Memory CTL Research Articles

Comparable in vivo efficacy of CD28/B7, ICOS/GL50, and ICOS/GL50B costimulatory pathways in murine tumor models: IFNγ-dependent enhancement of CTL priming, effector functions, and tumor specific memory CTL

Increasing evidence suggests that B7/CD28 interactions are important in clonal expansion and effector function of naı̈ve CD4 + T cells, whereas ICOS/GL50 interactions may optimize the responses of recently activated T H cells. In tumor models, it has been shown that engagement of ICOS, like CD28, by its ligands can be effective in enhancing tumor immunity. In this report, we have directly compared the in vivo efficacy of CD28 vs ICOS activation in the MethA fibrosarcoma and B16F1 melanoma tumor models. We studied the efficacy of systemic treatment of tumors with murine B7.2-IgG or GL50-IgG fusion proteins, and the therapeutic potential of B7.1 or GL50 vaccines given during various phases of the antitumor responses. In addition, we compare the efficacy of ICOS-ligand splice variants GL50 and GL50B in promoting tumor immunity. We find that each of these pathways is equally effective in promoting tumor immunity and that the efficacy of both GL50 and B7.1 vaccines is IFN-γ but not IL-10 dependent. Our results suggest that CD28 or ICOS costimulation-based strategies may be equally efficacious as adjuvants to conventional cancer treatment.

Hepatitis c virus-specific cytolytic t cell responses after antiviral therapy

Antigen-specific T cells are likely to provide a critical defense against hepatitis C virus (HCV) infection. However, their detection in blood is uncommon except in persons who undergo spontaneous recovery after acute HCV infection. We postulated that virological responses after antiviral interferon-α therapy may be associated with enhanced cytolytic T cell immunity. Peripheral blood memory CTL responses were quantified using short term limiting dilution culture, with cytolytic function detected by standard chromium release assay. In this cross-sectional study, 5 of 11 interferon-α or interferon-α plus ribavirin-treated subjects exhibited cytolytic T cell responses after therapy completion; 4 of these 5 subjects were HCV RNA negative at the time of assay. In contrast, only 1 of 9 untreated chronically viremic subjects had detectable HCV-specific cytolytic T cell responses. Although the requisite factors necessary to achieve sustained virologic response after therapy remain largely undefined, the findings presented here suggest that antiviral therapy-induced virological clearance may be associated with the induction, expansion, and/or recirculation of HCV antigen-specific cytolytic T cells, and may play a role in the maintenance of a nonviremic state.

Lessons learned from HIV treatment interruption: safety, correlates of immune control, and drug sparing.

Therapeutic approaches seeking to limit the exposure to antiretroviral drugs while retaining the benefits of continuous therapy have become an active area of investigation in HIV therapy research. Although early attempts to use interruptions of therapy as auto-vaccination strategies have shown little success, much has been discovered in regards to immunological correlates of viral control in acute and chronic infection, viral evolution, and the safety of single or multiple therapy interruptions in different patient sub-groups (acutely infected, chronically infected, and multi-drug resistant). Here we review safety data and candidate factors that may contribute to the striking differences observed between patients that undergo similar treatment interruption strategies but achieve different outcomes in controlling HIV replication. Differences between acute and chronic infection in the viral component (e.g. diversity of the viral pool) and the host immune system (e.g. low avidity CTL memory response), which may not be reversed by ART, may determine the potential for suppressive immune response upon therapy interruption. Consistent with goals of limiting toxicity and cost of antiretroviral drug regimens, safety outcomes to date indicate that intermittent therapy strategies may safely continue to be investigated in early and chronically infected patients. Based on ongoing research, we identify the topics to be targeted in future studies.

Epitope specificity is critical for high and moderate avidity cytotoxic T lymphocytes associated with control of viral load and clinical disease in horses with equine infectious anemia virus

Equine infectious anemia virus (EIAV) is a lentivirus that causes persistent infections in horses. We hypothesized that high-avidity CTL specific for nonvariable epitopes might be associated with low viral load and minimal disease in EIAV-infected horses. To test this hypothesis, memory CTL (CTLm) responses were analyzed in two infected horses with high plasma viral loads and recurrent disease (progressors), and in two infected horses with low-to-undetectable viral loads and mild disease (nonprogressors). High-avidity CTLm in one progressor recognized an envelope gp90 epitope, and the data documented for the first time in EIAV that viral variation led to CTL escape. Each of the nonprogressors had high-to-moderate avidity CTLm directed against epitopes within Rev, including the nuclear export and nuclear localization domains. These results suggested that the epitope specificity of high- and moderate-avidity CTLm was an important determinant for disease outcome in the EIAV-infected horses examined.

Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen.

Up-regulation of receptor-ligand pairs during interaction of an MHC-presented epitope on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In this study, we used replication deficient adenoviruses to introduce a model tumor-associated Ag (the E7 oncoprotein of human papillomavirus 16) and the T cell costimulatory molecule 4-1BBL into murine DCs, and monitored the ability of these recombinant DCs to elicit E7-directed T cell responses following immunization. Splenocytes from mice immunized with DCs expressing E7 alone elicited E7-directed effector and memory CTL responses. Coexpression of 4-1BBL in these E7-expressing DCs increased effector and memory CTL responses when they were used for immunization. 4-1BBL expression up-regulated CD80 and CD86 second signaling molecules in DCs. We also report an additive effect of 4-1BBL and receptor activator of NF-kappa B/receptor activator of NF-kappa B ligand coexpression in E7-transduced DC immunogens on E7-directed effector and memory CTL responses and on MHC class II and CD80/86 expression in DCs. Additionally, expression of 4-1BBL in E7-transduced DCs reduced nonspecific T cell activation characteristic of adenovirus vector-associated immunization. The results have generic implications for improved or tumor Ag-expressing DC vaccines by incorporation of exogenous 4-1BBL. There are also specific implications for an improved DC-based vaccine for human papillomavirus 16-associated cervical carcinoma.

CD30-governor of memory T cells?

CD30 is well recognized as a marker expressed by a heterogeneous group of lymphomas and in several immune and autoimmune disorders. However, the function of CD30 in theses diseases or in the normal immune response has remained unclear. Studying gene expression patterns induced by stimulating CD30 signals on a large granular lymphoma cell line, YT, with an agonistic anti-CD30 antibody, we found that CD30 signals affected proapoptotic and antiapoptotic genes, regulated cytotoxicity, and controlled molecules regulating T cell traffic. Creating CD30-L deficient mice and studying CD8 CTL activation and memory responses, it was observed that the absence of CD30-L resulted in diminished primary clonal expansion of CD8 cells. In addition the absence of CD30-L abolished clonal contraction after primary expansion and interfered with secondary expansion upon boosting. The studies suggest that CD30 regulates CD8 CTL function and survival during memory responses and is important for clonal contraction.

Eradication of established HPV16-transformed tumours after immunisation with recombinant Semliki Forest virus expressing a fusion protein of E6 and E7

Previously, we described the efficacy of immunisation with recombinant Semliki Forest virus (SFV), expressing the human papillomavirus 16 (HPV) oncoproteins E6 and E7, in inducing HPV-specific CTLs and anti-tumour responses. Recently, we developed a novel recombinant SFV construct encoding a relatively stable fusion protein of HPV16 E6 and E7 under control of a translational enhancer derived from the SFV capsid protein. In the present study we demonstrate that immunisation of tumour-bearing mice with this improved vector results in the regression and complete elimination of established tumours. We furthermore demonstrate that a long-term high level of CTL activity, up to 340 days, accompanies the anti-tumour response. Thus, immunisation with recombinant SFV particles encoding increased levels of a fusion protein of HPV16 E6 and E7 efficiently induces CTL activity and CTL memory resulting in a potent therapeutic anti-tumour effect.

Memory Cytotoxic T Cell Responses to Viral Tegument and Regulatory Proteins Encoded by Open Reading Frames 4, 10, 29, and 62 of Varicella-Zoster Virus

Cytotoxic T cell recognition of tegument and regulatory proteins encoded by open reading frames (ORFs) 4, 10, 29, and 62 of varicella-zoster virus (VZV) was evaluated using limiting dilution conditions to estimate the precursor frequencies of memory T cells specific for these proteins in immune subjects. Responder cell frequencies for ORFs 4, 10, and 62 gene products, which are virion tegument components and function as immediate early viral transactivating proteins, were equivalent. CTLp recognition of VZV proteins made in latently infected cells, which include ORF4 and ORF62 proteins, was not maintained preferentially when compared to ORF10 protein, which has not been shown to be expressed during latency. T cell recognition of ORF29 protein, the major DNA binding protein, which is expressed during replication but not incorporated into the virion tegument, was less common than responses to ORFs 4, 10, and 62 gene products. Older individuals had diminished numbers of memory CTLp that lysed autologous targets expressing IE62 protein; these responses were increased after immunization with live attenuated varicella vaccine to the range observed in younger adults. Adaptive immunity to VZV is characterized by a broad repertoire of memory CTL responses to proteins that comprise the virion tegument and regulate viral gene expression in infected cells.

A systematic comparison of methods to measure HIV-1 specific CD8 T cells

Several methods are now available to evaluate the frequencies of virus-specific CD8 T cells but require a systematic comparison to help at choosing the best strategy for evaluation. First, we compared the ELISpot-IFNγ assay, intracellular IFNγ staining and HLA class I tetramer-binding assay to quantify the HIV-specific CD8 T cells. Second, we determined the frequency of recognition of HIV antigens and evaluated whether the mode of antigen presentation might influence the results: We compared HIV antigen presentation in the same ELISpot-IFNγ assays by using recombinant vaccinia viruses (rVVs) encoding for HIV-LAI Gag, Pol, Env, Nef, Tat and Vif proteins, or a panel of 49 synthetic 8–11 amino acid length peptides tested either individually or pooled. Third, we compared the antigens recognized by memory CTL analysis using chromium release assay (CRA) on CTL lines and by effector CD8 cell analysis using ELISpot assay. Our results show that: (1) Flow cytometry and ELISpot assay measuring IFNγ production give the same frequency of HIV-specific CD8 T cells; (2) tetramer-binding assay detects more HIV-specific CD8 T cells than other methods; (3) pools of optimal peptides and sum frequencies of individual optimal peptides give similar results in ELISpot assay; (4) ELISpot assays using peptides are more sensitive than those using rVV; and (5) CRA and ELISpot assay when using rVV provide a comparable profile of HIV antigen recognition by memory CTLs (CRA) and effector CTLs (ELISpot) in two thirds cases. These results have important implications for the choice of immunological methods to evaluate CD8 T cells responses to vaccines.

Robust recall and long-term memory T-cell responses induced by prime-boost regimens with heterologous live viral vectors expressing human immunodeficiency virus type 1 Gag and Env proteins.

We investigated long-term memory and recall cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (VSVs) expressing Env and Gag. More than 7 months after a single vaccination with VSV-Env, approximately 6% of CD8(+) splenocytes stained with major histocompatibility complex class I tetramers containing the Env p18-I10 immunodominant peptide and showed a memory phenotype (CD44(Hi)). The level of tetramer-positive cells in memory was about 14% of the peak primary response. Recall responses elicited in these mice 5 days after boosting with a heterologous recombinant vaccinia virus expressing HIV-1 Env showed that 40 to 45% of CD8(+) splenocytes were tetramer positive and activated (CD62L(Lo)), and these cells produced gamma interferon after stimulation with Env peptide, indicating that they were functional. Five months after the boost, the long-term memory cell population (tetramer positive, CD44(Hi)) constituted 30% of the CD8(+) splenocytes. Recall responses to HIV-1 Gag were examined in mice primed with VSV recombinants expressing HIV-1 Gag protein and boosted with a vaccinia virus recombinant expressing Gag. Using this protocol, we found that approximately 40% of CD8(+) splenocytes were activated (CD62L(Lo)) and specific for a Gag immunodominant peptide (tetramer positive). The high-level Gag recall response elicited by the vaccinia virus-Gag was greater than that obtained by boosting with a VSV-Gag vector with a different VSV glycoprotein. The corresponding levels of CD44(Hi) memory cells were also higher long after boosting with vaccinia virus-Gag than after boosting with a glycoprotein exchange VSV-Gag. Our results show that VSV vectors elicit high-level memory CTL responses and that these can be amplified as much as six- to sevenfold using a heterologous boosting vector.

Selective expansion of perforin-positive CD8+ T cells by immature dendritic cells infected with live Bacillus Calmette-Guérin mycobacteria

Live, but not dead Bacillus Calmette-Guérin (BCG) is partially protective against infection by Mycobacterium tuberculosis, which causes a disease with high mortality in immune compromised individuals. We have shown that uptake of BCG induces maturation of immature dendritic cells (DCs) regardless of the viability of the bacteria. Importantly, when T cells are cocultured with live BCG-infected DCs, the proportion of CD45RA(-) perforin(+) CD8+ T cells is markedly expanded markedly; however, little expansion is seen when T cells are cocultured with DCs harboring heat-killed BCG. The direct contact of T cells with live BCG-infected DCs was required for the expansion of perforin(+) CD8+ T cells. These CD8+ T cells demonstrated a high level of killing activity against BCG-infected macrophages. There was little contribution of cytokines, including IFN-gamma, TNF-alpha, and IL-12, to the expansion of CD8+ T cells by live BCG-infected DCs. We found that the interaction between BCG-infected DCs and CD8+ T cells through CD40/CD40L was crucial for the expansion and maturation of CD8+ T cells, the process of which was CD4-independent. In contrast, blocking the CD58/CD2 but not the CD40/CD40L interaction reduced production of IFN-gamma without affecting the maturation of CD8+ T cells. This indicates that the production of IFN-gamma and perforin by CD8+ T cells is mediated by distinct signals delivered from BCG-infected DCs. Thus, BCG-specific CD8+ CTL memory cells may be maintained for a long period of time in BCG-vaccinated hosts, and these cells could mature rapidly into effectors through the potent antigen-presenting function of DCs upon mycobacterial infection.

Generation of CD8(+) T cell memory in response to low, high, and excessive levels of epitope.

The magnitude of a virus-specific memory CTL population can dramatically influence the outcome of secondary infections, yet little is known about the determinants of memory size. We investigated the impact of epitope levels on CTL memory generation by using a recombinant vaccinia virus system that allows for a broad range of epitope expression with the same infectious dose of virus. The size of the memory pool was examined using MHC class I/peptide tetramer staining and IFN-gamma ELISPOT analysis following priming with viruses expressing low, high, or excessive epitope levels. The size of the epitope-specific CD8(+) T cell memory population correlates with Ag dose at the low and high levels of epitope expression. However, at excessive epitope levels, the number of functional, IFN-gamma-producing, epitope-specific memory cells is significantly reduced compared with the number of tetramer(+) cells. These results demonstrate that the level of epitope expressed during an acute viral infection in vivo can dramatically influence CTL memory size. Furthermore, when epitope is overexpressed, the quality of the response can be adversely affected. Therefore, epitope expression level is an important consideration when developing approaches to optimize CTL memory induction.

Recombinant canarypox vaccine-elicited CTL specific for dominant and subdominant simian immunodeficiency virus epitopes in rhesus monkeys.

Since virus-specific CTL play a central role in containing HIV replication, a candidate AIDS vaccine should generate virus-specific CTL responses. In this study, the ability of a recombinant canarypox virus expressing SIV Gag-Pol-Env (ALVAC/SIV gag-pol-env) was assessed for its ability to elicit both dominant and subdominant epitope-specific CTL responses in rhesus monkeys. Following a series of five immunizations, memory CTL responses specific for a dominant Gag epitope could be demonstrated in the peripheral blood of vaccinated monkeys. Memory CTL responses to a subdominant Pol epitope were undetectable in these animals. Following challenge with SIVmac251, the experimentally vaccinated animals developed high frequency CTL responses specific for the dominant Gag epitope that emerged in temporal association with the early containment of viral replication. Interestingly, the experimentally vaccinated, but not the control vaccinated animals, developed CTL responses to the subdominant Pol epitope that were detectable only after containment of early viremia. Thus, recombinant canarypox vaccination elicited low frequency, but durable memory CTL populations. The temporal association of the emergence of the dominant epitope-specific response with early viral containment following challenge suggests that this immune response played a role in the accelerated clearing of early viremia in these animals. The later emerging CTL response specific for the subdominant epitope may contribute to the control of viral replication in the setting of chronic infection.

The Dual Role of CD4 T Helper Cells in the Infection Dynamics of HIV and Their Importance for Vaccination

Given the role of the CD4 T helper cells in the development of memory CTL precursors, it seems beneficial to boost the CD4 T helper response in the context of vaccination against the human immunodeficiency virus (HIV). However, CD4 T cells are also the preferred targets of infection by HIV. Here, we address the question as to whether it is advantageous to stimulate the CD4 T helper cell response, as this will increase the pool of potential target cells of infection. To do so we formulated a mathematical model describing the interactions between virus-infected cells, susceptible cells, HIV-specific CD4 helper T cells, and CTL precursor (CTLp) and effector cells (CTLe). The effect of increased initial CD4 helper and CTLp numbers on the outcome of infection, as well as the effect on viral set point of increased CD4 T helper growth rate, CTL responsiveness and the rate at which CTLp and CTLe are produced were studied. We found that only when the virus has a low basic reproductive number does the number of CTLp and CD4 T helper cells at the moment of infection influence the outcome of infection. In this situation, high initial T helper and CTL numbers can switch the outcome from full-blown infection to virus control. However, this holds for virus with infectivity in a limited range, and current estimates of virus infectivity suggest that it is higher. In that case, only a vaccination protocol that increases CTL responsiveness, ideally in combination with the rate of production of CD4 T helper cells, may offer a solution as it can reduce the viral set point considerably. If brought under a certain level, the viral population might be unable to replicate any further. However, changing these parameters of the immune response is only beneficial when infection is controlled by CTL in the long term. When a CD4 lymphoproliferative response is mounted but the CTL response is not maintained, increasing the CD4 T helper growth rate is deleterious.

Perforin-independent regulation of dendritic cell homeostasis by CD8+ T cellsin vivo: implications for adaptive immunotherapy

We investigated here the effects of perforin on CTL responses during interaction of dendritic cells (DC) with cytotoxic T lymphocytes in vivo. Using MHC class I tetramers complexed with the immunodominant CTL epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP33), we followed the kinetics of DC-induced CTL responses. GP33-presenting DC induced rapid primary expansion of both perforin-competent and -deficient CTL with similar kinetics. Secondary CTL responses in perforin-deficient and normal control mice after DC-booster immunization were more rapid than the primary responses, but never reached the high initial levels, suggesting that reactivated memory CTL eliminated the antigen-presenting DC and thereby limited the booster effect. Whereas killingof DC in vitro was strictly dependent on perforin, elimination of GP33-presenting DC by CTL in vivo was largely independent of perforin and Fas. Taken together, these results suggest that control of DC homeostasis by CTL, i. e. elimination of DC by the effector cells they had elicited, is controlled via multiple and probably redundant signals and represents an important fail-safe mechanism to avoid exaggerated CTL responses.

Perforin-independent regulation of dendritic cell homeostasis by CD8(+) T cells in vivo: implications for adaptive immunotherapy.

We investigated here the effects of perforin on CTL responses during interaction of dendritic cells (DC) with cytotoxic T lymphocytes in vivo. Using MHC class I tetramers complexed with the immunodominant CTL epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP33), we followed the kinetics of DC-induced CTL responses. GP33-presenting DC induced rapid primary expansion of both perforin-competent and -deficient CTL with similar kinetics. Secondary CTL responses in perforin-deficient and normal control mice after DC-booster immunization were more rapid than the primary responses, but never reached the high initial levels, suggesting that reactivated memory CTL eliminated the antigen-presenting DC and thereby limited the booster effect. Whereas killingof DC in vitro was strictly dependent on perforin, elimination of GP33-presenting DC by CTL in vivo was largely independent of perforin and Fas. Taken together, these results suggest that control of DC homeostasis by CTL, i. e. elimination of DC by the effector cells they had elicited, is controlled via multiple and probably redundant signals and represents an important fail-safe mechanism to avoid exaggerated CTL responses.

Differential expression of leukocyte receptor complex-encoded Ig-like receptors correlates with the transition from effector to memory CTL.

The human leukocyte receptor complex (LRC) on chromosome 19q13.4 encodes Ig superfamily receptors expressed on hemopoietic cells. Killer Ig-like receptors (KIR) are expressed in cytotoxic lymphocytes but other LRC molecules (Ig-like transcript(ILT)/leukocyte Ig-like receptor (LIR)) are more ubiquitous. We investigated expression of the ILT2/LIR1 inhibitory receptor compared with the related KIR. Both ILT2/LIR1 and KIR were expressed by peripheral CD8(+) T cells with a memory/effector phenotype. ILT2/LIR1(+) T cells demonstrated diverse TCRBV repertoires in contrast to KIR(+) T cells, while numbers of peripheral ILT2/LIR1(+) T cells were greater than KIR(+) T cells and the majority of ILT2/LIR1(+) T cells did not coexpress KIR. Analysis of CD8(+) T cells with specific HLA class I tetramers confirmed this pattern of expression, indicating differential regulation of LRC gene expression in T lymphocytes. Only a minor proportion of ILT2/LIR1(+) KIR(-) clones survived in vitro cloning, were more susceptible to anti-CD3 or cognate peptide induced cell death than KIR(+) T cells and exhibited lower levels of the Bcl-2 survival molecule. Our results indicate a sequential program of LRC-encoded receptor expression with initial ILT2/LIR1 expression in effector T cells and KIR gene transcription in the minor proportion of expanded clones which survives activation-induced cell death to become long term memory T cells.

Expansion of HBV-specific memory CTL primed by dual HIV/HBV genetic immunization during SHIV primary infection in rhesus macaques

We have previously shown the induction of humoral and cytotoxic responses specific for human immunodeficiency virus (HIV) and hepatitis B virus (HBV) antigens, following genetic immunization of rhesus macaques with a plasmid encoding both the third variable domain of the HIV-1 external envelope glycoprotein and the pseudo-viral particle of hepatitis B surface antigen (HBsAg) as presenting molecules. The DNA-immunized primates and two control animals were then challenged with a chimeric simian/human immunodeficiency virus (SHIV). They were all infected. Significant frequencies of SHIV specific cytotoxic T lymphocyte precursors (CTLp) were detected early in peripheral blood. But, in all DNA-immunized macaques, HBV envelope specific CTLp were detected during the primary infection, and they were correlated with the peak of SHIV viremia. Furthermore, HBV or SHIV specific cytotoxicity corresponded in part to CD8 + T cells presenting a memory phenotype. Several mechanisms could account for this cellular response. But our results suggest that an expansion of memory cytotoxic CD8 + cells, not restricted to SHIV specific effectors, could occur in peripheral blood during SHIV primary infection.

Predicting the dynamics of antiviral cytotoxic T-cell memory in response to different stimuli: cell population structure and protective function.

This paper examines the numerical and functional consequences of various stimuli on antiviral CD8+ T-cell memory using a mathematical model. The model is based upon biological evidence from the murine model of infection with lymphocytic choriomeningitis virus (LCMV) that the phenotype of immunological memory represents low-level responses driven by various stimuli, and the memory CTL population is partitioned between resting, cycling and effector cells. These subpopulations differ in their lifespan, their potential to mediate antiviral protection and in the stimuli needed for their maintenance. Three types of maintenance stimuli are examined: non-antigen-specific (bystander) stimulation, persisting antigen stimulation and reinfection-mediated stimulation. The modelling predicts that: (i) stable persistence of CTL memory requires the presence of either bystander or antigen-specific stimulation above a certain threshold depending on the sensitivity of memory CTL to stimulation and their life-span; (ii) a relatively low level of stimuli (approximately 10(4) fold less on a per CTL basis compared to acute infection) is needed to stabilize the expanded memory CTL population; (iii) the presence of CTL subsets in the memory pool of different activation states and lifespans ensures the robustness of memory persistence in the face of temporal variation in the low-level stimuli and; (iv) an 'optimal' population structure of the memory CTL pool, in terms of immediate protection, requires the presence of both activated cycling and effector CTL. For this, persisting antigen alone or synergistically with bystander signals provide the appropriate stimulation, so that the stimuli equivalent to approximately 30 p.f.u. of LCMV in the spleen are sufficient to maintain approximately 10(5)-10(6) specific CTL in the memory pool. These observations are relevant both to our understanding of natural protective immunity and to vaccine design.

Granulysin expression is a marker for acute rejection and steroid resistance in human renal transplantation

Differentiating etiologies of transplant dysfunction without biopsy and optimizing therapy for acute rejection by predicting steroid resistance will reduce patient morbidity. Granulysin is a cytolytic molecule released by CTL and NK cells and coexpressed with effectors of acute allograft rejection, like perforin and granzymes. Granulysin mRNA and protein expression were studied in peripheral blood lymphocytes (PBL; n = 61 total, n = 10 with intercurrent infections) and biopsy tissue from adult and children renal transplant recipients ( n = 97) by competitive quantitative-reverse transcriptase-PCR (QC-RT-PCR) and immunohistochemistry. Differences in cell phenotypes were studied in steroid sensitive and resistant acute rejection biopsies. Granulysin was studied in phytohemagglutinin (PHA) stimulated cell lines (donor PBL and CD45RO + T cells) by FACS, Western blotting, and RT-PCR after pretreating with cyclosporine A (CSA), azathioprine, mycophenolic acid, and steroids. Granulysin mRNA was significantly increased in patient PBL and transplant biopsies during acute rejection ( p < 0.0001) and infection ( p < 0.001). Rejecting biopsies alone ( n = 53) had mononuclear cell granulysin staining. Steroid resistant biopsies ( n = 25) had denser granulysin staining (>2 cells/high power field) and CD45RO + lymphocytes, when compared with steroid sensitive ( n = 28) rejecting tissue. Granulysin levels were unchanged after azathioprine and mycophenolic acid treatment, decreased after treating activated PBL with steroids and cyclosporine A (CSA), and paradoxically, increased ( p < 0.05) after treating CD45RO + CTL with CSA. Elevated PBL granulysin is a peripheral marker for acute rejection and infection and dense granulysin staining a tissue marker for steroid resistance. Memory CTL abound in steroid resistant grafts and may have a markedly different response to CSA immunotherapy, suggesting a possible mechanism for steroid resistance.