Lack of anti-tumour reactivity despite enhanced numbers of circulating natural killer T cells in two patients with metastatic renal cell carcinoma.

Natural killer T (NK T) cells play a central role as intermediates between innate and adaptive immune responses important to induce anti-tumour reactivity in cancer patients. In two of 14 renal cell carcinoma (RCC) patients, treated with interferon (IFN)-α, we detected significantly enhanced numbers of circulating NK T cells which were typed phenotypically and analysed for anti-tumour reactivity. These NK T cells were T cell receptor (TCR) Vα24/Vß11(+), 6B11(+) and bound CD1d tetramers. No correlation was observed between NK T frequencies and regulatory T cells (T(regs)), which were also enhanced. NK T cells expressed CD56, CD161, CD45RO and CD69 and were predominantly CD8(+), in contrast to the circulating T cell pool that contained both CD4(+) and CD8(+) T cells, as is found in healthy individuals. It is unlikely that IFN-α triggered the high NK T frequency, as all other patients expressed low to normal NK T numbers. A parallel was observed in IFN-α-related increase in activation of NK T cells with that in conventional T and non-T cells. Normal interleukin (IL)-7, IL-12 and IL-15 plasma levels were found. In one of the patients sporadic NK T cells were detected at the tumour site. α-Galactosylceramide (αGalCer) stimulation of peripheral blood mononuclear cells or isolated NK T cell lines from both patients induced IFN-γ, but no IL-4 and no response towards autologous tumour cells or lysates. The clinical course of disease in both patients was not exceptional with regard to histological subtype and extent of metastatic disease. Therefore, despite a constitutive high peripheral frequency and in vitroαGalCer responsiveness, the NK T cells in the two RCC patients did not show anti-tumour responsiveness.

Reinfusion of autologous lymphocytes with granulocyte-macrophage colony-stimulating factor induces rapid recovery of CD4+ and CD8+ T cells after high-dose chemotherapy for metastatic breast cancer.

PURPOSE: Repeated high-dose chemotherapy (HDCT) followed by peripheral-blood progenitor cell (PBPC) transplantation can induce a complete remission in patients with metastatic breast cancer sensitive to standard chemotherapy (CT), but the majority of patients relapse within 1 to 2 years. The immune system is seriously compromised after HDCT, which precludes the development of effective immunotherapy. We investigated whether autologous lymphocytes, reinfused after HDCT, could induce a rapid recovery of T cells. PATIENTS AND METHODS: Three patients were monitored for immune recovery without reinfusion of lymphocytes. In the next 11 patients, stem cells were harvested after CT + granulocyte colony-stimulating factor (G-CSF) and lymphocytes were harvested after CT + granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2. These patients received stem cells and G-CSF after the first HDCT; stem cells, G-CSF, and lymphocytes after the second; and stem cells, GM-CSF, and lymphocytes after the third HDCT. RESULTS: Patients not receiving lymphocyte reinfusion had a very slow recovery of lymphocytes. In particular, CD4 counts remained low (< 200/microL for 9 months). Lymphocyte reinfusion had a significant effect on the recovery of lymphocytes, T cells, and CD8+ T cells (normalized on day 25). Recovery of CD4+ T cells was significantly accelerated by lymphocyte reinfusion and GM-CSF, leading to counts of 500/microL at 25 days. CONCLUSION: Lymphocyte reinfusion with G-CSF had a significant effect on the recovery of CD8+ T cells, whereas rapid recovery of CD4+ T cells required lymphocyte reinfusion and GM-CSF, which possibly acts as a survival factor through activation of antigen presenting cells. Whether the rapid recovery of CD4+ and CD8+ T cells prevents or delays relapse of the disease should be further investigated.

Idiotypic immunoglobulin secretion by human B cell non-Hodgkin's lymphomas is related to the expression of the interleukin-2 receptor.

Tumor cells from 5 human B cell non-Hodgkin's lymphoma (B-NHL) patients were investigated for proliferative activity and idiotypic (Id+) immunoglobulin (Ig) secretion in serum-free medium without deliberate addition of B cell growth or differentiation factors (BCDF). These data were compared with cell surface marker expression, notably of activation antigens such as 4F2 and interleukin-2 (IL-2) receptor. Cells from all patients became 4F2 positive at the end of the 6-day culture period. Freshly drawn cells from 3 out of 5 patients expressed the IL-2 receptor (CD25; Tac antigen) or acquired this marker during culture in vitro and secreted relatively high levels of Id+ Ig in vitro. This correlated with elevated serum Id levels (greater than or equal to 0.5 micrograms/ml in vitro versus greater than or equal to 20 micrograms/ml in vivo). In the 2 CD25 (Tac)- B-NHL patients serum Id levels were below the detection limit and the amount of Id+ Ig secreted in vitro did not surpass 50 ng/ml. Only the B-NHL cells from a single patient were initially CD25 (Tac) positive and only these cells proliferated in serum-free culture. To test whether IL-2 receptor expression in the 3 CD25 (Tac)+ patients was functional, recombinant IL-2 (rIL-2) either alone or in conjunction with BCDF and recombinant IL-4 (rIL-4) was added to the cultures. In 2 out of 3 CD25 (Tac)+ patients rIL-2 was capable of enhancing proliferation or Ig secretion. In addition rIL-2 was found to enhance BCDF-mediated but not rIL-4 mediated responses. The third CD25 (Tac)+ B-NHL population was resistant to any of these lymphokines. Thus, this serum-free culture system may accurately reflect patient serum Id levels. IL-2 appears to regulate not only the in vitro but also the in vivo Ig secretion by neoplastic B cells.

Functional and molecular characterization of B cell line derived interleukin-1 alpha.

The cytokine secreted by a human hybrid B cell line (STS 25) obtained by fusion of the B lymphoblastoid cell line WI-L2-729-HF2 with neoplastic B cells from a patient with B cell non-Hodgkin's lymphoma (B-NHL) was characterized as IL-1 alpha. STS 25 cells express the idiotypic (Id+) immunoglobulin (Ig) specific for the neoplastic B cells of the B-NHL patient. STS 25 cells are weakly positive for surface mu delta kappa and in addition express the surface markers CD19, CD20, CD23, HLA class I and II, and the 4F2 activation antigen. STS 25 cells are also Epstein-Barr nuclear antigen positive but do not secrete viral particles. Serum-free culture supernatant from STS 25 cells (STS 25 SUP) does not show activity in assays for interleukin-2 (IL-2), -4 (IL-4), -6 (IL-6), interferon or tumor necrosis factor, but is active in the thymocyte costimulation assay and the D10.G4.1 T helper clone proliferation assay for interleukin-1 (IL-1). The IL-1 character of the STS 25 SUP activity was confirmed in inhibition studies with three different poly- or monoclonal anti-IL-1 antibodies (31, 88, and 94% inhibition in thymocyte costimulation assay, respectively). Furthermore, complete blocking of D10.G4.1 cell proliferation mediated by STS 25 SUP was observed by including anti-IL-1 alpha specific antibody in the assay, whereas anti-IL-1 beta antibody had no effect. These results indicate that this STS 25 SUP activity can be attributed to the presence of IL-1 alpha in the supernatant. Northern blot analysis of total STS 25 cellular RNA using IL-1 alpha or IL-1 beta specific probes revealed the constitutive expression of IL-1 alpha messenger RNA by STS 25 cells. In contrast, no IL-1 beta message was detectable, not even after treatment of the cells with phorbol ester or cycloheximide, which resulted in approximately 5-fold enhancement of IL-1 alpha mRNA expression. Binding studies with radiolabeled recombinant (r) IL-1 alpha indicated the presence of high numbers of IL-1 receptors on STS 25 cells (1,170 per cell, Kd = 392 pM). Although both IL-1 alpha and IL-1 beta bound to these IL-1 receptors, no indication was found for IL-1 mediated regulation of STS 25 cell growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Phase I trial of combined immunotherapy with subcutaneous granulocyte macrophage colony-stimulating factor, low-dose interleukin 2, and interferon alpha in progressive metastatic melanoma and renal cell carcinoma.

The purpose of our study was to determine the maximally tolerated dose (MTD) and DLT of combined administration of granulocyte macrophage colony-stimulating factor (GM-CSF), low-dose interleukin 2 (IL-2) and IFN-alpha in patients with progressive metastatic melanoma or renal cell carcinoma (RCC). In addition, the activation and expansion of effector cells were measured. Cohorts of three patients were treated with increasing doses of IL-2 (1, 4, and 8 MIU/m2) and GM-CSF (2.5 and 5 microg/kg) with a constant dose of IFNalpha (5 million units) s.c. for 12 days every 3 weeks. An additional six patients were treated at the MTD. Immune activation was monitored during the first cycle. Response was evaluated after two cycles. The MTD was found to be 2.5 microg/kg GM-CSF, 4 MIU/m2 IL-2, and 5 mega units of IFNalpha. DLT was grade 4 fever, chills with hypotension, grade 3 fatigue/malaise, and fluid retention. Dose reduction of IL-2 to 2 MIU/m2 was necessary in three of nine patients who initially received the MTD. Treatment was initiated in the hospital but could be continued at home after 3-4 days. Significant increases in lymphocytes, (activated) T cells (CD4+ and CD8+), NK cells, monocyte DR expression, neutrophils, and eosinophils were found. CD8+ T-cell activation (sCD8) and NK cell expansion was mainly present in patients receiving 2 or 4 MIU/m2 IL-2. Of eight patients with progressive metastatic RCC after nephrectomy, three achieved a complete remission, and 1 of 7 patients with metastatic melanoma achieved a partial remission. In our study, the MTD of combined immunotherapy with GM-CSF, IL-2, and IFNalpha was established; DLT was: (a) grade 4 fever with hypotension needing i.v. fluid support; and (b) grade 3 fluid retention and/or fatigue/malaise. The scheme resulted in considerable expansion and/or activation of various effector cells. The complete responses in RCC patients are promising but need to be confirmed in Phase II studies.

Phenotypic and functional characterization of HTLV positive neoplastic T cells cultured with interleukin-2--I. Retention of morphology, phenotype and selective cytotoxic properties in long term culture.

T cells from the peripheral blood of a T-cell chronic lymphocytic leukemia (T-CLL) patient, cultured in the presence of interleukin-2 (IL-2), were found to express the p19 structural core protein of the human T-cell leukemia/lymphoma virus (HTLV) and to release type C virus particles. Comparison of the T-CLL cell line with the original leukemic T cells revealed that both the fresh and the proliferating T-CLL cells were pleomorphic cells that showed a convoluted nucleus and formed rosettes with sheep erythrocytes (E-rosettes). They were reactive with the monoclonal antibodies OKT1, OKT4 and OKT11, but not with OKT3, OKT6 or OKT8, indicating that they were mature T cells but that they differed from normal T cells in their lack of reactivity with OKT3. In addition they did not bind peanut agglutinin or OKM-1, and were negative for Epstein-Barr nuclear antigen, surface immunoglobulin, non-specific esterase activity of Fc- or complement receptors. Part of the fresh T-CLL cells reacted with a monoclonal antibody recognizing HLA-DR antigens (p29, 34) (36%) and with anti-Tac (62%), a monoclonal antibody directed at the IL-2 receptor, indicating that the T-CLL cells were partially activated already in vivo. After culture in vitro all proliferating T-CLL cells expressed HLA-DR and Tac antigens. The fresh T-CLL cells were found to be defective in cell-mediated lympholysis (CML) generated in mixed lymphocyte culture (MLC), antibody-dependent cellular cytotoxicity (ADCC) and lectin-dependent cellular cytotoxicity (LDCC). In addition they failed to exhibit natural killer (NK) cell activity against targets that are usually very susceptible to lysis, such as K562, but were able to kill two tumor-derived cell lines, the melanoma NKI-4 and the neuroblastoma CHP-100. The same pattern of selective killing was observed using the proliferating T-CLL cells as effectors, or cloned T-CLL cultures obtained from them by limiting dilution procedures. Therefore, it was concluded that the T-CLL cells represented a clonal expansion of neoplastic T cells that retained their phenotype and cytotoxic properties after culture in vitro.

Phenotypic and functional characterization of HTLV positive neoplastic T cells cultured with interleukin-2--II. Inhibition of lymphoproliferative responses.

T cells from the peripheral blood of a T-cell chronic lymphocytic leukemia (T-CLL) patients, were expanded by culture in the presence of interleukin-2 (IL-2). In contrast to the original leukemic T cells that showed a complete lack of suppressor activity, the proliferating T-CLL cells were extremely potent inhibitors of mitogen, antigen and alloantigen induced lymphoproliferative responses. Compared to the fresh T-CLL cells, the proliferating T-CLL cells showed an enhanced expression of the human T-cell leukemia/lymphoma virus (HTLV) p19 antigen and released type C virus particles into their culture supernatant. A direct involvement of the virus particles in the suppression was however unlikely, since the inhibition was found to be mediated by a soluble inhibitor in the culture supernatant of proliferating T-CLL cells. This inhibitor was very hydrophobic and was inactivated by treatment with trypsin, by heating to 56 degrees C for 2 h and by storage at -70 degrees C for more than 14 weeks. It could be excluded that the suppression of lymphoproliferation was due to competition for IL-2, to toxic effects, to nutrient depletion or to a shift in the kinetics of the target cell responses. Furthermore, it could not be attributed to suppressor inducer activity of the OKT4+ proliferating T-CLL cells, since normal T cells enriched for OKT4+ T cells and depleted for OKT8+ T cells were also inhibited in their proliferation. Since other hemopoietic cell lines, not of OKT4+ T lymphocyte origin, also were suppressed to proliferate, it is concluded that the proliferating T-CLL cells represent a neoplastic T-cell clone that had differentiated into suppressor effector T cells after prolonged culture in the presence of IL-2.

Prevalence of HTLV-specific antibodies in Surinam emigrants to The Netherlands.

Sera of 98 participants in a methadone maintenance programme, all recent Surinam emigrants to the Netherlands, were examined for antibodies to disrupted HTLV using the ELISA technique. Twelve per cent of the donors possessed HTLV-specific antibodies with a range of titre from 41 to 20,000. Sera of 26 control Dutch drug users lacked such antibodies with the exception of one female who subsequently was found to reside with a Surinam male. Intravenous drug use was not a factor in these studies. These data indicate that HTLV circulates within the Surinam community in the Netherlands. More broadly, these results show that the region of the Caribbean endemic for HTLV extends as far south as Surinam. Furthermore, an antibody prevalence of 12% for clinically healthy donors with non-malignant disease suggests that the Caribbean has an incidence of HTLV infection approaching, if not equal to, that of southwestern Japan.

Enhanced expression of human T-cell leukemia/lymphoma virus in neoplastic T cells induced to proliferate by phorbol ester and interleukin-2.

Peripheral blood T cells from a patient with T-cell chronic lymphocytic leukemia (T-CLL) failed to respond to mitogenic lectins or alloantigens but could be induced to proliferate by the addition of exogenous interleukin-2 (IL-2). The T-CLL cells also proliferated in response to 12-O-tetradecanoyl phorbol-13-acetate (TPA), or to TPA in combination with phytohemagglutinin (PHA). These TPA-mediated proliferative responses were transient, and only small but significant amounts of IL-2 activity were generated. In contrast, no IL-2 activity was produced after the T-CLL cells had been stimulated with PHA only. The T-CLL cells that were induced to proliferate with PHA and exogenous IL-2 could be maintained in continuous culture by the addition of exogenous IL-2 at regular intervals. These continuously proliferating T-CLL cells failed to produce IL-2 constitutively. However, they could be induced to produce IL-2 activity by stimulation with TPA or TPA plus PHA. Irradiation of the proliferating T-CLL cells prior to incubation with TPA or TPA plus PHA resulted in a 9-fold increase in IL-2 activity, suggesting that the proliferating T-CLL cells were able to consume the IL-2 they produced. Studies on the presence of human T-cell leukemia/lymphoma virus (HTLV) in the fresh and proliferating T-CLL cells revealed that 12% of the fresh cells expressed the HTLV p19 structural core protein. HTLV p19 expression was strongly enhanced in the T-CLL cells induced to proliferate by TPA (66%) and in the continuously growing IL-2-dependent T-CLL cells (82%). In the latter culture, but not in the fresh T-CLL cells, type-C virus particles were observed. These results indicate that HTLV expression correlates with T-CLL cell proliferation but not with IL-2 production by these cells.

Induction of IL-2 production, IL-2 receptor expression and proliferation of T3- T-PLL cells by phorbol ester.

Leukemic T cells from the peripheral blood of a patient with T-prolymphocytic leukemia (T-PLL) were investigated for their potential to differentiate in vitro upon exposure to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The T-PLL cells, identified as typical PLL cells by nuclear morphology, were typed as E+slg-OKT1+3-4+6-8-11+ cells lacking reactivity with OKI-1 or OKM-1. In addition, between 3% and 10% of the cells reacted with monoclonal antibodies against T10. In contrast to normal T cells, the T-PLL cells could not be induced to proliferate by mitogenic lectins or alloantigens in the presence or absence of human interleukin-1 (IL-1), interleukin-2(IL-2) or allogeneic monocytes and did not produce IL-2. They also failed to proliferate in response to TPA or TPA in the presence of phytohemagglutinin (PHA), but under these conditions T-PLL cells secreted high levels of IL-2 activity. Incubation in the presence of PHA + TPA or TPA for 48 h induced T-PLL cells to become blasts exhibiting enhanced protein synthesis, and induced a 10-fold increase in the percentage of cells reactive with monoclonal antibodies against T10. At the same time, about 15% of the cells developed receptors for IL-2 as monitored by their reactivity with anti-Tac monoclonal antibody. Washing of these T-PLL cells to remove TPA resulted in the induction of proliferation upon subsequent culture in the presence of IL-2 or in medium only. Since proliferating T-PLL cells still failed to express T3 antigens, it was concluded that these leukemic cells represent a T-cell differentiation stage or a T-cell subset which can be activated via a T3-independent pathway.

Redundancy of direct priming and cross-priming in tumor-specific CD8+ T cell responses.

Against a subset of human cancers, vigorous tumor-specific CD8+ T cell responses can develop either spontaneously or upon allogeneic transplantation. However, the parameters that determine the induction of such pronounced anti-tumor immunity remain ill defined. To dissect the conditions required for the induction of high magnitude T cell responses, we have developed a murine model system in which tumor-specific T cell responses can be monitored directly ex vivo by MHC tetramer technology. In this model, tumor challenge of naive mice with Ag-bearing tumor cells results in a massive Ag-specific T cell response, followed by CD8+ T cell-dependent tumor rejection. We have subsequently used this model to assess the contribution of direct priming and cross-priming in the induction of tumor immunity in a well-defined system. Our results indicate that direct priming of T cells and Ag cross-priming are redundant mechanisms for the induction of tumor-specific T cell immunity. Moreover, T cell responses that arise as a consequence of Ag cross-presentation can occur in the absence of CD4+ T cell help and are remarkably robust.

Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue.

Functional interactions between B and T lymphocytes are known to depend on the expression of co-stimulatory molecules B7.1/CD80, B7.2/CD86 and their counter-receptors CD28 and CTLA4, as well as CD40 and its ligand CD40L. To study the role of these molecules in situ, an immunohistochemical analysis was carried out on normal human lymphoid tissue. In the germinal centers (GC), B7.1 and B7.2 were differentially expressed. In the dark zone, centroblasts were predominantly B7.1+, while centrocytes in the light zone were B7-2+, resulting in reversed gradients of both markers in GC. Follicle mantle cells were negative for B7.1 and B7.2. Macrophages and interdigitating dendritic cells (IDC) in T cell zones both expressed B7.1 and B7.2. Moreover, clusters of B7.2+ T cells were demonstrated in interfollicular areas. Intrafollicular CD4+ T cells in GC, predominantly in the apical light zone, expressed CD28 and CTLA4, as did the majority of interfollicular T cells. CTLA4 showed a striking excentric cytoplasmic staining, which was also seen on T cells activated in vitro. CD40 was expressed on all B cells and more strongly on macrophages and IDC. Moreover, small clusters of T cells in a rim outside the GC showed CD40 expression. CD40L was expressed both on intrafollicular CD4+ T cells as well as on T cells in T cell zones. The differential distribution of co-stimulatory molecules in different compartments of normal human lymphoid tissue in situ indicates that these interactions play a distinctive role in different stages of B cell differentiation and in the immune response.

Expression of pTalpha mRNA in a committed dendritic cell precursor in the human thymus.

We have characterized dendritic cell precursors (pre-DC) in the human thymus. These CD1a(-)CD3(-)CD4(+)CD8(-) cells express high levels of interleukin-3Ralpha (IL-3Ralpha) on the membrane and are able to develop into mature DC upon culture with IL-3 and CD40 ligation. The DC precursors are predominantly located in the thymic medulla. Interestingly, the pre-DC express pTalpha mRNA, which is also present in CD1a(+)CD3(-)CD4(+)CD8(-) pre-T cells. Yet, the pre-DC lack expression of recombination activating gene-1 mRNA and fail to develop into T cells in appropriate assays. The thymic pre-DC are very similar to the recently characterized pre-DC found in the T cell areas of the tonsil, and it is suggested that these pre-DC populations are of lymphoid origin.

Antibodies against human T-cell leukemia/lymphoma virus (HTLV) and expression of HTLV p19 antigen in relatives of a T-cell leukemia patient originating from Surinam.

Serum and peripheral blood cell samples from eleven relatives of a T-cell leukemia patient with human T-cell leukemia/lymphoma virus (HTLV)-associated disease, were investigated for the presence of HTLV antibody and antigen expression. In addition to the patient, three family members were seropositive and a wide range in HTLV antibody titer was observed. The father of the patient showed the highest titer (173,000) and carried HTLV p19+ cells in his peripheral blood. Upon induction of proliferation of these cells by short-term culture in the presence of phytohemagglutinin (PHA) and 12-O-tetradecanoyl phorbol-13-acetate (TPA), an increase from 1% to 28% HTLV p19+ cells was observed confirming previous findings that HTLV p19 expression was correlated with proliferative activity of the host cells. In none of the other seronegative or seropositive relatives of the patient HTLV p19 expression was revealed when tested in freshly isolated mononuclear cells, upon short-term incubation in PHA + TPA or after prolonged culture for 2 or 3 passages in medium containing T-cell growth factor. The results from HLA typing studies did not provide any evidence for HTLV related abnormalities in haplotype expression. Our data confirmed the earlier notion of a prevalence of HTLV infection within families of patients with HTLV-associated disease. It is furthermore obvious from our results that a normal individual may possess high titer HTLV antibody and circulating HTLV p19+ cells without showing signs of disease.

Functional expression of adhesion receptors and costimulatory molecules by fresh and immortalized B-cell non-Hodgkin's lymphoma cells.

Peripheral blood lymphocytes of a patient with follicular B-cell non-Hodgkin's lymphoma (B-NHL) were immortalized in vitro by Epstein-Barr virus (EBV). Eight cell lines were obtained (termed BNS1, BNS2-1 through BNS2-7), which showed a pattern of idiotypic (id) Ig surface expression and Ig JH and kappa gene rearrangement, identical to that of the parent cells (termed NS), confirming their neoplastic origin. Induction of allogeneic T-cell proliferation by NS cells was mediated by HLA-DR, leukocyte function-associated antigen-1 (LFA-1), LFA-3, B7-1/CD80, and CTLA4 and resulted in the upregulation (LFA-3, intercellular adhesion molecule-1 [ICAM-1], CD40) and induction (B7-1/CD80, B7-2/CD86, L16/activated LFA-1) of accessory molecules on NS cells. In turn, responder T lymphocytes were induced to express B7-1/CD80, B7-2/CD86, CD40 ligand (CD40L), ICAM-1, L16/activated LFA-1, and HLA-DR, reflecting bidirectional signaling between T lymphocytes and B-NHL cells. Preactivation of NS cells by EBV transformation or CD40 engagement resulted in enhanced expression of accessory molecules and abolished the requirement for accessory cells during allostimulation. These resting and activated clonal B cells will be useful in further dissecting the requirements for B-NHL costimulation.

Regulatory effect of interleukin-4 (IL-4) on the expression and function of lymphocyte adhesion receptors involved in IL-2-induced cell aggregation.

Human recombinant interleukin-4 (rIL-4) was studied for its capacity to inhibit rIL-2-induced lymphoid cell aggregation. In contrast to rIL-2, rIL-4 was unable to induce cluster formation by itself. However, when added simultaneously with rIL-2 to cultures of freshly isolated peripheral blood lymphocytes (PBL), rIL-4 inhibited cell aggregation in a dose-dependent way. In contrast, PBL, preactivated by a 4-day culture in the presence of 500 U/ml rIL-2, were not inhibited in their adhesive capacity by rIL-4. Inhibition of cell aggregation was most prominent at 24 hr and virtually lost after 72 hr of culture. Phenotypical analysis revealed that rIL-4, with similar kinetics, decreased the rIL-2-mediated up-regulation of the CD2, CD54 and CD49e adhesion molecules. In addition, it was observed that up-regulation of the activation epitope on CD11a recognized by the mAb NKI-L16, was prevented. During 24hr of culture rIL-4 itself did not alter the expression of these antigens. Blocking experiments with mAb directed against adhesion structures did not reveal a direct role for CD49e, but obviously demonstrated involvement of CD11a/CD18-CD54 and CD2-CD58 interactions in the rIL-2-induced adhesion. Therefore, rIL-4 appears to inhibit the early phase of rIL-2-induced aggregation by preventing the up-regulation of CD54 and CD2 antigens and by inhibiting the generation of the activated state of the CD11a/CD18 receptor.

Isolation of human thymocytes differing in maturation state and function by centrifugal elutriation.

A modified centrifugal elutriation technique was used to separate (up to 3 X 10(9)) human thymocytes, according to size in 6 different fractions. Eighty percent of the unfractionated thymocytes were recovered in fractions 1 and 2. The majority of these thymocytes appeared to be small and phenotypically immature as was determined by the high percentage of cells reacting with the monoclonal antibodies OKT-6, Mas-036 and peanut agglutinin. In addition, a relatively low percentage of the cells reacted with a monoclonal antibody directed against HLA-A, B and C determinants (Mas-015). The immaturity of these thymocytes was confirmed by their failure to respond to phytohemagglutinin (PHA) and their negligible responder capacity in mixed leukocyte cultures. Fractions 3-6, representing 20% of the unfractionated thymocytes, were collected arbitrarily and contained thymocytes of various maturation stages as judged by their phenotype. The PHA responsiveness and responder capacity in mixed leukocyte cultures of the thymocytes in these fractions were, in general, considerably higher than those of the unfractionated thymocytes. Our data indicate that centrifugal elutriation is a fast and reproducible method to separate large quantities of functionally inactive and phenotypically immature thymocytes from the more mature and functionally active thymocytes.

Role of LFA-1/ICAM-1 in interleukin-2-stimulated lymphocyte proliferation.

Major adhesion routes between lymphoid cells involve the receptor/ligand pairs LFA-1/ICAM-1 and CD2/LFA-3, in addition to VLA or CD44 molecules. In this study we evaluated the role of these adhesion receptors in the proliferative response of lymphoid cells to interleukin-2 (IL-2). Blocking studies were performed with a panel of monoclonal antibodies (mAb) directed against these adhesion molecules. Selective inhibition of recombinant (r)IL-2-induced cell proliferation was observed with mAb directed against the alpha or beta subunit of LFA-1 or to its ligand ICAM-1. Interestingly, rIL-2-induced proliferation was also inhibited by NKI-L16, and anti-1 alpha antibody known to enhance cell-cell interaction. Resting lymphocytes were preferentially susceptible to the inhibition, particularly in an early phase of culture and when stimulated with a relatively low dose of rIL-2. By using mAb that specifically could block distinct rIL-2 activation pathways, LFA-1/ICAM-1 interaction was found to be required for p55 IL-2 receptor (IL-2R)-mediated interaction of rIL-2 with its high-affinity receptor, but not for p75 IL-2R-mediated responses. Furthermore, it was shown that the rIL-2 response of T lymphocytes, but not of natural killer cells, was dependent on LFA-1/ICAM-1 interaction. This suggests that LFA-1/ICAM-1 interaction is required for an optimal rIL-2 response of cells capable of IL-2 secretion. Our data provide evidence for the hypothesis that adhesion receptor-directed release of IL-2 may result in a locally high concentration of IL-2 that triggers high-affinity IL-2R signaling and up-regulates p55 IL-2R to enhance cytokine responsiveness.