Cutting edge: CCR7+ and CCR7- memory T cells do not differ in immediate effector cell function

It has been proposed that expression of the chemokine receptor CCR7 represents a defining factor for nonpolarized central (CCR7(+)) and polarized effector memory (CCR7(-)) T cells. In this study, we have tested this hypothesis using in vivo-activated T cells from P14 and SMARTA TCR-transgenic (tg) mice specific for MHC class I- and II-restricted epitopes of the lymphocytic choriomeningitis virus (LCMV) glycoprotein. CCR7 cell surface expression on TCR-tg cells was monitored with a CC chemokine ligand 19-Ig fusion protein. CC chemokine ligand 19-Ig staining separated TCR-tg cells activated by LCMV infection into CCR7(-) and CCR7(+) effector/memory T cell populations. Nonetheless, both T cell populations isolated from spleen and liver produced identical amounts of IFN-gamma after short-term Ag stimulation. Furthermore, CCR7(+) and CCR7(-) CD8 TCR-tg cells from LCMV-infected mice exhibited similar lytic activity against LCMV peptide-coated target cells. These results question the proposed concept of differential effector cell function of CCR7(+) and CCR7(-) memory T cells.     

Functional expression of the chemokine receptor CCR5 on virus epitope-specific memory and effector CD8+ T cells

Because the chemokine receptor CCR5 is expressed on Th1 CD4(+) cells, it is important to investigate the expression and function of this receptor on other T cells involved in Th1 immune responses, such as Ag-specific CD8(+) T cells, which to date have been only partially characterized. Therefore, we analyzed the expression and function of CCR5 on virus-specific CD8+ T cells identified by HLA class I tetramers. Multicolor flow cytometry analysis demonstrated that CCR5 is expressed on memory (CD28+CD45RA-) and effector (CD28-CD45RA- and CD28-CD45RA+) CD8+ T cells but not on naive (CD28+CD45RA+) CD8+ T cells. CCR5 expression was much lower on two effector CD8+ T cells than on memory CD8+ T cells. Analysis of CCR7 and CCR5 expression on the different types of CD8+ T cells showed that memory CD8+ T cells have three phenotypic subsets, CCR5+CCR7-, CCR5+CCR7+, and CCR5-CCR7+, while naive and effector CD8+ T cells have CCR5-CCR7+ and CCR5+CCR7- phenotypes, respectively. These results suggest the following sequence for differentiation of memory CD8+ T cells: CCR5-CCR7+-->CCR5+CCR7+-->CCR5+CCR7-. CCR5+CD8+ T cells effectively migrated in response to RANTES, suggesting that CCR5 plays a critical role in the migration of Ag-specific effector and differentiated memory CD8+ T cells to inflammatory tissues and secondary lymphoid tissues. This is in contrast to CCR7, which functions as a homing receptor in migration of naive and memory CD8+ T cells to secondary lymphoid tissues.     

Immune surveillance and effector functions of CCR10(+) skin homing T cells

Skin homing T cells carry memory for cutaneous Ags and play an important sentinel and effector role in host defense against pathogens that enter via the skin. CCR10 is a chemokine receptor that is preferentially expressed among blood leukocytes by a subset of memory CD4 and CD8 T cells that coexpress the skin-homing receptor cutaneous lymphocyte Ag (CLA), but not the gut-homing receptor alpha(4)beta(7). Homing and chemokine receptor coexpression studies detailed in this study suggest that the CLA(+)/CCR10(+) memory CD4 T cell population contains members that have access to both secondary lymphoid organ and skin compartments; and therefore, can act as both "central" and "effector" memory T cells. Consistent with this effector phenotype, CLA(+)/CCR10(+) memory CD4 T cells from normal donors secrete TNF and IFN-gamma but minimal IL-4 and IL-10 following in vitro stimulation. Interactions of CCR10 and its skin-associated ligand CC ligand 27 may play an important role in facilitating memory T cell entry into cutaneous sites during times of inflammation.     

Differentiation of CD8+ T cells from tumor-invaded and tumor-free lymph nodes of melanoma patients: role of common gamma-chain cytokines

Differentiation of CD8(+) T cells at the tumor site toward effector and memory stages may represent a key step for the efficacy of antitumor response developing naturally or induced through immunotherapy. To address this issue, CD8(+) T lymphocytes from tumor-invaded (n = 142) and tumor-free (n = 42) lymph nodes removed from the same nodal basin of melanoma patients were analyzed for the expression of CCR7, CD45RA, perforin, and granzyme B. By hierarchical cluster analysis, CD8(+) T cells from all tumor-free lymph nodes and from 56% of the tumor-invaded lymph node samples fell in the same cluster, characterized mainly by CCR7(+) CD45RA(+/-) cytotoxic factor(-) cells. The remaining three clusters contained only samples from tumor-invaded lymph nodes and showed a progressive shift of the CD8(+) T cell population toward CCR7(-) CD45RA(-/+) perforin(+) granzyme B(+) differentiation stages. Distinct CD8(+) T cell maturation stages, as defined by CCR7 vs CD45RA and by functional assays, were identified even in melanoma- or viral Ag-specific T cells from invaded lymph nodes by HLA tetramer analysis. Culture for 7 days of CCR7(+) perforin(-) CD8(+) T cells from tumor-invaded lymph nodes with IL-2 or IL-15, but not IL-7, promoted, mainly in CCR7(+)CD45RA(-) cells, proliferation coupled to differentiation to the CCR7(-) perforin(+) stage and acquisition of melanoma Ag-specific effector functions. Taken together, these results indicate that CD8(+) T cells differentiated toward CCR7(-) cytotoxic factor(+) stages are present in tumor-invaded, but not in tumor-free, lymph nodes of a relevant fraction of melanoma patients and suggest that cytokines such as IL-2 and IL-15 may be exploited to promote Ag-independent maturation of anti-tumor CD8(+) T cells.     

Induction of cytotoxic granules in human memory CD8+ T cell subsets requires cell cycle progression

Memory CD8(+) T cell responses are thought to be more effective as a result of both a higher frequency of Ag-specific clones and more rapid execution of effector functions such as granule-mediated lysis. Murine models have indicated that memory CD8(+) T cells exhibit constitutive expression of perforin and can lyse targets directly ex vivo. However, the regulated expression of cytotoxic granules in human memory CD8(+) T cell subsets has been underexplored. Using intracellular flow cytometry, we observed that only a minor fraction of CD45RA(-)CD8(+) T cells, or of CD8(+) T cells reactive to EBV-HLA2 tetramer, expressed intracellular granzyme B (GrB). Induction of GrB-containing cytotoxic granules in both CD45RA(+) and CD45RA(-) cells was achieved by stimulation with anti-CD3/anti-CD28 mAb-coated beads, required at least 3 days, occurred after several rounds of cell division, and required cell cycle progression. The strongest GrB induction was seen in the CCR7(+) subpopulations, with poorest proliferation being observed in the CD45RA(-)CCR7(-) effector-memory pool. Our results indicate that, as with naive T cells, induction of cytotoxic granules in human Ag-experienced CD8(+) T cells requires time and cell division, arguing that the main numerical advantage of a memory T cell pool is a larger frequency of CTL precursors. The fact that granule induction can be achieved through TCR and CD28 ligation has implications for restoring lytic effector function in the context of antitumor immunity.     

CC chemokine receptor 7 expression by effector/memory CD4+ T cells depends on antigen specificity and tissue localization during influenza A virus infection

The lung is an important entry site for respiratory pathogens such as influenza A virus. In order to combat such invading infectious agents, effector/memory T cells home to the lung and other peripheral tissues as well as lymphoid organs. In this process, chemokines and their receptors fulfill important roles in the guidance of T cells into such organs and specialized microenvironments within tissues. In this study, we determined if CD4(+) T cells residing in different lung compartments and draining lymph nodes of influenza A virus-infected and na?ve mice express receptors allowing their recirculation into secondary lymphoid tissues. We found high levels of l-selectin and CC chemokine receptor 7 (CCR7) expression in lung-derived CD4(+) T cells, similar to that detected on T cells in secondary lymphoid organs. Upon influenza A virus infection, the bulk of gamma interferon-positive (IFN-gamma(+)) and IFN-gamma(-) CD4(+) T cells recovered from lung parenchyma retained functional CCR7, whereas virus-specific IFN-gamma-producing T cells were CCR7(-). In contrast, a majority of virus-specific IFN-gamma(+) T cells in the lung draining lymph node were CCR7(+). Independent of infection, CD4(+) T cells obtained from the lung airways exhibited the lowest expression level of l-selectin and CCR7, indicating that T cells at this anatomical site represent the most differentiated effector cell type, lacking the ability to recirculate. Our results suggest that effector/memory T cells that enter inflammatory sites retain functional CCR7 expression, which is lost only upon response to viral antigen and after localization to the final effector site.     

CCR2 identifies a stable population of human effector memory CD4+ T cells equipped for rapid recall response

Because T cells act primarily through short-distance interactions, homing receptors can identify colocalizing cells that serve common functions. Expression patterns for multiple chemokine receptors on CD4(+) T cells from human blood suggested a hierarchy of receptors that are induced and accumulate during effector/memory cell differentiation. We characterized CD4(+)CD45RO(+) T cells based on expression of two of these receptors, CCR5 and CCR2, the principal subsets being CCR5(-)CCR2(-) (～70%), CCR5(+)CCR2(-) (～25%), and CCR5(+)CCR2(+) (～5%). Relationships among expression of CCR5 and CCR2 and CD62L, and the subsets' proliferation histories, suggested a pathway of progressive effector/memory differentiation from the CCR5(-)CCR2(-) to CCR5(+)CCR2(-) to CCR5(+)CCR2(+) cells. Sensitivity and rapidity of TCR-mediated activation, TCR signaling, and effector cytokine production by the subsets were consistent with such a pathway. The subsets also showed increasing responsiveness to IL-7, and the CCR5(+)CCR2(+) cells were CD127(bright) and invariably showed the greatest response to tetanus toxoid. CCR5(+)CCR2(+) cells also expressed the largest repertoire of chemokine receptors and migrated to the greatest number of chemokines. By contrast, the CCR5(+)CCR2(-) cells had the greatest percentages of regulatory T cells, activated/cycling cells, and CMV-reactive cells, and were most susceptible to apoptosis. Our results indicate that increasing memory cell differentiation can be uncoupled from susceptibility to death, and is associated with an increase in chemokine responsiveness, suggesting that vaccination (or infection) can produce a stable population of effector-capable memory cells that are highly enriched in the CCR5(+)CCR2(+) subset and ideally equipped for rapid recall responses in tissue.     

Identification of naive or antigen-experienced human CD8(+) T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8(+) T cell response

Human CMV (HCMV) infection provides an informative model of how long term human CD8(+) T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8(+) T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8(+) T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8(+) clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45RO(high) cells and the subpopulation of CD45RA(high) cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8(+) T cell memory, we found that in healthy virus carriers highly purified CD28(-)CD45RA(high)CCR7(-) cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8(+) T cells are predominantly CD28(-)CD45RO(high)CCR7(-). During convalescence, an increasing proportion of pp65-specific CD8(+) T cells were CD28(-)CD45RA(high)CCR7(-). We conclude that naive human CD8(+) T cells are CD28(+)CD45RA(high), express CCR7 but not CCR6, and are predominantly CD27(+) and L-selectin CD62 ligand-positive. The phenotype CD27(+)CD45RA(high) should not be used to identify naive human CD8(+) T cells, because CD27(+)CD45RA(high) cells also contain a significant subpopulation of CD28(-)CD27(+) Ag-experienced expanded clones. Thus CD8(+) T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45RO(high) and CD28(-)CD45RA(high) T cell pools.     

Three memory subsets of human CD8+ T cells differently expressing three cytolytic effector molecules

Multicolor flow cytometric analysis for the expression of three effector molecules, i.e., perforin (Per), granzyme A (GraA), and granzyme B (GraB), in human CD8(+) T cells demonstrated that they included five subpopulations, implying the following pathway for the differentiation of CD8(+) T cells: Per(-)GraA(-)GraB(-)-->Per(-)GraA(+)GraB(-)-->Per(low)GraA(+)GraB(-)--> Per(low)GraA(+)GraB(+)-->Per(high)GraA(+)GraB(+). The analysis of the expression of these molecules in the subsets classified by the combination of the expression of CCR7 and CD45RA or by that of CD27, CD28, and CD45RA showed that functional CD8(+) T cell subsets could be partially identified by these phenotypic classifications. However, the functional subsets could be precisely identified by the classification using five cell surface markers or three cell surface markers and three cytolytic molecules. Per(-)GraA(-)GraB(-) and Per(-/low)GraA(+)GraB(-) cells were predominantly found in CCR5(-)CCR7(+) and CCR5(high/low)CCR7(-) subsets, respectively, of CD8(+) T cells expressing the CD27(+)CD28(+)CD45RA(-) phenotype, whereas Per(low)GraA(+)GraB(+) cells were found in the CCR5(low)CCR7(-) subset of those expressing this phenotype and in a part of the CCR5(-/low)CCR7(-) subset of those expressing the CD27(-/low)CD28(-)CD45RA(-/+) phenotype. Ex vivo EBV-specific CD8(+) T cells, which were Per(low/-)GraA(+)GraB(-/+) cells, hardly or very weakly killed the target cells, indicating that these were not effector T cells. These findings suggest that the Per(-)GraA(-)GraB(-), Per(-/low)GraA(+)GraB(-), and Per(low)GraA(+)GraB(+) cells were central memory, early effector memory, and late effector memory T cells, respectively. Per(-/low)GraA(+)GraB(-) cells gained GraB expression after TCR stimulation, indicating that early effector memory T cells could differentiate into late effector and effector T cells. The present study showed the existence of three memory subsets and the pathway for their differentiation.     

Proliferation requirements of cytomegalovirus-specific,effector-type human CD8+ T cells

Two prototypic types of virus-specific CD8(+) T cells can be found in latently infected individuals: CD45R0(+)CD27(+)CCR7(-) effector-memory, and CD45RA(+)CD27(-)CCR7(-) effector-type cells. It has recently been implied that CD45RA(+)CD27(-)CCR7(-) T cells are terminally differentiated effector cells and as such have lost all proliferative capacity. We show in this study, however, that stimulation of CMV-specific CD45RA(+)CD27(-)CCR7(-) T cells with their cognate peptide in concert with either CD4(+) help or IL-2, IL-15, or IL-21 in fact induces massive clonal expansion. Concurrently, these stimulated effector T cells change cell surface phenotype from CD45RA to CD45R0 and regain CCR7, while effector functions are maintained. Our data imply that CD45RA(+)CD27(-)CCR7(-) effector-type T cells contribute to immunity not only by direct execution of effector functions, but also by yielding progeny in situations of viral reinfection or reactivation.     

Phenotypical and functional analysis of memory and effector human CD8 T cells specific for mycobacterial antigens

Mycobacterium tuberculosis infects one-third of the global population and claims two million lives every year. Because memory CD8 T cells exhibit a high heterogeneity in terms of phenotype and functional characteristic, we investigated the frequency, phenotype, and functional properties of Ag85A epitope-specific HLA-A*0201 CD8 T cells in children affected by tuberculosis (TB) before and 4 mo after chemotherapy and healthy contact children. Using Ag85A peptide/HLA-A*0201 pentamer, we found a low frequency of blood peptide-specific CD8 T cells in tuberculous children before therapy, which consistently increased after therapy to levels detected in healthy contacts. Ex vivo analysis of the expression of CD45RA and CCR7 surface markers indicated a skewed representation of Ag85A epitope-specific CD8 T cells during active TB, with a predominance of T central memory cells and a decrease of terminally differentiated T cells, which was reversed after therapy. Accordingly, pentamer-specific CD8 T cells from tuberculous patients produced low levels of IFN-gamma and had low expression of perforin, which recovered after therapy. The finding of an elevated frequency of pentamer-specific CD8 T cells with T effector memory and terminally differentiated phenotypes in the cerebrospinal fluid of a child with tuberculous meningitis strongly indicates compartmentalization of such CD8 effectors at the site of disease. Our study represents the first characterization of Ag-specific memory and effector CD8 T cells during TB and may help to understand the type of immune response that vaccine candidates should stimulate to achieve protection.     

Breast milk-derived antigen-specific CD8+ T cells: an extralymphoid effector memory cell population in humans

Although mouse studies have demonstrated the presence of an effector memory population in nonlymphoid tissues, the phenotype of human CD8(+) T cells present in such compartments has not been characterized. Because of the relatively large number of CD8(+) T cells present in breast milk, we were able to characterize the phenotype of this cell population in HIV-infected and uninfected lactating women. CMV, influenza virus, EBV, and HIV-specific CD8(+) T cells as measured by the IFN-gamma ELISPOT and MHC class I tetramer staining were all present at greater frequencies in breast milk as compared with blood. Furthermore, a greater percentage of the breast milk CD8(+) T cells expressed the intestinal homing receptor, CD103, and the mucosal homing receptor CCR9. Breast milk T cells were predominantly CD45RO(+)HLADR(+) and expressed low levels of CD45RA, CD62L, and CCR7 consistent with an effector memory population. Conversely, T cells derived from blood were mainly characterized as central memory cells (CCR7(+)CD62L(+)). These results demonstrate a population of extralymphoid CD8(+) T cells with an effector memory phenotype in humans, which could contribute to enhanced local virologic control and the relative lack of HIV transmission via this route.     

Langerhans cells derived from genetically modified human CD34+ hemopoietic progenitors are more potent than peptide-pulsed Langerhans cells for inducing antigen-specific CD8+ cytolytic T lymphocyte responses

Sustained Ag expression by human dendritic cells (DCs) is an attractive means of optimizing Ag presentation for stimulating durable cellular immunity. To establish proof of principle, we used Langerhans cell (LC) progeny of retrovirally transduced CD34(+) hemopoietic progenitor cells to stimulate responses against the HLA-A*0201-restricted influenza matrix peptide (fluMP). Retroviral transduction of CD34(+) hemopoietic progenitor cells, during pre-expansion by thrombopoietin, c-kit ligand, and FLT-3 ligand, on recombinant fibronectin, but in the absence of FCS, resulted in gene expression by 20-30% of the LCs. Expression persisted at least 28 days, with little decline (<30%) over that time. Retroviral transduction did not alter the phenotype or potent immunogenicity of normal mature DCs. FluMP-transduced LCs stimulated a 130-fold expansion of T cells reactive with HLA-A*0201-fluMP tetramers, even at LC:T cell ratios of 1:100-150 and lower, whereas fluMP-pulsed LCs stimulated only a 30-fold expansion. FluMP-transduced LCs also stimulated higher IFN-gamma secretion (100-123 spot-forming cells/10(5) CD8(+) T cells) than did fluMP-pulsed LCs (10-91 spot-forming cells/10(5) CD8(+) T cells). CD8(+) T cells stimulated by transduced LCs did not react preferentially with retrovirally transduced targets, indicating that the responses targeted only the immunizing influenza and not the retroviral vector Ags, even though these could have provided nonspecific helper epitopes presented by the transduced LCs. These data demonstrate that gene-transduced LCs maintain the activated phenotype as well potent immunogenicity typical of mature DCs. LCs genetically modified to express fluMP are also more potent stimulators of Ag-specific CD8(+) T cell responses than are peptide-pulsed LCs.     

Differentiation of human CD8(+) T cells from a memory to memory/effector phenotype

Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA(-)CD8(+) T cells with different CD28/CD27 phenotypes showed that CD28(+)CD45RA(-)CD8(+) and CD27(+)CD45RA(-)CD8(+) T cells have characteristics of memory T cells, whereas CD28(-)CD45RA(-)CD8(+) and CD27(-)CD45RA(-)CD8(+) T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8(+) T cells into memory/effector CD8(+) T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8(+) T cells. Three subsets of CD45RA(-)CD8(+) T cells were observed in both total CD8(+) T cells and EBV- or HCMV-specific CD8(+) T cells: CD27(+)CD28(+), CD27(+)CD28(-), and CD27(-)CD28(-). A significant number of the CD27(-)CD28(+) subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8(+) T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-)-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). This was supported by the observation that the frequency of CCR5(+) cells and CCR7(+) cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27(+)CD28(+) memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5(-)CCR7(+), CCR5(+)CCR7(+), and CCR5(+)CCR7(-). These findings suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-) (CCR5(-)CCR7(+)-->CCR5(+)CCR7(+)-->CCR5(+)CCR7(-))-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). The presence of a CD27(-)CD28(+) subset with a CCR5(+)CCR7(-) phenotype implies a specialized role for this subset in the differentiation of CD8(+) T cells.     

Deficient anti-listerial immunity in the absence of perforin can be restored by increasing memory CD8+ T cell numbers

Compared with wild-type (WT) mice, Listeria monocytogenes (LM)-vaccinated perforin-deficient (PKO) mice have elevated levels of CD8(+) T cell memory, but exhibit reduced levels of protection against virulent LM. In this study, Ag-specific CD8(+) T cells from LM-vaccinated WT and PKO mice were used in adoptive transfer assays to determine the contribution of perforin-dependent cytolysis in protective immunity to LM. Perforin deficiency resulted in an approximately 5-fold reduction in the per-cell protective capacity of Ag-specific memory CD8(+) T cells that was not caused by differences in memory cell quality as measured by CD62L/CD27 expression, TCR repertoire use, functional avidity, differences in expansion of Ag-specific cells upon infection, or maintenance of memory levels over time. However, perforin-deficient CD8(+) T cells exhibited reduced in vivo cytotoxic function compared to WT CD8(+) T cells. Consistent with the existence of perforin-independent effector pathways, double-vaccinated PKO mice were as resistant to challenge with LM as single-vaccinated WT mice. Thus, increasing the number of memory CD8(+) T cells can overcome diminished per-cell protective immunity in the absence of perforin.     

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T(EMRA) cells in early infection are linked to control of HIV-1 viremia and predict the subsequent viral load set point

CD8(+) T cells are believed to play an important role in the control of human immunodeficiency virus type 1 (HIV-1) infection. However, despite intensive efforts, it has not been possible to consistently link the overall magnitude of the CD8(+) T-cell response with control of HIV-1. Here, we have investigated the association of different CD8(+) memory T-cell subsets responding to HIV-1 in early infection with future control of HIV-1 viremia. Our results demonstrate that both a larger proportion and an absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(+) effector memory T cells (T(EMRA) cells) were associated with a lower future viral load set point. In contrast, a larger absolute number of HIV-1-specific CD8(+) CCR7(-) CD45RA(-) effector memory T cells (T(EM)) was not related to the viral load set point. Overall, the findings suggest that CD8(+) T(EMRA) cells have superior antiviral activity and indicate that both qualitative and quantitative aspects of the CD8(+) T-cell response need to be considered when defining the characteristics of protective immunity to HIV-1.     

Antigen-specific central memory CD4+ T lymphocytes produce multiple cytokines and proliferate in vivo in humans

The function of Ag-specific central (T(CM)) and effector (T(EM)) memory CD4+ T lymphocytes remains poorly characterized in vivo in humans. Using CD154 as a marker of Ag-specific CD4+T cells, we studied the differentiation of memory subsets following anti-hepatitis B immunization. Hepatitis B surface Ag (HBs)-specific memory CD4+T cells were heterogeneous and included T(CM) (CCR7+CD27+) and T(EM) (CCR7(-)CD27(+/-)). HBs-specific T(CM) and T(EM) shared the capacity to produce multiple cytokines, including IL-2 and IFN-gamma. Several years postimmunization, approximately 10% of HBs-specific memory CD4+ T cells were in cycle (Ki67+) and the proliferating cells were CCR7+. These results suggest that the model of functional specialization of T(CM) and T(EM) cannot be applied to protein vaccine Ags and support the concept that T(CM) are capable of self-renewal and contribute to maintain the pool of memory cells.     

Human tumor-derived heat shock protein 96 mediates in vitro activation and in vivo expansion of melanoma- and colon carcinoma-specific T cells

Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8(+) T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8(+) T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-gamma and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A(27-35) in three of five HLA-A*0201 melanoma patients, and of CEA(571-579) and EpCAM(263-271) in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8(+) T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients.