Subsets of CD8 +, CD57+ cells in normal,healthy individuals: correlations with human cytomegalovirus (HCMV) carrier status,phenotypic and functional analyses

Two different subsets of CD8 ⁺, CD57⁺ cells have been defined, one expressing high levels (CD8_high⁺(CD57⁺)), the other expressing low levels of surface CD8 (CD8_low⁺(CD57⁺)). Increased numbers of CD8_high⁺(CD57⁺) cells correlated with previous HCMV infection. By three-colour fluorescence analysis, the CD8_high⁺(CD57 ⁺) population expressed T cell markers such as CD3 and CD5, and most were αβ T cell receptor (αβ TCR)-positive. A significant proportion also expressed CD71 (transferrin receptor) and MHC class II, although little if any CD25 (IL-2R-p55). Some (≥ 40%) co-expressed CD45RA and CD45RO. The CD8_low⁺ (CD57⁺) population expressed classical natural killer (NK) cell markers—CD2, CD16 and CD56. The two subsets were also functionally distinct; CD8_high⁺ (CD57⁺) cells suppressed pokeweed mitogen (PWM)-driven, but not phytohaem-agglutinin (PHA)-driven proliferation and immunoglobulin production; CD8_low⁺ (CD57⁺) cells exhibited NK cytotoxic activity which was not increased by interferon-alpha (IFN-α). Supernatant from cultured CD8_high⁺ (CD57⁺) cells suppressed PWM-driven immunoglobulin production, but not proliferation, and this effect was abrogated by physical separation with tissue culture inserts. Thus, a T cell subset expressing activation and memory T cell markers with direct non-specific suppressor activity was present in peripheral blood mononuclear cells (PBMC) of healthy subjects with asymptomatic HCMV infection.     

Proliferation and interleukin 5 production by CD8hi CD57+ T cells

CD8hi CD57+ T cells have previously been described as effector memory T cells with minimal expansion capacity and high susceptibility to activation-induced cell death. In contrast, we demonstrate here that CD8hi CD57+ T cells are capable of rapid expansion using multiple techniques including [(3)H]thymidine uptake, flow cytometric bead-based enumeration and standard haemocytometer counting. Previous reports can be explained by marked inhibition of activation-induced expansion and increased 7-amino-actinomycin D uptake by CD8hi CD57+ T cells following treatment with CFSE, a dye previously used to measure their proliferation, combined with specific media requirements for the growth of this cell subset. The ability of CD8hi CD57+ T cells to further differentiate is highlighted by a distinct cytokine profile late after activation that includes the unexpected release of high levels of interleukin 5. These data indicate that CD8hi CD57+ T cells should not be considered as "end-stage" effector T cells incapable of proliferation, but represent a highly differentiated subset capable of rapid division and exhibiting novel functions separate from their previously described cytotoxic and IFN-gamma responses.     

Alloreactive cytotoxic T cells can develop and function in mice lacking both CD4 and CD8

Using the technique of homologous recombination in embryonic stem cells, a mouse strain without functional CD4 and CD8 genes has been generated. Surprisingly, these mice contain significant numbers of αβ T cells. Although mice deficient for CD8 only do not show any cytotoxic response when their T cells are stimulated with either alloantigen or viral antigen, the CD4^?8^? mice do generate alloreactive cytotoxic T cells. These cytotoxic T cells bear the αβ T cell receptor and recognize major histocompatibility complex class I antigens. In addition fully allogeneic skin transplants were rejected but skin transplants expressing only minor transplantation antigens were not. Virus-specific cytotoxic T lymphocytes were also not detected. It seems that alloreactive cytotoxic T cells can be induced and exert their effector function in vitro and in vivo in the absence of CD8, and that they can develop and mature in vivo without the CD8 molecule or the signals it might provide.     

An inhibitor of cytotoxic functions produced by CD8+CD57+ T lymphocytes from patients suffering from AIDS and immunosuppressed bone marrow recipients

An inhibitor of the cytotoxic functions (ICF) mediated by human immunodeficiency virus (HIV)- or HLA-specific cytotoxic T lymphocytes, natural killer and lymphokine-activated killer (LAK) cells is secreted by CD8⁺CD57^? T lymphocytes, a subset expanded during infection with HIV and after bone marrow transplantation. We previously showed an apparent molecular mass of 20–30 kDa for this soluble glycosylated concanavalin A-binding inhibitor which is distinct from known cytokines. Here, we report a characterization of the mechanism of action of this CD8⁺CD57⁺ ICF. We show that the ICF-induced inhibition of LAK cell cytolytic activity is transient, with a spontaneous recovery of cytolytic potential after 18 h. When testing interactions of ICF with a large set of cytokines we found that the ICF-mediated inhibition of cytotoxic functions is antagonized by two cytokines: recombinant interleukin (rIL)-4 and recombinant interferon (rIFN)-γ. Finally, we show that ICF acts at the level of cytolytic effector cells, where it induces a significant increase of cyclic AMP (cAMP) level. In contrast, no modification of either cell surface antigen expression or of target/effector cell conjugate formation could be evidenced. Addition of rIL-4 and rIFN-γ reverses such an increase of cAMP levels and in parallel restores the cytolytic activity. Altogether, these data demonstrate that the glycoprotein ICF produced by CD8⁺CD57⁺ cells (1) inhibits cell-mediated cytotoxicity by sensitizing cytolytic effector cells to the cAMP pathway, and (2) is part of a cytokine network controlling cell-mediated cytotoxic functions.     

Lysis of CD4+ lymphocytes by non-HLA-restricted cytotoxic T lymphoe from HIV-infected individuals

Individuals infected with HIV have elevated numbers of total and activated CD8⁺ lymphocytes in peripheral blood. CD8⁺ lymphocytes from HIV-infected individuals have been shown to mediate non-human histocompatibility-linked antigen (HLA)-restricted suppression of viral replication, HLA-restricted killing of cells expressing HIV antigens, and killing of uninfected lymphocytes. We studied CD8⁽ T lymphocytes that lysed autologous CD4+ lymphocytes, hetcrologous CD4¹ lymphocytes from HIV-infected individuals and uninfected CD4⁺ lymphocytes. Killing in all cases required T cell receptor (TCR)-mediated recognition or triggering. However, these CD8 cytotoxic T lymphocytes (CTL) killed HLA class I mismatched CD4* lymphocytes and CD4⁴ lymphocytes treated with a MoAb against HLA-A, B and C antigens (PA2.6) which blocks HLA class I-restricted killing. HLA class H-negativc CD4* T lymphoma cells (CEM.NK^R) were also killed by anti-CD3 inhibited CTL. Stimulation of peripheral blood lymphocytes (PBL) from HIV-infected individuals, but not uninfected controls, with concanavalin A (Con A) and IL-2, induced non-HLA-restricted TCR aft¹, CD8^f CTL which lysed CD4⁺ lymphocytes. Activation ofCD4’lymphocytes increased their susceptibility to CD8^f CTL-mediated lysis. In HIV infection, a population of non-HLA-restricted CTL which lyse activated CD4⁺ lymphocytes is expanded. The expansion of CTL with unusual characteristics is interesting, because the stimulus for this expansion is unknown. CTL which recognize activated CD4⁺ cells could play a role in immune regulation and the pathogenesis of A IDS.     

Interactions between peripheral blood CD8 T lymphocytes and intestinal epithelial cells (iEC)

Intestinal intraepithelial lymphocytes (iIEL) are primarily CD8 cells and most of them have a CD28^? phenotype, the phenotype of effector cytotoxic T cells. We asked whether the predominance of CD8⁺ CD28^? T cells in the gut may result from peripheral blood T cells preferentially migrating to the iIEL compartment and adhering to iEC. Compared with CD4 cells, adhesion of resting CD8⁺ T cells to iEC cell lines was significantly higher. Adhesion could be blocked with a MoAb to gp180, a molecule expressed on iEC which is known to interact with CD8/lck. No significant difference in the level of adhesion was observed between CD8⁺ CD28⁺ and CD8⁺ CD28^? T cells. Thus CD8 cells may preferentially migrate to the iIEL compartment, but loss of CD28 expression could occur in situ after migration. Consistent with this hypothesis, the CD8⁺ CD28^? cells became enriched after co-culturing T cells with iEC cell lines and primary iEC. Induction of the CD8⁺ CD28^? phenotype in cord blood and adult T cells was observed in co-cultures with iEC and also with mitogens and superantigens. In the latter case, CD28 down-modulation was seen specifically in the Vβ subset targeted by the superantigen, indicating that loss of CD28 expression is a direct result of T cell receptor (TCR)-mediated stimulation. The combined results suggest that CD8⁺ CD28^? T cells are antigen experienced T cells, and that they may have a survival advantage in the presence of gut epithelial cells in vitro. This may contribute to the predominance of CD8⁺ CD28^? T cells in the iIEL compartment.     

T cell receptor and CD8-dependent tyrosine phosphorylation events in cytotoxic T lymphocytes: activation of p56lck by CD8 binding to class I protein

Tyrosine phosphorylation of proteins plays a central role in T cell activation. Mitogens or anti-receptor antibodies have been employed to study these signaling events, but the extent to which these mimic receptor interactions with native ligands is unclear. Cytotoxic T lymphocytes can be activated for functional responses using purified, native class I ligands presented on a surface. Previous work showed that stimulation with fluid-phase anti-T cell receptor (TCR) monoclonal antibody (mAb) activates CD8 to mediate adhesion to class I proteins and that activated CD8 generates a co-stimulatory signal upon binding to class I. Changes in tyrosine phosphorylation of substrates and activity of the p56^lck kinase have now been examined in this two-step process. The observed changes are small in comparison to those found using more potent nonphysiological stimuli, but may more accurately reflect the events required for activation of functional responses. Fluid-phase anti-TCR mAb caused increased tyrosine phosphorylation of a discrete subset of cellular substrates. Increased phosphorylation of additional substrates occurred upon CD8 binding to class I, resulting in a phosphorylation pattern comparable to that found in cells stimulated with class I alloantigen. Anti-TCR mAb alone caused increased tyrosine phosphorylation of p56^lck. When CD8 bound to class I, phosphorylation of p56^lck decreased to below the basal level found in unstimulated cells, accompanied by a substantial increase in kinase activity. These results are consistent with the two-step model for TCR activation of CD8/class I interactions and directly demonstrate that CD8 binding to class I leads to up-regulation of p56^lck activity.     

CD8+ CD28- and CD8+ CD57+ T cells and their role in health and disease

Chronic antigenic stimulation leads to gradual accumulation of late-differentiated, antigen-specific, oligoclonal T cells, particularly within the CD8(+) T-cell compartment. They are characterized by critically shortened telomeres, loss of CD28 and/or gain of CD57 expression and are defined as either CD8(+) CD28(-) or CD8(+) CD57(+) T lymphocytes. There is growing evidence that the CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population plays a significant role in various diseases or conditions, associated with chronic immune activation such as cancer, chronic intracellular infections, chronic alcoholism, some chronic pulmonary diseases, autoimmune diseases, allogeneic transplantation, as well as has a great influence on age-related changes in the immune system status. CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population is heterogeneous and composed of various functionally competing (cytotoxic and immunosuppressive) subsets thus the overall effect of CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell-mediated immunity depends on the predominance of a particular subset. Many articles claim that CD8(+) CD28(-) (CD8(+) CD57(+)) T cells have lost their proliferative capacity during process of replicative senescence triggered by repeated antigenic stimulation. However recent data indicate that CD8(+) CD28(-) (CD8(+) CD57(+)) T cells can transiently up-regulate telomerase activity and proliferate under certain stimulation conditions. Similarly, conflicting data is provided regarding CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell sensitivity to apoptosis, finally leading to the conclusion that this T-cell population is also heterogeneous in terms of its apoptotic potential. This review provides a comprehensive approach to the CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population: we describe in detail its origins, molecular and functional characteristics, subsets, role in various diseases or conditions, associated with persistent antigenic stimulation.     

Generation of a novel CD8+ cytotoxic T lymphocyte that requires soluble factor to lyse autologous antigen-presenting cells

We reported the existence of high and low responders to the streptococcal cell wall antigen (SCW) in the human population. To analyze the mechanism of the low responsiveness to SCW at the cellular level, we established SCW-specific CD4⁺ T cell lines. During the course of generation of a SCW-specific CD4⁺ T cell line restricted by HLA-DQ from a low responder, we obtained autoreactive CD8⁺ cytotoxic T lymphocytes as a cell line (HYCD8). They proliferated in the presence of autologous monocytes and IL-2, without SCW. HYCD8 lysed autologous monocytes and Epstein-Barr virus-transformed B lymphoblastoid cell line (BLCL). This cytotoxic activity was specifically inhibited by an anti-HLA class I framework monoclonal antibody and restricted by HLA-B52 or B54 specificity, as judged by killing activity against panel cells and HLA class I-transfected BLCL. It was unique to HYCD8 that the HLA class I-restricted cytotoxicity was observed only in the presence of soluble factor with low molecular mass (< 10⁴ Da) produced mainly by B cells, which could not be replaced by known cytokines and their mixtures. We thus describe novel HLA class I-restricted cytotoxic CD8⁺ T cells that kill antigen-presenting cells in a soluble factor-dependent manner.     

CD8 lymphocytosis in primary cytomegalovirus (CMV) infection of allograft recipients: expansion of an uncommon CD8+ CD57- subset and its progressive replacement by CD8+ CD57+ T cells.

Allograft recipients undergoing cytomegalovirus infection present increased proportions of circulating CD8+ lymphocytes. A longitudinal study of 11 kidney and five liver allograft recipients with primary CMV infection but no other etiological factor of graft dysfunction revealed selective imbalances of peripheral blood CD8+ T cell subsets. Initially, CMV viraemia is associated with elevated CD8+bright T cell numbers and T cell activation. Activation markers fall to normal when viral cultures become negative (before the end of the first month). During the second to sixth month, most (12/16) patients keep up high CD8+ T cell counts (1050-2900 CD8+ cells/mm3), comprising an uncommon CD8+ T cell subset, as 45-73% of CD8+bright lymphocytes were CD3+ and TCR alpha beta+, but were not stained by anti-CD28, CD11b, CD16, CD56, and CD57 antibody. Unexpectedly, CD8+CD57+ T cells, a hallmark of CMV infection, do not appear until the second to sixth month of primary CMV infection, and their numbers increase progressively thereafter. They become the predominant CD8+ T cell subset after 6 months of infection and their persistence for several (up to 4) years is strongly correlated (r = 0.87) with expansion of CD8+ cells. By analysis with MoAbs, there was no bias towards the use of particular TCR-V beta gene families at any time of primary CMV infection. Persistence of CD8 lymphocytosis is thus directly related to the rate of expansion of an uncommon CD8+CD57- subset and its progressive replacement by CD8+CD57+ T cells that are chronically elicited by CMV.     

NK相关抗原在HIV/AIDS患者CD8+T淋巴细胞上的表达

目的　探讨NK相关抗原CD5 6、CD16在HIV AIDS患者CD8 T淋巴细胞上的表达。方法　取外周血细胞 ,用标记荧光的抗体进行染色 ,以CD8强阳 淋巴细胞为门 ,用流式细胞仪分析CD8 T淋巴细胞上CD5 6、CD16的表达。结果　HIV AIDS患者CD8 T淋巴细胞表达CD5 6 、CD5 6 CD16 - 、CD5 6 CD16 均明显低于HIV抗体阴性健康对照组 (P <0 .0 5 ) ;经高效抗逆转录病毒疗法 (HAART)治疗后CD8 T淋巴细胞表达的CD5 6 、CD5 6 CD16 - 、CD5 6 CD16 呈逐渐升高趋势。HIV AIDS患者表达CD5 6 CD16 - 的CD8 T淋巴细胞亚群绝对数与CD4 T淋巴细胞绝对数呈正相关 ,r=0 .393,P <0 .0 5 ;表达CD5 6 - CD16 的CD8 T淋巴细胞百分数与CD4 T细胞绝对数呈负相关 ,r=- 0 .32 4 ,P <0 .0 5。结论　表达CD5 6的CD8 T淋巴细胞在HIV AIDS患者中明显缺失 ,HAART治疗可恢复缺失。CD8 T淋巴细胞上CD5 6的表达是HIV感染中值得关注的重要指标之一 ,对评价抗病毒疗效具有指导意义。     

Resistance of normal, unstimulated, CD8+ T cells to lysis by cytotoxic granules from cloned T cell lines

Previous work has shown that both long-term cloned cytotoxic T lymphocyte (CTL) lines and primary CD8+ CTL are resistant to lysis by the toxic granules purified from long-term cloned CTL cell lines. We show here that normal, unstimulated CD8+ spleen and thymus cells are also relatively resistant to lysis by these granules. Using flow cytometric analysis we demonstrate that CD8+ T cells in normal spleen and thymus cell populations are enriched by subjecting the total cell population to lysis by cytolytic granules. Similar enrichment was not seen when the total cell populations were subjected to lysis by melittin, an unrelated, cytolytic, pore-forming polypeptide.     

Characterization of bronchoalveolar lavage T cell subsets in sarcoidosis on the basis of CD57, CD4 and CD8

T cells expressing CD57 (a natural killer cell marker) with interferon-gamma (IFN-gamma) producing capacity increase under various conditions. CD57+ T cells are also present in the bronchoalveolar lavage fluid (BALF) of sarcoidosis, and several phenotypical and functional analyses of these cells have been reported. In the present study, BALF T cells obtained from 52 patients with sarcoidosis were classified further into CD4+CD57+ T cells, CD4+CD57- T cells, CD8+CD57+ T cells and CD8+CD57- T cells and their phenotypes and functional characteristics were assessed. Substantial proportions of these T cell subsets expressed natural killer cell markers CD161 and CD122. The biased expansion of Vbeta2 T cells was observed in both CD4+CD57+ T cells and CD4+CD57- T cells in BALF from most patients, while the expansion of other Vbeta T cells was also observed in some patients. Unexpectedly, the biased expansion of certain Vbeta T cells was also seen in either CD8+CD57+ T cells or CD8+CD57- T cells, while the expanded Vbeta T cells in CD8+ T cells differed substantially among individuals. BALF T cells showed a remarkably lower T cell receptor (TCR) intensity than that of peripheral blood T cells. Both CD8+ T cell subsets in BALF of sarcoidosis expressed the intracellular perforin/granzyme B, while all four subsets expressed intracellular IFN-gamma after in vitro activation, and CD4+ T cells, especially CD4+CD57+ T cells, expressed tumour necrosis factor-alpha. These findings indicate that CD57+ T cells as well as CD57- T cells in the BALF are phenotypically and functionally different from peripheral blood T cells and may play an important role in the Th1 dominant state and inflammation in pulmonary sarcoidosis.     

Successful in vitro generation of Epstein-Barr virus-specific cytotoxic T lymphocytes from severe chronic active EBV patients

Severe chronic active Epstein-Barr virus (EBV) infection (SCAEBV) is a rare but life-threatening disorder. Poor cytotoxic activity against the virus is widely believed to contribute to the development of this disease. We wished to determine whether it is possible to generate autologous EBV-specific cytotoxic T cells (CTLs) in vitro that can be infused back into the patient to treat his/her viremia. To do this, we first had to establish autologous EBV-transformed B cells (EBCL) as antigen-presenting cells, which is known to be difficult to do with B cells from SCAEBV patients. In one patient, the standard method of incubating B cells with EBV-containing B95-8 supernatant was sufficient. In a second patient, however, the B cells apoptosed too rapidly in culture to permit transformation. However, apoptosis could be blocked by the presence of CD40 ligand-transfectant cells, and EBV transformation was successful when performed with this transfectant. Indicating a native immune response to EBV, peripheral blood lymphocytes from both patients proliferated in response to autologous EBCL. Furthermore, patient T cells had higher frequencies of IFN-γ-producing CD8⁺ cells after stimulation with autologous EBCL than sero-positive healthy controls. EBV-specific CTLs could be generated from both patients after repeated stimulation with autologous EBCL. These CTL lines were predominantly composed of CD4⁺ cells, and autologous EBCL killing was largely inhibited by an antibody against HLA-DR. These findings support the possibility of adoptive immune therapy to treat SCAEBV patients. Received: 4 October 2000     

癌灶CD4~+CD25~+和CD8~+ T细胞亚群与卵巢浆液性癌患者生存期相关

检测卵巢浆液性癌患者癌组织中CD4+CD25+及CD8+T细胞的数目,探讨其两种T细胞介导的免疫功能对疾病发展及预后的影响。免疫组织化学双标及单标的染色方法检测41例卵巢浆液性癌患者手术切除癌组织标本中CD4+CD25+和CD8+T细胞的数目。结果显示,癌灶中CD4+CD25+T淋巴细胞为(19.95±11.50)个/10HPF,CD8+T淋巴细胞为(43.46±16.69)个/10HPF。生存分析发现高CD4+CD25+T细胞组患者总生存期较低CD4+CD25+T细胞组缩短,差异有显著性(P<0.05);而高CD8+T细胞组患者总生存期与低CD8+T细胞组相比延长,且差异有显著性(P<0.05),此外两种T细胞数目与患者年龄、病理分级、临床分期、腹水细胞学及淋巴结转移等临床病理因素均无关(P>0.05)。结果表明,卵巢浆液性癌中高CD4+CD25+T细胞提示患者预后不良,可能与CD4+CD25+T细胞介导的免疫抑制导致肿瘤免疫逃逸有关;癌组织中高CD8+T细胞提示患者预后较好,两种T细胞对卵巢浆液性癌预后的评估有重要的价值,同时可以通过阻断CD4+CD25+T细胞的免疫抑制作用改善卵巢浆液性癌患者的预后,为卵巢癌治疗提供靶目标。     

B细胞对T细胞及其亚群CD25、CD30表达的影响

目的 探讨Ｂ淋对Ｔ淋巴细胞的抗原提呈作用方法 分离外周血Ｔ、Ｂ细胞及ＣＤ４￣ＣＤ８Ｔ淋巴细胞,分别用ＬＰＳ、ＰＰＤ、ＰＷＭ刺激Ｂ细胞,选去刺激原后与Ｔ细胞及亚群共同培养。最后用流式细胞仪检测Ｔ细胞及其亚群ＣＤ２５、ＣＤ３０表达水平。结果 ＰＰＤ刺激组Ｔ细胞ＣＤ２５、ＣＤ３０表达水平明显升高,而ＰＷＭ、ＬＰＳ刺激组与对照组间无差异。ＰＰＤ刺激组ＣＤ４＾Ｔ细胞ＣＤ２５、ＣＤ３０表达与对照组间有显著差异     

Does the age-related change in CD44-defined T-cell subsets have functional significance for cytotoxic T lymphocyte generation?

CD44 or Pgp-1 is a transmembrane leukocyte adhesion-related glycoprotein which is often expressed in greater density on the membranes of memory T lymphocytes (CD44^hi) compared to naive T cells (CD44^lo). The proportion of Pgp^hi or CD44^hi cells among T cells is increased with advancing age. We examined the relevance of this alteration for the age-related decrease in the generation of allospecific CTL activity. The findings confirm the age-related increase in the frequency of CD44^hi cells in spleens of aged mice of several strains, but also show inter-strain variability in the magnitude of the increase (bm1> C57BL/6> BALB/c). In contrast, we found that after allo-stimulation, the proportion of cells bearing the memory phenotype is decreased in cells from aged mice, particularly within the CD8⁺ T cell subset. To determine if these observations reflected an alteration in the frequency or responsiveness of naive T cells, enriched populations of spleen cells depleted of CD44^hi cells were prepared from spleen cells of young and aged mice, and stimulated in mixed lymphocyte culture. Enrichment for cells expressing the naive phenotype did not restore the ability of T cells from aged mice to generate allospecific CTL. Together, these findings suggest that (1) the age-related increase in frequency of splenic T cells expressing memory phenotype and concordant decrease in phenotypically naive cells, does not explain the age-related decrease in the ability to generate primary allo-CTL, and (2) naive cells from aged mice exhibit intrinsically compromised ability to generate CTL in response to primary alloantigenic stimulation.     

Absence of Epstein-Barr virus-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes in infectious mononucleosis.

Cytotoxic T lymphocytes (CTL) with the CD4+ phenotype that recognize major histocompatibility complex (MHC) class II antigens are detectable very frequently in cultures of human alloreactive or virus-specific T cells. The significance of these CD4+ CTL for an immune reaction in vivo is not clear. Since Epstein-Barr virus (EBV) transformed B cells express HLA-class I and class II antigens equally well both CD8+ and CD4+ CTL should be stimulated during an acute EBV infection. We analysed the MHC specificity and the phenotype of EBV-specific CTL from patients with infectious mononucleosis (IM). When tested directly without any previous culture, T cells from patients in the acute phase of IM showed specific MHC-restricted cytotoxicity against the autologous B cell line. Addition of a HLA class I specific monoclonal antibody (MoAb) but not of a HLA class II specific MoAb resulted in a complete blocking of the lytic activity. Cell sorting revealed that the entire cytotoxic activity was present in the CD8+ fraction whereas no specific CTL were detectable in the CD4+ fraction. The absence of cytotoxicity in CD4+ cells was not due to a lack of activation of these cells since both CD8+ and CD4+ cells were activated in situ, showing spontaneous growth in interleukin-2 (IL-2) and expressing the activation marker TP103. Frequency estimation revealed that 1/300-1/600 CD8+ but only 1/2000-1/4000 CD4+ T cells gave rise to a specific CTL colony after 10 days. If CD4+ colonies were tested repeatedly for cytotoxicity we found that CD4+ CTL acquired their cytotoxicity during in vitro culture. In addition, we isolated EBV-specific CD4+ T cell clones able to lyse their stimulator cells in the presence but not in the absence of lectin, even after a long period of culture. Taken together our results show that cytotoxicity mediated by CD4+ T cells does not play a role in an anti-viral immune response.     

Assessment of alloreactive T cell subpopulations of aged mice in vivo. CD4+ but not CD8+ T cell-mediated rejection response declines with advanced age

The present investigation explored age-related alterations in T cell populations mediating allospecific responses in vivo. Healthy aged and young H-2^b and H-2^bxH-2^k mice were engrafted with major histocompatibility complex (MHC) class II-disparate bm12 skin, rejection of which requires CD4⁺ T cells, and MHC class I-disparate bm1 skin, rejection of which requires CD8⁺ T cells. Aged mice of both genders exhibited prolonged survival of bm12 skin grafts relative to their young counterparts but rejected bm1 skin grafts at a rate equivalent to that of young mice. Consistent with prolonged survival of bm12 skin grafts, markedly diminished levels of Ia^bm12 CTL activity were elicited from T cells of aged mice in vitro. However, no such decline was observed in the level of K^bm1 CTL from T cells of aged mice. The alterations in Ia^bm12 allospecific responses were not attributable to quantitative changes in CD4⁺ T cells of aged mice, and addition of soluble T cell helper factors to response cultures of aged mice did not augment Ia^bm12 cytotoxic T lymphocytes activity. These data demonstrate that aging fundamentally affects CD4⁺ T cell-mediated allospecific responses particularly in vivo, and that deficient generation of soluble T cell helper factors alone cannot explain this deficit.