Age-related decrement in cytotoxic T lymphocyte (CTL) activity is associated with decreased levels of mRNA encoded by two CTL-associated serine esterase genes and the perforin gene in mice

The age-related decline in cytotoxic T lymphocyte (CTL) activity has been recognized for many years. Age-related alterations in several immunologic events have been suggested to be partly or completely responsible for this decline. We had previously demonstrated (Bloom et al., Cell. Immunol. 1988. 144: 440) in mice that a deterioration in the lytic mechanism may be at least in part responsible for the decline in CTL activity. We now report that this decline correlates with an age-related decrease in serine esterase activity released into the supernatant medium in the process of generating CTL. Northern analyses were then used to examine the effect of age on expression of genes encoding for perforin and two CTL-associated serine esterases. The products of all three of these genes have all been postulated to play roles in CTL-mediated lysis. We show that the expression of all three of these genes appears to decline with age in the process of generating allogeneic CTL. These alterations in gene expression correlated both with diminished cytolytic and released esterase activities generated by mixed leukocyte culture in spleen cells of old mice compared to young. The age-related decline in gene expression could not be attributed to shifts in T cell subsets, but CD8+ cells generated by allogeneic stimulation of nylon wool-passed spleen cells from old mice expressed significantly less cytolytic activity than those from young. This report is the first demonstration of an age-related decrease in expression of a functionally related group of genes. In addition, these findings are compatible with the suggested roles for perforin and serine esterase release in CTL-mediated target cell lysis.     

Corrective effects of interleukin-12 on age-related deficiencies in IFN-gamma production and IL-12Rbeta2 expression in virus-specific CD8+ T cells.

Interleukin-12 receptor beta2 (IL-12Rbeta2) has been shown to be selectively expressed on Th1 T cell subsets, and we have previously shown that influenza-specific CD8+ cytotoxic T lymphocyte (CTL) deficiency in old mice was associated with deficient Th1 (interferon-gamma [IFN-gamma]) cytokine production. This study tested whether IL-12Rbeta2 expression was also deficient in CD8+ CTL from old mice and the effect of IL-12 treatment on these responses. Splenic lymphocytes from influenza-primed old and young BALB/c mice were stimulated with influenza virus in vitro with and without IL-12 and then enriched for CD8+ T cells. IFN-gamma was significantly reduced, whereas IL-4 and IL-12p40 (an antagonist of IL-12 function) were evaluated in old when compared with young mice. This was true for secreted protein measured by ELISA and for mRNA levels quantitated by RT-PCR. IL-12Rbeta2 mRNA expression in CD8+ CTL was also significantly reduced in old mice. IL-12 treatment in vitro caused significant upregulation of IFN-gamma and IL-12Rbeta2 and downregulation of IL-4 in CD8+ T cells from old mice and young mice. The present demonstration of an age-related downregulation in IL-12Rbeta2 expression and our previous data showing reduced IFN-gamma and elevated IL-4 production provide strong evidence that CD8+ CTL deficiency in aging results from a Th1/Th2 cytokine production switch. Agents that increase IL-12Rbeta2 expression and redirect Th2 to Thl immune responses are likely to enhance CD8+ CTL-mediated control of viral infections in aging.     

Pore-forming protein in individual cytotoxic T lymphocytes: the effect of senescence provides a probe for understanding the lytic mechanism.

The senescent decline of cytolytic T lymphocyte (CTL) activity was examined (a) to learn more about the effect of aging on the immune system, and (b) to probe the mechanism of cell-mediated cytolysis. The effect of age on the generation of pore-forming protein (Pfp) was examined at the cellular level in a murine model using CTL stimulated in allogeneic mixed lymphocyte culture (MLC). Pfp expression was analyzed by immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). Immunocytochemical analyses of Pfp in MLC-stimulated splenic T cells from a large number of mice revealed that although stimulated cells from aged mice exhibited fewer Pfp-producing cells than those from young, the diminution in the proportion of Pfp+ cells was small compared to the age-related decrease in lytic activities (approximately 2-fold vs. approximately 7.4-fold, respectively). Time-course analysis disclosed similar kinetics for the generation of Pfp+ cells among responding cells from young and aged mice. No significant age-related difference in the proportion of Pfp+ cells was observed in MLC-stimulated lymph node cells despite a large and significant difference in lytic activity (approximately 6.5-fold). Purified CD8+ T cells demonstrated a large age-related difference in CTL activity (approximately 3-11-fold) and accounted for virtually all the Pfp. Although little difference in the proportion of Pfp+ CD8+ T cells could be detected between age groups, stimulated CD8+ cells or whole splenic T cells from old mice consistently exhibited a striking reduction in both the intensity of Pfp staining and the apparent numbers of granules per cell. This difference in Pfp was examined by ELISA and total Pfp levels were found to be approximately 12-fold greater in CTL generated from splenic T cells of young compared to aged mice. The results demonstrate that Pfp levels are reduced in CTL from aged compared to young mice at the level of the individual cells and suggest the possibility that a threshold level of Pfp may be required for potency of effector cell function.     

Impairment of CD3-dependent and CD3-independent activation pathways in CD4+ and in CD8+ T cells from old CBA/RIJ mice

Stimulation of T cells from old mice with anti-CD3 antibodies resulted in a high variability of proliferative responses, which were 2- to 8-fold lower than the responses by T cells from young mice, even in the presence of exogenous rIL-2. Moreover, the CD4+ T cells from these old mice displayed a diminished capacity to produce IL-2 in response to anti-CD3. A partial explanation was found in the observation that T cells from the majority of old mice displayed a diminished expression of CD3 of variable intensity. However, after stimulation of the T cells with the combination of phorbol-12-myristate-13-acetate (PMA) and ionomycin to bypass CD3, 3 out of 6 old mice still exhibited 2-fold lower proliferative responses than T cells from young mice; IL-2 production by the CD4+ T cells was lower in all old mice tested. Comparison of CD4+ T cells and CD8+ T cells from old mice revealed a defective PMA/ionomycin response in both subsets, although this defect seemed more pronounced in CD4+ T cells when compared with the young counterparts. The diminished response of CD8+ T cells was accompanied by a diminished expression of the IL-2R alpha-chain. In contrast, old CD4+ T cells expressed rather higher levels of IL-2R alpha-chain than young CD4+ T cells. Altogether, multiple defects which are not necessarily the same in CD4+ and CD8+ T cells are responsible for defective T cell responses in old mice.     

Relationship of large and small CD3- CD56+ lymphocytes mediating NK-associated activities.

We have defined a population of CD3-, CD56+ small lymphocytes (SLs) that exhibit the same phenotype and lytic capacity as natural killer (NK) cells. NK cells characteristically express the surface markers CD16 and CD56, mediate non-major histocompatibility complex (MHC)-restricted lysis, and have been equated with CD3- large granular lymphocytes (LGLs). In the present study we extended the observation that CD3-, CD56+ SLs can mediate NK- and antibody-dependent cellular cytotoxicity activity by studying the activation signals and lytic mechanisms that might be utilized by CD3-, CD56+ SLs in comparison to CD3- CD56+ LGLs. Our results show that CD3- SLs, similar to CD3- LGLs, exhibited activated killing in response to interleukin-2 (IL-2). In addition, after IL-2 activation, the CD3- SLs exhibited morphologic changes, including increases in size and granularity, and both morphologically and phenotypically became virtually indistinguishable from CD3- LGLs. Similar to CD3- LGLs, CD3- SLs could be directly activated by IL-2 alone to secrete significant quantities of interferon-gamma (IFN-gamma) and to express IL-2 receptor (IL-2R) p55. Examination of serine esterases and pore-forming protein (PFP) demonstrated that these cells exhibited a cytoplasmic distribution of perforin, which, unlike that of CD3- LGLs, was not associated with dense cytoplasmic azurophilic granules. Serine esterase levels were similar. However, after IL-2 activation PFP was concentrated in dense cytoplasmic granules, similar or identical to the situation in CD3-, CD56+ LGLs. These CD3-, CD56+ subsets appear to represent a continuum of activated cells that might represent various states of maturation of NK cells.     

Age-associated decrease in virus-specific CD8+ T lymphocytes during primary influenza infection

The mechanism of the age-associated decrease in CD8+ T cell response of mice to virus infection was examined in young adult (6 months) and aged (22 months) C57BL/6 mice during primary pulmonary influenza A virus infection. A significant age-associated decrease in both the percentage (P<0.0001) and number (P<0.05) of CD8+ T cells binding MHC Class I tetramers containing influenza A nucleoprotein (NP) epitope and in virus-specific CTL activity (P<0.05) was observed with pulmonary lymphocytes. The percentage of NP+CD8+ cells of individual mice strongly correlated with NP-specific cytotoxic activity (r(2)=0.77, P<0.02) and with the percentage of CD8+ cells that produced interferon-gamma (r(2)=0.86, P<0.002) in both young and aged mice. Comparable expression of the CD28, CD25, and the memory CD44(hi)/CD62L(lo) phenotype was detected on NP+CD8+ lymphocytes from mice of both age groups. There was a delay in the maximal expansion of NP+CD8+ cells in aged compared to young mice that paralleled a delay in maximal cytotoxic activity and in virus clearance. These data suggest that the age-related impairment of CD8+ lymphocyte activity during a primary influenza A infection is due to a defect in the expansion, rather than in effector activity, of influenza-specific CD8+ T cells.     

Differential age-change in the numbers of CD4+CD45RA+ and CD4+CD29+ T cell subsets in human peripheral blood.

Peripheral blood mononuclear cells were obtained from people ranging in age from newborn to 102 years old and analyzed by dual color flow cytometer in terms of number and percentage of various subsets of T cells, B cells and natural killer cells (CD3, 4, 5, 8, 11b, 19, 20, 21, 25, 29, 45RA and 56). Numbers of T cells (CD3+ or CD5+ cells) significantly declined at the 3rd decade as compared with those of younger people, stayed at a relatively constant level between the 3rd and the 7th decade and gradually declined thereafter. In T cell subsets, both CD4 and CD8 positive positive cells decreased with age, but a decrease was more pronounced in the latter, showing an age-related increase of CD4/CD8 ratio. The most interesting finding was a contrasting age-change in two subsets of CD4+ T cells; i.e. a subset of suppressor inducer T cells (CD4+CD45RA+ naive cells) decreased with age, while a subset of helper inducer T cells (CD4+CD29+ memory cells) increased with age. CD20+ B cells also decreased with age in a manner similar to that observed in T cells. Natural killer cells (CD56) showed an increase in numbers with age. The relationship between these changes in various subsets of peripheral blood leukocytes and the age-related decline in immune functions has been discussed.     

Expression of perforin in murine natural killer cells and cytotoxic T lymphocytes in vivo.

We have previously detected perforin expression in a subpopulation of asialo GM1+ natural killer (NK) cells and CD8+ T lymphocytes in murine spleen cells by immunocytochemical staining with an anti-perforin monoclonal antibody. In the present study, more detailed analyses of perforin expression in murine cytotoxic lymphocyte subpopulations were performed. The expression of perforin in asialo GM1+ spleen cells was predominantly confined to the NK1.1+ subset, where all NK activity also resided. Perforin expression was also studied on alloreactive cytotoxic T lymphocyte (CTL) induced in vivo. The cells expressing perforin in peritoneal exudate lymphocytes predominantly resided in the CD8+ T cell subpopulation co-expressing asialo GM1 where an allospecific CTL activity also resided. Furthermore, the percentage of perforin-positive cells in this population was greatly reduced after stimulation with anti-CD3 or anti-T cell receptors antibodies, which induce serine esterase release from the cytoplasmic granules. These findings highly suggest that perforin is involved in in vivo NK cell- and CTL-mediated cytolysis.     

Cooperation of human tumor-reactive CD4+ and CD8+ T cells after redirection of their specificity by a high-affinity p53A2.1-specific TCR

Efficient immune attack of malignant disease requires the concerted action of both CD8+ CTL and CD4+ Th cells. We used human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic mice, in which the mouse CD8 molecule cannot efficiently interact with the alpha3 domain of A2.1, to generate a high-affinity, CD8-independent T cell receptor (TCR) specific for a commonly expressed, tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the human p53 tumor suppressor protein. Retroviral expression of this CD8-independent, p53-specific TCR into human T cells imparted the CD8+ T lymphocytes with broad tumor-specific CTL activity and turned CD4+ T cells into potent tumor-reactive, p53A2.1-specific Th cells. Both T cell subsets were cooperative and interacted synergistically with dendritic cell intermediates and tumor targets. The intentional redirection of both CD4+ Th cells and CD8+ CTL by the same high-affinity, CD8-independent, tumor-specific TCR could provide the basis for novel broad-spectrum cancer immunotherapeutics.     

Homeostatic regulation of CD8+ T cells by perforin.

To prevent uncontrolled expansion, the massive proliferation of T cells during an acute immune response has to be followed by controlled deletion. Here we show that similar to Fas, perforin is not only an important effector molecule of cytotoxic T lymphocytes (CTL) but also involved in down-regulating peripheral T cells. Mice deficient for both the CTL effector molecule perforin and the apoptosis-inducing Fas ligand spontaneously develop infiltration of highly activated CD8(+) T cells in kidney and liver and die between 5 and 12 weeks of age. Injection of staphylococcal enterotoxin B (SEB) into perforin-deficient mice results in dramatically increased selective expansion and prolonged persistence of CD8(+), but not CD4(+), SEB-reactive T cells. Also, secondary immunization of TCR transgenic perforin-deficient mice with the lymphocytic choriomeningitis virus glycoprotein-derived epitope peptide leads to an increased proliferation of transgenic CD8(+) T cells, that is not explained by failure to deplete professional antigen-presenting cells. These results point to a novel mechanism of T cell homeostasis in which the acquisition of perforin-dependent cytotoxic activity regulates the expansion and persistence of CD8(+) effector T cells in vivo.     

Age-related changes in lymphokine production related to a decreased number of CD45RBhi CD4+ T cells.

The ability of CD4+ T cells from CBA/Rij mice to produce interleukin (IL) 2 after stimulation with anti-CD3, concanavalin A, or the combination of phorbol 12-myristate 13-acetate and ionomycin declines during aging. This phenomenon was accompanied by an increased production of IL 4 and interferon-gamma. These age-related changes in lymphokine production correlated with the decrease in the percentage of CD45RBhi CD4+ T cells from about 80% in 2-month-old to about 40% in 27-month-old mice. This phenotypic shift was responsible for the decline in IL 2 production, because in young and in old mice CD45RBhi CD4+ T cells were more potent IL 2 producers than CD45RBlo cells. Moreover, old CD45RBhi CD4+ T cells produced less IL 2 than their young counterparts. Proliferative responses by T cells from old mice were lower than those of young mice, regardless whether the cultures were supplemented with IL 2, IL 4 or both lymphokines. As far as CD4+ T cells were concerned, this hyporesponsiveness was found in the CD45RBlo as well as in the CD45RBhi CD4+ T cell population.     

CD4+CD25+FoxP3+ regulatory T cells enhance the allogeneic activity of endothelial-specific CD8+/CD28-CTL

Numerous data indicate that CD4+CD25+FoxP3+ regulatory T cells (Treg cells) can attenuate alloresponses of conventional T lymphocytes against professional antigen-presenting cells and thus qualify for clinical use in various transplant settings. However, it is unknown whether Treg cells also influence T cell-endothelial cell interactions. CD8+ PBMC (CD8+ PBMC, CTL) from healthy human donors were stimulated for 7 days with an allogeneic microvascular endothelial cell line (CDC/EU. HMEC-1, an immortalized human microvascular endothelial cell line, further referred to as HMEC) and additional endothelial cell types and analysed for their lytic activity against these target cells in the presence or absence of Treg cells. Addition of Treg cells (1:1:1) to the CTL/HMEC co-cultures in the efferent immune phase (day -1 prior to the assay) led to an increased cytotoxicity against HMEC. In contrast, Treg cells alone did not lyse HMEC. Treg cell-mediated enhancement of CTL activity was endothelial cell specific since lysis of HLA-matched Epstein-Barr virus-transformed B lymphoblastoid cells (B-LCL) was not influenced by the addition of Treg cells. Further analysis of CD28-positive and CD28-negative CTL sub-populations revealed that only the CD28-negative CTL showed an increased activity against HMEC after Treg cell co-culture. Although there is no doubt about the potential therapeutic efficacy of Treg cells to ameliorate outcome of allogeneic transplants, the endothelium might require additional protective interventions to prevent endothelial cell type-specific alloreactivity.     

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.     

Age-dependent variation in the proportion and number of intestinal lymphocyte subsets, especially natural killer T cells, double-positive CD4+ CD8+ cells and B220+ T cells, in mice

The age-dependent variation in the proportion and number of lymphocyte subsets was examined at various extrathymic sites, including the liver, small intestine, colon and appendix in mice. In comparison with young mice (4 weeks of age), the number of total lymphocytes yielded by all tested organs was greater in adult (9 weeks) and old (40 weeks) mice. The major lymphocyte subset that expanded with age was interleukin-2 receptor (IL-2R) beta+ CD3int cells (50% of them expressed NK1.1) in the liver, whereas it was CD3+ IL-2Rbeta- NK1.1- cells at all intraepithelial sites in the intestine. Although NK1.1+ CD3+ cells were present at intraepithelial sites in the intestine, the proportion of this subset was rather low. The ratio of CD4 to CD8 tended to decrease among natural killer T (NKT) cells and T cells at all intraepithelial sites in the intestine with age. A unique population of double-positive CD4+ CD8+ cells in the small intestine increased in old mice. B220+ T cells were found mainly in the appendix and colon, and the proportion of these T cells decreased in old mice. Conventional NKT cells were very few in Jalpha281-/- and CD1d-/- mice in the liver, while NKT cells which existed in the appendix remained unchanged even in these mice. This was because unconventional CD8+ NKT cells were present in the intestine. The present results suggest that despite the fact that both the liver and intraepithelial sites in the intestine carry many extrathymic T cells, the distribution of lymphocyte subsets and their age-associated variation are site-specific.     

Switch from perforin-expressing to perforin-deficient CD8+ T cells accounts for two distinct types of effector cytotoxic T lymphocytes in vivo

Although CD8⁺ cytotoxic T lymphocytes (CTL) exhibit both Fas ligand (FasL) -based and perforin-based lytic activities, the accepted hallmark of a fully active CTL remains its perforin killing machinery. Yet the origin, rationale for possessing both a slow-acting (FasL) and a fast-acting (perforin) killing mechanism has remained enigmatic. Here we have investigated perforin expression in CTL directly involved in acute tumour (i.e. leukaemias EL4 and L1210) allograft rejection occurring within the peritoneal cavity. We show that at the height of the immune response, the majority of conjugate-forming CD8⁺ CTL express high levels of perforin messenger RNA and protein, and kill essentially via perforin. Later however, coinciding with complete rejection, fully cytocidal CTL emerge which exhibit a stark decrease in perforin and now kill preferentially via constitutively expressed FasL. Although late in emergence, and persistent, these powerful CTL are neither effector-memory nor memory CTL. This finding has implications for the monitoring of anti-transplant responses in clinical settings, based on assessing perforin expression in graft infiltrating CD8⁺ T cells. The results show that as the immune response progresses in vivo, targeted cellular suicide mainly prunes high perforin-expressing CD8⁺ cells, resulting in the gradual switch in effector CTL, from mostly perforin-based to largely Fas/FasL-based killers. Hence, two kinds of CD8⁺ CTL have two killing strategies.     

In vivo responses of CD4+ and CD8+ cells to bacterial superantigens.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex (MHC) class II molecules and specifically activates T cells bearing V beta 8 T cell receptor domains. We have compared several aspects of the response of CD4+ and CD8+ T cell subsets to SEB in vivo. V beta 8+ cells in both subsets proliferated to a similar extent upon SEB injection. Furthermore, mRNA for interferon-gamma was induced in both subsets with similar kinetics and SEB dose-response. Finally CD8+ (but not CD4+) T cells from SEB-injected mice exhibited SEB-specific lysis of MHC class II-bearing target cells. Collectively, these data indicate that the CD4: MHC class II interaction confers no detectable selective advantage to CD4+ cells in the in vivo response to SEB. The observed effector functions of both subsets may contribute to SEB-induced immunopathology.     

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.     

Apparent requirement for CD4+ T cells in primary anti-herpes simplex virus cytotoxic T-lymphocyte induction can be overcome by optimal antigen presentation.

Mice depleted in vivo of either CD4+ or CD8+ T cells were used to define the requirement for interaction between the two T subsets for the induction and maturation of a herpes simplex virus (HSV) cytotoxic T-lymphocyte (CTL) response. Whereas C3H mice generated normal CD8+ CTL in the absence of CD4+ T cells, responses were undetectable in BALB/c mice. However, the role of CD4+ T cells appeared to be to supply helper factors for CTL maturation, as the numbers of CTL precursors in CD4-depleted mice were similar to those in nondepleted animals. Moreover, CTL responses were demonstrable if CD4-depleted primed populations were stimulated with antigen or supplied with a source of helper factors. The optimal means of presenting antigen appeared to be via dendritic cells. Our results indicated that CD8+ cells alone were fully capable of differentiating into CTL provided they were appropriately stimulated with antigen. Possibly, the environment necessary for this to occur in vivo is usually lacking, accounting for the fact that in the mouse, it usually is not possible to demonstrate HSV-specific CTL unless cells are cultured or restimulated in vitro.     

The critical role of CD40/CD40L in the CD4-dependent generation of CD8+ T cell immunity

Control of virus infections and eradication of tumors usually involves the lytic activity of CD8+ cytotoxic T lymphocytes (CTLs). The induction of effective CTL immunity relies on several factors, one of the most important of which is CD4+ T cell help. Numerous studies have demonstrated the dependence of CTL priming on the presence of CD4+ T cells, but until recently little was known of the mechanisms regulating this process. Based on reports that CD4+ and CD8+ T cells must recognize antigen on the same antigen-presenting cell (APC), help was originally thought to be provided through the delivery of short-range, CD4+ T cell-secreted cytokines. However, the results of subsequent studies favor an alternative mechanism, whereby CD4+ T cells modify the APC, converting it into a stimulatory cell for CD8+ T cell priming. It is important that CD40 and its ligand, CD40L, have been implicated in the provision of this help and, in particular, the generation of long-lasting CTL memory.