Suppression of lymphokine-activated killer cell generation by tumor-infiltrating lymphocytes

A functional analysis of tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma (RCC) and malignant melanoma was performed. TILs were expanded in recombinant interleukin-2 (50 U/ml) in Iscoves medium. Phenotypic and functional (cytolytic vs regulatory) analyses were carried out with the fresh and expanded TIL populations after 4 weeks in culture. Only one TIL population from an RCC case (out of six cases studied) was CD8+ and demonstrated MHC class I-restricted tumor-specific cytotoxicity against the autologous RCC target. TIL populations from the other five cases became predominantly CD4+ and they neither killed the respective autologous tumor cells nor killed the NK-sensitive target K-562 cells. When studied for other functions, two CD4+ TIL populations were found to suppress the lymphokine-activated killer cell response by peripheral blood lymphocytes (PBL) in coculture. Of these two, a TIL population from an RCC case (MJ TIL) was used to study the cellular and molecular mechanisms of suppression. The MJ TIL synthesized a supernatant factor that blocked activation of resting PBL as measured by the induction of high-affinity IL-2 receptor (IL-2R) when stimulated by phytohemagglutinin but did not down-regulate the fully expressed IL-2R on activated T cells. The suppression of high-affinity IL-2R induction on T cells did not result from tumor necrosis factor-alpha and beta or from transforming growth factor-beta as these cytokines were not detected in the cell-free supernatant from the MJ TIL culture. The supernatant factor also suppressed IL-2-mediated enhancement of cytotoxicity by natural killer (NK) cells without demonstrating direct toxic effect on the NK cells. Thus, when TIL are used for adoptive immunocytotherapy, it may be useful to fully characterize them functionally, in vitro.     

Early-appearing tumour-infiltrating natural killer cells play a crucial role in the generation of anti-tumour T lymphocytes.

Natural killer (NK) cells that infiltrated into the primary tumour site at an early stage of tumour development, were examined for their participation in the generation of anti-tumour cytotoxic T lymphocytes (CTL). NK cells, which were detected by anti-NK1.1 monoclonal antibody (mAb), increased in the peritoneal exudate cells (PEC) on days 3 and 7 after an intraperitoneal (i.p.) inoculation of syngeneic B16 melanoma cells. These tumour-infiltrating NK cells showed a high level of cytotoxic activity against NK-sensitive YAC-1 cells and an increased expression of interferon-gamma (IFN-gamma) mRNA and interleukin-2 (IL-2) mRNA. The in vivo depletion of NK cells with anti-NK1.1 mAb, prior to i.p. inoculation of B16 melanoma cells, resulted in an increased number of tumour cells in the PEC compared to NK cell non-depleted mice. Interestingly, the differences in tumour cell number between both groups were more prominent on days 7 and 14 than on day 3, which strongly suggested that early-infiltrating NK cells have a large influence on the subsequent anti-tumour response. The in vivo depletion of NK cells prior to immunization with melanoma cells abrogated the capacity of the spleen cells to generate CD8+ tumour-specific CTL after in vitro restimulation. This inability of generating anti-tumour CTL was partially restored by additional i.p. injections of recombinant IL-2 and/or IFN-gamma simultaneously with the immunization of melanoma cells. The in vitro depletion of NK cells prior to the in vitro restimulation with melanoma cells partially impaired the anti-tumour CTL generation from the spleen cells of the immunized mice. Lastly, the in vivo depletion of NK cells prior to immunization with melanoma cells abolished the protective immunity against melanoma cells at the rechallenge. Overall, these results indicate that early-appearing tumour-infiltrating NK cells not only participate in the anti-tumour early defence by themselves, but also play a crucial role in the generation of anti-tumour CTL.     

中国HIV感染者细胞毒性淋巴细胞内穿孔素的差异性表达

目的 了解中国HIV感染者细胞毒性相关的NK细胞及CD8^＋T细胞内穿孔素表达水平，探讨HIV感染过程中穿孔素表达与机体免疫功能的关系。方法 采集31例未经抗病毒治疗的HIV感染者和经过高效抗逆转录病毒疗法（HAAS）治疗的17例HIV／AIDS患者以及15例健康对照的抗凝全血，应用流式细胞仪胞内染色法检测CD56^＋／CD3^-、CD3^-／CD16^＋NK细胞及CD8^＋／CD3^＋内穿孔素表达的百分数，分析其与NK细胞绝对值、NK细胞百分数、CD4^＋T、CD8^＋T淋巴细胞绝对值及血浆病毒载量的相关性。结果 中国HIV感染者的NK细胞CD56^＋／CD3^-及CD3^-／CD16^＋亚群穿孔素表达百分数（平均13．17％，平均24．05％）高于CD8^＋T细胞穿孔素表达百分数（平均9．03％）；NK细胞内穿孔素表达低于健康对照（P〈0．05，P〈0．05），CD8^＋T细胞内穿孔素表达高于健康对照（P〈0．05）；NK细胞及CD8^＋T细胞内穿孔素表达水平与其绝对计数显著相关，与疾病进展不相关。HAART治疗组NK细胞内穿孔素表达升高，CD8T^＋细胞内穿孔素表达无显著变化。结论 中国HIV感染者NK细胞内穿孔素表达降低，抗病毒治疗后升高；CD8^＋T细胞内穿孔素表达升高，抗病毒治疗后无显著变化。     

The emergence of non-cytolytic NK1.1+ T cells in the long-term culture of murine tumour-infiltrating lymphocytes: a possible role of transforming growth factor-beta.

The mechanism by which murine tumour-infiltrating lymphocytes (TIL) decreased their anti-tumour activity during an in vitro culture with interleukin-2 (IL-2) was investigated. A phenotype analysis revealed that the TIL cultured for 7 days (TIL-d7) were exclusively NKI.1- CD4- CD8+ CD3+ cells and that this population was replaced by natural killer (NK)1.1+ CD4- CD8 CD3+ cells by day 27 (TIL-d27) during the culture of TIL. The TIL-d7 cells showed a cytolytic activity against B16 melanoma, whereas the TIL-d27 cells had lost this activity, suggesting that the decrease in the anti tumour effect of TIL during the culture with IL-2 was due to their populational change. Analysis on the characteristics of the TIL-d27 cells revealed that they expressed skewed T-cell receptor (TCR) V beta 5 and increased mRNA expression of V alpha 14. In addition, they expressed transforming growth factor beta (TGF-beta) mRNA. Interestingly, TGF-beta augmented the proliferation of TIL-d27 cells under the presence of IL-2, but suppressed that of TIL-d7 cells. Moreover, the proliferation of TIL-d27 cells was suppressed by anti-TGF-beta monoclonal antibody. Collectively, these results suggest that, in contrast to its suppressive effect on anti-tumour effector T cells. TGF-beta could be an autocrine growth factor for NKL1.1+ T cells and thereby induce non-cytolytic NK1.1+ T cells in the long-term culture of TIL.     

CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients.

In humans, NK receptors are expressed by natural killer cells and some T cells, the latter of which are preferentially alphabetaTCR+ CD8+ cytolytic T lymphocytes (CTL). In this study we analyzed the expression of nine NK receptors (p58.1, p58.2, p70, p140, ILT2, NKRP1A, ZIN176, CD94 and CD94/NKG2A) in PBL from both healthy donors and melanoma patients. The percentages of NK receptor-positive T cells (NKT cells) varied strongly, and this variation was more important between individual patients than between individual healthy donors. In all the individuals, the NKT cells were preferentially CD28-, and a significant correlation was found between the percentage of CD28- T cells and the percentage of NK receptor+ T cells. Based on these data and the known activated phenotype of CD28- T cells, we propose that the CD28- CD8+ T cell pool represents or contains the currently active CTL population, and that the frequent expression of NK receptors reflects regulatory mechanisms modulating the extent of CTL effector function. Preliminary results indicate that some tumor antigen-specific T cells may indeed be CD28- and express NK receptors in vivo.     

Phenotyping of lymphocytes expressing regulatory and effector markers in infiltrating ductal carcinoma of the breast

Dysfunction of the host immune system in cancer patients can be due to a number of reasons including suppression of tumour associated antigen reactive lymphocytes by regulatory T (Treg) cells. In this study, we used flow cytometry to determine the phenotype and relative abundance of the tumour infiltrating lymphocytes (TILs) from 47 enzymatically dissociated tumour specimens from patients with infiltrating ductal carcinoma (IDC) of the breast. The expression of both effector and regulatory markers on the TILs were determined by using a panel of monoclonal antibodies. Analysis revealed CD8(+) T cells (23.4+/-2.1%) were predominant in TILs, followed by CD4(+) T cells (12.6+/-1.7%) and CD56(+) natural killer cells (6.4+/-0.7%). The CD4(+)/CD8(+) ratio was 0.8+/-0.9%. Of the CD8(+) cells, there was a higher number (68.4+/-3.5%) that expressed the effector phenotype, namely, CD8(+)CD28(+) and about 46% of this subset expressed the activation marker, CD25. Thus, a lower number of infiltrating CD8(+) T cells (31.6+/-2.8%) expressed the marker for the suppressor phenotype, CD8(+)CD28(-). Of the CD4(+) T cells, 59.6+/-3.9% expressed the marker for the regulatory phenotype, CD4(+)CD25(+). About 43.6+/-3.8% CD4(+)CD25(+) subset co-expressed both the CD152 and FOXP3, the Treg-associated molecules. A positive correlation was found between the presence of CD4(+)CD25(+) subset and age (> or =50 years old) (r=0.51; p=0.045). However, no significant correlation between tumour stage and CD4(+)CD25(+) T cells was found. In addition, we also found that the CD4(+)CD25(-) subset correlated with the expression of the nuclear oestrogen receptor (ER)-alpha in the tumour cells (r=0.45; p=0.040). In conclusion, we detected the presence of cells expressing the markers for Tregs (CD4(+)CD25(+)) and suppressor (CD8(+)CD28(-)) in the tumour microenvironment. This is the first report of the relative abundance of Treg co-expressing CD152 and FOXP3 in breast carcinoma.     

Suppression of natural killer cells in the presence of CD34+ blood progenitor cells and peripheral blood lymphocytes.

Abstract Mobilization of CD34 + peripheral blood progenitor cells (PBPCs) with granulocyte-colony stimulating factor (G-CSF) may induce functional alterations in peripheral blood lymphocyte (PBL) subsets. We and others have shown that natural killer (NK) cells from PBPC collections are less expandable in vitro than those obtained during steady-state hematopoiesis. We show here that the extent of this proliferation deficit is related to the number of circulating CD34 + cells in vivo at the time of PBPC apheresis. Likewise, addition of autologous CD34 + cells to unseparated PBL reduced the expansion of the NK-cell subset by 22.2% +/- 6.0% (n = 10; P <.005). In contrast, when using purified NK cells, their proliferation remained unimpaired by autologous CD34 + cells. Supernatants from CD34 + cells cultured with autologous PBLs had an inhibitory effect on proliferation of purified NK cells (n = 16; P =.03), indicating that an interaction between CD34 + cells and lymphocytes is essential for the suppressive effect on NK cells. To investigate the role of T cells in this interaction, intracellular cytokines were determined in T cells cultured for 7 days with or without autologous CD34 + cells. When cultured with CD34 + cells, the frequency of IL-2-producing CD4 + and CD8 + T cells was reduced by 19% and 24%, respectively, compared with T cells cultured alone (n = 7; P =.016). Interferon-gamma-producing T cells were slightly reduced ( P = not statistically significant [ns]). Finally, the influence of T cells and NK cells on the recovery of myeloid colony-forming cells (CFU-GMs) from purified CD34 + cells was examined. In the presence of T cells, 16% +/- 6% of the input CFU-GM recovered after 7 days, compared with 5% +/- 4% in the presence of NK cells (n = 5; P = ns). Our findings point to an inhibition of NK-cell proliferation mediated by an interaction of CD34 + cells and T cells occurring during PBPC mobilization with G-CSF.     

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/Vβ11(+), 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.     

Expression of p75 chain of IL-2 receptor in the early immunological reconstitution after allogeneic bone marrow transplantation.

Expression of p55 and p75 chains of IL-2 receptor (IL-2R) on the surface of both T and natural killer (NK) circulating lymphocytes was investigated in 14 paediatric patients given allogeneic bone marrow transplantation (BMT) from HLA-identical sibling or partially matched family donors. IL-2-induced proliferative and cytotoxic responses were also studied and all analysis was performed within 45 days from transplant. We found that, early after transplant, the percentage of p55+ and of p75+ peripheral blood lymphocytes (PBL) was not significantly different in patients who had received HLA-identical BMT (p55+ 8.04 +/- 4.87%; p75+ 28.27 +/- 18.85%) compared with healthy controls (p55+ 7.26 +/- 6.17%; p75+ 19.42 +/- 10.49%), while recipients of T cell-depleted marrow included a remarkably high percentage (76-90%) of p75+ PBL, which were mostly CD3- and co-expressed CD56 molecule. Comparable values of p55+ lymphocytes were observed in all patients and controls. However, in contrast to the other two groups, in recipients of T cell-depleted BMT the majority of these cells co-expressed p75 chain and CD56 antigen. IL-2-induced proliferation and lymphokine-activated killer (LAK) activity were detectable in all patients, and their values did not correlate with expression of p55 or p75 chains. Our data suggest that expansion of NK subsets expressing IL-2R chains after T cell-depleted BMT may be related to early reconstitution of natural immunity in the presence of allogeneic stimuli.     

Human T cells in hu-PBL-SCID mice proliferate in response to Daudi lymphoma and confer anti-tumour immunity.

In vitro culture of human peripheral blood lymphocytes (PBL) with Daudi (Burkitt lymphoma) cells results in selective proliferation of V gamma 9/V delta 2 T cells with high cytotoxicity against Daudi cells. After adoptive transfer into severe combined immunodeficient (SCID) mice, these cells exert specific anti-tumour activity against Daudi lymphoma. To test whether cytotoxic V gamma 9/V delta 2 T cells are induced in SCID mice, human PBL injected intraperitoneally were stimulated with irradiated Daudi cells (PBL/Daudi-SCID). After 7-14 days, PBL/Daudi-SCID had a significantly higher percentage of human gamma delta T cells in their peritoneal cavity, lymph nodes and blood than controls (PBL-SCID). DNA content analysis of T cell subsets from PBL/Daudi-SCID showed a significantly higher percentage of cells in S + G2 + M phases of the cell cycle in the TCR-gamma delta-1+ than in CD3+ cell population. Human cells recovered from PBL/Daudi-SCID showed specific cytotoxicity against Daudi cells. PBL/Daudi-SCID inoculated with a lethal dose of Daudi lymphoma survived significantly longer than controls. This protection was specific for Daudi cells and was not mediated by murine natural killer (NK) cells. Thus human peripheral blood T cells grafted in SCID mice proliferate in response to antigen and confer specific immunity.     

Activation of regulatory T cells instigates functional down-regulation of cytotoxic T lymphocytes in human breast cancer

Regulatory T (Treg) cells are a subpopulation of T cells with the ability to control the responses of both CD4+ and CD8+ T cells. A case–control study was conducted in order to determine the functional attributes of Treg cells within the breast cancer milieu. Triple-color flow cytometry was utilized to study the phenotype expression of CD4+CD25+ Treg cells and CD8+ T cells in autologous tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) derived from 33 patients with stage I–III breast cancer. The prevalence of CD4+CD25+ T cells was significantly higher in TILs than in PBLs. The expressions of FOXP3 and GITR in CD4+CD25+ Treg cells were lower in PBLs than in TILs. Functional studies showed that both granzyme B and perforin were barely expressed in peripheral Treg cells but were highly expressed in Treg cells in the tumor microenvironment. On the contrary, down-regulation of both granzyme B and perforin expressed in the CD8+ cytotoxic T lymphocytes was significantly lower in TILs than in PBLs. Further functional assays demonstrated that Th1 cytokines and cytotoxic molecules were synchronously up-regulated in CD8+ cytotoxic T cells. The in vitro kinetic study showed that adequate activation of TILs derived from breast cancer tissue could restore the appropriate antitumor immune response.     

Cytotoxic phenotype of tumor infiltrating lymphocytes in medullary carcinoma of the breast.

Medullary carcinoma (MC) of the breast is considered to carry a more favorable prognosis than other subtypes of infiltrating ductal carcinoma This is a biological paradox because its clinical behavior contrasts with its anaplastic morphology. MC is characterized by a dense lymphocytic infiltrate. In this study, we determined the cytotoxic potential and activity of tumor infiltrating lymphocytes (TILs) in MC by CD3, CD8, TIA-1, and granzyme B immunostaining on paraffin-embedded sections. Fourteen cases of typical MC (TMC) and 15 cases of atypical MC (AMC) classified according to Ridolfi criteria, and 19 cases of poorly differentiated infiltrating ductal carcinoma (PDC) were studied. TILs were quantified separately into two groups: cells infiltrating tumor nests and cells within stroma The number of CD8+ and TIA-1+ cells infiltrating tumor cell nests were markedly increased in TMC and AMC, as opposed to the PDC subgroup (159.6+/-132.8; 77.4+/-59.3; 9.4+/-10.5 and 171.2+/-152.4; 72.3+/-55.0; 10.8+/-12.7 per high power field, respectively). The number of tumor infiltrating granzyme B+ cells was significantly greater in TMC and AMC, as compared with the PDC subgroup (82.1+/-64.9, 33.9+/-19.7, and 3.1+/-5.1, respectively). Although no significant difference was found between the number of stromal CD3+ and CD8+ lymphocytes among the three subgroups, stromal granzyme B+ cells were significantly elevated in TMC and AMC as compared with the PDC subgroup. Finally, the relative proportion of granzyme B+ as opposed to CD3+ intraepithelial and stromal lymphocytes was greater in TMC and AMC as compared with the PDC subgroup (0.52+/-0.29; 0.47+/-0.31; 0.19+/-0.18 and 0.18+/-0.11; 0.13+/-0.11; 0.06+/-0.05, respectively). The presence of increased numbers of activated cytotoxic lymphocytes in MC of the breast may be a key mechanism active in the host versus tumor response leading to improved prognosis.     

Localization of T cell receptor (TCR)-gamma delta + T cells into human colorectal cancer: flow cytometric analysis of TCR-gamma delta expression in tumour-infiltrating lymphocytes.

We analysed TCR-gamma delta expression in tumour-infiltrating lymphocytes (TIL) obtained from 13 patients with colorectal cancer and simultaneously isolated the T lymphocytes from normal intestinal tissue (IL) to compare the frequencies of TCR-gamma delta expression in TIL, IL, and peripheral blood lymphocytes (PBL) in the same patient. Flow cytometric analysis showed that the frequency of TCR-gamma delta expression in TIL (2.75 +/- 1.84%) was significantly lower than that in IL (15.28 +/- 9.45%, P < 0.01). However, a larger quantity of TIL was separated than IL per unit weight of specimen, so the total number of gamma delta T cells obtained per unit weight was not different between tumour tissue and normal intestine. In addition, phenotypic analysis revealed that about half of the TCR-gamma delta + TIL were CD8+ (CD4+, 3.0 +/- 3.1%; CD8+, 54.7 +/- 19.9%, mean +/- s.d. of five patients), and a very similar result was obtained in TCR-gamma delta + IL (CD4+, 2.7 +/- 2.4%; CD8+, 53.1 +/- 17.4%). In contrast, most TCR-gamma delta + PBL were double-negative (CD4+, 3.2 +/- 3.0%; CD8+, 20.6 +/- 7.4%). These results indicated that TCR-gamma delta + CD8+ T cells selectively and consistently localized in colorectal tumour tissue, similarly to normal intestinal epithelium.     

Isolation, phenotyping, and functional analysis of lymphocytes from human liver.

The isolation of mononuclear cells from human liver tissues was achieved by a simple method consisting of enzymatic and mechanical dissociation, density gradient centrifugation, and adherence to plastic. The method was optimized to obtain a nearly complete recovery of different lymphoid subpopulations. The mononuclear cells recovered from "normal" liver tissues consisted of 33 +/- 9% (mean +/- SD) small lymphocytes, 44 +/- 6% large granular lymphocytes, 9 +/- 2% monocytes/macrophages, 9 +/- 3% granulocytes, and 5 +/- 2% other cells as determined by microscopic analysis of May-Grünwald-Giemsa-stained cytocentrifuge smears. Phenotypic analysis of the liver-infiltrating lymphocytes (LIL) by two-color flow cytometry showed that CD3-CD56+ NK cells represented 43 +/- 6% (mean +/- SD), CD3+CD56- T cells, 30 +/- 9%, and CD19+ B cells 3 +/- 1%. The predominant phenotype of the liver-infiltrating NK cells was CD3-CD56+CD16- in contrast to that of circulating NK cells, which are largely CD3-CD56+CD16+. The alpha/beta TCR+ T cells were 42 +/- 14% and gamma/delta T cells were infrequent (4 +/- 4%). The proportions of the three major lymphoid populations (T, NK, and B cells) recovered from liver tissues of 10 patients with different liver diseases were altered. Tissue sections from the same specimens were stained by the immunoperoxidase method to compare the in situ cellular composition with that determined by flow cytometry. LIL recovered from normal (control) and virally infected (non-A, non-B hepatitis) liver tissues had high NK activity (up to 1,000 LU/10(7) cells) as measured against K-562 targets in 4-hr 51Cr-release assays. NK activity was significantly lower (P less than 0.05) in LIL recovered from other liver diseases. LIL had spontaneous cytotoxicity against NK-resistant Daudi targets which was significantly higher (P less than 0.05) in control and virally infected than in other liver tissues. Our results indicated that human LIL recovered from normal and diseased liver tissues contained a high proportion of functionally active NK cells and that NK and lymphokine-activated killer activities but not the percentages of CD56+ cells were decreased in end-stage primary biliary cirrhosis and primary sclerosing cholangitis. In contrast, the proportions of NK cells and NK activity remain high in non-A, non-B hepatitis.     

Detection of cell-adhesion molecules on human liver-associated lymphocytes.

Liver-associated lymphocytes (LAL) from human liver are phenotypically and functionally different from peripheral blood lymphocytes (PBL). Phenotypically, they are mainly represented by the CD3+/-CD56+ phenotype and functionally they spontaneously possess lymphokine-activated killer (LAK) activity. In this study we evaluated the expression of cell-adhesion molecules (CAM) which could be involved in LAL contacts with other sinusoidal cells and/or be responsible for the LAK activity. The LAL population was isolated by sinusoidal high-pressure lavage from partial hepatectomies obtained from patients operated on for benign liver disease (n = 6). Surface expression of the beta 2 integrin chains (CD18, CD11a, CD11b, CD11c), as well as that of members of the immunoglobulin superfamily (CD2, CD54, CD56, CD58), were analysed by one or two-colour flow cytometry. Quantitative and qualitative differences were observed in the expression of CAM between LAL and PBL. LAL were characterized by an increase in the percentages of CD11b+, CD54+, CD56+ and CD58+ cells and a decrease in the percentage of CD2+ cells compared to PBL. Fluorescence intensity values for CD2 and CD56 were higher in LAL than in PBL. Moreover, CD11a/CD18 cells presented a bimodal distribution (dim and bright) in both PBL and LAL; whereas these two subpopulations were equally represented in PBL, the number of bright cells was significantly greater (> 80%) in LAL. The increase in CAM expression (percentage of positive cells and intensity of fluorescence) on LAL combined with their increase in natural killer (NK) and LAK activities already reported, support the idea that, at least some, LAL might be, compared to PBL, in an activated state in vivo.     

IgE Fc receptor positive T and B lymphocytes in patients with the hyper IgE syndrome.

The percentages of peripheral blood lymphocytes (PBL), bearing Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R) were determined in four patients with the hyper IgE syndrome by a rosette assay employing IgE and IgG coated fixed ox erythrocytes. The patients had 8 +/- 3% Fc epsilon R+ and 13 +/- 8% Fc gamma R+ PBL, compared to 1.2 +/- 1% Fc epsilon R+ and 17 +/- 4% Fc gamma R+ PBL for control donors. T cells were isolated by rosetting with neuraminidase treated sheep erythrocytes (EN). Indirect immunofluorescence with Lyt 3 monoclonal antibody (MoAb) to the sheep erythrocyte receptor, followed by rosetting for Fc epsilon R and Fc gamma R showed that the patients' T cells contained less than 0.1% Fc epsilon R+ and 1.4 +/- 0.2% Fc gamma R+ cells; T cells from the control subjects contained less than 0.1% Fc epsilon R+ and 11 +/- 4% Fc gamma R+ cells. The non-T (EN rosette depleted) cells of the patients included 56 +/- 18% sIgM+/sIgD+, 45 +/- 9% Fc epsilon R+ and 35 +/- 27% Fc gamma R+ cells. Indirect immunofluorescence with MoAb to IgM, IgD, and NK cells (antibody B73.1) followed by rosetting for Fc epsilon R and Fc gamma R, indicated that 92 +/- 2% of the Fc epsilon R+ cells and 9 +/- 7% of the Fc gamma R+ cells were B cells (mu+/delta+), while 3 +/- 4% of the Fc epsilon R+ and 30 +/- 23% of the Fc gamma R+ cells were NK cells (B73.1+). Thus, most of the Fc epsilon R+ non-T cells were B cells, and only a small fraction appeared to be NK cells. On the other hand, Fc gamma R+ B cells were outnumbered by Fc gamma R+ NK cells (B73.1+) by three to one. The data indicate that patients with the hyper IgE syndrome have increased numbers of Fc gamma R+ PBL, most of them being B cells, whereas their T cells contain less than 0.1% Fc epsilon R+ cells.     

T cell receptor gamma/delta expression on lymphocyte populations of breast cancer patients.

The quantitative distribution and phenotype of gamma/delta lymphocytes in the peripheral blood (PBL), tumour draining lymph node (LNL) and tumour infiltrating lymphocytes (TIL) from breast carcinoma patients were determined by one- and two-colour flow cytometry. The TCR-gamma/delta + cells generally expressed the T cell lineage antigen CD3. The proportions of such cells were variable but generally small from all the three sources. Phenotypic analysis revealed that the CD8 marker was consistently and predominantly observed on gamma/delta + CD3+ cells in the tumour infiltrate, whereas CD4 expression, while generally low, was noted on a significant percentage (median 10%) of LNL gamma/delta + lymphocytes. In both PBL and LNL the predominant gamma/delta cell population was CD4-8-.     

Peripheral blood CD4+CD8+ lymphocytes in cynomolgus monkeys are of resting memory T lineage

In this study, we analyzed peripheral blood CD4+CD8+ double-positive (DP) lymphocytes in adult cynomolgus monkeys (Macaca fascicularis). Forty of 55 monkeys had > 5% of the peripheral blood DP subpopulation (9.3 +/- 5.9%; mean +/- SD) in peripheral blood lymphocytes (PBL) in contrast to a low percentage of peripheral blood DP cells in humans and mice. In a cross-sectional study, the peripheral blood DP cells were found to increase in proportion with age. To clarify whether peripheral blood DP lymphocytes were immature precursors released from thymus without prior differentiation, the expressions of CD8 chains and CD1b on peripheral blood DP lymphocytes were compared with those on thymocytes. The peripheral blood DP lymphocytes were CD8 alpha + beta- and CD1b-, while thymic DP lymphocytes were CD8 alpha + beta + and CD1b +, suggesting that the peripheral blood DP cells are extrathymic T lymphocytes. Furthermore, the peripheral blood DP lymphocytes exhibited a resting memory T cell phenotype with CD2hiCD3+CD28-CD29hiCD49dhiCD69- CD80lo. Taken together, adult cynomolgus monkeys possess a unique peripheral blood DP T cell subpopulation which expresses a resting memory T cell phenotype. In addition, similar phenotypic properties of DP lymphocytes were distributed in the spleen and lymph nodes, although the proportion was less in the spleen and much less in lymph nodes than in PBL.      

Expression of Inhibitory Receptors in Natural Killer (CD3CD56) Cells and CD3CD56 Cells in the Peripheral Blood Lymphocytes and Tumor Infiltrating Lymphocytes in Patients with Primary Hepatocellular Carcinoma

The cytolytic responses of NK (CD3(-)CD56(+)) and CD3(+)CD56(+) cells are inhibited by the engagement of the killer inhibitory receptors (p58.1, p58.2, and CD94) with respective ligands on the target cell. The expression of these receptors in peripheral blood lymphocytes (PBLs) (n = 18) and tumor infiltrating lymphocytes (TILs) (n = 7) was examined in patients with primary hepatocellular carcinoma (HCC). There were no differences in the expression of the three inhibitory receptors by both NK and CD3(+)CD56(+) PBLs in patients with HCC compared to that of control NK and CD3(+)CD56(+) PBLs, respectively (all P = NS). However, the expression of p58.1 by NK TILs and by CD3(+)CD56(+) TILs in patients with HCC was significantly decreased compared to that of hepatic lymphocytes of the control subjects (8.9% vs 37.85%, P = 0.047; 4.1% vs 25.2%, P = 0.049, respectively). The expression of p58.2 by CD3(+)CD56(+) TILs and CD94 by NK TILs was also decreased compared to that of hepatic lymphocytes of the control subjects (16.9% vs 73.1%, P = 0.047; 21% vs 49.95%, P = 0.037, respectively). These changes were limited to hepatic TILs, and this observation may reflect an adaptive anti-tumor phenomenon occurring in the microenvironment of HCC.     

Intracellular expression of MICA in activated CD4 T lymphocytes and protection from NK cell-mediated MICA-dependent cytotoxicity

MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously, we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here, we show that IL-2, IL-4, and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs), as assessed by Western blot. IL-2 effect involved Jak3/STAT5, p38 MAPK, p70(56) kinase, Lck/fyn kinases, and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However, surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot, but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells, and susceptibility was increased when HLA class I molecules were blocked. Also, cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However, the participation of MICA in these responses, if any, was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory, virus-infected, or tumor microenvironment, where NK and activated CD4+ T cells are recruited.