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Previous studies on lymphocyte cytotoxicity against autologous human hepatocytes had been studied using Terasaki plates, in which dead hepatocytes after incubation with lymphocytes were counted visually. No studies with 51Cr-labeled human hepatocytes as targets have been reported, although it can give us more objective results. In the present study, we established procedures for labeling human hepatocytes with 51Cr and for measuring cytotoxicity of freshly isolated liver-infiltrating lymphocytes (LIL) against 51Cr-labeled human autologous hepatocytes. Hepatocytes were isolated from diseased and 'normal' liver tissues, cultured overnight, and labeled with 51Cr 'in situ' in the wells of 96-well round bottom plates. Human hepatocytes isolated from either 'normal' or diseased liver tissues labeled well with 51Cr, and mean spontaneous 51Cr release was less than 15% in the presence of 5% fetal bovine serum or human AB serum. Freshly isolated LIL obtained from chronic viral hepatitis but not from other liver diseases showed cytotoxicity against 51Cr-labeled autologous hepatocytes in 4 h 51Cr release assays, and the percent specific lysis was linearly related to the E:T cell ratio. LIL from viral hepatitis were able to mediate natural killer (NK) activity against K562 targets, lymphokine-activated killer-like activity against Daudi cells, and lectin-dependent cellular cytotoxicity. Two-color flow cytometry analysis showed that these LIL contained both CD3+ T cells and CD3-CD56+ NK cells. This is the first report which examined the cytotoxicity of liver-infiltrating lymphocytes against 51Cr-labeled human hepatocytes, and it will be useful in assessing local immune response against autologous hepatocytes in chronic liver diseases.

In vitro effects of human recombinant IL-6 (1-1000 U/ml) on highly enriched human NK CD3-CD56+ cells (94% +/- 2; mean +/- SEM; n = 8), obtained from PBL were studied. IL-6 induced low levels of NK cell proliferation (7- to 30-fold during 6-day incubation), which was IL-2-independent, because IL-6 did not induce detectable IL-2 production by NK cells. Two-color flow cytometry analysis demonstrated that incubation of NK cells with IL-6 at the optimal concentration of 250 U/ml for 6 days significantly increased the proportion of NK cells expressing the following activation Ag: CD25 (26% +/- 17, mean +/- SEM vs 4% +/- 1 in control, n = 5), CD54 (44% +/- 17 vs 9% +/- 3), HLA-DR (29% +/- 13 vs 12% +/- 4), CD69 (45% +/- 7 vs 12% +/- 3), and CD71 (34% +/- 17 vs 6% +/- 2). The mean fluorescence intensity of these activation Ag was increased as well. IL-6 induced expression of CD49b (alpha-chain of VLA-2, 20% +/- 11 vs 2% +/- 1) and CD49c (alpha-chain of VLA-3, 43% +/- 17 vs 5% +/- 3), which are not expressed on resting NK cells. IL-6 also enhanced the fluorescence intensity of beta 1 integrins, CD49d, CD49e, and CD49f, expressed on NK cells. IL-6-stimulated NK cells showed significantly increased integrin-mediated adhesion to fibronectin- or laminin-coated plates (26 +/- 3 mean % cells adhering +/- SEM vs 15 +/- 4 in control for FN and 19 +/- 1 vs 11 +/- 1 for LM, p0.05 for both) as determined in a 3 h binding assay. As assessed by inhibition of adhesion using mAb to the VLA-2, -3, -4, -5, and -6, NK cell adhesion to fibronectin was mediated by VLA-4 and 5, and their adhesion to laminin by VLA-3 and -6. NK cells incubated in the presence of IL-6 were found to produce a factor cytostatic to WEHI-164 clone 13 target cells. This effect was partly, although significantly, blocked by neutralizing antibodies to TNF-alpha or TNF-beta. Our data demonstrate that IL-6 can directly activate human NK cells, but is a less potent NK cell activator, for all activation and functional parameters studied, than IL-2.

Cytolytic T lymphocytes play an important role in host defense against viral infections, including human immunodeficiency virus (HIV). In a phase I clinical trial (protocol 080 of the AIDS Clinical Trials Group), generation of CD8+ effector cells from peripheral blood of patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC) or AIDS and safety of autologous adoptive transfer of these cells were evaluated. For therapeutic infusions, CD8+ T cells were purified by positive selection on anti-CD8 monoclonal antibody-coated flasks from leukapheresed peripheral blood of seven patients. These CD8+ T cells were cultured in the presence of interleukin-2 and phytohemagglutinin for up to 3 weeks to obtain cells sufficient for therapeutic infusions (10(8) to 10(10)). All 31 cell cultures established from the seven patients and used for therapy were highly enriched in CD8+ (mean, 97%), CD8+HLA-DR+ (50%), cytotoxic CD8+CD11b- (82%), and memory CD29+ (78%) T lymphocytes. In vitro expanded CD8+ cells had excellent cytotoxic function at the time they were used for therapy, including HIV-specific activity against autologous targets infected with vaccinia vectors expressing HIV-IIIb antigens, gag, pol, and env. Anti-HIV activity of cultured CD8+ cells was significantly higher than that of autologous fresh peripheral blood lymphocytes. Our results show that CD8+ T lymphocytes obtained from peripheral blood of symptomatic HIV-infected patients can be purified, cultured to obtain large numbers of cells with enhanced anti-HIV activity, and safely infused into patients with AIDS as a form of immunotherapy.

The regulation of human natural killer (NK) activity by IgG described previously by us depends on the ability of cytophilic molecules of monomeric IgG (mIgG) to inhibit the subsequent killing of NK-sensitive targets. Highly purified NK cells obtained from human peripheral blood are able to directly bind mIgG as well as antigen-complexed IgG through its Fc region. The demonstration that NK cells bear labile cytophilic IgG, a property which usually has been attributed to L cells, indicates that mIgG-induced inhibition of NK activity is mediated by direct interactions between the inhibitory ligand and cytotoxic effector cells. The Fc receptor (FcR) mediating downregulation of NK cytotoxicity appeared to be FcR gamma III, previously found to be selectively expressed on NK cells and granulocytes. In studies of unidirectional cross-inhibition of mIgG binding to NK cells by various anti-CD16 monoclonal antibodies, binding characteristics of mIgG or complexed IgG were similar. Thus, the FcR gamma III for mIgG appear to be indistinguishable from receptors responsible for binding of polymeric IgG on human NK cells. The negative regulation of NK activity by mIgG was not attributable to inhibition of conjugate formation between effector cells and K532 targets, but rather to inhibition of a post-binding event involved in killing of conjugated targets. The data presented suggest that the Fc gamma RIII on human NK cells can either mediate killing against IgG antibody-coated target cells or, upon interaction with cytophilic monomeric ligand in soluble form, induce inhibition of NK activity.

Human cytotoxic T-lymphocyte (CTL) lines with specificity restricted for autologous squamous cell carcinoma of the head and neck (SCCHN) were established from peripheral blood lymphocytes obtained at the time of surgery and again at two different times after surgery from a patient with cancer of the tongue. The CTL lines were cultured in the presence of interleukin (IL) 2, IL4, and autologous tumor (AuTu) cell monolayers. All three lines were CD3+CD8+CD11b-HLA-DR+ T-cell receptor alpha/beta+. They were tested in 4-h51Cr release assays against SCCHN cell lines (n = 5) and a variety of nonsquamous human tumor (n = 5) and normal (n = 5) cell targets and was found to lyse only AuTu (PCI-50) and three allogenic SCCHN cell lines. Lysis of AuTu and the three allogenic SCCHN targets by the established CTL lines appeared to be major histocompatibility complex class I restricted, since it was blocked by monoclonal antibodies to class I histocompatibility complex antigens. The CTL lines proliferated in vitro in response to autologous PCI-50 or an allogenic SCCHN cell line (PCI-1). The lines have been maintained in culture in the presence of AuTu monolayers and retained cytotoxicity against AuTu for over 20 weeks. The AuTu (PCI-50) cell line was tested for in vitro sensitivity to cytotoxic or cytostatic effects of various effector cells, including the CTL lines. PCI-50 targets were resistant to lysis by resting human mononuclear cells but sensitive to IL2-activated natural killer cells in 4-h 51Cr release assays. In comparison with IL2-activated natural killer cells, the CTL line mediated lower levels of lysis against AuTu. Growth of PCI-50 cells in culture was significantly inhibited by a combination of gamma-interferon and IL2 or by high concentrations of tumor necrosis factor alpha. While supernants of IL2-activated natural killer cells were growth inhibitory, those of the CTL line were not. On the other hand, lysis of AuTu targets by the CTL line was increased by preincubation of the tumor cells with tumor necrosis factor alpha or gamma-interferon. These cytokines augmented expression of HLA-class I, HLA-class II, and intercellular adhesion molecule I, but not squamous cell carcinoma-associated antigens, E7 and A9, on PCI-50 cells. The CTL lines described are the first with restricted specificity for autologous SCCHN ever reported and their availability will facilitate studies of the AuTu T-cell response in head and neck cancer.

Natural killer (NK) cells selected by IL-2-induced rapid adherence to plastic and called A-NK cells represent a phenotypically and functionally distinct subset of mature peripheral blood NK cells. To further characterize this subset of NK cells functionally, their potential to express mRNA for the IL-2R and various cytokines after IL-2 activation was examined. Highly purified normal human peripheral blood resting NK (R-NK) cells were obtained by negative immunoselection using OKT3 mAb and magnetic beads coated with goat anti-mouse Ig. By two-color flow cytometry, > 90% of these R-NK cells were either CD3-CD56+CD16+ or - or CD3-CD56-CD16+. R-NK cells were activated in the presence of 6000 IU/ml (22 nM) of IL-2 for different periods of time. After 1, 3, 5, or 24 h, plastic-adherent (A) and nonadherent (NA) NK cells were separated and compared for the expression of the IL-2R or cytokine mRNA by in situ hybridization, using 35[S]-cDNA probes. Only low proportions of R-NK cells expressed genes for IL-2Rp55 (16%) or cytokines IL-2 (20%), IFN-gamma (18%), TNF-alpha (16%), and TGF-beta (7%). Thus, the genes for the IL-2Rp55 and these cytokines were not constitutively expressed by most human R-NK cells, and there was no indication that the NK cells used in these experiments were activated in vivo or during the purification procedure. However, larger proportions of R-NK cells showed expression of mRNA for IL-1-beta (35%) and IL-6 (40%), which indicates that genes for these cytokines may be constitutively expressed in a substantial proportion of normal human circulating NK cells. When R-NK cells were incubated in the presence of 22 nM of IL-2 for 1 to 24 h and separated into A-NK cells and NA-NK cells, a large proportion of A-NK cells became positive for IL-2R and cytokine gene expression. In contrast, the proportion of mRNA-positive NA-NK cells was similar or lower than that observed for R-NK cells, with the exception of an increase in TGF-beta.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin 2 (IL2) was injected peritumorally and intranodally in 36 patients with unresectable squamous cell carcinoma of the head and neck enrolled in an Eastern Cooperative Oncology Group-sponsored phase Ib trial (EST P-Z388). Groups of 6 patients received escalating doses(200, 2 x 10(3), 2 x 10(4), 2 x 10(5), 2 x 10(6), and 4 x 10(6) units) of IL2 daily 5 times/week for 2 weeks. Tumor biopsies were obtained before and after IL2 therapy. Tumor tissue was provided for histology, and the remaining fresh tissue was divided for snap-freezing in -75 degrees C and for separation of tumor-infiltrating lymphocytes (TIL) and tumor cells. Immunophenotyping of TIL performed on cryostat sections of paired pre- and post-IL2 biopsy tissues showed increases after IL2 therapy in the number of T-cells (P = 0.005), natural killer (NK; CD16+) cells (P = 0.0001), CD25+ cells (P = 0.004), and HLA-DR+ cells (P = 0.001) accumulating in the tumor stroma. In the tumor parenchyma, NK cells (P = 0.0001) and HLA-DR+ cells (P = 0.003) were increased after IL2 therapy. The T:NK cell ratios in the tumor stroma and parenchyma were decreased after therapy, suggesting selective accumulation of NK cells. By flow cytometry, TIL recovered from post-IL2 biopsy tissues were enriched (P0.05) in CD3-CD56+ (NK) cells. In situ hybridization with [35S] cDNA probes for cytokines and IL2 receptors indicated that the numbers of cells expressing mRNA for IL2, tumor necrosis factor alpha, IL1-beta, gamma-interferon, transforming growth factor beta, and IL2 receptor p55 or p70 were increased in post-IL2 biopsy tissues as compared to pre-IL2 tissues. Cytolytic activity of TIL isolated from post-IL2 tissues was also increased, as determined in 4-h 51Cr release assays against K562 targets (12 +/- 3 mean lytic units/10(7) cells +/- SEM pre-IL2 versus 46 +/- 13 post-IL2; n = 16) and against autologous tumor (13 +/- 8 versus 68 +/- 26; n = 9). Fresh TIL of one clinical responder showed relatively high levels (195 lytic units) of autotumor cytotoxicity after IL2 therapy versus no activity prior to therapy. In the blood, NK and lymphokine-activated killer cell activity, and percentages of CD3-CD56+ NK cells and of activated (CD25+) T-lymphocytes were increased for all doses of IL2.(ABSTRACT TRUNCATED AT 400 WORDS)

Peritumoral injection of human IL-2-activated natural killer cells into nude mice consistently induced regression of xenografts of human squamous cell carcinoma of the head and neck (SCCHN). To determine the mechanisms responsible for the tumor regression, the lymphoid cells infiltrating the tumor stroma at 24 to 48 h after adoptive immunotherapy were examined by in situ hybridization for the presence of mRNA for cytokines or IL-2R. Numerous lymphoid cells expressing cytokine or IL-2R genes were observed in these tumors, whereas the cultured IL-2-activated NK cells used for therapy were negative. Thus, it appeared that the transferred NK cells became activated in situ after coming into proximity with the tumor cells. To analyze this phenomenon, fresh or cultured human NK cells were coincubated in vitro with irradiated human SCCHN cell line, PCI-1, with or without the presence of IL-2. Expression of mRNA for IL-2R, perforin, and various cytokines was observed within 5 h. Contact with the tumor cells stimulated NK cells to proliferate, secrete IFN-gamma, TNF-alpha, and soluble IL-2R, up-regulate cell surface expression of IL2R p55 and p75 as well as CD16 Ag, and mediate higher levels of antitumor activity in 51Cr-release assays. In addition, supernatants of in vitro-activated NK cells significantly inhibited proliferation of SCCHN cell lines. By examining the effects of neutralizing mAb to various cytokines, this inhibitory activity was shown to be partially attributable to IFN-gamma. To determine the possible in vivo role of soluble factors produced by activated human NK cells, the supernatants (0.2 ml) or rIFN-gamma (10(5) U) were injected perilesionally each day for 2 wk into 3-day SCCHN established in immunosuppressed nude mice. These treatments caused significant (p less than 0.02) inhibition of tumor growth. The results of our studies indicate that human NK cells are strongly activated by SCCHN cells and that the consequent release of cytokines contribute to the regression of SCCHN growing in nude mice.

Development of effective local immune responses depends on the ability of lymphocytes to extravasate and migrate into nonlymphoid solid tissues. Different lymphocyte subpopulations seem to vary in their abilities to extravasate. In this review, recent advances made in understanding lymphocyte extravasation, interactions between lymphocytes and vascular endothelial cells, receptors on lymphocytes which are involved in their movement through the extracellular matrix, and cytokines, which regulate lymphocyte mobility through tissue, are described. In pathologic conditions, lymphocyte extravasation may be compromised, and delivery of immune effector cells to the site of injury is an important therapeutic end point. Adoptive therapy of solid tumors with antitumor effector cells depends on their successful delivery to the tumor. Although parameters which determine this delivery are not yet defined, considerable progress has been made in studies of the mechanisms involved in lymphocyte movement through tissues.

Three autotumor-reactive T-cell clones have been established from tumor-infiltrating lymphocytes isolated from a metastatic lesion of human gastric carcinoma in the liver. The clones all were shown to be CD3+, CD8+, CD4-, CD16-, T-cell receptor alpha/beta +, and T-cell receptor gamma/delta-, and they have retained both their autotumor reactivity and the same phenotype for over a year in culture. Each clone had a different rearrangement of the T-cell receptor gamma chain genes as indicated by Southern blot analysis. Tested against a panel of 18 tumor cell targets, the clones preferentially lysed autologous tumor (AuTu) cells, but each clone also showed weak cytotoxicity against one allogeneic cholangiocarcinoma cell line. At the same time, each clone showed appreciable cytotoxicity against K562 targets. In blocking experiments, anti-CD3, anti-WT31, anti-CD8, or anti-HLA class I monoclonal antibodies blocked AuTu cytotoxicity but not cytotoxicity against K562. In contrast, allocytotoxicity against the cholangiocarcinoma was blocked only by anti-CD3 monoclonal antibody. All 10 subclones of one T-cell clone had high levels of AuTu cytotoxicity but variable levels of anti-K562 cytotoxic activity. Proliferation of the T-cell clones was significantly stimulated by the addition of irradiated autologous but not allogeneic tumor cells. Preincubation of cultured AuTu cells with tumor necrosis factor alpha or gamma-interferon (IFN-gamma), but not with IFN-alpha, increased their susceptibility to lysis by the T-cell clones; however, it increased resistance of AuTu to lysis by interleukin 2-activated natural killer cells. The expression of an adhesion molecule, intercellular adhesion molecule 1, on the surface of AuTu was also up-regulated by tumor necrosis factor alpha or IFN-gamma, but not by IFN-alpha. All three cytokines up-regulated HLA-class-I antigens on AuTu. Pretreatment of K562 targets or allogeneic cholangiocarcinoma cells with the same cytokines increased their resistance to lysis by the T-cell clones. Overall, the results indicate that these T-cell clones show specificity for AuTu but also independently recognize a limited number of allogeneic tumor targets and lyse K562 targets. The mechanisms involved in the recognition by the T-cell clones of autologous, allogeneic, and K562 tumor targets appeared to be distinct.

Immunotherapy with tumor-infiltrating lymphocytes (TIL) activated in the presence of recombinant interleukin 2 (IL2) in vitro and adoptively transferred to patients with metastatic melanoma or renal cell carcinoma has resulted in complete or partial responses in some cases. These results have generated a wave of optimism and expectations, which may be premature. Much has been learned about TIL biology and their functional characteristics recently, but only few clinical trials have been completed to date. In this review, therapeutic potential of human TIL is evaluated based on limited knowledge of the antitumor mechanisms involved and imperfect understanding of events which occur during systemic administration of TIL. Limitations and advantages of TIL therapy are discussed and approaches to optimizing this form of therapy which are likely to be implemented in the future are summarized.

Liver-derived lymphocytes were isolated from 40 human livers with end-stage liver disease that were removed at the time of orthotopic liver transplantation. In addition, 10 resection specimens or whole livers removed from patients with liver cancer and seven normal livers (unused donor organs) were studied as controls. Liver-derived lymphocytes were isolated from enzymatically digested tissue by gradient centrifugation and adherence to plastic. Their phenotypical characteristics were studied by two-color flow cytometry, and effector cell function was determined in 4-hr 51Cr-release assays against a natural killer-sensitive target, K562 (natural killer activity), natural killer-resistant Daudi line (lymphokine-activated killer activity) and by P815 line with or without phytohemagglutinin to assess lectin-dependent cellular cytotoxicity. Liver-derived lymphocytes isolated from normal liver contained equal proportions of T and natural killer lymphocytes (mean natural killer/T ratio = 0.7). CD3-CD56+CD16- natural killer cells were the main natural killer subset present in liver-derived lymphocytes, in contrast to the predominant natural killer phenotype in the circulation (CD56+CD16+). Control liver-derived lymphocytes had levels of cytotoxicity significantly greater than those of the normal peripheral-blood lymphocytes against all three targets. In contrast, liver-derived lymphocytes isolated from organs with advanced liver disease differed markedly in the natural killer/T cell ratio and levels of liver-derived lymphocyte cytotoxicity. Liver-derived lymphocytes obtained from hepatocellular carcinoma or rejecting allografts treated by immunosuppressive therapy had virtually no cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

To study antitumor functions of T-lymphocyte subpopulations in the blood [peripheral blood lymphocytes (PBLs)] and tumor-draining lymph nodes (LNs) of patients (n = 26) with squamous cell carcinoma of the head and neck (SCCHN), antibody-coated devices were used to positively select CD8+ or CD4+ cells. The mean percentage of CD8+ cells captured on antibody-coated flasks from PBLs was 92% and that captured from lymph node lymphocytes (LNLs) was 98%. The initial enrichment in CD4+ T-cells was comparable. CD8+ T-lymphocytes captured from PBLs proliferated as well as unseparated lymphocytes in both patients with SCCHN and normal donors, while captured CD4+ PBLs of the patients showed significantly lower expansion than those of normal volunteers. Unseparated LNLs proliferated as well as PBLs, but captured CD4+ or CD8+ LNLs failed to proliferate in the presence of interleukin 2 (100 units/ml) and phytohemagglutinin (5 micrograms/ml). The addition to captured LNL cultures of irradiated autologous or allogeneic feeder cells significantly improved expansion of CD8+ LNLs but not CD4+ LNLs. During 15-day culture of captured CD8+ PBLs or CD8+ LNLs in the presence of feeder cells, a significant (P less than 0.05) enrichment in CD8+ T-cells was maintained [94 +/- 5% (mean +/- SEM) or 99.5 +/- 0.1%, respectively, on day 15]. Capture of CD8+ LNLs and their expansion resulted in the outgrowth of CD8+CD11b- effectors which had no or little cytotoxicity against Daudi, low cytotoxicity against K562, and very high levels of cytotoxicity against 4 different natural killer cell-resistant SCCHN targets, as measured in 4-h 51Cr release assays. Such significant enrichment in SCCHN-restricted cytotoxicity could be obtained with LNLs from tumor-uninvolved LNs but not from tumor-involved LNs. Captured and cultured CD4+ LNLs had no preferential anti-SCCHN cytotoxicity. The addition of irradiated autologous tumor cells to captured CD8+ PBLs did not result in improved proliferation or antitumor function of the effector cells. Positive selection on antibody-coated flasks of CD8+ T-lymphocytes from tumor-uninvolved LNs of patients with SCCHN led to the enrichment in SCCHN-restricted but the major histocompatibility complex-unrestricted effector cells in 15-day cultures. Thus, CD8+ lymphocytes separated from tumor-draining LNs in patients with head and neck cancer contained cytolytic T-cell precursors capable of developing into effectors with preferential activity against SCCHN targets.

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation.

The efficacy of local adoptive immunotherapy with human lymphokine-activated killer cells and recombinant interleukin 2 (rIL-2) in growth inhibition of established squamous cell carcinoma of the head and neck (SCCHN) was evaluated in a nude mouse model. The model of xenografted SCCHN was established by s.c. injections of in vitro maintained tumor cells (2-10 x 10(6) cells/mouse) into the flank of splenectomized animals pretreated with cyclophosphamide (200 mg/kg). The SCCHN line used was tumorigenic in 95% of the appropriately conditioned nude mice. Inhibition of tumor growth by locally administered effector cells was the end point of the study, since the tumors did not metastasize within 6 weeks of tumor challenge. Either i.p. or local administration of rIL-2 alone (1000 units/day) to the tumor site daily for 2 weeks resulted in a significant inhibition of tumor growth. In the absence of detectable natural killer activity in these mice, a modest dose of rIL-2 had a direct antitumor effect on SCCHN cells in vivo. In addition, complete inhibition of tumor growth was achieved with 3 times weekly injections of 5-10 x 10(6) lymphokine-activated killer cells delivered to the tumor site and 1000 units of rIL-2 administered locally every day for 2 weeks. Our data indicate that local or systemic immunotherapy with rIL-2 alone or local adoptive immunotherapy with an adequate dose of lymphokine-activated killer cells plus rIL-2 may be effective in preventing the growth of established SCCHN tumors in vivo.

The human natural killer (NK) cell has long been known to mediate spontaneous, non-major histocompatibility complex-restricted cytotoxicity and antibody-dependent cellular cytotoxicity. It is only fairly recently, however, that the role NK cells play in other functions of the immune system has been realized. The NK cell participates either directly or indirectly in multiple developmental, regulatory and communication networks of the immune system and thus is important in human health and disease. The NK cell has distinct morphologic, phenotypic and ultrastructural characteristics that distinguish it from T and B lymphocytes. Human NK cells are heterogeneous, and functionally different subpopulations of NK cells can be distinguished. NK activity may be regulated by soluble products of hematopoietic as well as non-hematopoietic cells and by a wide variety of exogenous biological response modifiers. Both lymphocytes and monocytes are capable of regulating of NK cell growth and activity. Populations enriched in human NK cells can be obtained utilizing the property of adherence to plastic and subsequent expansion in the presence of IL-2. The adherent lymphokine activated killer (A-LAK) cells represent populations of CD3-CD56+ (up to 98%) IL-2 activated NK cells. They are highly effective in eliminating tumor cells both in vitro and in vivo in animal models of tumor metastasis. A-LAK cells are being used for therapy in the phase I clinical trial in patients with metastatic melanoma and renal cell carcinoma. The studies described emphasize the biologic importance of the NK cell, its therapeutic potential, and a need for more extensive monitoring of NK activity in human disease.

Monoclonal antibodies to antigens on the surfaces of mononuclear cells (MNC) were used to characterize lymphocyte subpopulations infiltrating portal areas and parenchyma of livers in 31 patients with chronic active hepatitis (CAH). The distribution and numbers of infiltrating lymphocytes were determined in serial sections immunostained by the avidin-biotin-peroxidase complex method. T lymphocytes were the major component of inflammatory cells in the portal tracts. In the peripheral blood and portal areas, T helper-inducer (T4+) cells were the more numerous subpopulation. However, the hepatic lobules and areas of "piecemeal" necrosis always contained more T suppressor-cytotoxic (T8+) cells. The latter were demonstrated in contact with HBsAg-containing hepatocytes in tissues of patients with HBsAg-positive CAH. The mean numbers of T lymphocytes infiltrating the portal and periportal areas of livers from patients with HBsAg-negative and HBsAg-positive CAH were not different. Large numbers of B cells forming distinct follicles were seen in tissues from patients with HBsAg-positive CAH. The presence of increased numbers of portal T and B lymphocytes correlated with progressive liver damage as observed in two patients studied at yearly intervals.

Tumor-infiltrating lymphocytes (TIL) were obtained from 22 humans with solid tumors. In three cases only, one colon and two lung carcinomas, TIL which contained from 3 to 10% of T cells expressing the interleukin 2 receptor (IL 2R) were obtained, and these proliferated in the presence of exogenous IL 2. In most TIL preparations, however, the T lymphocytes did not express the IL 2R and failed to proliferate in response to IL 2. In contrast, TIL were able to proliferate in response to irradiated allogeneic spleen cells in mixed lymphocyte culture. Proliferative responses of autologous PBL were not inhibited by the addition of TIL. In most tumors, the TIL showed no response or had significantly lower (p less than 0.01) responses to PHA, Con A, and the phorbol ester TPA than did autologous peripheral blood lymphocytes (PBL). A limiting-dilution microculture system which allows clonal growth of every T cell was used to demonstrate decreased responses of the TIL to PHA at a single-cell level. In contrast to normal PBL-T with proliferating frequencies from 0.46 to 1.0, those for T cells in three TIL preparations were zero, 0.005, and 0.01. Normal PBL exposed in vitro to tumor cells or their supernatants lost the ability to respond to mitogens and to clone normally (e.g., proliferating frequency of 0.147 vs 0.863 in control). The TIL isolated from solid tumors resemble normal PBL exposed in vitro to tumor cells or their supernatants in terms of decreased responses to mitogens and poor clonogenicity in the PHA-dependent microculture system. It is possible that tumor cells may inhibit certain functions of the TIL in human solid tumors.

Human peripheral blood lymphocytes grown in vitro were stimulated with nonspecific mitogens and in mixed lymphocyte culture. The presence of IgM and thymus (T) surface markers on large and small lymphocytes was investigated by immunofluorescence and correlated with spontaneous rosette formation. All stimulated large lymphocytes formed spontaneous rosettes and all except pokeweed mitogen (PWM)-stimulated large lymphocytes had IgM and T markers. IgG, IgA, and light chain determinants were only detected on PWM-induced large lymphocytes. Thus, surface markers expressed on activated human lymphocytes may differ for different mitogens. IgM was always present on large cells which had the T markers, and it cannot be used to identify a lymphocyte as a bone marrow-derived (B) cell. Due to the overlap of surface markers, the classification into B and thymus-derived (T) cells ought to be restricted to functional phenomena of antibody-production or cell-mediated immunity.

Twenty failed human liver allograft specimens obtained at the time of retransplantation procedures were studied using a panel of monoclonal antibodies (T11, T4, T8, NK, B1, OKM1, OKM5, Ia, DR). A clinicopathologic analysis was used to distinguish between graft failures secondary to rejection (n = 10) and those due, at least in part, to other causes (n = 10). T lymphocytes constituted the major infiltrating cellular population in the liver in rejection cases, but significant numbers of B cells and monocytes/macrophages were present also. Following transplantation, but not before, the bile duct epithelium, as well as portal and central vein and hepatic artery endothelium expressed DR/Ia antigens. These structures are preferential targets of the rejection reaction. The selective destruction of bile ducts in livers undergoing rejection was manifested in these patients by striking elevations of serum gamma glutamyl transpeptidase (GGTP) activity, a marker of biliary epithelial damage. The induced expression of DR/Ia antigens on structures targeted for immune destruction may be an important event in the pathogenesis of liver allograft rejection.

The distribution and numbers of human natural killer (NK) cells in different lymphoid tissues and several tumors were determined with the use of monoclonal antibody to the Leu-7 antigen and immunohistologic methods. Anti-Leu-7 reacts with large granular lymphocytes containing most of NK activity in the peripheral blood. The Leu-7+ cells were found mainly in the germinal centers of secondary follicles in lymph nodes, spleens, and tonsils. The mean number of Leu-7+ cells in 40 germinal centers was 223 +/- 103 SD. Only rare cells were found in the thymus. In the follicles studied in serial sections, the distribution of the Leu-7+ cells was distinctly different from that of T cells, B cells and Ia+ cells. The Leu-7+ cells did not react with OKT 6 antibody specific for immature (control) thymocytes and Langerhans' cells. By double staining techniques, two populations of Leu-7+ cells were identified in lymphoid tissues: those that did and did not express the Ia-like antigen. The numbers and localization of the Leu-7+ cells varied in different tumors, but often the Leu-7+ cells surrounded the neoplastic nodules or were seen in contact with individual cancer cells. Anti-Leu-7 may be a useful reagent for monitoring the presence and localization of NK cells in normal and malignant human tissues.

The feasibility of in vitro interleukin 2 (IL-2) activation and expansion of mononuclear cells (MNCs) derived from adult patients with acute myelogenous leukemia (ANLL) was studied. Patients' natural killer (NK) and lymphokine-activated killer (LAK) cell activity was compared with that of normal donors in terms of: (a) cytolytic activity (four- hour 51Cr release assay) against an NK-sensitive target (K562), NK- resistant targets (Raji/Daudi), and fresh/cryopreserved autologous and allogeneic leukemic blasts; (b) proliferation and expansion in culture with 1,000 U/mL recombinant IL 2 (rIL 2); and (c) the cell surface phenotype of the cultured cells. In 21 of 24 patients with active disease (AP) MNCs derived from the peripheral blood (PBL) or bone marrow (BM) could be cultured and expanded in the presence of rIL 2. These cultures initially contained between 30% and 50% blasts, and during 2 to 4 weeks of culture destruction of blasts and enrichment of up to 60% in cells with the morphology of large granular lymphocytes (LGLs) was observed. Expansion in culture varied between two- and 100- fold. MNCs from all patients in remission (RP) could be activated by rIL 2 and expanded up to 30-fold after 1 to 3 weeks in culture. NK activity of fresh PBLs from AP was significantly lower than in normal controls, whereas NK activity of RP was within the normal range. High levels of postactivation NK and LAK activity on K562/Raji/Daudi and on fresh/cryopreserved leukemic blasts was generated in approximately 50% of cases of AP and in most RP. Cell surface phenotype studies showed that cultured cells derived from ANLL patients were significantly enriched (up to 40%) in NKH-1 (Leu 19) positive cells, with RP LAK cells also expressing a high proportion of CD16 positive cells (up to 40%). This study has shown that it is feasible to activate and significantly expand killer cells derived from active disease and remission ANLL patients during 1 to 3 weeks culture with IL 2 with good maintenance of cytolytic activity. Both initial NK activity and LAK generation was optimal in remission patients. Based on data from this study, a clinical protocol has been developed for treatment of early relapse ANLL patients with LAK cells cultured for 1 to 3 weeks and systemic IL 2.

The colony-stimulating factors (CSFs) promote the proliferation and differentiation of hematopoietic precursors and more recently have been shown to amplify the functions of mature phagocytes in vitro. In this study recombinant human granulocyte/macrophage colony-stimulating factor (rGM-CSF) was administered to cancer patients to determine whether the cytotoxic and secretory activity of their blood monocytes could be enhanced. Patients with refractory neoplastic disease were treated with rGM-CSF either as a single bolus or as a constant infusion for 14 days at either 100 or 500 micrograms/m2 per day. As has been reported by others, the number of peripheral blood monocytes and granulocytes rose markedly in a dose-response fashion during infusion with rGM-CSF. The functional capacity of monocytes was increased by rGM- CSF, since the cytotoxicity of monocytes against antibody-coated xenogeneic cells was increased during the constant infusion compared to baseline. In addition, monocytes harvested during the constant infusion and stimulated with lipopolysaccharide (LPS) in vitro secreted increased quantities of tumor necrosis factor alpha (TNF-alpha) and interferon (IFN). These data indicate that rGM-CSF can enhance both the number and the function of peripheral blood monocytes in vivo.

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.

Lymphocyte subpopulations in the intestinal tissues of seven patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) were studied by immunohistologic technics at two different locations, the small bowel and the rectum. Intraepithelial and lamina propria lymphocyte subsets stained with monoclonal antibodies T11, T4, T8, and B1 were enumerated in the patients and different normal and patient control groups. Intraepithelial T11+ cells were decreased (P less than 0.05) in the small bowel of AIDS and ARC patients, primarily because of the near complete absence of T4+ lymphocytes. In the lamina propria of these patients, a depletion of T4+ cells (P less than 0.05), an increase in T8+ cells (P less than 0.05), and a reversal of the T4/T8 ratio were observed (e.g., the small bowel ratio was 0.1 +/- 0.02 vs. the normal ratio of 2.3 +/- 0.2 and the rectal ratios were 0.2 +/- 0.06 vs. normal 2.6 +/- 0.3). The T-lymphocytes in the intestine of AIDS and ARC patients did not express the receptor for interleukin-2 (IL-2). A near complete absence of T4+ lymphocytes was also seen in lymphoid follicles in the rectum. B1+ cells were not depleted. The reversal of the T4/T8 ratio, which is a hallmark of AIDS, occurs not only in the circulation but also in the gastrointestinal tissues of patients with AIDS and ARC.

Tumor-infiltrating lymphocytes (TIL) were obtained from 22 humans with solid tumors. In three cases only, one colon and two lung carcinomas, TIL which contained from 3 to 10% of T cells expressing the interleukin 2 receptor (IL 2R) were obtained, and these proliferated in the presence of exogenous IL 2. In most TIL preparations, however, the T lymphocytes did not express the IL 2R and failed to proliferate in response to IL 2. In contrast, TIL were able to proliferate in response to irradiated allogeneic spleen cells in mixed lymphocyte culture. Proliferative responses of autologous PBL were not inhibited by the addition of TIL. In most tumors, the TIL showed no response or had significantly lower (p less than 0.01) responses to PHA, Con A, and the phorbol ester TPA than did autologous peripheral blood lymphocytes (PBL). A limiting-dilution microculture system which allows clonal growth of every T cell was used to demonstrate decreased responses of the TIL to PHA at a single-cell level. In contrast to normal PBL-T with proliferating frequencies from 0.46 to 1.0, those for T cells in three TIL preparations were zero, 0.005, and 0.01. Normal PBL exposed in vitro to tumor cells or their supernatants lost the ability to respond to mitogens and to clone normally (e.g., proliferating frequency of 0.147 vs 0.863 in control). The TIL isolated from solid tumors resemble normal PBL exposed in vitro to tumor cells or their supernatants in terms of decreased responses to mitogens and poor clonogenicity in the PHA-dependent microculture system. It is possible that tumor cells may inhibit certain functions of the TIL in human solid tumors.

Immunologic characterization of the neoplastic cells in the circulation of patients with CLL suggests these cells show significant differences in membrane characteristics from normal B lymphocytes. Although the leukemic cells bear a homogenous membrane-associated immunoglobulin, they also react with an anti-human T cell serum. In all patients studied, 60-90% of the cells, were stained by this antiserum. This suggests that the leukemic cells share antigenic determinants with T lymphocytes. CLL cells, unlike normal B cells, showed a marked increase in mouse-complement receptors. No increase in receptors for guinea pig complement was observed in the leukemic cells. The population of SIg-bearing lymphocytes was significantly greater than that of complement-receptor bearing lymphocytes. The total number of E-rosetting cells was increased in all CLL patients. Mitogenic responses of the leukemic cells were depressed and delayed. These results suggest that neoplastic lymphocytes cannot be classified as T- or B-derived on the basis of criteria used to define normal lymphocytes.

A xenograft model of local adoptive immunotherapy would be useful in evaluating the therapeutic effects both cytokines with and without added effector cells have on human head and neck tumors implanted into nude mice. Human squamous cell carcinomas of the head and neck lend themselves to local intervention and, since conventional radio- and/or chemotherapy often fail to arrest tumor growth, new therapeutic modalities, including immunotherapy, must be considered and local immunotherapy may have the advantage of minimal side effects. In the present study the ability of local adoptive immunotherapy with human lymphokine-activated killer cells (LAKc) and recombinant interleukin-2 (IL-2) to inhibit the growth of established squamous cell carcinoma of the head and neck was evaluated in nude mice. The model was established by subcutaneous injections of in vitro maintained tumor cells into the flank splenectomized animals pretreated with cyclophosphamide and anti-asialo GM1. The line used was tumorigenic in 95% of the nude mice and neither metastasized nor killed the animals. The local daily administration of IL-2 to the tumor site for two weeks resulted in a significant inhibition of tumor growth. Complete inhibition of tumor growth was achieved with three times weekly injections of 5 to 10 x 10(6) LAKc delivered to the tumor site and 1000 U of IL-2 administered locally every day for two weeks. The data obtained indicate that local adoptive immunotherapy with IL-2 and an adequate dose of LAKc may be effective in preventing the in vivo growth of established squamous cell carcinomas of the head and neck.

Tumor-infiltrating lymphocytes (TIL) from 16 squamous cell carcinomas of head and neck (SCCHN) and four nonsquamous cell carcinomas were studied. By immunoperoxidase staining in situ, the tumors studied were found to be infiltrated mainly by CD2+CD3+ cells, and 30-50% of the T-lymphocytes were HLA-DR positive and transferrin-receptor positive. They also contained scarce NKH1+ cells. When TIL as well as autologous peripheral blood lymphocytes (A-PBL) were cultured in 1,000 U/ml of recombinant interleukin 2 (rIL2), TIL proliferated in all but three cases, and A-PBL proliferated in all but two cases. Frequently, but not always, TIL expanded better than A-PBL. The median expansion for TIL was 100-fold and that for A-PBL was 31-fold in long-term cultures maintained for up to 88 days. TIL obtained from untreated primary SCCHN were initially delayed for up to 20 days in their proliferative response to rIL2, but then grew well. In contrast, TIL and A-PBL from metastatic SCCHN either did not proliferate or were delayed in their proliferative response for up to 40 or 50 days. A-PBL, when tested early (days 10-20 in culture), showed the highest cytotoxic activity against cultured and fresh tumor-cell targets, whereas TIL were most active later in culture (days 20-30). On a per culture basis, TIL achieved higher antitumor cytotoxicity than A-PBL. By day 80, lytic activities of most TIL cultures declined to undetectable levels. CD3+Leu19- T-lymphocytes were the major expanding cell population in most TIL cultures. However, these cells were poor mediators of antitumor cytotoxicity in TIL or A-PBL cultures as shown in cell sorting experiments. The antitumor effector cells expressed CD3-Leu19+ and/or CD3+Leu19+ phenotypes. On Giemsa-stained smears, these two types of IL2-expanded effector cells had the morphology of large granular lymphocytes. Our results indicate that TIL from human SCCHN could be expanded and reach high levels of antitumor effector function in long-term cultures with rIL2.

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.

A procedure for enrichment in recombinant interleukin-2 (rIL2)-activated natural killer (NK) cells was developed and used for in vitro generation of antitumor effector cells from the peripheral blood of 20 patients with central nervous system (CNS) tumors. In comparison to the patients' unseparated mononuclear cells and nonadherent lymphocytes cultured in the presence of 1000 U/ml of rIL2 for up to 3 weeks, interleukin-2-stimulated lymphoid cells, when purified by adherence to plastic, proliferated better (up to 6,720-fold expansion) and achieved up to five times higher levels of antitumor activity against K562 cell targets and NK-resistant glioblastoma cell targets. Two-color flow cytometry analysis showed that cultures of cells purified by adherence to plastic which had the best proliferation contained 10% or less of CD3+Leu19- T-lymphocytes, while the unseparated lymphokine-activated killer cell cultures which proliferated poorly contained up to 85% of CD3+Leu19- T-cells. Cultures of adherent lymphocytes which reached the highest antitumor cytotoxicity were enriched in CD3+Leu19+ effectors (60-80%); the proportion of CD3-Leu19+ NK-cells was not greater than 25% in these cultures. Thus, using the technique of 24- or 48-h activation in rIL2 and adherence to plastic, and in contrast to the results obtained with cells from normal donors, it was not possible to enrich in activated NK cells from the blood of patients with CNS tumors. Instead of activated NK cells, a population enriched in non-major histocompatibility complex-restricted cytotoxic T-cells (CD3+Leu19+) was obtained in cultures from most but not all patients. Low NK cell activity and elevated numbers of circulating CD3+Leu11+ cells seen in the blood of these patients, previously treated by surgery/radiation/chemotherapy and maintained on steroids, could be responsible for the preferential adherence and subsequent expansion to plastic of IL2-activated non-major histocompatibility complex restricted cytotoxic T-lymphocytes.

Typing at the HLA-DR locus was performed in 125 North American white patients with progressive systemic sclerosis (PSS). We could not confirm associations of PSS with the HLA-DR5 antigen or the HLA-B8/DR3 haplotype. A weak association of the DR1 antigen and PSS with diffuse scleroderma (27.5% vs 11.5% in local controls (corrected p less than 0.05) was observed. HLA-DR1 was significantly associated with the presence of anticentromere antibodies (p less than 0.05), and this combination was found more frequently in individuals with the CREST syndrome variant of PSS. This study failed to identify a clear-cut association between PSS and a genetic factor linked to the major histocompatibility complex.

Carcinoembryonic antigen (CEA) determinations were performed in 86 malignant and 103 benign serous fluids submitted for routine cytologic examination. Cytology alone identified 54 (63%) of the malignant fluids. The remaining 32 (37%) had elevated CEA levels but no detectable malignant cells. In nearly half of 67 effusions with CEA greater than 2.5 ng/ml and negative cytology, elevated CEA concentrations were confirmed to be associated with malignancy. Most (92.5%) cytologically positive serous fluids also had elevated levels of CEA. Elevated CEA assay may raise the suspicion of malignancy in serous fluids that are negative cytologically. Increased CEA concentrations alone, however, cannot be used for confirming malignancy because CEA levels greater than 2.5 ng/ ml (mean, 10.5 +/- 8.2) were found in 50% of the fluids proven to be nonmalignant. Fluid CEA was more consistently elevated in tumors of gastrointestinal tract (greater than 25 ng/ml), lung (11.2 +/- 8.9) and breast (11.6 +/- 7.7) than in lymphomas or sarcomas (3.6 +/- 1). Paired plasma and fluid CEA levels may be particularly helpful in confirming a clinical diagnosis of malignancy in cases with negative fluid and biopsy findings.

The T-lymphocyte subpopulations identified by the Fc receptors for IgG (TG cells) and IgM (TM cells) in 12 patients with B-cell chronic lymphocytic leukemia (CLL) were quantitated and studied for functional capabilities in an in vitro assay. The TG cells in patients were elevated in relation to age- and sex-matched normal controls (P less than 0.05) resulting in an altered TM/TG ratio of 1.8 +/- 0.76 in CLL versus 5.9 +/- 3.7 in controls (mean +/- SD, P less than 0.001). Despite this altered of B cells was found to be normal as reflected by the mean helper-suppressor score of 0.77 +/- 0.13 (+/- SEM) obtained for both patients and controls. This unimpaired capacity of the T cells from CLL patients to help normal B cells mature into immunoglobulin-secreting cells indicated that hypogammaglobulinemia and monoclonal serum immunoglobulins in these patients may be a result of either an intrinsic defect in the B lymphocytes or their replacement by a neoplastic clone rather than a defect in the immunoregulatory T cells.

A limiting dilution analysis (LDA) was used to assess the functional profiles of tumor-infiltrating lymphocytes (TIL) recovered from 15 human solid tumors. The microculture system applied in this study has been shown to allow virtually all normal peripheral blood T lymphocytes (PBL-T) to undergo clonal proliferation and was applied to obtain estimates of the frequency of both proliferating and cytolytic cells among the TIL population. A total of 624 microcultures proliferating in the presence of irradiated allogeneic spleen cells and interleukin 2 (IL 2) were expanded for clonal analysis. These TIL microcultures were assessed for surface antigen phenotype, IL 2 production (helper function) and for their cytolytic capabilities against the human erythroleukemic line K562 (natural killer (NK)-like activity) and P815, a mouse mastocytoma line, in the presence of phytohemagglutinin (PHA), i.e., lectin-dependent cell cytotoxicity (LDCC) which allows the detection of cytolytic activity irrespective of the antigenic specificity of the effector cells. Whenever feasible, cytolytic activity against autologous and allogeneic tumor cells was tested. LDA first demonstrated that the proliferative potential was decreased in T lymphocytes infiltrating human solid tumors (approximately 1 in 50 to 1 in 2 proliferating T lymphocyte precursors (PTL-P) in this series) as compared to normal PBL-T (1 in 2 to 1 in 1 PTL-P). The growth pattern in the titration cultures showed a remarkable agreement with the single-hit Poisson model implying that third party cells are unlikely to be involved in the reduced proliferative potential. Quantitative estimates of functional precursors showed that, in spite of reduced proliferative potential, cytolytic T lymphocyte precursors (CTL-P) against unknown antigens (LDCC-reactive) accounted for a considerable part of the microcultures in many cases. The precursor frequency of T lymphocytes with NK-like activity was usually low in situ (with the exception of glioma), whereas it was in the normal range in the patient's autologous PBL-T. In four evaluable cases, quantitative assessment showed that 1 in 200 to 1 in 1000 T lymphocytes from TIL was cytolytic against allogeneic tumor cells, which is in the range of alloreactive cytolytic T lymphocytes (CTL) generated in the mixed lymphocyte culture from normal PBL. Cytolytic activity against autologous target cells could not be quantitatively estimated but out of 88 clones from 4 patients, 3 clones originating from 2 glioma patients showed high lytic values against autologous tumor.(ABSTRACT TRUNCATED AT 400 WORDS)

Lymphocytes from human peripheral blood incubated with interleukin-2 (IL2) develop lymphokine-activated killer (LAK) activity with the ability to kill a wide variety of tumor cells in a non-major histocompatibility complex-restricted manner. Adoptive immunotherapy with LAK cells and IL2 has been reported to lead to a regression of solid tumors in some patients with advanced malignancies. Aiming to improve the effectiveness of clinical adoptive immunotherapy, we developed a procedure for selective enrichment from human blood mononuclear cells (MNC) of IL2-activated antitumor effector cells. These cells, termed adherent LAK (A-LAK) cells because of their characteristic property of adherence to plastic, demonstrated both higher proliferative potential and greater antitumor cytotoxicity than unseparated MNC. Human A-LAK cells represented only 1 to 4% of IL2-activated MNC at 24 h but expanded from 130- to 1100-fold in 20 days. They comprised a population highly enriched in CD3-Leu19+ effector cells with antitumor activity against fresh human solid tumor cells and established cell lines. A-LAK cells retained antitumor activity for up to 14 days when cultured in the presence of IL2. They also mediated antibody-dependent cytotoxicity. Large-scale generation of A-LAK cells from the blood of patients with cancer proved feasible and should yield populations that are effective in vivo at lower doses than those required with unseparated LAK cells. This offers the potential for improving the antitumor effects, reducing the toxicity, and facilitating the administration of adoptive immunotherapy in humans. A Phase I/II clinical trial utilizing A-LAK cells and IL2 in patients with melanoma and renal cell carcinoma is now in progress.

Specific anti-human T-cell serum was prepared in rabbits by multiple subcutaneous injections of human brain homogenates in incomplete Freund's adjuvant. The serum was exhaustively absorbed with human RBCs, lyophilized human liver, lyophilized normal human serum, and peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (CLL). Specificity of the antiserum for human T lymphocytes was tested by indirect immunofluorescence. It stained 70 to 80% of lymphocytes in circulation, 95% of thymus, 27 to 35% of spleen, 5 to 10% of tonsil lymphocytes, and over 90% of phytohemagglutinin-stimulated lymphocytes in vitro. Only T-dependent areas of cryostat-sectioned human lymph nodes stained with the antiserum. It did not stain circulating lymphocytes which formed HEAC rosettes, plasma cells in marrows of multiple myeloma patients or macrophages. After removal of HEAC rosettes by centrifugation in Ficoll-Hypaque, 75% of interface cells formed E rosettes and 65 to 75% stained with the antiserum. The antiserum was used in studies of lymphocytes in chronic and acute lymphocytic leukemias, lymphomas, and other lymphoproliferative diseases. Numbers and distribution in the circulation, spleen and nodes of lymphocytes bearing the T marker were significantly altered in patients with these disorders.

Mononuclear cells (MNC) present in the dermis of the forearm and in the blood of patients with progressive systemic sclerosis (PSS) were quantified and analyzed for subsets using monoclonal antibodies. The findings were correlated with cutaneous and systemic features of the disease. Total T lymphocytes and their subsets, B cells, and macrophages were enumerated in the skin samples of 21 patients with PSS. The dermal MNC infiltrates consisted mostly of activated T lymphocytes with a mean T helper/T suppressor (T4/T8) ratio of 2.4 +/- 1.3 SD. Few B1-positive or T6-positive cells (macrophages) were observed. There was no correlation between the skin or blood T4/T8 ratios and the degree of skin thickening. On histologic examination, 58 of 115 (50%) untreated patients with PSS had prominent dermal MNC infiltration. Significant correlations between the degree of MNC infiltration and both the degree (P less than 0.05) and progression (P less than 0.05) of skin thickening were observed. No correlations with other systemic disease features of PSS were noted. These results suggest that cutaneous T lymphocytes may play a role in mediating dermal sclerosis in PSS.

TIL were isolated from human solid tumors and cultured in rIL 2. Long-term TIL lines from primary tumors showed a shorter lag period, better expansion, and higher anti-tumor activity in vitro than those from metastatic tumors. The anti-tumor effector cells in TIL and A-PBL cultures had CD3+ Leu 19+ and CD3- Leu19+ phenotypes and morphology consistent with LGL/LAK cells. These cells mediated lysis of both autologous and allogeneic fresh tumor cell targets, while CD3+ Leu19- cells had little or no antitumor cytotoxicity.