Herpes simplex virus antigens directly activate NK cells via TLR2, thus facilitating their presentation to CD4 T lymphocytes

NK cells infiltrate human herpetic lesions, but their role has been underexplored. HSV can stimulate innate immune responses via surface TLR2, which is expressed on monocyte-derived dendritic cells (DCs) and NK cells. In this study, UV-inactivated HSV1/2 and immunodominant HSV2 glycoprotein D peptides conjugated to the TLR2 agonist dipalmitoyl-S-glyceryl cysteine stimulated CD4 T lymphocyte IFN-γ responses within PBMCs or in coculture with monocyte-derived DCs. NK cells contributed markedly to the PBMC responses. Furthermore, NK cells alone were activated directly by both Ags, also upregulating HLA-DR and HLA-DQ and then they activated autologous CD4 T lymphocytes. Using Transwells, Ag-stimulated NK cells and CD4 T lymphocytes were shown to interact through both cell-to-cell contact and cytokines, differing in relative importance in different donors. A distinct immunological synapse between Ag-stimulated NK cells and CD4 T lymphocytes was observed, indicating the significance of their cell-to-cell contact. A large proportion (57%) of NK cells was also in contact with CD4 T lymphocytes in the dermal infiltrate of human recurrent herpetic lesions. Thus, NK cells stimulated by TLR2-activating HSV Ags can present Ag alone or augment the role of DCs in vitro and perhaps in herpetic lesions or draining lymph nodes. In addition to DCs, NK cells should be considered as targets for adjuvants during HSV vaccine development.     

Induction of activation antigens on human natural killer cells mediated through the Fc-gamma receptor

NK cells, defined here as lymphocytes bearing the CD16 Ag found on the NK cell Fc-gamma receptor (FcR), are known to enter a proliferative and activated state in response to stimulation with IL-2 as assessed by clonal expansion, short-term DNA synthesis, and de novo expression of lymphocyte-associated activation Ag. We have found that the FcR of NK cells acts as a signaling pathway through which IL-2-dependent activation may be greatly enhanced, allowing for more rapid induction of activation Ag and recruitment of an increased percentage of cells expressing surface markers of cellular activation. FcR-interactive agents, such as solid phase immobilized immune complexes or cross-linked CD16-specific mAb, work synergistically with rIL-2 to elicit a rapid expression of IL2R and transferrin receptors on greater than 50% of purified NK cells as early as day 3 of culture. IL-2 or FcR-interactive stimuli alone were weak or ineffective stimulators by comparison. In contrast to the induction of de novo activation Ag, DNA synthesis was elicited by IL-2 alone, but was not substantially or consistently enhanced by the subsequent addition of FcR-interactive stimuli.     

Clonal analysis of human CD4-CD8-CD3- thymocytes highly purified from postnatal thymus.

Human triple-negative (CD4-CD8-CD3-) thymocytes purified from postnatal thymus by the use of magnetic bead columns and cell sorting were cultured in bulk or cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells, and PHA. Triple-negative thymocytes proliferated well under these culture conditions, and after 12 days in bulk culture they remained triple negative. Limiting dilution experiments revealed that the frequency of clonogenic cells in fresh triple-negative thymocytes was less than 1%. Of 40 clones obtained in a representative experiment, 37 were triple negative and 3 were CD4+ TCR-alpha beta+. No TCR-gamma delta+ clones were isolated. Some of the triple-negative clones expressed CD16 and were apparently NK cells. Seven representative CD16-triple-negative clones were expanded and characterized in detail. These clones shared the common cell surface phenotype of CD1-CD2+CD3-CD4--CD8-CD5-CD7+CD16-CD56+. One of them expressed cytoplasmic CD3 delta and CD3 epsilon Ag, but these Ag were not detected in any peripheral blood-derived CD16- NK clones examined for comparison. The seven CD16- thymus-derived clones exhibited significant cytolytic activity against K562. The clone that expressed cytoplasmic CD3 Ag was shown to have the germ-line configuration of the TCR-beta and TCR-gamma genes. Thus, it is suggested that in vitro culture of triple-negative thymocytes can give rise to NK-like cells, including those that express cytoplasmic CD3 Ag. In contrast to previous reports, our results gave no evidence of differentiation of triple-negative thymocytes into TCR-alpha beta+ or TCR-gamma delta+ T cells.     

Helper activity in antigen-specific antibody production mediated by CD4+ human cytotoxic T cell clones directed against herpes simplex virus

Three HSV type 1 (HSV-1) and HSV type 2 (HSV-2) common ("HSV-type common") and three HSV-1 specific CTL clones, which were CD3+, CD4+, CD8-, 4B4+, and 2H4-, were established. These clones proliferated in response to stimulation with HSV in the presence of autologous APC. The HSV type specificity of the proliferative response was identical with that of the cytotoxic activity of the clones. The cytotoxic activity and the proliferative response were both inhibited by addition of anti-HLA-DR mAb to the culture. After culture of these CTL clones with autologous B cells and macrophages followed by HSV Ag stimulation, anti-HSV antibody was detected in the culture supernatant. The HSV type specificity of the helper function for antibody production was identical with that of the cytotoxicity, i.e., HSV-type common clones, upon stimulation with either HSV-1, or HSV-2, and HSV-1-specific clones, upon stimulation with HSV-1 but not with HSV-2, showed helper activity for anti-HSV antibody production by autologous B cells. Moreover, it was found that these clones produced humoral factors which help autologous B cells to produce antibody. The helper factors were produced by T cell clones in an HSV-type-specific manner. These data suggest that some CD4+ T cells can simultaneously manifest both specific cytotoxicity and helper activity for Ag-specific antibody production by B cells, and that these multifunctional T cells might play an important role in protection against viral infection.     

The human MHC-restricted cellular response to herpes simplex virus type 1 is mediated by CD4+, CD8- T cells and is restricted to the DR region of the MHC complex

The nature of the in vitro human cytotoxic T-cell responder population to HSV type 1 (HSV-1) was studied. In 5-day HSV-1-stimulated cultures that contained MHC-restricted activity, two phenotypically distinct populations of cells were present that were capable of lysing HSV-1-infected B cell lines in a 5-h 51Cr-release assay. The first was CD4+, CD8-, CD16- cell typical of class II-restricted T cells, whereas the other population bore a CD4-, CD8-, CD16+ NK-cell phenotype. Elimination of the NK cell fraction from bulk cultures by using anti-CD16 plus C frequently resulted in cell populations that killed in an Ag-specific, HLA-DR-restricted fashion. In some cases the anti-CD16-pretreated cultures retained a killing population that was unrestricted to MHC products. In no instance were any cytotoxic T cells that were restricted to class I Ag in evidence. Limiting dilution analysis of precursor frequency indicated that about 1 in 4000 to 1 in 8000 cells from peripheral blood are specific for HSV-1 in seropositive individuals. Comparisons of HLA class I-matched and HLA class II-matched targets with the autologous target by using limiting dilution analysis yielded results entirely consistent with those obtained in the bulk culture assay system.     

Induction of natural killer effectors from human thymus with recombinant IL-2

The NKH1 Ag is expressed on all cells in human peripheral blood capable of mediating spontaneous non-MHC restricted cytolytic function (i.e., natural killing). The majority of NK cells do not express CD3 Ag and do not express TCR gene products. However, approximately 20 to 25% of NKH1+ cells coexpress CD3 and TCR proteins. Both NKH1+CD3+ and NKH1+CD3- effectors can proliferate in response to IL-2 which also results in enhancement of cytolytic function. In the present studies, we examined thymocytes after incubation with rIL-2 for the presence of NKH1+ cells and for the development of non-MHC restricted cytolytic function. NKH1+ cells and NK activity could not be detected in fresh thymus. After culture with rIL-2 only, NK activity appeared in 3 days, reached a maximum after 7 days, and was effective against a panel of NK-sensitive targets. NK activity was correlated with the expression of NKH1 on the surface of in vitro proliferating thymocytes and immunofluorescent cell sorting demonstrated that almost all cytolytic activity was mediated by NKH1+ cells. As expected given the thymic origin of these cells, the majority of NKH1+ cells in culture expressed CD3. However, all cultures contained NKH1+CD3- effector cells which represent 15 to 40% of the NKH1+ population. As in peripheral blood, both NKH1+CD3- and NKH1+CD3+ exhibited non-MHC-restricted cytotoxicity, but only CD3+ effectors could be inhibited by anti-T3 mAb. These findings demonstrate that rIL-2 alone can induce subpopulations of thymocytes to proliferate, to express the NKH1 marker and become NK active in vitro. Furthermore, they suggest that the thymus which plays a role in the differentiation of NKH1+CD3+ NK effectors may also play a role in the differentiation or maturation of NKH1+CD3- NK effectors.     

The role of IL-4 in proliferation and differentiation of human natural killer cells. Study of an IL-4-dependent versus an IL-2-dependent natural killer cell clone

The role of IL-4 in proliferation and differentiation of human NK cells was studied using newly established sublines of an IL-4-dependent NK cell clone (IL4d-NK cells) and an IL-2-dependent NK cell clone (IL2d-NK cells) derived from a parental conditioned medium-dependent NK cell clone (CM-NK cells). IL-4 induced the higher proliferation of CM-NK cells, but abolished their NK activity and decreased CD16 and CD56 Ag expression. In contrast, IL-2 induced the higher NK activity and increased CD16 and CD56 Ag expression. Addition of anti-IL-4 antibody to the culture of CM-NK cells with CM inhibited the proliferation, but slightly increased NK activity, and largely increased CD56 Ag expression. Addition of anti-IL-2 antibody to the culture of CM-NK cells with CM inhibited both proliferation and cytotoxicity. Proliferation of IL4d-NK cells, which is totally dependent on rIL-4, is greater than that of IL2d-NK cells, which was greater than parental CM-NK cells. Morphologically, IL4d-NK cells are small and round, whereas IL2d-NK cells are large and elongated. Anti-IL-4 antibody inhibited proliferation of IL4d-NK but not IL2d-NK cells, whereas anti-IL-2 antibody inhibited that of IL2d-NK but not IL4d-NK cells. IL-2 was not detected in the supernatant from IL4d-NK cells, nor was IL-2-mRNA expressed in IL4d-NK cells. In contrast, IFN-gamma production and protein expression in IL4d- and IL2d-NK cells were detected. NK cell activation markers (CD16 and CD56) were expressed on IL2d-NK cells but not IL4d-NK cells. IL4d-NK cells were not cytotoxic to any tumor cells tested, whereas IL2d-NK cells displayed potent NK activity and lymphokine-activated killer activity. IL4d-NK cells failed to bind K562 tumor cells, whereas one-third of the IL2d-NK cells did. IL4d-NK cells responded to rIL-2, proliferated, and differentiated into cytotoxic NK cells, whereas IL2d-NK cells failed to respond to rIL-4 and died. These results raise a possibility that IL4d-NK cells or IL2d-NK cells primarily represent the immunologic properties of immature or activated types of human NK cells, respectively. Our results provide the first evidence of the capability of IL-4 to support continuous proliferation of a lymphocyte clone with immature NK cell characteristics and to stimulate IFN-gamma production in the clone. IL-4 is suggested as a potential growth factor for certain types of human NK cell progenitors.     

Heteroconjugate antibodies enhance cell-mediated anti-herpes simplex virus immunity

Impaired cell-mediated immunity predisposes individuals to severe systemic HSV infections. A potential approach for enhancing antiviral immunity is to alter the specificity of T cells and NK cells so that they become cytotoxic against HSV. We describe here the use of heteroconjugate antibodies to augment the killing of HSV-infected cells. Two different types of heteroconjugate antibodies were used: 1) CD3-specific mAb, covalently linked to HSV-specific mAb (e.g., anti-CD3 x anti-HSV-1 glycoprotein C); 2) FcR-specific mAb linked to HSV-specific mAb (e.g., anti-Fc gamma RIII x anti-HSV-1 glycoprotein D). Whereas freshly isolated, PBL were not cytotoxic against HSV-infected target cells in a 5-h 51Cr-release assay, co-incubation with either heteroconjugate resulted in significant cytotoxicity. In vitro activated PBL (anti-CD3 + IL-2) also became more potent killers of HSV-infected cells in the presence of each heteroconjugate. The specificity of anti-CD3 x anti-HSV-1 and anti-Fc gamma RIII x anti-HSV-1 gD for enhancing T cell and NK cell immunity, respectively, was confirmed by using cloned, homogeneous human T cell and NK cell lines as effectors. Kinetic analysis demonstrated that as soon as the infected cells began to express HSV glycoproteins on their surface they became susceptible to this enhanced killing. Prolonged culture of HSV-infected cells with heteroconjugate antibodies and effector cells also decreased the amount of viral replication that occurred, as measured in a plaque inhibition assay. These results suggest that heteroconjugate antibodies are potent immunotherapeutic tools that enhance anti-HSV immunity.     

A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor-gamma/delta+ T lymphocytes. I. Inhibition of the IL-2-dependent proliferation by anti-Kp43 monoclonal antibody.

In the present study we describe a novel functional cell surface molecule, designated as Kp43, which is expressed among leukocytes by NK cells, TCR-gamma/delta + T lymphocytes, and some CD8+ CD56+TCR-alpha/beta + T cell clones. The Kp43 Ag is a 70-kDa disulfide-linked dimer, which migrates in SDS-PAGE under reducing conditions as a single 43-kDa band. Two-color immunofluorescence staining of fresh PBL revealed that only a fraction of CD16+, and of TCR-gamma/delta + T lymphocytes expressed the Ag. The analysis of TCR-alpha/beta + T cell clones showed that a small proportion (2 out of 20) weakly expressed Kp43 together with the CD8 and CD56 molecules. By immunoperoxidase staining of different tissues the anti-Kp43, reactivity was detected exclusively in lymphoid organs, where a minority of scattered cells was stained, and in some liver sinusoidal cells. Essentially all NK cells acquired Kp43 when stimulated with a B lymphoblastoid cell line. By contrast, the pattern of distribution of Kp43 remained stable upon in vitro culture of T-gamma/delta lymphocytes, thus delineating two subsets according to its expression. In lymphokine-activated killer populations, obtained by culturing either PBL or NK cells with high concentration of IL-2, most CD16+ and CD56+ cells became Kp43+. The Kp43-specific mAb inhibited the IL-2-dependent proliferative response of cultured NK and TCR-gamma/delta + T cells without affecting their non-MHC-restricted cytotoxicity. The partial inhibitory effect, which was mediated as well by pepsin digested F(ab')2 fragments, was lost upon reduction to Fab. The anti-Kp43 mAb did not interfere with the specific binding of IL-2 to its surface receptors. Altogether the data point out that the Kp43 dimer is involved in the regulation of the IL-2-dependent proliferative response of NK cells and a subset of TCR-gamma/delta + T lymphocytes.     

Monocyte-induced down-modulation of CD16 and CD56 antigens on human natural killer cells and its regulation by histamine H2-receptors.

Human natural killer (NK) cells carry CD16/FcR and CD56 cell-surface Ag but lack the T-cell marker CD3. Here we show that incubation of resting human NK cells with CD3-/16+/56+ phenotype with autologous monocytes induced the disappearance of CD16 and CD56 cell-surface Ag on NK-cells but did not affect CD2 or CD3 Ag expression on T-cells. Monocyte-induced down-modulation of NK-cell-surface Ag was cell-contact dependent and induced only by freshly isolated monocytes, recovered from peripheral blood by counter-current centrifugal elutriation. Adherence of monocytes abrogated the capacity to induce down-modulation of NK-cell-surface Ag. The biogenic amine histamine dose-dependently reversed the monocyte-induced down-modulation of CD16 and CD56 on CD3- NK-cells. The effect of histamine was mediated by H2-type receptors on monocytes. The data presented are suggestive of a cell-cell-mediated interaction between monocytes and NK-cells which modulates surface expression of NK-cell Ag and its histaminergic regulation.     

CD94/NKG2A expression is associated with proliferative potential of CD8 T cells during persistent polyoma virus infection

Memory CD8 T cells comprise a critical component of durable immunity because of their capacity to rapidly proliferate and exert effector activity upon Ag rechallenge. During persistent viral infection, memory CD8 T cells repetitively encounter viral Ag and must maintain a delicate balance between limiting viral replication and minimizing immunopathology. In mice infected by polyoma virus, a natural mouse pathogen that establishes long-term persistent infection, the majority of persistence-phase antiviral CD8 T cells express the inhibitory NK cell receptor CD94/NKG2A. In this study, we asked whether CD94/NKG2A expression is associated with Ag-specific recall of polyoma virus-specific CD8 T cells. During the persistent phase of infection, polyoma virus-specific CD8 T cells that express CD94/NKG2A were found to preferentially proliferate; this proliferation was dependent on cognate Ag both in vitro and in vivo. In addition, CD94/NKG2A(+) polyoma-specific CD8 T cells have a markedly enhanced capacity to produce IL-2 upon ex vivo Ag stimulation compared with CD94/NKG2A(-) polyoma-specific CD8 T cells. Importantly, CD94/NKG2A(+) anti-polyoma virus CD8 T cells appear to be essential for Ag-specific recall responses in mice persistently infected by polyoma virus. Because of its higher proliferative potential and capacity to produce IL-2, we propose that the CD94/NKG2A(+) subpopulation represents a less differentiated state than the CD94/NKG2A(-) subpopulation. Identification of proliferation-competent subpopulations of memory CD8 T cells should prove valuable in designing therapeutic vaccination strategies for persistent viral infections.     

Activation of cloned human natural killer cells via Fc gamma RIII

The Fc gamma RIII (CD16) Ag on human NK cells involved in antibody-dependent cellular cytotoxicity has been demonstrated to be an important activation structure. The present studies were carried out to further characterize the functional role of the CD16 Ag and the mechanisms whereby cytotoxicity is activated by using human NK clones. In phenotypic studies Fc gamma RIII was found to be expressed heterogeneously on various human cloned NK cells. Expression on CD3- and CD3+ clones varied with the donor and mAb used for detection. Functional data demonstrated that cytotoxicity against NK-resistant target cells can be induced in CD3-CD16+ NK clones and CD3+CD16+ clones with NK activity when various CD16 mAb were used. CD16 antibodies but not reactive isotype control antibodies induced cytotoxicity. In contrast to complete CD16 antibodies F(ab')2 fragments were not able to activate the cytotoxic mechanism. Both an antibody against FcR on the target cell (Fc gamma RII) and a CD11a antibody blocked induction of cytotoxicity. These results suggest that three steps are critical for activation of CD16+ cells via Fc gamma RIII: 1) specific binding of CD16 antibodies to Fc gamma RIII on effector cells irrespective of the epitope recognized; 2) cross-linking of effector cell CD16 Ag through binding of the Fc site of CD16 antibodies via corresponding FcR on the target cell membrane; and 3) interaction of CD11a/18 molecules with the target cell membrane.     

In vivo effects of recombinant IL-2. I. Isolation of circulating Leu-19+ lymphokine-activated killer effector cells from cancer patients receiving recombinant IL-2

This study was designed to isolate and phenotypically characterize lymphokine-activated killer (LAK) cells generated in vivo during administration of high dose rIL-2 to cancer patients. The development of circulating LAK effector cells in these patients was demonstrated by the ability of fresh PBL to exhibit lytic activity against the NK-resistant Daudi cell line and fresh tumor cells without prior in vitro culture with rIL-2. Kinetic studies demonstrated that circulating LAK effector cells are detectable 4 to 6 wk after the initiation of rIL-2 therapy. Cells isolated by FACS revealed that circulating LAK cells are Leu-19+, Leu-17+ but CD5-. We have previously reported that circulating Leu-19+ cells are heterogeneous with regard to the expression of CD16 and CD8. Since sorting of cells expressing Leu-19 and either low quantities of CD8 or CD16 resulted in cytolytic activity in both the positive and negative fractions, these latter two markers do not identify subpopulations of Leu-19+ cells with or without LAK cytolytic activity. Although all LAK cells generated in vivo were Leu-19+, we generated LAK cells from the Leu-19- subpopulation after in vitro culture with rIL-2, suggesting that at least some of in vitro generated LAK cells are derived from Leu-19- precursor cells. These LAK cells did not, however, express the Leu-19 surface marker. Based on the functional data reported in this paper, we conclude that circulating LAK effector cells are a phenotypically heterogeneous population that express surface Ag in association with NK cells and not T lymphocytes.     

IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production

The common gamma-chain cytokine, IL-21, is produced by CD4(+) T cells and mediates potent effects on a variety of immune cells including NK, T, and B cells. NKT cells express the receptor for IL-21; however, the effect of this cytokine on NKT cell function has not been studied. We show that IL-21 on its own enhances survival of NKT cells in vitro, and IL-21 increases the proliferation of NKT cells in combination with IL-2 or IL-15, and particularly with the CD1d-restricted glycosphingolipid Ag alpha-galactosylceramide. Similar to its effects on NK cells, IL-21 enhances NKT cell granular morphology, including granzyme B expression, and some inhibitory NK receptors, including Ly49C/I and CD94. IL-21 also enhanced NKT cell cytokine production in response to anti-CD3/CD28 in vitro. Furthermore, NKT cells may be subject to autocrine IL-21-mediated stimulation because they are potent producers of this cytokine following in vitro stimulation via CD3 and CD28, particularly in conjunction with IL-12 or following in vivo stimulation with alpha-galactosylceramide. Indeed, NKT cells produced much higher levels of IL-21 than conventional CD4 T cells in this assay. This study demonstrates that NKT cells are potentially a major source of IL-21, and that IL-21 may be an important factor in NKT cell-mediated immune regulation, both in its effects on NK, T, and B cells, as well as direct effects on NKT cells themselves. The influence of IL-21 in NKT cell-dependent models of tumor rejection, microbial clearance, autoimmunity, and allergy should be the subject of future investigations.     

NK cells regulate CD4 responses prior to antigen encounter

NK cells not only respond rapidly to infection, shaping subsequent adaptive immunity, but also play a role in regulating autoimmune disease. The ability of NK cells to influence adaptive immunity before Ag exposure was examined in a gender-dependent model of preferential Th1 and Th2 activation. The inability of young adult male SJL mice to activate Th1 cells was reversed via depletion of NK1.1(+) cells, whereas the presence or the absence of NK1.1(+) cells did not alter responses in age-matched females. Consistent with a gender-dependent role in regulating adaptive immunity, significantly more NK1.1(+) cells were present in males compared with females, and this difference was reversed by castration. In contrast to NK1.1(+) cells derived from C57BL/6 mice, no spontaneous cytokine secretion was detected in NK1.1(+) cells derived from either male or female SJL mice, although an increased frequency of IL-10-secreting NK1.1(+) cells was observed in males vs females following in vitro stimulation. Direct evidence that NK1.1(+) cells in males influence CD4(+) T cell activation before Ag exposure was demonstrated via the adoptive transfer of APC from control and NK1.1-depleted males. The absence of a functional NK T cell population in SJL mice suggests that NK cells influence adaptive immunity before Ag exposure via alterations in APC activity.     

Functional and biochemical analysis of CD16 antigen on natural killer cells and granulocytes

CD16 Ag is associated with the low affinity FcR for IgG expressed on human NK cells and granulocytes. In this study, we demonstrate that NK cells specifically lyse murine anti-CD16 hybridoma cell lines, but do not lyse hybridomas against other cell surface differentiation Ag expressed on NK cells. Moreover, the CD18 structure is involved in the CD16-specific xenogeneic interaction between human effector cells and murine hybridoma target cells. Although interaction with anti-CD16 hybridomas or antibodies triggers the cytolytic mechanism of NK cells, this interaction does not induce cellular proliferation. In contrast to NK cells, CD16+ granulocytes do not lyse anti-CD16 hybridoma cell targets and do not mediate ADCC against antibody-coated human tumor cell targets. These findings indicate a fundamental difference in the antibody-dependent cellular cytotoxicity mechanisms of NK cells and granulocytes. Comparative biochemical analysis of CD16 on NK cells and granulocytes revealed significant differences in the size of the polypeptides obtained after removal of N-linked carbohydrate residues with endo-F and N-glycanase digestion.     

Lineage-specific telomere shortening and unaltered capacity for telomerase expression in human T and B lymphocytes with age

Age effects on telomere length and telomerase expression in peripheral blood lymphocytes were analyzed from 121 normal individuals age newborn to 94 years and revealed several new findings. 1) Telomere shortening was observed in CD4+ and CD8+ T and B cells with age. However, the rate of telomere loss was significantly different in these populations, 35 +/- 8, 26 +/- 7, and 19 +/- 7 bp/year for CD4+ and CD8+ T and B cells, respectively. In addition, CD4+ T cells had the longest average telomeres at all ages, followed by B cells, with CD8+ T cell telomeres the shortest, suggesting that these lymphocyte populations may have different replicative histories in vivo. 2) Telomerase activity in freshly isolated T and B cells was indistinguishably low to undetectable at all ages but was markedly increased after Ag and costimulatory receptors mediated stimulation in vitro. Furthermore, age did not alter the magnitude of telomerase activity induced after stimulation of T or B lymphocytes through Ag and costimulatory receptors or in response to PMA plus ionomycin treatment. 3) The levels of telomerase activity induced by in vitro stimulation varied among individual donors but were highly correlated with the outcome of telomere length change in CD4+ T cells after Ag receptor-mediated activation. Together, these results indicate that rates of age-associated loss of telomere length in vivo in peripheral blood lymphocytes is specific to T and B cell subsets and that age does not significantly alter the capacity for telomerase induction in lymphocytes.