Lectin-dependent Cellular Cytotoxicity Research Articles

Role of regulatory T cells and checkpoint inhibition in hepatocellular carcinoma.

Immune checkpoint inhibition suggests promising progress for the treatment of advanced hepatocellular carcinoma (HCC). However, the underlying cellular mechanisms remain unclear because liver cancer cells apparently do not upregulate inhibitory checkpoint molecules. Here, we analysed whether regulatory T cells (Tregs) can alternatively trigger checkpoint inhibition pathways in HCC. Using flow cytometry we analysed expression of checkpoint molecules (PD-1, PD-L1, CTLA-4, GITR, Tim-3) on peripheral CD4+CD25+Foxp3+ Tregs and their secretion of inhibitory mediators (IL-10, IL-35, TGF-beta, galectin-9) in 116 individuals (50 patients with HCC, 41 non-tumour bearing liver disease controls, 25 healthy controls). Functional activity of Tregs on T effector cells (IFN-gamma production, cytotoxicity) was characterized in vitro using a lectin-dependent cellular cytotoxicity (LDCC) assay against checkpoint inhibitor-negative P815 target cells. Unlike liver patients without malignancy and healthy controls, the frequency of checkpoint inhibitor-positive Tregs inversely correlated to age of patients with HCC (PD-L1, p = 0.0080; CTLA-4, p = 0.0029) and corresponded to enhanced numbers of Tregs producing IL-10 and IL-35 (p < 0.05 each). Tregs inhibited IFN-gamma secretion and cytotoxicity of CD8+ T cells when added to LDCC against P815 cells. Treg-induced inhibition of IFN-gamma secretion could be partially blocked by neutralizing PD-1 and PD-L1 antibodies specifically in HCC patients. In HCC peripheral Tregs upregulate checkpoint inhibitors and contribute to systemic immune dysfunction and antitumoural activity by several inhibitory pathways, presumably facilitating tumour development at young age. Blocking PD-L1/PD-1 interactions in vitro selectively interfered with inhibitory Treg -T effector cell interactions in the patients with HCC and resulted in improved antitumoural activity also against checkpoint inhibitor-negative tumour cells.

Curative potential of foremost mitogen applications.

This paper is presented as a sequel to the Mitogen Information Summaries article, representing a condensation of salient features involved with facilitating the curative potential of the more important mitogen applications. Following is a resumé of the critical attributes of mitogen therapy relative to the management of malignant tumors: (1) An inherent capability to recognize and destroy mutated or damaged tissues without altering those that are normal; (2) The capacity to induce global immunostimulation by the nonspecific activation of CD4+/- and CD8+/- cells with balanced production of a variety of cytokines able to stimulate B cell, NK cell, and macrophage pathways, at the same time augmenting myeloproliferation; (3) The ability to afford protection and accelerated recovery from the immunosuppressive and myelosuppressive effects of tumors, infections, GvH reactions, and autoimmune states along with the surgery, irradiation, chemotherapeutic agents, antibiotics, and suppressive factors used in their management; (4) Berke's in vitro data from the lectin-dependent cellular cytotoxicity (LDCC) system showing that systemic administration of mitogens such as PHA-L4 should indeed prove destructive to virtually any type of malignancy, leaving normal tissues undamaged; and (5) The potential of these activities to reconstitute the immune competence so vital to lasting cures. The potential role of L4 immunotherapy for infections may be defined by the following criteria: those, including drug-resistant infections, not satisfactorily treatable otherwise; those in which a rapid response is essential; those that are subclinical, latent, recurrent, chronic, persistent, highly lethal, or opportunistic; those in patients with impaired immune responses; and most importantly, those that are not likely to be adversely affected by immunostimulation. Certain paths of administration such as the intralesional (for granulomas), intrapleural (for pleurisy, empyema), and intraperitoneal (for peritonitis) routes might be tested as supplements to intravenous administration in treating infections using dosages comparable to tumor therapy. Intradermal or dermal applications might be used alone for localized viral or fungal skin lesions. The comparative advantages of each mitogen are listed as follows: PHA-L4, longest experience and most complete, including use in humans; Fraction IV PHA, nonagglutinating, otherwise comparable to L4; PWM, nonagglutinating in the broad mitogenic range, high potency that reduces dosage requirements, noncommercial product preferable for experimental studies and for potential advancement to human use.

Effect of branched-chain amino acids on the composition and cytolytic activity of liver-associated lymphocytes in rats.

Although branched-chain aminoacids (BCAA) are reported to be effective in prolongation of the mean survival time of patients with liver cirrhosis, it is not clear whether BCAA could influence the immune function in those patients. Branched-chain amino acids were given as a supplement to carbon tetrachloride-induced cirrhotic rats, and an aminogram of the liver and kinetics of liver-associated lymphocytes (LAL) were then analysed. Liver cirrhosis was established at the 12th week, and glutathione S-transferase placental form (GST-P)-positive lesions, which are known to be pre-neoplastic lesions, occupied 1.72+/-0.84% of the liver at the 16th week in the controls. At this time the LAL showed an increase in the number of CD5-, CD8- and CD18-positive cells and augmentation of lectin-dependent cellular cytotoxicity (LDCC) activity. Furthermore, supplementation of BCAA increased the number of LAL, especially CD8-positive cells and natural killer cells, and augmented LDCC activity of LAL at the 16th week. The number of LAL was positively correlated with the valine concentration in the plasma and liver, and the area of GST-P-positive lesions tended to be decreased in the BCAA group. These findings suggest that BCAA administration has stimulatory effects on the local immune systems of the liver, which may have a potential to inhibit hepatocarcinogenesis. Moreover, among all amino acids valine might be an important amino acid for enhancing the immune function of LAL.

レクチンによる抗ＣＤ３抗体添加ｒＩＬ‐２誘導ＬＡＫ細胞の抗腫よう活性増強についての検討

Interleukin-2 (IL-2) with anti-CD 3 antibody can induce large numbers of lymphokine activated killer (CD3LAK) cells. However, CD3LAK cells are less cytotoxicity than LAK cells treated with IL-2 alone. Therefore, enhancement by lectins of the cytotoxicity of activated lymphocytes is regarded as lectin-dependent cellular cytotoxicity (LDCC). We studied the enhancement of cytotoxicity by PHA or PWM against PBMC (peripheral blood mononuclear cell)-CD3LAK cells derived from peripheral blood mononuclear cells from a normal donor as well as PBMC-CD3LAK and TIL (tumor infiltrated lymphocyte)-CD3LAK cells obtained from two patients with tongue cancer (stage II). The treatment with PHA or PWM was given to two groups: one (additional group) was treated with PHA or PWM in the effector phase. Another (pretreatment group) was treated with the lectins, described above, and then excess lectin was washed from the surface of the CD3LAK cells. Cytotoxicity in the above two groups was estimated with a 51Cr release assay for four hours. We found that the NK activity of the PBMC-CD3LAK cells derived from a normal donor was significantly enhanced by PHA or PWM, with the exception of NK activity in the pretreatment group. LAK activity of the above cells was enhanced significantly only by PWM in the additional group. In contrast, LAK activity of PBMC-CD3LAK cells derived from case 2 was significantly enhanced by PWM in both groups. The NK activity of TIL-CD3LAK cells derived from case 1 was enhanced by PHA or PWM, and the LAK activity of these cells was also enhanced significantly by PWM in both groups. The LAK activity of TIL-CD3LAK cells derived from case 2 was enhanced by PWM in both groups. As for cytotoxicity against the autologous tumor, PBMC-CD3LAK cells derived from case 2 were also slightly enhanced by PHA or PWM in both groups, but TIL-CD3LAK cells were enhanced significantly more than the PBMC-CD3LAK cells in all studies. These results suggest differences between PBMC-CD3LAK and TIL-CD3LAK cells in repertoire and in the sugar chain expressed on the tumor cell surface. In addition, PHA or PWM induced high levels of TNF-α in both PBMC-CD3LAK and TIL-CD3LAK cells, but this cytokine did not contribute to enhanced cytotoxicity

Arlee line of rainbow trout (Oncorhynchus mykiss) exhibits a low level of nonspecific cytotoxic cell activity

Nonspecific cytotoxic cell (NCC) activity was assessed in the peripheral blood of four isogenic lines of rainbow trout (Oncorhynchus mykiss) which were derived by the chromosome set manipulation technique of androgenesis. In these fish, whose isogenicity was previously confirmed by multilocus DNA fingerprint analysis, NCC activity was studied by the release of 51Cr from YAC-1 targets. Two groups of trout (the homozygous Arlee 12 line and the heterozygous hybrid of the Arlee 63 and Arlee 12 lines) had significantly lower levels of NCC activity in peripheral blood than either outbred rainbow trout or other lines with Hot Creek or hybrid Arlee × Hot Creek ancestry. The low NCC activity in the Arlee line appears to be inherited as a recessive trait. Peripheral blood cells of the trout mediated lectin dependent cellular cytotoxicity (LDCC) with the addition of phytohemagglutirain to co-cultures of effector cells and YAC-1 cells. The low NCC activity in the peripheral blood of these fish is not due to a condition analogous to the NCC-deficient Chediak-Higashi syndrome of man or the beige mutation of mice.

Functional ambivalence of the Kp43 (CD94) NK cell-associated surface antigen.

We originally characterized the Kp43 (CD94) surface Ag, whose expression is restricted to human NK cells and a minor T lymphocyte subset. As shown in the present study, anti-Kp43 mAbs but not their F(ab')2 fragments markedly inhibited the cytolytic activity of polyclonal-activated NK cells in a redirected lysis assay against the murine P815 cell line. Furthermore, anti-Kp43 mAbs also down-regulated the CD16-dependent redirected killing and PHA-induced lectin-dependent cellular cytotoxicity. However, the intensity of the inhibitory effect was variable and no significant down-regulation of cytotoxicity was substantiated in NK cell populations from some individuals. A similar variability in the responsiveness to anti-Kp43 mAb was noticed when fresh CD3- lymphocyte populations were tested: in some donors we observed the induction of redirected lysis, whereas in other samples the Kp43-specific mAb inhibited cytotoxicity. The analysis of single cell-derived microcultures provided a clue to interpret the variable responsiveness of polyclonal cell populations; remarkably, the cytolytic activity of some NK clones was inhibited, whereas that of others was either stimulated or unaffected. The pattern of responsiveness in the cytolytic assay correlated with TNF production upon stimulation with solid phase-bound anti-Kp43 mAbs. The different types of clones could be derived from the same individual, although their relative proportions varied from donor to donor. Moreover, Kp43 appeared to be coupled differently to signal transduction pathways, because (Ca2+)i mobilization in the presence of the Kp43-specific mAbs was substantiated only in clones that were activated in the redirected lysis assay.

15Cr-labeled human hepatocytes as target cells for cytotoxicity mediated by freshly isolated liver-infiltrating lymphocytes

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.

Mechanism of interferon gamma-induced protection of human gliosarcoma cells from lymphokine-activated killer lysis: division of lymphokine-activated killer cells into natural killer- and T-like cells.

The mechanism by which interferon gamma (IFN-gamma) decreases the susceptibility of the established cultured gliosarcoma line Gl-1 to lymphokine-activated killer (LAK) lysis was analyzed. The results of monolayer depletion and lectin-dependent cellular cytotoxicity assays by LAK cells revealed that the resistance to LAK lysis of IFN-gamma-treated Gl-1 cells is manifested at the stage of LAK cell target recognition alone. We have also divided LAK cells into populations of phenotypically natural killer (NK)- and T-like cells with monoclonal antibodies and complement, respectively. We have used these cells to examine the mechanism of IFN-gamma-induced protection of Gl-1 cells from LAK lysis in cold target inhibition, monolayer depletion, and direct binding assays. The results revealed that NK-like cells do not recognize IFN-gamma-treated Gl-1 cells as efficiently as they do untreated targets, whereas T-like cells show the opposite tendency. In conclusion, we have demonstrated that the IFN-gamma induced protection of tumor cells from LAK lysis is predominantly regulated by the target recognition of NK-like cells. On the other hand, IFN-gamma-treated tumor cells may bind to T-like cells but fail to trigger them to initiate further stages for lysis as effectively as NK-like cells.

Resistance of Different Tumor Cells to Lysis by Lymphokine Activated Killer Cells Can Be Mediated by Distinct Mechanisms

Lymphokine activated killer (LAK) cells have been shown to exert a potent cytotoxic effect on many histologically different tumors and virally infected targets. Most normal cells but very few tumors have proven resistant to LAK lysis. The availability of two LAK resistant tumors, P815r, a murine mastocytoma, and SNUC-1, a human colon carcinoma, allowed us to study the phenomenon of LAK lysis. We examined the role of surface molecules on targets, which mediate binding to LAK cells, by cold target competition experiments and lectin dependent cellular cytotoxicity assays.The results showed that in the murine system, P815r cells do not compete for lysis of the LAK sensitive target B16 whereas other LAK sensitive murine targets compete. Alternatively, in the human system, SNUC-1 cells compete for lysis of the LAK sensitive target SNUC-4 as do other LAK sensitive human tumor cells. Furthermore, inducing binding of target and effector cells with lectin reverted the resistance of P815r but not SNUC-1 targets to lysis by LAK cells. These results imply that distinct stages of the lytic pathway might be involved in the resitance of different tumors to killing by LAK cells. The murine cell line is resistant to lysis because it cannot bind LAK cells. The human target, which does bind LAK, was insensitive to the effects of tumor necrosis factor alpha (TNF-α), a lymphokine released by LAK effectors and possibly involved in their lysis. Resistance to TNF-α was not mediated by the presence of endogenous short-lived proteins in the SNUC-1 targets. The elucidation of mechanisms of resistance may provide a tool to improve current protocols of adoptive immunotherapy as well as insights as to how tumor cells are or are not killed by LAK effectors.

Cytotoxic T cells isolated from the central nervous systems of mice infected with Theiler's virus.

Intracerebral inoculation of resistant mice (C57BL/10SNJ) with Theiler's murine encephalomyelitis virus (TMEV) results in acute encephalitis followed by subsequent clearance of virus from the central nervous system (CNS). In contrast, infection of susceptible mice (SJL/J) results in virus persistence and chronic immune-mediated demyelination. Both resistance and susceptibility to TMEV-induced disease appear to be immune mediated, since immunosuppression results in enhanced encephalitis in resistant mice but diminished demyelination in susceptible mice. The purpose of these experiments was to determine whether anti-TMEV cytotoxic T lymphocytes (CTLs) are generated during acute and chronic TMEV infection. Nonspecific lectin-dependent cellular cytotoxicity was used initially to detect the cytolytic potential of lymphocytes infiltrating the CNS irrespective of antigen specificity. Using TMEV-infected targets, H-2-restricted TMEV-specific CTLs of the CD8+ phenotype were demonstrated in lymphocytes from the CNS of susceptible and resistant mice, arguing against the hypothesis that the ability to generate CD8+ CTLs mediates resistance. In chronically infected SJL/J mice, TMEV-specific CTL activity was detected in the CNS as late as 226 days postinfection. These experiments demonstrate that virus-specific CTLs are present in the CNS during both acute and chronic TMEV infection. Anti-TMEV CTLs in the CNS of chronically infected SJL/J mice may play a role in demyelination through their ability to lyse TMEV-infected glial cells.

Natural killer activity of human liver-derived lymphocytes in various liver diseases

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)

Murine epidermal γ/δ T cells express Fcγ receptor II encoded by the FcγRα α gene

Short-term bulk cultures and some long-term clones and lines of murine T cell receptor (TcR) gamma/delta-bearing epidermal T cells (dEC) were found to express an Fc gamma receptor II (Fc gamma RII), as revealed by reactivity with the monoclonal antibody 2.4G2. Northern blot analysis showed that the Fc gamma RII expressed on dEC is encoded solely by the Fc gamma R alpha gene. While all the various cultured dEC cell populations analyzed exhibit lectin-dependent cellular cytotoxicity, only those which expressed Fc gamma R alpha were also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). These results in combination with the previous demonstration of Fc gamma R alpha on mouse natural killer cells support an essential role for Fc gamma R alpha in ADCC and extend an analogy with surface CD16 (Fc gamma RIII) expression and ADCC in human natural killer cells and peripheral TcR gamma/delta T cells.

Lymphocyte activation and serine-esterase induction following recombinant interleukin-2 infusion for lymphomas and acute leukaemias.

In this study we investigated the pattern of T lymphocyte changes in 16 adult patients with acute myeloid leukaemia (8), non-Hodgkin's lymphoma (4) and Hodgkin's disease (4) treated with continuous infusion of recombinant interleukin-2 (rIL-2). Effects indicative of lymphocyte activation occurred even prior to any rIL-2 therapy in these patients, being most prominent in patients with active diseases. Following each course of cytokine therapy, there were further changes in these parameters. Significant rebound lymphocytoses occurred with a concomitant increase in the cytotoxic functions and induction of the cytotoxicity-linked cytoplasmic serine esterase. Hence, both the natural killer and lectin-dependent cellular cytotoxicity activities were up-regulated. There were also increases in the serum sIL-2 receptor, sCD4 and sCD8 levels. More CD3+ lymphocytes, especially cells bearing CD4, were also recruited to the pool of potential effector cells, as demonstrated by the greater proportion of cells expressing the cytoplasmic serine esterase.

Cellular cytotoxic function and potential in acute myelogenous leukaemia

Twenty-seven adult AML patients (13 with active disease and 14 in complete remission) were investigated for their cellular cytotoxic potential and function. All AML patients, whether with active disease or in complete remission, showed increased percentage of CD3+ lymphocytes expressing the cytotoxicity-linked cytoplasmic serine esterase, suggesting a higher than normal cytotoxic potential. However, when the cytotoxic function in these patients were analysed in terms of the natural killer and lectin-dependent cellular cytotoxicity, all AML patients, whether with active disease or in complete remission, had impaired target cell lytic activity. This paradox of cytotoxicity is most likely due to the immunosuppressive effect of the serum factor elaborated by leukaemia myeloblasts.

X-linked codominant genes control all types of non-major histocompatibility complex-restricted cytotoxicity

In most individuals, natural killer (NK) activity is abolished after lymphocyte irradiation with 3,000 cGy, while lymphocytes from a minority of males retain 100% NK activity and lymphocytes from some females retain 50% NK activity after this dose. Radiation sensitivity of NK activity is controlled by X-linked codominant genes. The frequency of the allel that imparts resistance is 7%. We studied a unique family in which both parents have the resistant allele such that the father is completely resistant and the mother is partially resistant. The three offspring of this couple were one sensitive male, one partially resistant female, and one completely resistant female. The radiation sensitivity of nonspecific cytotoxic functions mediated by various types of effector cells from all five family members were evaluated in order to determine whether other cytotoxic functions were controlled by the same set of genes. The cytotoxic functions investigated were: NK and lymphokine-activated killing, anomalous killing and lectin-dependent cellular cytotoxicity, and antibody-dependent cell-mediated cytotoxicity. Our data indicate that the radiation sensitivity of all types of nonspecific cytotoxic cells is under the same genetic control.

Differences in Non-MHC Restricted Cytotoxic Activities of Human Peripheral Blood Lymphocytes after Transfusion with Allogeneic Leukocytes or Platelets Possessing Class I and/or Class II MHC Molecules

MHC-unrestricted cytotoxic activity of peripheral blood lymphocytes (PBL) from 4–6 healthy donors was investigated before and after transfusion with allogeneic leukocytes or platelets. Natural killer and lectin-dependent cellular cytotoxicity (LDCC) of PBL was tested against K562 and Raji target cells in a 4-h and 16-h 51Cr-release assay, respectively. After allotransfusion with leukocytes, we found increased cytotoxic activity of each donor's PBL against all the three targets on day 3 or 7. The highest non-specific cytotoxic activity was detected against the relatively NK resistant Raji target cells. The increase of cytotoxic activity was lowest against the LDCC target (PHA-treated Raji) cells. On the contrary, no changes in cytotoxic activity against any targets were observed after allotransfusion with platelets (possessing class I HLA antigens but no HLA class 11 molecules). Our results suggest that HLA class II molecules, presumably by inducing immune responses, are essential for activation/generation of non-specific killing of tumor targets after leukocyte transfusion. Thrombocytes, known to be less immunogenic than leukocytes, are not effective in in vivo enhancing of non-specific cytotoxicity. Cellular activation of PBL following leukocyte allotransfusion was confirmed by detection of elevated serum neopterin and β-2-micro globulin levels on day 3. This was not the case after platelet allotransfusion. In addition, the expression of ICAM-1 antigen (as a molecule involved directly in MHC-unrestricted cytotoxicity) was also found to be increased in two donors' PBL on day 3 after leukocyte transfusion in contrast to transfusion with platelets.

Immunoselection of a human melanoma resistant to specific lysis by autologous tumor-infiltrating lymphocytes. Possible mechanisms for immunotherapeutic failures.

Intratumoral heterogeneity has been proposed as a possible basis for immunotherapeutic failure when tumor-specific agents such as tumor infiltrating lymphocytes (TIL) are employed for cancer therapy. To examine this issue, highly specific oligoclonal MHC class I-restricted cytolytic TIL grown in bulk culture from patient 397 were used to immunoselect a TIL-resistant variant tumor from the autologous cultured melanoma line 397-mel. Four cycles of immunoselection produced tumor 397-R4, a variant completely resistant to 397 TIL but not to allogeneic LAK cell lysis in 4-h 51Cr release assays. By flow microfluorometry analysis, this tumor variant had not lost MHC molecules, adhesion molecules, or a variety of tumor-associated Ag expressed by the parent tumor but showed decreased expression of many Ag examined. Failure of 397-R4 to cold target inhibit TIL lysis of 397-mel suggested that cell-surface modification was at least one mechanism causing TIL resistance. The inherent lysability of 397-R4 was equal to 397-mel, as confirmed by lectin-dependent cellular cytotoxicity, lysis by non-MHC restricted allogeneic TIL, and lysis by a second line of 397 TIL grown independently from tumor 397. Treatment of 397-R4 with IFN-alpha or IFN-gamma, +/- TNF-alpha for 72 h before cytolytic assays enhanced TIL lysis of this target slightly, and enhanced surface expression of MHC class I and II molecules and the adhesion molecule ICAM-1. The resistant phenotype of 397-R4 was evident in all clones of 397-R4 examined and has been maintained in serial culture for over 13 mo and through passage in nude mice, suggesting that such stable tumor variants may provide an in vivo escape mechanism from specific immune reagents such as TIL. Evolving patterns of TIL culture clonality over time, as well as the spontaneous emergence of different clones in two long term TIL cultures grown under identical conditions from the same source of cryopreserved tumor, were documented by analyzing TCR gene rearrangements and suggest that TIL from different culture passages or lines may be used to overcome resistant tumor subpopulations.

Perforin Gene Expression in Granular Lymphocyte Proliferative Disorders

By Northern blot analysis using a cDNA clone of the perforin gene, we studied the levels of perforin mRNA in peripheral blood mononuclear cells from 11 cases of granular lymphocyte-proliferative disorders (GLPDs). The granular lymphocytes studied were characterized by morphologic, immunophenotypic, and immunogenotypic analyses. Cytolytic functions of the lymphocytes assayed included nonmajor histocompatibility complex-requiring cytotoxicity, anti-CD3-redirected cytotoxicity, antibody-dependent cellular cytotoxicity, and lectin-dependent cellular cytotoxicity. The results showed that in lymphocytes with strong cytolytic functions high levels of perforin mRNA existed, whereas in lymphocytes with weak or undetectable levels of cytolytic functions, low levels of perforin mRNA existed. Because the levels of perforin mRNA correlated with those of cytolytic functions, perforin is probably a mediator in cytolytic functions of granular lymphocytes in patients with GLPDs. When the lymphocytes were cultured for 1 day, however, the levels of cytolytic activity were increased, and those of perforin mRNA were decreased. Therefore, we cannot rule out the possibility that factors other than perforin protein are involved in the cytolytic functions of granular lymphocytes.

Interaction of bluetongue virus with bovine lymphocytes

Freshly isolated, and established, cultures of bovine peripheral blood mononuclear leukocytes (PBMLs) were exposed to bluetongue virus (BTV) for the purpose of defining potential lymphotropism. PBML cultures were established in the presence of interleukin 2 (IL-2) and mitogen and maintained either as bulk culture or were cloned prior to infectivity studies. All cultures appeared to be of the T cell phenotype based on the following characteristics: binding of T lymphocyte-specific lectins (i.e. peanut agglutinin and Helix pomatia), rosetting of sheep erythrocytes, binding of a putative pan-T monoclonal antibody, and absence of surface immunoglobulin (Ig). T lymphocyte cultures were further characterized by their ability to elicit lectin-dependent cellular cytotoxicity (LDCC). Exposure of established lymphocyte cultures to BTV resulted in productive cytopathic and non-cytopathic infections. Non-cytopathic productive infections were observed in LDCC-negative cultures whereas cytopathic and non-cytopathic infections were observed in LDCC-positive cultures. Exposure of freshly isolated PBMLs to BTV resulted in minimal virus replication; addition of mitogen and IL-2 to such cultures did not augment virus replication. Addition of mitogen and IL-2 induced negligible blast transformation, whereas PBML viability was minimally affected. These studies establish a tropism of BTV for bovine T lymphocytes with virus replication being limited to those cells undergoing blastogenesis. Establishment of infected lymphocyte cultures, without loss of culture viability, suggest such an interaction may contribute to the long term viraemias associated with BTV infection of cattle.

Association of increased lytic effector cell function with high estrogen receptor levels in tumor-bearing patients with breast cancer

Tumor-bearing patients with breast cancer were assayed for their natural killer (NK) cell activity and for the function of activated cytotoxic T-cells, as assessed by lectin-dependent cellular cytotoxicity (LDCC). Tumor-bearing patients with breast cancer had a significant increase in NK activity and in LDCC, as compared with healthy control individuals. Although the enhanced NK cell activity and LDCC were closely associated with high levels (greater than 31 fmol/mg) of estrogen receptor (ER) content in the primary tumor, no other clinical or histologic correlation between the increase in either parameter of cytotoxic effector cell function could be found. Thus, ER levels greater than 31 fmol/mg might be associated with increased cytotoxic effector cell function in tumor-bearing patients with breast cancer.