Persistence and function of central and effector memory CD4+ T cells following infection with a gastrointestinal helminth

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62L(high) central memory T cells (T(CM)) and CD62L(low) effector memory T cells (T(EM)) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ T(CM) and T(EM) populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62L(high)CD4+ T(CM) were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ T(EM). Consistent with this theory, transfer of CD62L(low)CD4+ T(EM) into naive recipients resulted in the development of a population of protective CD62L(high)CD4+ T(CM). Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.     

Molecular signatures distinguish human central memory from effector memory CD8 T cell subsets

Memory T cells are heterogeneous in terms of their phenotype and functional properties. We investigated the molecular profiles of human CD8 naive central memory (T(CM)), effector memory (T(EM)), and effector memory RA (T(EMRA)) T cells using gene expression microarrays and phospho-protein-specific intracellular flow cytometry. We demonstrate that T(CM) have a gene expression and cytokine signaling signature that lies between that of naive and T(EM) or T(EMRA) cells, whereas T(EM) and T(EMRA) are closely related. Our data define the molecular basis for the different functional properties of central and effector memory subsets. We show that T(EM) and T(EMRA) cells strongly express genes with known importance in CD8 T cell effector function. In contrast, T(CM) are characterized by high basal and cytokine-induced STAT5 phosphorylation, reflecting their capacity for self-renewal. Altogether, our results distinguish T(CM) and T(EM)/T(EMRA) at the molecular level and are consistent with the concept that T(CM) represent memory stem cells.     

Strength of stimulus and clonal competition impact the rate of memory CD8 T cell differentiation

The developmental pathways of long-lived memory CD8 T cells and the lineage relationship between memory T cell subsets remain controversial. Although some studies indicate the two major memory T cell subsets, central memory T (T(CM)) and effector memory T (T(EM)), are related lineages, others suggest that these subsets arise and are maintained independently of one another. In this study, we have investigated this issue and examined the differentiation of memory CD8 T cell subsets by tracking the lineage relationships of both endogenous and TCR transgenic CD8 T cell responses after acute infection. Our data indicate that TCR transgenic as well as nontransgenic T(EM) differentiate into T(CM) in the absence of Ag. Moreover, the rate of memory CD8 T cell differentiation from T(EM) into the self-renewing and long-lived pool of T(CM) is influenced by signals received during priming, including Ag levels, clonal competition, and/or the duration of infection. Although some T(EM) appear to not progress to T(CM), the vast majority of T(CM) are derived from T(EM). Thus, long-lasting, Ag-independent CD8 T cell memory results from progressive differentiation of memory CD8 T cells, and the rate of memory T cell differentiation is governed by events occurring early during T cell priming.     

Suppression of memory CD8 T cell generation and function by tryptophan catabolism

Dendritic cell-derived indoleamine 2,3-dioxygenase (IDO) suppresses naive T cell proliferation and induces their apoptosis by catalyzing tryptophan, and hence is essential for the maintenance of peripheral tolerance. However, it is not known whether memory T cells are subject to the regulation by IDO-mediated tryptophan catabolism, as memory T cells respond more rapidly and vigorously than their naive counterparts and are resistant to conventional costimulatory blockade. In this study, we present the evidence that memory CD8+ T cells are susceptible to tryptophan catabolism mediated by IDO. We found that overexpression of IDO in vivo attenuated the generation of both central memory CD8+ T cells (T(CM)) and effector memory CD8+ T cells (T(EM)) while suppressing IDO activity promoted their generation. Moreover, IDO overexpression suppressed the effector function of T(CM) cells or T(CM) cell-mediated allograft rejection as well as their proliferation in vivo. Interestingly, T(CM) cells were resistant to apoptosis induced by tryptophan catabolism. However, IDO overexpression did not suppress the effector function of T(EM) cells or T(EM) cell-mediated allograft rejection, suggesting that T(EM) cells, unlike T(CM) cells, do not require tryptophan for their effector function once they are generated. This study provides insight into the mechanisms underlying the differential regulation of memory T cell responsiveness and has clinical implications for vaccination or tolerance induction.     

Effector CD8+ T cells mediate inflammation and airway hyper-responsiveness

Allergic asthma is a complex syndrome characterized by airway obstruction, airway inflammation and airway hyper-responsiveness (AHR). Using a mouse model of allergen-induced AHR, we previously demonstrated that CD8-deficient mice develop significantly lower AHR, eosinophilic inflammation and interleukin (IL)-13 levels in bronchoalveolar lavage fluid compared with wild-type mice. These responses were restored by adoptive transfer of antigen-primed CD8(+) T cells. Previously, two distinct populations of antigen-experienced CD8(+) T cells, termed effector (T(EFF)) and central memory (T(CM)) cells, have been described. After adoptive transfer into CD8-deficient mice, T(EFF), but not T(CM), cells restored AHR, eosinophilic inflammation and IL-13 levels. T(EFF), but not T(CM), cells accumulated in the lungs, and intracellular cytokine staining showed that the transferred T(EFF) cells were a source of IL-13. These data suggest an important role for effector CD8(+) T cells in the development of AHR and airway inflammation, which may be associated with their Tc2-type cytokine production and their capacity to migrate into the lung.     

A novel role of CD30/CD30 ligand signaling in the generation of long-lived memory CD8+ T cells

Memory CD8+ T cells can be divided into two subsets, central memory (T(CM)) and effector memory (T(EM)) CD8+ T cells. We found that CD30, a member of the TNFR-associated factor (TRAF)-linked TNFR superfamily, signaling is involved in differentiation of long-lived CD8+ T(CM) cells following Listeria monocytogenes infection. Although CD8+ T(EM) cells transiently accumulated in the nonlymphoid tissues of CD30 ligand (CD153-/-) mice after infection, long-lived memory CD8+ T(CM) cells were poorly generated in these mice. CCR7 mRNA expression was down-regulated in CD8+ T cells of the spleen of CD153-/- mice in vivo and the expression was up-regulated in CD8+ T(EM) cells by anti-CD30 mAb cross-linking in vitro. These results suggest that CD30/CD30 ligand signaling plays an important role in the generation of long-lived memory CD8+ T cells at least partly by triggering homing receptors for T(CM) cells.     

Differential requirements for OX40 signals on generation of effector and central memory CD4+ T cells

Memory T cells can be divided into effector memory (T(EM)) and central memory (T(CM)) subsets based on their effector function and homing characteristics. Although previous studies have demonstrated that TCR and cytokine signals mediate the generation of the two memory subsets of CD8(+) T cells, the mechanisms for generation of the CD4(+) T(EM) and T(CM) cell subsets are unknown. We found that OX40-deficient mice showed a marked reduction in the number of CD4(+) T(EM) cells, whereas the number of CD4(+) T(CM) cells was normal. Adoptive transfer experiments using Ag-specific CD4(+) T cells revealed that OX40 signals during the priming phase were indispensable for the optimal generation of the CD4(+) T(EM), but not the CD4(+) T(CM) population. In a different transfer experiment with in vitro established CD4(+)CD44(high)CD62L(low) (T(EM) precursor) and CD4(+)CD44(high)CD62L(high) (T(CM) precursor) subpopulations, OX40-KO T(EM) precursor cells could not survive in the recipient mice, whereas wild-type T(EM) precursor cells differentiated into both T(EM) and T(CM) cells. In contrast, T(CM) precursor cells mainly produced T(CM) cells regardless of OX40 signals, implying the dispensability of OX40 for generation of T(CM) cells. Nevertheless, survival of OX40-KO T(EM) cells was partially rescued in lymphopenic mice. During in vitro recall responses, the OX40-KO T(EM) cells that were generated in lymphopenic recipient mice showed impaired cytokine production, suggesting an essential role for OX40 not only on generation but also on effector function of CD4(+) T(EM) cells. Collectively, the present results indicate differential requirements for OX40 signals on generation of CD4(+) T(EM) and T(CM) cells.     

Dynamic differentiation of activated human peripheral blood CD8+ and CD4+ effector memory T cells

Two functionally different memory T cell subsets were originally defined based on their different CCR7 expression profile, but the lineage relationship between these subsets referred to as central memory T cells (T(CM)) and effector memory T cells (T(EM)), is not resolved. A prevalent model proposes a linear progressive differentiation from T(CM) to T(EM). Our results demonstrate that on activation, human CCR7-CD62L- peripheral blood CD8+ and CD4+ T(EM) cells exhibit a dynamic differentiation, involving transient as well as stable changes to T(CM) phenotype and properties. Whereas the larger fraction of T(EM) cells increases expression of effector molecules, such as perforin or IFN-gamma, a smaller fraction first acquires CCR7 expression. We demonstrate that this acquisition of lymph node homing potential is associated with strong proliferation similar to that of activated T(CM) cells. After proliferation, most of these cells lose CCR7 expression again and acquire effector functions (e.g., perforin production). A small proportion (approximately 6%), however, maintain phenotypic and functional T(CM) properties over a long time interval. These results suggest that T(EM) cells provide immediate effector function by a fraction of cells as well as self-renewal by others through up-regulation of CCR7 followed by either secondary peripheral effector function or long term maintenance of T(CM)-like properties.     

IL-15 transpresentation augments CD8+ T cell activation and is required for optimal recall responses by central memory CD8+ T cells

Although the adaptive immune system has a remarkable ability to mount rapid recall responses to previously encountered pathogens, the cellular and molecular signals necessary for memory CD8(+) T cell reactivation are poorly defined. IL-15 plays a critical role in memory CD8(+) T cell survival; however, whether IL-15 is also involved in memory CD8(+) T cell reactivation is presently unclear. Using artificial Ag-presenting surfaces prepared on cell-sized microspheres, we specifically addressed the role of IL-15 transpresentation on mouse CD8(+) T cell activation in the complete absence of additional stimulatory signals. In this study we demonstrate that transpresented IL-15 is significantly more effective than soluble IL-15 in augmenting anti-CD3epsilon-induced proliferation and effector molecule expression by CD8(+) T cells. Importantly, IL-15 transpresentation and TCR ligation by anti-CD3epsilon or peptide MHC complexes exhibited synergism in stimulating CD8(+) T cell responses. In agreement with previous studies, we found that transpresented IL-15 preferentially stimulated memory phenotype CD8(+) T cells; however, in pursuing this further, we found that central memory (T(CM)) and effector memory (T(EM)) CD8(+) T cells responded differentially to transpresented IL-15. T(CM) CD8(+) T cells undergo Ag-independent proliferation in response to transpresented IL-15 alone, whereas T(EM) CD8(+) T cells are relatively unresponsive to transpresented IL-15. Furthermore, upon Ag-specific stimulation, T(CM) CD8(+) T cell responses are enhanced by IL-15 transpresentation, whereas T(EM) CD8(+) T cell responses are only slightly affected, both in vitro and in vivo. Thus, our findings distinguish the role of IL-15 transpresentation in the stimulation of distinct memory CD8(+) T cell subsets, and they also have implications for ex vivo reactivation and expansion of Ag-experienced CD8(+) T cells for immunotherapeutic approaches.     

Bcl6 acts as an amplifier for the generation and proliferative capacity of central memory CD8+ T cells

Central memory CD8(+) T cells (T(CM)) are considered to be more efficient than effector ones (T(EM)) for mediating protective immunity. The molecular mechanism involved in the generation of these cells remains elusive. Because Bcl6 plays a role in the generation and maintenance of memory CD8(+) T cells, we further examined this role in the process in relation to T(CM) and T(EM) subsets. In this study, we show that T(CM) and T(EM) were functionally identified in CD62L(+) and CD62L(-) memory (CD44(+)Ly6C(+)) CD8(+) T cell subsets, respectively. Although T(CM) produced similar amounts of IFN-gamma and IL-2 to T(EM) after anti-CD3 stimulation, the cell proliferation capacity after stimulation and tissue distribution profiles of T(CM) differed from those of T(EM). Numbers of T(CM) were greatly reduced and elevated in spleens of Bcl6-deficient and lck-Bcl6 transgenic mice, respectively, and those of T(EM) were constant in nonlymphoid organs of these same mice. The majority of Ag-specific memory CD8(+) T cells in spleens of these mice 10 wk after immunization were T(CM), and the number correlated with Bcl6 expression in T cells. The proliferation of Ag-specific memory CD8(+) T cells upon secondary stimulation was dramatically up-regulated in lck-Bcl6 transgenic mice, and the adoptive transfer experiments with Ag-specific naive CD8(+) T cells demonstrated that some of the up-regulation was due to the intrinsic effect of Bcl6 in the T cells. Thus, Bcl6 is apparently a crucial factor for the generation and secondary expansion of T(CM).     

Functional blockade of the voltage-gated potassium channel Kv1.3 mediates reversion of T effector to central memory lymphocytes through SMAD3/p21cip1 signaling

The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (T(EM)) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (T(CM)) into T(EM). Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of T(EM) into T(CM), via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21(cip1) with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in T(EM)-mediated autoimmune diseases.     

Lack of ICAM-1 on APCs during T cell priming leads to poor generation of central memory cells

ICAM-1/LFA-1 interactions are known to enhance T cell/APC interactions and to promote T cell activation and cytokine secretion. We have analyzed the consequences of ICAM-1-mediated signaling on the generation of memory T cell subsets. We report that lack of ICAM-1 on APCs, but not on T cells, leads to poor T cell activation and proliferation in vitro and in vivo, and that the defect can be compensated by Ag dose, exogenous IL-2, additional costimulation, and by increasing responder T cell density on APCs. ICAM-1-null mice do not respond to immunization with OVA peptide, but immunization with OVA or with Salmonella typhimurium leads to good T cell proliferation 7-10 days later, and clearance of a challenge infection is equivalent to that of wild-type mice. However, when followed over time, recall proliferation and antibacterial immunity decay rapidly in ICAM-1-null mice, while recall cytokine responses are unaffected. The decline in immunity is not related to poor survival of T cells activated on ICAM-1-null APCs, or to poor generation of effectors in ICAM-1-null mice. Phenotypic analysis of T cells stimulated on ICAM-1-null APCs reveals preferential generation of CD44(high) CD62L(low) effector memory cells (T(EM)) over CD44(high) CD62L(high) central memory cells (T(CM)). Further, while the proportion of naive:memory T cells is similar in unmanipulated wild-type and ICAM-1-null mice, there is an accumulation of T(EM) cells, and a high T(EM):T(CM) ratio in aging ICAM-1-null mice. Together, the data indicate that signaling through LFA-1 during T cell activation may be involved in commitment to a proliferation-competent memory pool.     

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 effector memory CD4+ T cells directly recognize allogeneic endothelial cells in vitro and in vivo

The frequency of circulating alloreactive human memory T cells correlates with allograft rejection. Memory T cells may be divided into effector memory (T(EM)) and central memory (T(CM)) cell subsets, but their specific roles in allograft rejection are unknown. We report that CD4+ T(EM) (CD45RO+ CCR7- CD62L-) can be adoptively transferred readily into C.B-17 SCID/bg mice and mediate the destruction of human endothelial cells (EC) in vascularized human skin grafts allogeneic to the T cell donor. In contrast, CD4+ T(CM) (CD45RO+ CCR7+ CD62L+) are inefficiently transferred and do not mediate EC injury. In vitro, CD4+ T(EM) secrete more IFN-gamma within 48 h in response to allogeneic ECs than do T(CM). In contrast, T(EM) and T(CM) secrete comparable amounts of IFN-gamma in response to allogeneic monocytes (Mo). In the same cultures, both T(EM) and T(CM) produce IL-2 and proliferate in response to IFN-gamma-treated allogeneic human EC or Mo, but T(CM) respond more vigorously in both assays. Blockade of LFA-3 strongly inhibits both IL-2 and IFN-gamma secretion by CD4+ T(EM) cultured with allogeneic EC but only minimally inhibits responses to allogeneic Mo. Blockade of CD80 and CD86 strongly inhibits IL-2 but not IFN-gamma production by in response to allogeneic EC or Mo. Transduction of EC to express B7-2 enhances allogeneic T(EM) production of IL-2 but not IFN-gamma. We conclude that human CD4+ T(EM) directly recognize and respond to allogeneic EC in vitro by secreting IFN-gamma and that this response depends on CD2 but not CD28. Consistent with EC activation of effector functions, human CD4+ T(EM) can mediate allogeneic EC injury in vivo.     

The basis of distinctive IL-2- and IL-15-dependent signaling: weak CD122-dependent signaling favors CD8+ T central-memory cell survival but not T effector-memory cell development

Recent work suggests that IL-2 and IL-15 induce distinctive levels of signaling through common receptor subunits and that such varied signaling directs the fate of Ag-activated CD8(+) T cells. In this study, we directly examined proximal signaling by IL-2 and IL-15 and CD8(+) T cell primary and memory responses as a consequence of varied CD122-dependent signaling. Initially, IL-2 and IL-15 induced similar p-STAT5 and p-S6 activation, but these activities were only sustained by IL-2. Transient IL-15-dependent signaling is due to limited expression of IL-15Rα. To investigate the outcome of varied CD122 signaling for CD8(+) T cell responses in vivo, OT-I T cells were used from mouse models where CD122 signals were attenuated by mutations within the cytoplasmic tail of CD122 or intrinsic survival function was provided in the absence of CD122 expression by transgenic Bcl-2. In the absence of CD122 signaling, generally normal primary response occurred, but the primed CD8(+) T cells were not maintained. In marked contrast, weak CD122 signaling supported development and survival of T central-memory (T(CM)) but not T effector-memory (T(EM)) cells. Transgenic expression of Bcl-2 in CD122(-/-) CD8(+) T cells also supported the survival and persistence of T(CM) cells but did not rescue T(EM) development. These data indicate that weak CD122 signals readily support T(CM) development largely through providing survival signals. However, stronger signals, independent of Bcl-2, are required for T(EM) development. Our findings are consistent with a model whereby low, intermediate, and high CD122 signaling support T(CM) memory survival, T(EM) programming, and terminal T effector cell differentiation, respectively.     

Characterization of the functional properties of the voltage-gated potassium channel Kv1.3 in human CD4+ T lymphocytes

Previous studies have shown that central memory T (T(CM)) cells predominantly use the calcium-dependent potassium channel KCa3.1 during acute activation, whereas effector memory T (T(EM)) cells use the voltage-gated potassium channel Kv1.3. Because Kv1.3-specific pharmacological blockade selectively inhibited anti-CD3-mediated proliferation, whereas naive T cells and T(CM) cells escaped inhibition due to up-regulation of KCa3.1, this difference indicated a potential for selective targeting of the T(EM) population. We examined the effects of pharmacological Kv1.3 blockers and a dominant-negative Kv1.x construct on T cell subsets to assess the specific effects of Kv1.3 blockade. Our studies indicated both T(CM) and T(EM) CD4+ T cells stimulated with anti-CD3 were inhibited by charybdotoxin, which can block both KCa3.1 and Kv1.3, whereas margatoxin and Stichodactyla helianthus toxin, which are more selective Kv1.3 inhibitors, inhibited proliferation and IFN-gamma production only in the T(EM) subset. The addition of anti-CD28 enhanced proliferation of freshly isolated cells and rendered them refractory to S. helianthus, whereas chronically activated T(EM) cell lines appeared to be costimulation independent because Kv1.3 blockers effectively inhibited proliferation and IFN-gamma regardless of second signal. Transduction of CD4+ T cells with dominant-negative Kv1.x led to a higher expression of CCR7+ T(CM) phenotype and a corresponding depletion of T(EM). These data provide further support for Kv1.3 as a selective target of chronically activated T(EM) without compromising naive or T(CM) immune functions. Specific Kv1.3 blockers may be beneficial in autoimmune diseases such as multiple sclerosis in which T(EM) are found in the target organ.     

Stable CD8+ T cell memory during persistent Trypanosoma cruzi infection

CD8(+) T cell responses to persistent infections caused by intracellular pathogens are dominated by resting T effectors and T effector memory cells, with little evidence suggesting that a T central memory (T(CM)) population is generated. Using a model of Trypanosoma cruzi infection, we demonstrate that in contrast to the T effector/T effector memory phenotype of the majority of T. cruzi-specific CD8(+) T cells, a population of cells displaying hallmark characteristics of T(CM) cells is also present during long-term persistent infection. This population expressed the T(CM) marker CD127 and a subset expressed one or more of three other T(CM) markers: CD62L, CCR7, and CD122. Additionally, the majority of CD127(high) cells were KLRG1(low), indicating that they have not been repetitively activated through TCR stimulation. These CD127(high) cells were better maintained than their CD127(low) counterparts following transfer into naive mice, consistent with their observed surface expression of CD127 and CD122, which confer the ability to self-renew in response to IL-7 and IL-15. CD127(high) cells were capable of IFN-gamma production upon peptide restimulation and expanded in response to challenge infection, indicating that these cells are functionally responsive upon Ag re-encounter. These results are in contrast to what is typically observed during many persistent infections and indicate that a stable population of parasite-specific CD8(+) T cells capable of Ag-independent survival is maintained in mice despite the presence of persistent Ag.     

Homogenization of TCR repertoires within secondary CD62Lhigh and CD62Llow virus-specific CD8+ T cell populations

Influenza virus-specific CD8(+) T cell clonotypes generated and maintained in C57BL/6J mice after respiratory challenge were found previously to distribute unequally between the CD62L(low) "effector" (T(EM)) and CD62L(high) "central" (T(CM)) memory subsets. Defined by the CDR3beta sequence, most of the prominent TCRs were represented in both the CD62L(high) and CD62L(low) subsets, but there was also a substantial number of diverse, but generally small, CD62L(high)-only clonotypes. The question asked here is how secondary challenge influences both the diversity and the continuity of TCR representation in the T(CM) and T(EM) subsets generated following primary exposure. The experiments use single-cell RT-PCR to correlate clonotypic composition with CD62L phenotype for secondary influenza-specific CD8(+) T cell responses directed at the prominent D(b)NP(366) and D(b)PA(224) epitopes. In both the acute and long-term memory phases of the recall responses to these epitopes, we found evidence of a convergence of TCR repertoire expression for the CD62L(low) and CD62L(high) populations. In fact, unlike the primary response, there were no significant differences in clonotypic diversity between the CD62L(low) and CD62L(high) subsets. This "TCR homogenization" for the CD62L(high) and CD62L(low) CD8(+) populations recalled after secondary challenge indicates common origin, most likely from the high prevalence populations in the CD62L(high) central memory set. Our study thus provides key insights into the TCR diversity spectrum for CD62L(high) and CD62L(low) T cells generated from a normal, unmanipulated T cell repertoire following secondary challenge. A better understanding of TCR selection and maintenance has implications for improved vaccine and immunotherapy protocols.     

Long-Term Central and Effector SHIV-Specific Memory T Cell Responses Elicited after a Single Immunization with a Novel Lentivector DNA Vaccine

Prevention of HIV acquisition and replication requires long lasting and effective immunity. Given the state of HIV vaccine development, innovative vectors and immunization strategies are urgently needed to generate safe and efficacious HIV vaccines. Here, we developed a novel lentivirus-based DNA vector that does not integrate in the host genome and undergoes a single-cycle of replication. Viral proteins are constitutively expressed under the control of Tat-independent LTR promoter from goat lentivirus. We immunized six macaques once only with CAL-SHIV-IN⁻ DNA using combined intramuscular and intradermal injections plus electroporation. Antigen-specific T cell responses were monitored for 47 weeks post-immunization (PI). PBMCs were assessed directly ex vivo or after 6 and 12 days of in vitro culture using antigenic and/or homeostatic proliferation. IFN-γ ELISPOT was used to measure immediate cytokine secretion from antigen specific effector cells and from memory precursors with high proliferative capacity (PHPC). The memory phenotype and functions (proliferation, cytokine expression, lytic content) of specific T cells were tested using multiparametric FACS-based assays. All immunized macaques developed lasting peripheral CD8⁺ and CD4⁺ T cell responses mainly against Gag and Nef antigens. During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes. These responses contracted but then reemerged later in absence of antigen boost. Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI. Altogether, our study demonstrated that a single immunization with a replication-limited DNA vaccine elicited persistent vaccine-specific CM and EM CD8⁺ and CD4⁺ T cells with immediate and readily inducible effector functions, in the absence of ongoing antigen expression.