Four functionally distinct populations of human effector-memory CD8+ T lymphocytes

In humans, the pathways of memory and effector T cell differentiation remain poorly defined. We have dissected the functional properties of ex vivo effector-memory (EM) CD45RA-CCR7- T lymphocytes present within the circulating CD8+ T cell pool of healthy individuals. Our studies show that EM T cells are heterogeneous and are subdivided based on differential CD27 and CD28 expression into four subsets. EM(1) (CD27+CD28+) and EM(4) (CD27-CD28+) T cells express low levels of effector mediators such as granzyme B and perforin and high levels of CD127/IL-7Ralpha. EM(1) cells also have a relatively short replicative history and display strong ex vivo telomerase activity. Therefore, these cells are closely related to central-memory (CD45RA-CCR7+) cells. In contrast, EM(2) (CD27+CD28-) and EM(3) (CD27-CD28-) cells express mediators characteristic of effector cells, whereby EM(3) cells display stronger ex vivo cytolytic activity and have experienced larger numbers of cell divisions, thus resembling differentiated effector (CD45RA+CCR7-) cells. These data indicate that progressive up-regulation of cytolytic activity and stepwise loss of CCR7, CD28, and CD27 both characterize CD8+ T cell differentiation. Finally, memory CD8+ T cells not only include central-memory cells but also EM(1) cells, which differ in CCR7 expression and may therefore confer memory functions in lymphoid and peripheral tissues, respectively.     

Increased frequency of CCR4+ and CCR6+ memory T-cells including CCR7+CD45RAmed very early memory cells in granulomatosis with polyangiitis (Wegener's)

Introduction

Chemokine receptors play an important role in mediating the recruitment of T cells to inflammatory sites. Previously, small proportions of circulating Th1-type CCR5+ and Th2-type CCR3+ cells have been shown in granulomatosis with polyangiitis (GPA). Wondering to what extent CCR4 and CCR6 expression could also be implicated in T cell recruitment to inflamed sites in GPA, we investigated the expression of CCR4 and CCR6 on T cells and its association with T cell diversity and polarization.

Methods

Multicolor flow cytometry was used to analyze CCR4, CCR6, and intracellular cytokine expression of T cells from whole blood of GPA-patients (n = 26) and healthy controls (n = 20). CCR7 and CD45RA were included for phenotypic characterization.

Results

We found a significant increase in the percentages of circulating CCR4+ and CCR6+ cells within the total CD4+ T cell population in GPA. In contrast, there was no difference in the percentages of CD8+CCR4+ and CD8+CCR6+ T cells between GPA and healthy controls. CCR4 and CCR6 expression was largely confined to central (T_CM) and effector memory T cells (T_EM, T_EMRA). A significant increase in the frequency of CCR4+ and CCR6+ T_EMRA and CCR6+ T_CM was shown in GPA. Of note, we could dissect CCR4 and CCR6 expressing CCR7+CD45RA^med very early memory T cells (T_VEM) from genuine CCR7+CD45RA^high naïve T cells lacking CCR4 and CCR6 expression for peripheral tissue-migration within the CCR7+CD45RA+ compartment. The frequencies of CCR4+ and CCR6+ T_VEM were also significantly increased in GPA. An increased percentage of IL-17+ and IL-22+ cells was detected in the CCR6+ cell subsets and IL-4+ cells in the CRR4+ cell subset when compared with CD4+ cells lacking CCR4 and CCR6 expression.

Conclusions

Increased frequencies of circulating CCR4+ and CCR6+ memory T cell subsets including hitherto unreported T_VEM suggest persistent T cell activation with the accumulation of CCR4+ and CCR6+ cells in GPA. CCR4 and CCR6 could be involved in the recruitment of T cells including cytokine-producing subsets to inflamed sites in GPA.     

Identification of new cytokine combinations for antigen-specific T-cell therapy products via a high-throughput multi-parameter assay

Infusion of viral-specific T cells (VSTs) is an effective treatment for viral infection after stem cell transplant. Current manufacturing approaches are rapid, but growth conditions can still be further improved. To optimize VST cell products, the authors designed a high-throughput flow cytometry-based assay using 40 cytokine combinations in a 96-well plate to fully characterize T-cell viability, function, growth and differentiation. Peripheral blood mononuclear cells (PBMCs) from six consenting donors were seeded at 100 000 cells per well with pools of cytomegalovirus peptides from IE1 and pp65 and combinations of IL-15, IL-6, IL-21, interferon alpha, IL-12, IL-18, IL-4 and IL-7. Ten-day cultures were tested by 13-color flow cytometry to evaluate viable cell count, lymphocyte phenotype, memory markers and interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) expression. Combinations of IL-15/IL-6 and IL-4/IL-7 were optimal for the expansion of viral-specific CD3+ T cells, (18-fold and 14-fold, respectively, compared with unstimulated controls). CD8+ T cells expanded 24-fold in IL-15/IL-6 and 9-fold in IL-4/IL-7 cultures (P < 0.0001). CD4+ T cells expanded 27-fold in IL-4/IL-7 and 15-fold in IL-15/IL-6 (P < 0.0001). CD45RO+ CCR7– effector memory (CD45RO+ CCR7– CD3+), central memory (CD45RO+ CCR7+ CD3+), terminal effector (CD45RO– CCR7– CD3+), and naive (CD45RO– CCR7+ CD3+). T cells were the preponderant cells (76.8% and 72.3% in IL-15/IL-6 and IL-15/IL-7 cultures, respectively). Cells cultured in both cytokine conditions were potent, with 19.4% of CD3+ cells cultured in IL-15/IL-6 producing IFNγ (7.6% producing both TNFα and IFNγ) and 18.5% of CD3+ cells grown in IL-4/IL-7 producing IFNγ (9% producing both TNFα and IFNγ). This study shows the utility of this single-plate assay to rapidly identify optimal growth conditions for VST manufacture using only 10⁷ PBMCs.     

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.     

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.     

Age-associated change in the frequency of memory CD4+ T cells impairs long term CD4+ T cell responses to influenza vaccine

We investigated the relationship of memory CD4+ T cells with the evolution of influenza virus-specific CD4+ T cell responses in healthy young and elderly people. Elderly individuals had a similar frequency of CD69+CD4+ T cells producing IFN-gamma and TNF-alpha at 1 wk, but a lower frequency of these CD4+ T cells at 3 mo after influenza vaccination. Although the elderly had a higher frequency of central memory (CM; CCR7+CD45RA-) CD4+ T cells, they had a significantly lower frequency of effector memory (EM; CCR7-CD45RA-) CD4+ T cells, and the frequency of the latter memory CD4+ T cells positively correlated with the frequency of influenza virus-specific CD69+CD4+ T cells producing IFN-gamma at 3 mo. These findings indicate that the elderly have an altered balance of memory CD4+ T cells, which potentially affects long term CD4+ T cell responses to the influenza vaccine. Compared with the young, the elderly had decreased serum IL-7 levels that positively correlated with the frequency of EM cells, which suggests a relation between IL-7 and decreased EM cells. Thus, although the healthy elderly mount a level of CD4+ T cell responses after vaccination comparable to that observed in younger individuals, they fail to maintain or expand these responses. This failure probably stems from the alteration in the frequency of CM and EM CD4+ T cells in the elderly that is related to alteration in IL-7 levels. These findings raise an important clinical question about whether the vaccination strategy in the elderly should be modified to improve cellular immune responses.     

Direct stimulation of human T cells via TLR5 and TLR7/8: flagellin and R-848 up-regulate proliferation and IFN-gamma production by memory CD4+ T cells

TLRs are involved in innate cell activation by conserved structures expressed by microorganisms. Human T cells express the mRNA encoding most of TLRs. Therefore, we tested whether some TLR ligands may modulate the function of highly purified human CD4+ T lymphocytes. We report that, in the absence of APCs, flagellin (a TLR5 ligand) and R-848 (a TLR7/8 ligand) synergized with suboptimal concentrations of TCR-dependent (anti-CD3 mAb) or -independent stimuli (anti-CD2 mAbs or IL-2) to up-regulate proliferation and IFN-gamma, IL-8, and IL-10 but not IL-4 production by human CD4+ T cells. No effect of poly(I:C) and LPS, ligands for TLR3 and TLR4, respectively, was detected. We also observed that CD4+CD45RO+ memory T cell responses to TLR ligands were more potent than those observed with CD4+CD45RA+ naive T cells. Moreover, among the memory T cells, CCR7- effector cells were more sensitive to TLR ligands than CCR7+ central memory cells. These data demonstrate for the first time a direct effect of TLR5 and TLR7/8 ligands on human T cells, and highlight an innate arm in T cell functions. They also suggest that some components from invading microorganisms may directly stimulate effector memory T cells located in tissues by up-regulating cytokine and chemokine production.     

Bacillus Calmette-Guérin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles

The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-gamma, IL-2, and TNF-alpha in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-gamma and/or IL-2 and less TNF-alpha, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-gamma. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA-CCR7-CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-gamma+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-gamma production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.     

Interleukin-15 increases effector memory CD8+ t cells and NK Cells in simian immunodeficiency virus-infected macaques

Interleukin-15 (IL-15) in vitro treatment of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected individuals specifically enhances the function and survival of HIV-specific CD8+ T cells, while in vivo IL-15 treatment of mice preferentially expands memory CD8+ T cells. In this study, we investigated the in vivo effect of IL-15 treatment in 9 SIVmac251-infected cynomolgus macaques (low dose of IL-15, 10 microg/kg of body weight, n = 3; high dose of IL-15, 100 microg/kg, n = 3; control [saline], n = 3; dose administered twice weekly for 4 weeks). IL-15 treatment induced a nearly threefold increase in peripheral blood CD8+CD3- NK cells. Furthermore, CD8+ T-cell numbers increased more than twofold, mainly due to an increase in the CD45RA-CD62L- and CD45RA+CD62L- effector memory CD8+ T cells. Expression of Ki-67 in the CD8+ T cells indicated expansion of CD8+ T cells and not redistribution. IL-15 did not affect CD4+ T-cell, B-cell, and CD14+ macrophage numbers. No statistically significant differences in changes from baseline in the viral load were observed when control-, low-dose-, and high-dose-treated animals were compared. No clinical adverse effects were observed in any of the animals studied. The selective expansion of effector memory CD8+ T cells and NK cells by IL-15 further supports IL-15's possible therapeutic use in viral infections such as HIV infection.     

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.     

Phenotypic and functional characterization of CD4 T cells expressing killer Ig-like receptors

Killer Ig-like receptors (KIR) are commonly found on human NK cells, gammadelta T cells, and CD8 T cells. Although KIR(+) CD4 T cells are found in certain patients, their prevalence in healthy donors is controversial. We now provide definitive proof that such cells are present in most individuals, and report on their frequency, surface phenotype, cytokine profile, and Ag specificity. The number of KIR(+) CD4 T cells detected in peripheral blood increased with age. In contrast with regular KIR(-) CD4 T cells, the majority of KIR(+) CD4 T cells lacked surface expression of CD27, CD28, CCR4, and CCR7, but did express CD57 and 2B4. In addition, KIR were detected on approximately one-tenth of CD28(-) and CD57(+) memory CD4 T cells. In line with the absence of the Th2 marker CCR4, the KIR(+) CD4 cells produced mainly IFN-gamma and little IL-4, IL-10, or IL-17 upon TCR triggering. Furthermore, the KIR(+) population contained cells that responded to recall Ags in an HLA class II-restricted fashion. Together, our data indicate that KIR-expressing CD4 T cells are predominantly HLA class II-restricted effector memory Th1 cells, and that a significant, previously unrecognized fraction of effector memory Th1 cells expresses KIR.     

Prolonged (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells in macaques

Although phosphoantigen-specific Vgamma2Vdelta2 T cells appear to play a role in antimicrobial and anticancer immunity, mucosal immune responses and effector functions of these gammadelta T cells during infection or phospholigand treatment remain poorly characterized. In this study, we demonstrate that the microbial phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment of macaques induced a prolonged major expansion of circulating Vgamma2Vdelta2 T cells that expressed CD8 and produced cytotoxic perforin during their peak expansion. Interestingly, HMBPP-activated Vgamma2Vdelta2 T cells underwent an extraordinary pulmonary accumulation, which lasted for 3-4 mo, although circulating Vgamma2Vdelta2 T cells had returned to baseline levels weeks prior. The Vgamma2Vdelta2 T cells that accumulated in the lung following HMBPP/IL-2 cotreatment displayed an effector memory phenotype, as follows: CCR5+CCR7-CD45RA-CD27+ and were able to re-recognize phosphoantigen and produce copious amounts of IFN-gamma up to 15 wk after treatment. Furthermore, the capacity of massively expanded Vgamma2Vdelta2 T cells to produce cytokines in vivo coincided with an increase in numbers of CD4+ and CD8+ alphabeta T cells after HMBPP/IL-2 cotreatment as well as substantial perforin expression by CD3+Vgamma2- T cells. Thus, the prolonged HMBPP-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells may confer immunotherapeutics against infectious diseases and cancers.     

Differentiation of human gamma-delta T cells towards distinct memory phenotypes

Vgamma9/Vdelta2 T cells comprise a small population of peripheral T cells responding towards the low molecular weight antigen, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMB-PP). HMB-PP-stimulated Vgamma9/Vdelta2 T cells proliferated, expressed CCL5/RANTES, and upregulated markers like CD16, CD25, CD69, and CD94, in the presence of either IL-15 or IL-21. Vgamma9/Vdelta2 T cells grown in the presence of IL-15 differentiated into an effector/memory population characterized by production of TNF-alpha, expression of CD45RO and CCR5, and lack of CD62L, CD81, and CCR7. In contrast, Vgamma9/Vdelta2 T cells grown with IL-21 differentiated into putative central memory CD45RO(+) T cells that did not produce TNF-alpha, IFN-gamma, or IL-4, and maintained expression of CD62L, CD81, and CCR7.     

Human T cells that are able to produce IL-17 express the chemokine receptor CCR6

Some pathways of T cell differentiation are associated with characteristic patterns of chemokine receptor expression. A new lineage of effector/memory CD4+ T cells has been identified whose signature products are IL-17 cytokines and whose differentiation requires the nuclear receptor, RORgammat. These Th17 cells are critical effectors in mouse models of autoimmune disease. We have analyzed the association between chemokine receptor expression and IL-17 production for human T cells. Activating cord blood (naive) CD4+ T cells under conditions driving Th17 differentiation led to preferential induction of CCR6, CCR9, and CXCR6. Despite these data, we found no strong correlation between the production of IL-17 and expression of CCR9 or CXCR6. By contrast, our analyses revealed that virtually all IL-17-producing CD4+ T cells, either made in our in vitro cultures or found in peripheral blood, expressed CCR6, a receptor found on approximately 50% of CD4+ memory PBL. Compared with CD4+CD45RO+CCR6- cells, CD4+CD45RO+CCR6+ cells contained at least 100-fold more IL-17A mRNA and secreted 100-fold more IL-17 protein. The CCR6+ cells showed a similar enrichment in mRNA for RORgammat. CCR6 was likewise expressed on all IL-17-producing CD8+ PBL. CCR6 has been associated with the trafficking of T, B, and dendritic cells to epithelial sites, but has not been linked to a specific T cell phenotype. Our data reveal a fundamental feature of IL-17-producing human T cells and a novel role for CCR6, suggesting both new directions for investigating IL-17-related immune responses and possible targets for preventing inflammatory injury.     

CD127+CCR5+CD38+++ CD4+ Th1 effector cells are an early component of the primary immune response to vaccinia virus and precede development of interleukin-2+ memory CD4+ T cells

The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-gamma)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-gamma and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.     

IL-15 treatment during acute simian immunodeficiency virus (SIV) infection increases viral set point and accelerates disease progression despite the induction of stronger SIV-specific CD8+ T cell responses

In this study, we examined the effect of in vivo treatment of acutely SIV-infected Mamu-A*01+ rhesus macaques with IL-15. IL-15 treatment during acute infection increased viral set point by 3 logs and accelerated the development of simian AIDS in two of six animals with one developing early minimal lesion SIV meningoencephalitis. Although IL-15 induced a 2- to 3-fold increase in SIV-specific CD8+ T cell and NK cell numbers at peak viremia and reduced lymph node (LN) SIV-infected cells, this had no impact on peak viremia and did not lower viral set point. At viral set point, however, activated SIV-specific CD8+ T cells and NK cells were reduced in the blood of IL-15-treated animals and LN SIV-infected cells were increased. Week 30 LN from IL-15-treated animals had significantly increased Gag-specific CD8+ T cell numbers, whereas total cell, lymphocyte, and CD4+ T cell numbers were reduced. IL-15 treatment significantly reduced anti-SIV Ab concentrations at week 3 and viral set point. IL-15 increased Ki-67+CD4+ T cells at week 1 of treatment and reduced blood CCR5+ and CD45RA-CD62L- CD4+ T cells. The frequency of day 7 Ki-67+CD4+ T cells strongly correlated with viral set point. These findings suggest that CD4+ T cell activation during acute infection determines subsequent viral set point and IL-15 treatment by increasing such activation elevates viral set point. Finally, IL-15-treated acutely SIV-infected primates may serve as a useful model to investigate the poorly understood mechanisms that control viral set point and disease progression in HIV infection.     

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.     

Regulation of IL-17 in human CCR6+ effector memory T cells

IL-17-secreting T cells represent a distinct CD4(+) effector T cell lineage (Th17) that appears to be essential in the pathogenesis of numerous inflammatory and autoimmune diseases. Although extensively studied in the murine system, human Th17 cells have not been well characterized. In this study, we identify CD4(+)CD45RO(+)CCR7(-)CCR6(+) effector memory T cells as the principal IL-17-secreting T cells. Human Th17 cells have a unique cytokine profile because the majority coexpress TNF-alpha but not IL-6 and a minor subset express IL-17 with IL-22 or IL-17 and IFN-gamma. We demonstrate that the cytokines that promote the differentiation of human naive T cells into IL-17-secreting cells regulate IL-17 production by memory T cells. IL-1beta alone or in association with IL-23 and IL-6 markedly increase IL-17(+) CCR6(+) memory T cells and induce IL-17 production in CCR6(-) memory T cells. We also show that T cell activation induces Foxp3 expression in T cells and that the balance between the percentage of Foxp3(+) and IL-17(+) T cells is inversely influenced by the cytokine environment. These studies suggest that the cytokine environment may play a critical role in the expansion of memory T cells in chronic autoimmune diseases.     

CD4 T cell depletion at the cervix during HIV infection is associated with accumulation of terminally differentiated T cells

In blood, the accumulation of terminally differentiated (TD) T cells during HIV infection is associated with CD4 T cell loss and HIV disease progression. Here, we investigated the maintenance and functional characteristics of memory T cells at the cervix. We found that CD4 T cell depletion at the cervix mirrors CD4 depletion in blood. In all women, depletion of CD4 T cells at the cervix was associated with significant reductions in CD45RA- CCR7+ (central memory [CM]) T cells and the accumulation of CD45RA+ CCR7- (TD T cells). We determined whether inflammation in the genital tract was associated with the local differentiation of T cells at the cervix. In uninfected women, genital tract inflammation was associated with the accumulation of CD45RA- CCR7+ CM CD4 T cells and reduced frequencies of CD45RA+ CCR7- TD cells at the cervix. This finding may reflect the fact that, in the absence of HIV infection, TD T cells may be slowly lost in the presence of genital inflammation, while CD45RA- CCR7+ CM T cells are recruited to replenish the diminishing CD4 T cell pool. Following global stimulation with phorbol myristate acetate (PMA)-ionomycin, we noted a significant interleukin 2 (IL-2) deficit in both cervical and blood CD4 T cells from HIV-infected women compared to uninfected women, while gamma interferon (IFN-γ) production was similar, irrespective of HIV status. Few HIV-infected women had detectable IFN-γ and IL-2 HIV-specific T cell responses at the cervix, and these responses were significantly lower in magnitude than the corresponding responses in blood. These data suggest that CD4 depletion was associated with the accumulation of terminally differentiated T cell phenotypes at the cervical mucosa defective in their ability to produce IL-2. CD4 depletion and compromised immunity at the cervix may be accompanied by progressive decline of central memory-like T cells and development of T cells toward terminally differentiated phenotypes.     

Age related changes in T cell mediated immune response and effector memory to Respiratory Syncytial Virus (RSV) in healthy subjects

Respiratory syncytial virus (RSV) is the major pathogen causing respiratory disease in young infants and it is an important cause of serious illness in the elderly since the infection provides limited immune protection against reinfection. In order to explain this phenomenon, we investigated whether healthy adults of different age (20-40; 41-60 and > 60 years), have differences in central and effector memory, RSV-specific CD8+ T cell memory immune response and regulatory T cell expression status. In the peripheral blood of these donors, we were unable to detect any age related difference in term of central (CD45RA^-CCR7⁺) and effector (CD45RA^-CCR7^-) memory T cell frequency. On the contrary, we found a significant increase in immunosuppressive regulatory (CD4⁺25⁺FoxP3⁺) T cells (T_reg) in the elderly. An immunocytofluorimetric RSV pentamer analysis performed on these donors' peripheral blood mononuclear cells (PBMCs), in vitro sensitized against RSV antigen, revealed a marked decline in long-lasting RSV specific CD8+ memory T cell precursors expressing interleukin 7 receptor α (IL-7Rα), in the elderly. This effect was paralleled by a progressive switch from a Th1 (IFN-γ and TNF-α) to a Th2 (IL-10) functional phenotype. On the contrary, an increase in T_reg was observed with aging. The finding of T_reg over-expression status, a prominent Th2 response and an inefficient RSV-specific effector memory CD8+ T cell expansion in older donors could explain the poor protection against RSV reinfection and the increased risk to develop an RSV-related severe illness in this population. Our finding also lays the basis for new therapeutic perspectives that could limit or prevent severe RSV infection in elderly.