In vivo expression of perforin by natural killer cells during a viral infection. Studies on uveitis produced by herpes simplex virus type I.

A potent cytolytic pore-forming protein (PFP, perforin, or cytolysin) is associated with the cytoplasmic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The role of PFP/perforin in cytolytic reactions carried out in vivo is still unclear. Here, the authors performed immunohistochemical analysis using antibodies monospecific for perforin and made use of a murine uveitis model produced by intracameral inoculation of herpes simplex virus I (HSV-I). The main cell infiltrate found in the anterior segment of virus-inoculated eyes consisted of Thy-1+/asialo GM1+/CD8-/CD4- cells, presumably representing NK cells. Perforin staining was detected mainly in cells bearing this phenotype. Perforin was only detected in cells displaying the large granular lymphocyte morphology. A small number of perforin-positive cells (less than 5%) colabeled as CD8+, indicating that these cells could have belonged to the CTL lineage. These observations show for the first time the presence of perforin-containing NK cells in tissues of animals undergoing acute viral infections.     

Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.     

GD2 Ganglioside-Specific Monoclonal Antibody Reacts with Murine Cytotoxic T Lymphocytes Reactive with FBL-3N Erythroleukaemia

Ganglioside expression on cytotoxic T lymphocytes induced against the mouse erythroleukaemia line FBL-3N, was analysed, compared with that of naive T lymphocytes in the thymus, lymph nodes and spleen. Although normal uncultured and cultured T lymphocytes expressed no GD2, GD3 or GM2 gangliosides, cytotoxic T cells with CD4⁺ CD8⁻, CD4⁻CD8⁺, and CD4⁻CD8⁻ phenotypes reacted with anti-GD2 monoclonal antibody with various intensities. The reactivity with anti-GD2 was associated with the intensity of cytotoxic activity as analysed using cytotoxic T lymphocyte (CTL) clones established from the bulk CTLs of each phenotype. An increase of the mRNA expression of GM2/GD2 synthase gene was demonstrated by Northern blot hybridization using RNA extracted from thymocytes, spleen cells, Con A-treated spleen cells and various types of CTL cells. These results indicated that GD2 ganglioside expression might be associated with the functional differentiation of murine T lymphocytes.     

Expression of perforin and cytolytic potential of human peripheral blood lymphocyte subpopulations.

To verify the physiological role of the pore-forming protein perforin in vivo, its expression in subpopulations of human peripheral blood lymphocytes was examined by immunocytochemical staining and their cytolytic potentials compared. In addition to NK cells and gamma delta T cells, which uniformly expressed abundant perforin in their cytoplasmic granules, only a small subpopulation of CD8+ alpha beta T cells contained perforin, namely the CD11b+ subset. However, in vitro activation with an anti-CD3 antibody and IL-2 induced perforin expression in approximately 50% of the CD8+CD11b- T cells and also in a small subset of CD4+ T cells. A distribution of perforin in CD8+ and CD4+ T cells, similar to in vitro activated T cells, was observed in fresh peripheral blood lymphocytes from infectious mononucleosis patients. In all instances, the expression of perforin correlated with the cytolytic potential of these subpopulations. The results strongly suggest that perforin plays a role in the manifestation of cytotoxic activity in vivo.     

Expression of asialo GM1 by both Thy-1-positive and Thy-1-negative lymphocytes: evidence for modification of asialo GM1 by sialic acid

In order to determine the extent to which Thy-1-positive and Thy-1-negative lymphocytes express the asialo GM1 antigen, lymphocytes in normal spleen and lymph node were examined for simultaneous expression of the asialo GM1 and Thy-1.2 determinants. The results presented herein demonstrate that 55-57% of Thy-1.2-positive cells in spleen and 61-70% of Thy-1.2-positive cells in lymph node express asialo GM1. Furthermore, a significant frequency of Thy-1-negative cells in spleen (12-19%) and in lymph node (28-32%) also express asialo GM1. Since asialo GM1 has previously been shown to be absent on Ig-positive lymphocytes [9], these results establish that asialo GM1 is a marker shared by lymphocytes belonging to the T-cell lineage and lymphocytes apparently not committed to the T-cell to the T-cell lineage, most probably including natural killer (NK) cells. The implication of this finding as to the controversy regarding the possible relation of NK cells to T cells is discussed. Pretreatment of lymphocytes from spleen, thymus and lymph node with neuraminidase resulted in subsequent reactivity of 80-90% of these cells with anti-asialo GM1 anti-bodies. A smaller increase in asialo GM1 detection after neuraminidase treatment was seen with bone-marrow cells (65%). Protease treatment did not affect the subsequent reactivity of lymphocytes with anti-asialo GM1 antibodies. It is concluded that in situ enzymatic modification of asialo GM1 by the addition of sialic acid may be an important regulatory event in lymphocyte differentiation.     

Natural Killer Cells Contribute to Inflammation but Do Not Appear to be Essential for the Induction of Clinical Lymphocytic Choriomeningitis

The inflammatory exudate found in cerebrospinal fluid (CSF) of mice 6 days after intracerebral infection with lymphocytic choriomeningitis virus (LCMV) contains substantial populations of both cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Removal of NK cell activity by in vivo treatment with antibody to the asialo GM1 ganglioside and studies with NK-deficient bg/bg mice did not clearly determine whether NK cells contribute in any way to the development of clinical LCM. However, the LCM disease process induced in cyclophosphamide-suppressed, LCMV-infected recipients by the adoptive transfer of LCMV-immune spleen cells occurs in the absence of NK cell effector function in spleen, lymph nodes, or CSF of the recipients, though potent CTL populations are present in all of these sites. In this situation, NK cells are apparently not required for the induction of neurological symptoms that are indistinguishable from those of classical LCM.     

The CD45RA molecule is expressed in naive murine CTL precursors but absent in memory and effector CTL.

We have studied the expression of the CD45RA molecule in murine cytotoxic T lymphocytes (CTL) specific for the allogeneic H-2Kb molecule at different stages of differentiation. The CD45RA phenotype of naive H-2Kb-specific CTL precursors has been determined using primary in vitro CTL responses. For the analysis of memory CTL we have immunized mice in vivo followed by restimulation in vitro. We have also determined the CD45RA expression at the CTL effector stage. Our results show that among naive CD8+ T cells both the CD45RA+ and the CD45RA- subpopulations can mount Kb-specific CTL responses. In contrast, memory CTL responses are mediated only by the CD8+ CD45RA+ T cell subpopulation. Similarly, effector CTL are CD8+ CD45RA- while the CD8+ CD45RA+ subpopulation does not exhibit specific cytolytic activity. The data indicate that CD45RA expression changes during CTL differentiation and that memory as well as effector CTL lack this marker.     

Localization and identification of granzymes A and B-expressing cells in normal human lymphoid tissue and peripheral blood.

Cytoplasmic granules from activated natural killer (NK) and cytotoxic T lymphocytes (CTL) contain a pore-forming protein, perforin, and several homologous serine proteinases called granzymes. Expression of these proteins correlates with the cytolytic potential of cytotoxic lymphocytes. Using a panel of MoAbs specific for human granzyme A and B, respectively, expression of these proteinases in non-pathological lymphoid tissue and peripheral blood lymphocyte (PBL) subpopulations was investigated. Using immunohistochemistry and double stainings, the phenotype of granzyme-expressing cells in lymphoid tissue was investigated. Granzyme-positive cells were detected in all lymphoid tissues tested. No large differences in the number and distribution between granzyme A- and granzyme B-positive cells were observed. The highest number of positive cells was located in the red pulp of the spleen. Significant numbers were detected in tonsil, lymph nodes, liver and thymus. Low numbers were present in the lamina propria of non-inflamed stomach, small intestine and colon. Phenotypic analysis and cell sorting showed that most of the granzyme-positive cells in lymphoid tissue and PBL consisted of CD3-CD16+CD56+ lymphocytes. Hardly any granzyme-positive CD3+CD8+ CTL were present in peripheral blood. The synthesis of granzyme A as well as B by both CD3+CD16+CD56+ and CD3+CD8+ cells in peripheral blood was increased upon IL-2 stimulation. These results indicate that in normal lymphoid tissue the predominant cytolytic cell population is formed by the NK cells, and activated CTL are rare.     

A glycolipid on the surface of mouse natural killer cells

Cytotoxic treatment with rabbit antiserum raised against purified glycosphingolipid “asialo GM1” was capable of eliminating natural killer (NK) activity of spleen cells from different inbred mouse strains including CBA/J, C57BL/6, BALB/c, AKR, and athymic nude mice. The anti-asialo GM 1 antiserum showed little cross-reactivity with structurally related glycolipids, e.g. GM 1, GD 1 b and asialo GM 2 in the microflocculation test. The specific reactivity of this antiserum with NK cells was confirmed by the quantitative absorption of anti-NK activity with graded amounts of asialo GM 1 but not with other glycosphingolipids. The absorption of anti-brain-associated T cell antigen (anti-BAT) with asialo GM 1 also effectively diminished its anti-NK activity, leaving the ability to kill T cells intact. This suggests that the antibody to asialo GM 1 is responsible for the anti-NK activity contained in the anti-BAT antiserum. In contrast to the extreme sensitivity of NK cells to anti-asialo GM 1, alloreactive cytotoxic T killer cells generated in the mixed lymphocyte culture were not killed by anti-asialo GM 1 and complement. These results indicate that asialo GM 1 is expressed on mouse NK cells in a high concentration.     

Functional importance of CD4+ and CD8+ cells in cytotoxic lymphocytes activity and associated gene expression. Impact on the age-related decline in lytic activity.

Previously we found that the age-related decline in cytotoxic lymphocyte (CTL) activity in a murine allogeneic system is associated with declining expression of the perforin and two serine esterase genes by senescent CTL. To identify the cell subsets responsible for the age-related decrement in the generation of CTL, splenic T cells, subsets, and mixtures of subsets from aging and young BALB/c female mice were analyzed for their allo-responsiveness to C57BL/6 spleen cells. Depletion studies revealed that CD8+ cells expressed both the lytic activity and the CTL-associated genes, although both CD8+ and CD4+ cells were required for generation of optimal lytic and proliferative responses. Mixture experiments demonstrated that the reduced lytic activity generated by CD8+ cells from the spleens of old mice is a consequence of alterations in both CD8+ and CD4+ cells. The results of experiments in which CD4+ cells from young and old mice were mixed revealed that the alteration in CD4+ cells is consistent with a loss of function. These findings show that (a) the expression of the perforin and serine esterase genes in primary CTL is associated with CD8+ T cells in old and young mice, and exhibits an age-related decrement consistent with that for lytic activity, and (b) the senescent decline in CTL activity is a consequence of aging decreasing activity in both the CD4+ and CD8+ subsets.     

Differential expression of the ASGM1 antigen on anti-reovirus and alloreactive cytotoxic T lymphocytes (CTL)

Anti-reovirus cytotoxic effectors were found to be: (i) H-2 restricted; (ii) virus specific; (iii) non-lytic (in 4 h) for natural killer (NK)-sensitive YAC-1 cells; and (iv) positive for the Thy-1 and Lyt-2 lymphocyte markers. Thus, anti-reovirus cytotoxic effectors have the functional and phenotype characteristics of cytotoxic T lymphocyte (CTL). A significant fraction of anti-viral CTL, as well as alloreactive CTL, were also found to be positive for the asialo GM1 (ASGM1) cell surface antigen, generally considered to be a NK cell marker. ASGM1 expression on these CTL, as determined by sensitivity to antibody plus complement (C), appeared to be highly variable and dependent on two factors-the nature of the antigenic stimulus (viral vs. alloantigen), and the mouse strain from which the CTL originated. Thus, ASGM1 antigen expression on CTL appears to be regulated and may be under the control of lymphokines, development differentiation signals and/or other strain-dependent genetic factors.     

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。     

Recombinant interleukin-2 inhibits growth of human tumor xenografts in congenitally athymic mice

Administration of human recombinant interleukin-2 (RIL-2) into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of HeLa, HU 609T and T24B human carcinoma cells partially inhibited growth of the human tumor xenografts. In vitro activation of nu/nu spleen cells with human RIL-2 resulted in generation of killer cells showing in the 51Cr cytotoxicity assay similar levels of cytolysis as RIL-2-activated spleen cells from heterozygous (nu/+) mice. The RIL-2-activated (LAK) cells were cytotoxic for a variety of mouse and human tumors, reaching the peak of their cytotoxic activity after 3 days of cultivation in the RIL-2-containing medium. The cytotoxic activity of activated nu/nu spleen cells was significantly reduced by treatment with antibody against glycolipid asialo GM1, the differentiation antigen of natural killer (NK) cells. This finding suggests that in addition to the conventional, asialo GM1- LAK cells, asialo GM1+ activated NK cells participated in the cytotoxicity displayed by the IL-2-activated nu/nu killer spleen cells.     

Immunotherapeutic elimination of syngeneic tumors in vivo by cytotoxic T lymphocytes generated in vitro from lymphocytes from the draining lymph nodes of tumor-bearing mice

The draining lymph nodes (LN) of mice injected with viable syngeneic tumor cells contain populations of lymphoid cells which are capable of generating functional cytotoxic T lymphocytes (CTL) during an in vitro culture period without the need of secondary stimulation with irradiated tumor cells or addition of exogenous cytokines. Intravenous injection of as few as 2.5 x 10(6) tumor-reactive effector cells generated using this protocol effectively eliminated challenges of tumor cells at distal skin sites, whereas injection of cultured control lymphoid cells was ineffective at rejecting tumor challenges. The anti-tumor CTL generated from the draining LN of C57BL10 mice immunized with viable syngeneic tumor cells were found to be specific for the tumor used for immunization both in vitro and in vivo. Phenotypic analysis of the cells responsible for tumor elimination (both in vitro. and in vivo) demonstrated the anti-tumor effector cells to be CD4-CD8+ asialo GM1+NK1.1-. Finally, anti-tumor CTL generated during the short in vitro culture period were able to effectively reject pre-existing tumors in vivo in a systemic fashion. These data indicate that the generation of tumor-reactive CTL from the lymphoid cells contained in the LN draining the site of a tumor may provide a valuable source of effector cells which can be used in the immunotherapeutic treatment of cancer.     

In vivo rapid reduction of alloantigen-activated CD8+ mature cytotoxic T cells by inhibitors of acidification of intracellular organelles, prodigiosin 25-C and concanamycin B

Prodigiosin (PrG) 25-C and concanamycin B (CMB) are immunosuppressants that specifically inhibit the induction of cytotoxic T cells (CTL) without affecting the function of B cells and helper T cells in vivo. Both compounds inhibit acidification of intracellular organelles and induce destruction of cytotoxic granules and degradation of perforin in vitro. Here we show that a single intraperitoneal (i.p.) injection of PrG 25-C, and of CMB, into mice eliminates cytotoxic activity 7 days after alloantigen stimulation (when mature CTL activity has been detected in control mice), with minimal effect on the alloantigen-specific antibody titre in serum. FK506 did not suppress the cytotoxic activity with this administration schedule. Suppression was accompanied by a decrease in the CD8+ population and in perforin expression of spleen cells induced by alloantigen stimulation. The suppression of CTL activity and decrease in CD8+ cell number was detected as early as 7 hr after the injection of compounds. These results suggest that inhibitors of acidification of intracellular organelles suppress CTL activity in vivo by reducing the number of mature CD8+ CTL.     

Analysis of splenic lymphocytes with high electrophoretic mobility in adult and aged nude mice

Electrophoretic mobility (EPM) and surface markers of splenic lymphocytes in adult (8 weeks old) and aged (over 1 year old) nude mice were investigated. Splenic lymphocytes in nude mice showed a bimodal pattern consisting of low mobility lymphocytes (LML) corresponding to B cells and high mobility lymphocytes (HML). The HML of nude mice showed the following immunological characteristics: (1) surface Ig- cells; (2) asialo GM1+ cells; (3) an increase in natural killer (NK) activity after depletion of B cells; (4) abrogation of the HML peak and NK activity after treatment with anti-asialo GM1 and complement. These findings suggested that HML in nude mice were NK cells. The mobility of NK cells was slightly lower than that of T cells in normal mice, although their histograms greatly overlapped each other. In the spleen cells of nude mice, there was a significant increase in the numbers of Thy-1+ cells and a decrease in the intensity of asialo GM1 antigen as a function of age. The surface markers of HML were Thy-1+- asialo GM1++ in adult nude mice, but were Thy-1+ asialo GM1+ in aged nude mice. However, although HML in aged nude mice became Thy-1+, these had almost the same EPM as those in adult nude mice.     

Comparative susceptibility of peripheral blood leucocytes and related cell lines to killing by T-cell perforin.

The comparative susceptibility of lymphocyte subsets, monocytes and polymorphonuclear leucocytes (PMN) to killing by murine perforin was measured using physical separation techniques, cell-surface phenotyping and scatter characteristics to isolate cell types, together with propidium iodide (PI) uptake as a measure of cell death. In the majority of individuals, PMN were more resistant to perforin than other peripheral blood cells including natural killer (NK) cells and CD8+ lymphocytes. Among the lymphocytes, CD4+ cells were the most susceptible subset, followed by CD19+, CD8+ and CD56+ lymphocytes respectively. The human promyelocytic leukaemia cell line, HL-60, and the human histiocytic lymphoma cell line, U937, were readily killed by perforin. When HL-60 were differentiated to either macrophage- or neutrophil-like end cells, and U937 differentiated to macrophage-like end cells, there was no difference between differentiated and undifferentiated cells in their relative susceptibility to perforin. The relative resistance of PMN to perforin may be important in protecting them from damage in in vivo situations where both NK cells and neutrophils are localized in the same inflammatory areas.     

中国HIV感染者细胞毒性淋巴细胞与CD4~+CD25~+调节性T细胞相关性研究

目的：对中国HIV感染者NK细胞、CD8^＋T细胞及胞内穿孔素、颗粒酶-B表达与CD4^＋CD25^＋Foxp3^＋调节性T细胞水平的相关性进行研究,探讨调节性T细胞在HIV感染中的作用机制。方法：选取73名HIV/AIDS患者（长期不进展组、无症状HIV组、AIDS组）,应用流式细胞仪胞内染色技术检测NK细胞、CD8^＋T细胞数量及胞内穿孔素、颗粒酶-B表达水平,分析其与CD4^＋CD25^＋Foxp3^＋调节性T细胞水平的相关性。结果：CD4^＋CD25^＋Foxp3^＋调节性T细胞百分率与NK细胞、CD8^＋T淋巴细胞数量呈明显负相关（P〈0.01）,与CD8^＋T细胞内穿孔素、颗粒酶-B表达百分率呈明显正相关（P〈0.05）,与NK细胞内穿孔素、颗粒酶-B表达水平绝对值负相关（P〈0.01）,与CD8＋T细胞内颗粒酶-B表达绝对值呈明显负相关（P〈0.01）,与CD8^＋T细胞内穿孔素表达绝对值无明显相关性（P〉0.05）。CD4^＋CD25^＋Foxp3^＋调节性T细胞绝对值与CD8^＋T细胞内穿孔素、颗粒酶-B表达百分率呈明显负相关（P〈0.05）。结论：中国HIV感染者细胞毒性淋巴细胞数量功能的变化与调节性T细胞显著相关,提示高水平的调节性T细胞可能与细胞毒性淋巴细胞耗竭相关,可能为导致疾病进展的原因之一。     

In situ detection of activated cytotoxic cells in follicular lymphomas.

The presence of cytotoxic cells and their activation status were analyzed in tissue sections of 26 follicular lymphomas. To this end, expression of the perforin and granzyme B genes was studied by in situ hybridization experiments, and expression of the TIA-1 antigen was analyzed by immunohistochemistry. Cells expressing the granzyme B gene and the perforin gene were detected in all cases. Their density was, however, highly heterogeneous from case to case, ranging from 160 to 7,040 positive cells/cm2 of tissue sections. TIA-1-positive cells were also evidenced in the 10 follicular lymphomas tested. Virtually all cytotoxic cells were located in interfollicular areas. Double labeling immunochemical experiments showed that most cytotoxic cells belonged to the CD8+ T lymphocyte population, although few CD4+ T lymphocytes and CD56+ natural killer cells also expressed the TIA-1 antigen. These findings show that development of a malignant B lymphocyte proliferation is associated with a host-derived immune response involving intratumoral cytotoxic T lymphocytes. Further studies comparing the density of such cells with the final outcome are required to determine whether the intensity of this immune response has a prognostic value.     

Higher level expression of lymphocyte function-associated antigen-1 (LFA-1) on in vivo natural killer cells

Normal mouse spleen cells express low levels of lymphocyte function-associated antigen-1 (LFA-1) as well as other lymphoid cells. However, fractionation of spleen cells with Percoll discontinuous gradients resulted in the appearance of lymphocytes expressing high levels of LFA-1 molecule (LFA-1 high lymphocytes) in parallel with the enrichment of natural killer (NK) activity. Lower density spleen cells (fractions 1 and 2) expressed higher level of LFA-1 antigen than unfractionated spleen cells and showed a higher NK activity. In contrast, higher density spleen cells (fractions 3 and 4) expressed lower levels of LFA-1 antigen and revealed lower NK activity. LFA-1 high lymphocytes possessed a high level of asialo GM1, which was the cell surface marker for NK cells. Moreover, sorting of LFA-1 high lymphocytes from spleen cells caused a great enrichment of NK cells. These results demonstrated that in vivo NK cells expressed higher levels of LFA-1 molecule, which was an important adhesion molecule for NK cell-mediated cytotoxicity.