells or tumor cells by the same CD3-CD16+ effector cell has been investigated: 1 anti-A specific CD3-CD16+ clones lysed PHA blasts of three of six cancer patients, while they lysed fresh tumor cells (ovarian carcinoma) from all six patients. The type of inheritance of the character "susceptibility to lysis" was analyzed in representative families. This analysis revealed that the character is inherited in an autosomic recessive fashion, and it is therefore different from MHC. We further investigated the type of segregation of the opposite character "resistance to lysis" (which is inherited in a dominant mode). The finding that this character segregated in all donors expressing given MHC haplotypes indicated that the gene regulating the expression of the NK-defined alloantigen is present on chromosome 6.     

Functional and phenotypic characteristics of effusion-associated lymphoid cells cultured in the presence of either recombinant interleukin 2 or T-cell growth factor from malignant pleural and peritoneal effusions in patients with advanced carcinoma

Malignant pleural or peritoneal effusion-associated lymphoid (EAL) cells from 17 patients with advanced carcinoma were cultured with autologous carcinoma cells in the presence of either recombinant interleukin 2 (rIL 2) or T-cell growth factor (TCGF). Considerable cytolytic activity of the cells against allogeneic tumor cells, such as K562 and Daudi cells was induced by the cultivation. TCGF-activated EAL cells acquired higher anti-Daudi tumor cytotoxicity than rIL 2-activated EAL cells. The resultant TCGF-activated EAL cells from cancer patients significantly exceeded lytic activity of TCGF-activated EAL cells from patients with liver cirrhosis for control (p less than 0.01). Four of 6 cases examined also showed cytotoxic activity against autologous tumor. In facts, viable carcinoma cells co-cultured with EAL cells and TCGF mostly disappeared during 14 days. Similar phenomenon was not observed in rIL 2-activated EAL cells. Thus, it was suggested that more additional lymphokine other than IL 2 was necessary to generate cytotoxic activity against autologous tumor cells. The cell populations responsible for cytolytic activity to allogeneic and/or autologous tumor cells were investigated by two-color flow cytometry. The majority of killer-effector cells against allogeneic cells in rIL 2-activated EAL cells from cancer patients showed CD4+Leu8- phenotype at population level. In contrast, it was suggested that cytolytic activity against allogeneic and/or autologous tumor cells in TCGF-activated EAL cells might be mediated by CD8+ CD11- and CD8+ CD28+ effector cells.     

Cellular basis of skin allograft rejection across a class I major histocompatibility barrier in mice depleted of CD8+ T cells in vivo.

The present study was undertaken to define the cellular mechanisms involved in the rejection of major histocompatibility complex (MHC) class I disparate skin grafts by mice depleted of CD8+ T cells in vivo. Mice were effectively depleted of CD8+ T cells by adult thymectomy followed by in vivo administration of anti-CD8 monoclonal antibody (mAb) and then engrafted with allogeneic skin. We found that CD8 depleted mice did reject MHC class I disparate skin grafts, but only when the grafts also expressed additional alloantigens. Despite the marked depletion of CD8+ T cells in these mice, we found that their rejection of MHC class I disparate grafts was mediated by CD8+ cytolytic T lymphocyte (CTL) effectors that had escaped depletion. These CD8+ CTL effectors were unique in that: (a) their generation was dependent upon the injected anti-CD8 mAb and upon exposure to class I MHC alloantigens expressed on the engrafted skin, and (b) their effector function was resistant to blockade by anti-CD8 mAb. We observed that the additional alloantigens coexpressed on MHC class I disparate grafts that triggered graft rejection in CD8-depleted mice could be MHC-linked or not and that they functioned in these rejection responses to activate third party specific CD4+ T helper (Th) cells to provide helper signals for the generation of CD8+ anti-CD8 resistant CTL effector cells. Thus, mice depleted of CD8+ T cells by thymectomy and in vivo administration of anti-CD8 mAb harbor a unique population of anti-CD8 resistant, CD8+ effector cells that mediate anti-MHC class I responses in vivo and in vitro, but require help from third party specific Th cells to do so.     

Clonal analysis of cytotoxic and regulatory T cell responses against human melanoma

T cell-mediated immune response against autologous melanoma cells was analyzed, at population and clonal levels, in 31 patients with recurrent and/or metastatic disease. Fresh PBL and lymph node lymphocytes (LNL) from melanoma-involved nodes were not cytotoxic against the respective melanoma cells. When activated in in vitro coculture (IVC) against the autologous melanoma cells in the presence of IL-2, a majority of the activated PBL and LNL became cytotoxic against the autologous targets. The activated effector cells were cloned in limiting dilution microcultures, and growing clones were phenotypically defined and were functionally characterized for cytotoxicity and for potential regulatory function. Functional T cell clones were obtained from 15 of 31 cases. Of these, CTL responses exhibiting cytotoxicity restricted against the autologous melanoma were seen in four cases. All four CTL clones were CD3+, CD8+, and CD4-. Three of these four CTL clones were studied extensively. All three of these CTL clones expressed MHC class I-restricted cytotoxicity. mAb anti-CD3 blocked cytotoxicity in two and enhanced cytotoxicity in the other. Neither autologous sera nor autologous nonactivated fresh PBL modulated the cytotoxic functions of the CTL clones at the effector phase. T cell lines exhibiting regulatory function were obtained in 11 cases. The regulatory T cell lines were CD3+, CD4+, and CD8-. In three cases CD4+ clones amplified the cytotoxic response in the PBL in coculture, while in eight other cases the T cell lines downregulated the cytotoxic responses. Such T cell-mediated down-regulations were either restricted to the autologous system, induced by D/DR antigens expressed by the autologous or allogeneic melanoma cells, or induced by stimulus other than D/DR antigens. Taken together, these findings clearly demonstrate the existence of T cell-mediated cytotoxic and regulatory responses against human melanoma.     

CD4-CD8- T cell receptor alpha beta T cells: generation of an in vitro major histocompatibility complex class I specific cytotoxic T lymphocyte response and allogeneic tumor rejection

The generation of an in vitro major histocompatibility complex class I specific response of CD4-CD8- T cell receptor (TCR) alpha beta cytotoxic T lymphocytes (CTL) and their allogeneic tumor rejection were investigated. Inocula of BALBRL male 1 were rejected in C57BL/6 (B6) mice treated with minimum essential medium (MEM) (control), anti-L3T4 (CD4) monoclonal antibody (mAb) or anti-Lyt-2.2 (CD8) mAb and CTL against the tumor were generated in vitro. No rejection and no induction of CTL were observed in B6 mice treated with anti-L3T4 (CD4) plus anti-Lyt-2.2 (CD8) mAb. CTL with the classical Thy-1+ CD3+CD4-CD8+ TCR alpha beta phenotype were generated in mixed lymphocyte tumor cell culture (MLTC) spleen cells from B6 mice treated with MEM (control) or anti-L3T4 (CD4) mAb, whereas CTL with an unusual Thy-1+CD3+CD4-CD8- TCR alpha beta phenotype were generated in MLTC spleen cells from anti-Lyt-2.2 (CD8) mAb-treated B6 mice. Both types of CTL were reactive with both H-2Kd and Dd (Ld) class I antigen. These findings suggest that when CD4+ cells were blocked by anti-L3T4 (CD4) mAb, CD8+ CTL mediated rejection, and when CD8+ cells were blocked by anti-Lyt-2.2 (CD8) mAb, CD4+ cells were capable of mediating rejection, although less efficiently than CD8+ cells, by inducing CD4-CD8- TCR alpha beta CTL. The finding that adoptive transfer of CD4 and CD8-depleted MLTC spleen cells, obtained from anti-Lyt-2.2 (CD8) mAb-treated B6 mice that had rejected BALBRL male 1, resulted in rejection of BALBRL male 1 inoculated into B6 nu/nu mice confirmed the above notion. CTL clones with the CD4-CD8- TCR alpha beta phenotype specific for Ld were established.     

Dendritic cells stimulate primary human cytolytic lymphocyte responses in the absence of CD4+ helper T cells

Cytotoxic lymphocytes are typically generated from unfractionated suspensions of human lymphocytes by stimulating with heterogeneous APCs and exogeneous growth factors. We have found that human blood dendritic cells can directly stimulate allogeneic human CD8+ T cells to proliferate and express antigen-specific cytotoxic activity. These primary responses, which are accompanied by the release of T cell growth factor(s), are induced in the absence of CD4+ helper T cells and are not inhibited by anti-CD4 mAb. Both antigen-specific CTL as well as nonspecific NK cells can be elicited by dendritic cells. The NK cell response can be depleted at the precursor level by panning with an anti-CD11b mAb, which removes a CD11b+/CD28-, CD16+ subset from the starting CD4- responders. Allogeneic blood monocytes are neither stimulatory nor inhibitory of these primary CD4- MLRs, even though monocytes present alloantigen in such a way as to be recognized as specific targets for CTL that have been sensitized by dendritic cells. The number of CD8+ cells that are blast transformed and express an activated phenotype (i.e., HLA DR/DQ+, CD25/IL-2R+, CD45R-) reaches 30-40% of the culture at day 4-5, the peak of the helper-independent response. We conclude that antigen-presentation by dendritic cells is sufficient in itself to prime cytolytic precursors. We speculate that using dendritic cell stimulators and CD4- responders in MLRs may be more efficient than standard tissue typing approaches for the detection of subtle, but important class I MHC-restricted histoincompatibilities in human transplantation.     

Intraepithelial lymphocytes. Anatomical site, not T cell receptor form, dictates phenotype and function

The function and structure of the TCR proteins of intraepithelial lymphocytes (IEL) were examined using a panel of mAbs specific for TCR-gamma/delta. Three subsets of TCR-gamma/delta+ IEL could be detected with five mAbs, termed GL1-GL5. The mAbs were able to trigger lysis via crosslinking of the IEL TCR and all of the subsets were constitutively cytolytic. Immunoprecipitation of IEL TCR proteins revealed that the GL2 mAb reacted only with gamma, delta heterodimers containing high Mr delta chains, while the other mAbs precipitated all of the observed gamma and delta proteins. Two-color fluorescence analysis showed that the GL2+ subset was contained within the larger GL1+ subset. The GL3 and GL4 mAbs appear to be specific for all TCR-gamma/delta while GL2 was V delta 4 specific. Analysis of IEL for TCR-alpha/beta expression demonstrated that approximately 20% of B6 IEL were TCR-alpha/beta+. Interestingly, this population of IEL contained Thy-1- and CT1+ cells, indicating that the unique phenotype of IEL was not restricted to TCR-gamma/delta+ cells. Moreover, the TCR-alpha/beta+ IEL were also constitutively cytolytic, suggesting that the intestinal milieu was controlling the functional programming of IEL regardless of TCR type. The mAbs reported here as well as the ability to exploit the distinct phenotype of IEL should prove useful in determining the function of IEL and the TCR-gamma/delta.     

Distinct molecular forms of human T cell receptor gamma/delta detected on viable T cells by a monoclonal antibody

A second type of TCR molecule has been identified on human and murine T lymphocytes, which involves the protein products of the gamma and delta genes. T lymphocytes bearing this receptor may constitute a separate cell lineage with a distinct immune function. We have produced an mAb, which specifically detects human TCR-gamma/delta in native as well as denatured states, this in contrast to previously used anti-gamma chain peptide sera, which only reacted with denatured protein. The receptor occurs in different molecular forms, with or without interchain disulphide bonds, in which a delta chain may or may not be detected by cell surface iodination. The mAb is reactive with all these receptor forms. Therefore, this antibody could be used to determine the expression of TCR-gamma/delta on viable human T lymphocytes. In normal individuals, TCR-gamma/delta was found on a subset composing 2-7% of CD3+ lymphocytes in peripheral blood and 0.1-1.0% in thymus. The majority of these cells do not express the CD4 or CD8 antigens, although a significant percentage of CD8+ cells was found. TCR-gamma/delta+ cells in peripheral blood are resting lymphocytes, as judged by ultrastructural analysis. T cell clones with different receptor types can display MHC-nonrestricted cytolytic activity, which is shown to be induced by the culture conditions, most likely by growth factors such as IL-2. This strongly suggests that TCR-gamma/delta does not play a role in target cell recognition in MHC-nonrestricted cytotoxicity. The anti-TCR-gamma/delta antibody can specifically induce cytotoxic activity in clones expressing the receptor, but in addition inhibit growth factor induced cytotoxicity, which indicates a regulatory role of the TCR-gamma/delta/CD3 complex in MHC-nonrestricted cytotoxicity.     

Melanoma cells and normal melanocytes share antigens recognized by HLA- A2-restricted cytotoxic T cell clones from melanoma patients

HLA-A2-restricted, CD3+, CD8+, alpha/beta+ cytotoxic T cell (CTL) clones were isolated from peripheral blood (PBL) or tumor infiltrating lymphocytes (TIL) of two HLA-A2+ melanoma patients (9742 and 5810), to evaluate the possible recognition of autologous melanoma and of allogeneic HLA-A2-matched normal melanocytes. These CTL clones lysed not only fresh and cultured autologous melanoma cells, but also allogeneic HLA-A2+, but not HLA-A2-, normal melanocytes. The lysis of autologous neoplastic cells and of melanocytes could be inhibited by an anti-HLA-A2 monoclonal antibody (mAb). Lysis of the normal melanocytes was not dependent on the presence of human or fetal calf serum in the culture medium. HLA-A2-restricted CTL clones recognized not only proliferating melanocytes cultured in complete melanocyte medium, but also melanocytes made quiescent by culture for up to 6 d in a basal medium devoid of exogenous factors such as phorbol ester (O- tetradecanoyl phorbol 13-acetate [TPA]), epidermal growth factor, insulin, and pituitary extracts. Analysis of specificity of four CTL clones (A75, A83, A94, and 119) from patient 9742, performed on a panel of 39 targets, indicated that the three HLA-A2-restricted CTL (A75, A83, and A94) lysed all but one of nine allogeneic melanomas expressing the HLA-A2 molecule with no reactivity on nine HLA-A2- allogeneic melanomas. Only a few instances of borderline reactivity were seen by the same effectors on 21 targets of nonmelanocyte lineage, including 12 carcinomas of different histology, four Epstein-Barr virus-transformed B cells (lymphoblastoid cell lines [LCL]), including the autologous LCL, four lines of normal fibroblasts, and normal kidney cells. Lack of reactivity on allogeneic targets of nonmelanocyte lineage occurred in spite of expression of HLA-A2 on 14 of these targets as determined by conventional tissue typing and cytofluorimetric analysis with four different anti-HLA-A2 mAb. These data indicate that tissue-related antigens can be expressed on normal and neoplastic cells of the melanocyte lineage and can be recognized in association with HLA-A2 by CTL clones from melanoma patients.