MHC I tetramer staining tends to overestimate the number of functionally relevant self-reactive CD8 T cells in the preimmune repertoire.

Previous studies that used peptide-MHC (pMHC) tetramers (tet) to identify self-specific T cells have questioned the effectiveness of thymic-negative selection. Here, we used pMHCI tet to enumerate CD8 T cells specific for the immunodominant gp33 epitope of lymphocytic choriomeningitis virus glycoprotein (GP) in mice transgenically engineered to express high levels of GP as a self-antigen in the thymus. In GP-transgenic mice (GP+ ), monoclonal P14 TCR+ CD8 T cells that express a GP-specific TCR could not be detected by gp33/Db -tet staining, indicative of their complete intrathymic deletion. By contrast, in the same GP+ mice, substantial numbers of polyclonal CD8 T cells identifiable by gp33/Db -tet were present. The gp33-tet staining profiles of polyclonal T cells from GP+ and GP-negative (GP- ) mice were overlapping, but mean fluorescence intensities were ∼15% lower in cells from GP+ mice. Remarkably, the gp33-tet+ T cells in GP+ mice failed to clonally expand after lymphocytic choriomeningitis virus infection, whereas those of GP- mice did so. In Nur77GFP -reporter mice, dose-dependent responses to gp33 peptide-induced TCR stimulation revealed that gp33-tet+ T cells with high ligand sensitivity are lacking in GP+ mice. Hence, pMHCI tet staining identifies self-specific CD8 T cells but tends to overestimate the number of truly self-reactive cells.

The Inhibitory Receptor NKG2A Sustains Virus-Specific CD8⁺ T Cells in Response to a Lethal Poxvirus Infection.

CD8(+) T cells and NK cells protect from viral infections by killing virally infected cells and secreting interferon-γ. Several inhibitory receptors limit the magnitude and duration of these anti-viral responses. NKG2A, which is encoded by Klrc1, is a lectin-like inhibitory receptor that is expressed as a heterodimer with CD94 on NK cells and activated CD8(+) T cells. Previous studies on the impact of CD94/NKG2A heterodimers on anti-viral responses have yielded contrasting results and the in vivo function of NKG2A remains unclear. Here, we generated Klrc1(-/-) mice and found that NKG2A is selectively required for resistance to ectromelia virus (ECTV). NKG2A functions intrinsically within ECTV-specific CD8(+) T cells to limit excessive activation, prevent apoptosis, and preserve the specific CD8(+) T cell response. Thus, although inhibitory receptors often cause T cell exhaustion and viral spreading during chronic viral infections, NKG2A optimizes CD8(+) T cell responses during an acute poxvirus infection.

Memory vs memory-like: The different facets of CD8+ T-cell memory in HCV infection.

Memory CD8+ T cells are essential in orchestrating protection from re-infection. Hallmarks of virus-specific memory CD8+ T cells are the capacity to mount recall responses with rapid induction of effector cell function and antigen-independent survival. Growing evidence reveals that even chronic infection does not preclude virus-specific CD8+ T-cell memory formation. However, whether this kind of CD8+ T-cell memory that is established during chronic infection is indeed functional and provides protection from re-infection is still unclear. Human chronic hepatitis C virus infection represents a unique model system to study virus-specific CD8+ T-cell memory formation during and after cessation of persisting antigen stimulation.

NK1.1+ innate lymphoid cells in salivary glands inhibit establishment of tissue-resident memory CD8+ T cells in mice.

NK1.1+ cells found in salivary glands (SG) represent a unique cell population of innate lymphoid cells (ILC) with characteristics of both conventional NK cells and ILC1. Here, we demonstrate that these NK1.1+  cells limit the accumulation and differentiation of virus-specific tissue-resident memory CD8+ T cells (TRM  cells) in SG of mice infected with lymphocytic choriomeningitis virus (LCMV). The negative regulation of LCMV-specific CD8+ TRM  cells by NK1.1+  cells in SG is independent of NKG2D, NKp46, TRAIL, and perforin. Moreover, analysis of NKp46iCre+ Eomesfl/fl mice revealed that Eomes-dependent conventional NK cells are dispensable for negative regulation. Since the SG are prone to autoimmune reactions, regulation of TRM  cells by tissue-resident ILC may be particularly important to prevent immunopathology in this organ.

Trigger-dependent differences determine therapeutic outcome in murine primary hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is a hyperinflammatory syndrome affecting patients with genetic cytotoxicity defects. Perforin-deficient (PKO) mice recapitulate the full clinical picture of FHL after infection with lymphocytic choriomeningitis virus (LCMV). Hyperactivated CD8+ T cells and IFN-γ have been identified as the key drivers of FHL and represent targets for therapeutic interventions. However, the response of patients is variable. This could be due to trigger-dependent differences in pathogenesis, which is difficult to address in FHL patients, since the trigger frequently escapes detection. We established an alternative FHL model using intravenous infection of PKO mice with murine CMV (MCMV)Smith . PKO mice developed acute FHL after both infections and fulfilled HLH diagnostic criteria accompanied by excessive IFN-γ production by disease-inducing T cells, that enrich in the BM. However, direct comparison of the two infection models disclosed trigger-dependence of FHL progression and revealed a higher contribution of CD4 T cells and NK cells to IFN-γ production after MCMV infection. Importantly, therapeutic intervention by IFN-γ neutralization or CD8+ T-cell depletion had less benefit in MCMV-triggered FHL compared to LCMV-triggered FHL, likely due to MCMV-induced cytopathology. Thus, the context of the specific triggering viral infection can impact the success of targeted immunotherapeutic HLH control.

Enhancing immunity prevents virus-induced T-cell-mediated immunopathology in B cell-deficient mice.

Hyper-activated or deviated immune responses can result in immunopathological diseases. Paradoxically, immunodeficiency represents a frequent cause of such immune-mediated pathologies. Immunopathological manifestations are commonly treated by immunosuppression, but in situations in which immunodeficiency is the basis of disease development, enhancing immunity may represent an alternative treatment option. Here, we tested this counterintuitive concept in a preclinical model using infection of mice with lymphocytic choriomeningitis virus (LCMV). Firstly, we demonstrate that infection of B-cell-deficient (B-/- ) but not of wild-type (WT) mice with the LCMV strain Docile induced a rapid and fatal CD8+ T-cell-mediated immunopathological disease. Similar to WT mice, LCMV-infected B-/- mice generated a potent, functional LCMV-specific CD8+ T-cell response but exhibited prolonged viral antigen presentation and increased vascular leakage in liver and lungs. Secondly, we were able to prevent this virus-induced immunopathology in B-/- mice by active or passive T-cell immunizations or by treatment with LCMV-specific virus neutralizing or non-neutralizing monoclonal antibodies (mAb). Thus, boosting antiviral immunity did not aggravate immunopathology in this model, but prevented it by decreasing the formation of target structures for damage-causing CD8+ T cells.

Residual LCMV antigen in transiently CD4+ T cell-depleted mice induces high levels of virus-specific antibodies but only limited B-cell memory.

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8+ T cells independently of CD4+ T cell help. Residual viral antigen may, however, persist for a prolonged time. Here, we demonstrate that mice that had been transiently depleted of CD4+ T cells during acute LCMV Arm infection generated high levels of virus-specific IgG antibodies (Ab) after viral clearance. Robust induction of LCMV-specific IgG after transient CD4+ T cell depletion was dependent on Fcγ receptors but not on the complement receptors CD21/CD35. In contrast to the potent production of LCMV-specific IgG, the generation of LCMV-specific isotype-switched memory B cells after transient CD4+ T cell depletion was considerably reduced. Moreover, mice depleted of CD4+ T cells during acute infection were strongly impaired in generating a secondary LCMV-specific B cell response upon LCMV rechallenge. In conclusion, our data indicate that LCMV antigen depots after viral clearance were capable of inducing high levels of virus-specific IgG. They failed, however, to induce robust virus-specific B cell memory revealing a previously unappreciated dichotomy of specific Ab production and memory cell formation after priming with residual antigen.

Expression of IL-7Rα and KLRG1 defines functionally distinct CD8+ T-cell populations in humans.

During acute viral infections in mice, IL-7Rα and KLRG1 together are used to distinguish the short-lived effector cells (SLEC; IL-7Rαlo KLRGhi ) from the precursors of persisting memory cells (MPEC; IL-7Rαhi KLRG1lo ). We here show that these markers can be used to define distinct subsets in the circulation and lymph nodes during the acute phase and in "steady state" in humans. In contrast to the T cells in the circulation, T cells derived from lymph nodes hardly contain any KLRG1-expressing cells. The four populations defined by IL-7Rα and KLRG1 differ markedly in transcription factor, granzyme and chemokine receptor expression. When studying renal transplant recipients experiencing a primary hCMV and EBV infection, we also found that after viral control, during latency, Ki-67-negative SLEC can be found in the peripheral blood in considerable numbers. Thus, combined analyses of IL-7Rα and KLRG1 expression on human herpes virus-specific CD8+ T cells can be used to separate functionally distinct subsets in humans. As a noncycling IL-7Rαlo KLRG1hi population is abundant in healthy humans, we conclude that this combination of markers not only defines short-lived effector cells during the acute response but also stable effector cells that are formed and remain present during latent herpes infections.

NK-cell responses are biased towards CD16-mediated effector functions in chronic hepatitis B virus infection.

BACKGROUND & AIMS: Phenotypic and functional natural killer (NK)-cell alterations are well described in chronic hepatitis B virus (cHBV) infection. However, it is largely unknown whether these alterations result from general effects on the overall NK-cell population or the emergence of distinct NK-cell subsets. Human cytomegalovirus (HCMV) is common in cHBV and is associated with the emergence of memory-like NK cells. We aimed to assess the impact of these cells on cHBV infection. METHODS: To assess the impact of memory-like NK cells on phenotypic and functional alterations in cHBV infection, we performed in-depth analyses of circulating NK cells in 52 patients with cHBV, 45 with chronic hepatitis C virus infection and 50 healthy donors, with respect to their HCMV serostatus. RESULTS: In patients with cHBV/HCMV+, FcεRIγ- memory-like NK cells were present in higher frequencies and with higher prevalence than in healthy donors with HCMV+. This pronounced HCMV-associated memory-like NK-cell expansion could be identified as key determinant of the NK-cell response in cHBV infection. Furthermore, we observed that memory-like NK cells consist of epigenetically distinct subsets and exhibit key metabolic characteristics of long-living cells. Despite ongoing chronic infection, the phenotype of memory-like NK cells was conserved in patients with cHBV/HCMV+. Functional characteristics of memory-like NK cells also remained largely unaffected by cHBV infection with the exception of an increased degranulation capacity in response to CD16 stimulation that was, however, detectable in both memory-like and conventional NK cells. CONCLUSIONS: The emergence of HCMV-associated memory-like NK cells shapes the overall NK-cell response in cHBV infection and contributes to a general shift towards CD16-mediated effector functions. Therefore, HCMV coinfection needs to be considered in the design of immunotherapeutic approaches that target NK cells in cHBV. LAY SUMMARY: In chronic hepatitis B virus infection, natural killer (NK)-cell phenotype and function is altered. In this study, we demonstrate that these changes are linked to the emergence of a distinct NK-cell subset, namely memory-like NK cells. The emergence of these memory-like NK cells is associated with coinfection of human cytomegalovirus that affects the majority of patients with chronic hepatitis B.

Immunological tolerance to LCMV antigens differently affects control of acute and chronic virus infection in mice.

Cytotoxic T lymphocytes (CTLs) play a key role in the control of lymphocytic choriomeningitis virus (LCMV) infection. In C57BL/6 mice (H-2b ), the CTL response is mainly directed against epitopes from the LCMV glycoprotein (GP) and the nucleoprotein (NP) which represent the two major viral proteins. The role of GP- versus NP-derived epitopes for viral clearance was examined using transgenic (tg) mice ubiquitously expressing LCMV GP and NP, respectively. These mice lack GP- or NP-specific CTLs and show decreased levels of GP- or NP-specific antibodies as a result of tolerance induction. During acute LCMV infection, CTLs specific for GP- and NP-derived epitopes are generated with similar frequencies. Nonetheless, we found that lack of GP- but not of NP-specific CTLs abolished control of acute LCMV infection. In contrast, after high-dose or chronic LCMV infection, virus elimination was delayed to a similar extent in GP- and NP-tg mice. Thus, immunological tolerance to LCMV antigens differently affects virus clearance in acute and chronic infection settings. In addition, our data reveal that immunodominance of H-2b -restricted LCMV-specific CTL epitopes and their antiviral activity do not strictly correlate.

α4 ß1 integrin promotes accumulation of tissue-resident memory CD8+ T cells in salivary glands.

The salivary glands (SGs) of virus-immune mice contain substantial numbers of tissue-resident memory CD8+ T cells (TRM cells) that can provide immunity to local infections. Integrins regulate entry of activated T cells into nonlymphoid tissues but the molecules that mediate migration of virus-specific CD8+ T cells to the SGs have not yet been defined. Here, we found that polyinosinic-polycytidylic acid (poly(I:C)) strongly promoted the accumulation of P14 TCR-transgenic CD8+ TRM cells in SGs in an α4 ß1 integrin-dependent manner. After infection with lymphocytic choriomeningitis virus, accumulation of P14 TRM cells in SGs and intestine but not in kidney was also α4 integrin dependent. Blockade of α4 ß7 by monoclonal antibodies (mAbs) inhibited lymphocytic choriomeningitis virus-induced accumulation of P14 TRM cells in the intestine but not in SGs. In conclusion, our data reveal that α4 ß1 integrin mediates CD8+ TRM accumulation in SGs and that poly(I:C) can be used to direct activated CD8+ T cells to this organ.

CMV drives the expansion of highly functional memory T cells expressing NK-cell receptors in renal transplant recipients.

Cytomegalovirus (CMV) is a common opportunistic infection encountered in renal transplant recipients (RTRs) and may be reactivated without symptoms at any time post-transplant. We describe how active and latent CMV affect T-cell subsets in RTRs who are stable on maintenance therapy. T-cell responses to CMV were assessed in RTRs (n = 54) >2 years post-transplant, and healthy controls (n = 38). Seven RTRs had CMV DNA detectable in plasma. CMV antibody and DNA aligned with increased proportions of CD8+ T cells and reduced CD4/CD8 ratios. This paralleled an expansion of effector memory T-cell (TEM ), terminally differentiated T-cell (TEMRA ) and CD57+ TEMRA cell populations. Expression of NK-cell receptors, LIR-1 and KLRG1 on CD4+ and CD8+ CD57+ TEM and TEMRA cells correlated with elevated interferon-γ and cytotoxic responses to anti-CD3 and increased cytotoxic responses to CMV phosphoprotein (pp) 65 in RTRs who carried CMV DNA. CD8+ T cells from all CMV seropositive RTRs responded efficiently to CMV immediate early (IE) -1 peptides. The data show that latent and active CMV infection can alter T-cell subsets in RTRs many years after transplantation, and up-regulate T-cell expression of NK-cell receptors. This may enhance effector responses of CD4+ and CD8+ T cells against CMV.

Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.

The cytolytic activity of natural killer (NK) cells is regulated by inhibitory receptors that detect the absence of self molecules on target cells. Structural studies of missing self recognition have focused on NK receptors that bind MHC. However, NK cells also possess inhibitory receptors specific for non-MHC ligands, notably cadherins, which are downregulated in metastatic tumors. We determined the structure of killer cell lectin-like receptor G1 (KLRG1) in complex with E-cadherin. KLRG1 mediates missing self recognition by binding to a highly conserved site on classical cadherins, enabling it to monitor expression of several cadherins (E-, N-, and R-) on target cells. This site overlaps the site responsible for cell-cell adhesion but is distinct from the integrin alpha(E)beta(7) binding site. We propose that E-cadherin may coengage KLRG1 and alpha(E)beta(7) and that KLRG1 overcomes its exceptionally weak affinity for cadherins through multipoint attachment to target cells, resulting in inhibitory signaling.

KLRG1 impairs regulatory T-cell competitive fitness in the gut.

Immune homeostasis requires the tight, tissue-specific control of the different CD4+ Foxp3+ regulatory T (Treg) cell populations. The cadherin-binding inhibitory receptor killer cell lectin-like receptor G1 (KLRG1) is expressed by a subpopulation of Treg cells with GATA3+ effector phenotype. Although such Treg cells are important for the immune balance, especially in the gut, the role of KLRG1 in Treg cells has not been assessed. Using KLRG1 knockout mice, we found that KLRG1 deficiency does not affect Treg cell frequencies in spleen, mesenteric lymph nodes or intestine, or frequencies of GATA3+ Treg cells in the gut. KLRG1-deficient Treg cells were also protective in a T-cell transfer model of colitis. Hence, KLRG1 is not essential for the development or activity of the general Treg cell population. We then checked the effects of KLRG1 on Treg cell activation. In line with KLRG1's reported inhibitory activity, in vitro KLRG1 cross-linking dampened the Treg cell T-cell receptor response. Consistently, lack of KLRG1 on Treg cells conferred on them a competitive advantage in the gut, but not in lymphoid organs. Hence, although absence of KLRG1 is not enough to increase intestinal Treg cells in KLRG1 knockout mice, KLRG1 ligation reduces T-cell receptor signals and the competitive fitness of individual Treg cells in the intestine.

Myocardial ischemia and reperfusion leads to transient CD8 immune deficiency and accelerated immunosenescence in CMV-seropositive patients.

RATIONALE: There is mounting evidence of a higher incidence of coronary heart disease in cytomegalovirus-seropositive individuals. OBJECTIVE: The aim of this study was to investigate whether acute myocardial infarction triggers an inflammatory T-cell response that might lead to accelerated immunosenescence in cytomegalovirus-seropositive patients. METHODS AND RESULTS: Thirty-four patients with acute myocardial infarction undergoing primary percutaneous coronary intervention were longitudinally studied within 3 months after reperfusion (Cohort A). In addition, 54 patients with acute myocardial infarction and chronic myocardial infarction were analyzed in a cross-sectional study (Cohort B). Cytomegalovirus-seropositive patients demonstrated a greater fall in the concentration of terminally differentiated CD8 effector memory T cells (TEMRA) in peripheral blood during the first 30 minutes of reperfusion compared with cytomegalovirus-seronegative patients (-192 versus -63 cells/µL; P=0.008), correlating with the expression of programmed cell death-1 before primary percutaneous coronary intervention (r=0.8; P=0.0002). A significant proportion of TEMRA cells remained depleted for ≥3 months in cytomegalovirus-seropositive patients. Using high-throughput 13-parameter flow cytometry and human leukocyte antigen class I cytomegalovirus-specific dextramers, we confirmed an acute and persistent depletion of terminally differentiated TEMRA and cytomegalovirus-specific CD8(+) cells in cytomegalovirus-seropositive patients. Long-term reconstitution of the TEMRA pool in chronic cytomegalovirus-seropositive postmyocardial infarction patients was associated with signs of terminal differentiation including an increase in killer cell lectin-like receptor subfamily G member 1 and shorter telomere length in CD8(+) T cells (2225 versus 3397 bp; P<0.001). CONCLUSIONS: Myocardial ischemia and reperfusion in cytomegalovirus-seropositive patients undergoing primary percutaneous coronary intervention leads to acute loss of antigen-specific, terminally differentiated CD8 T cells, possibly through programmed cell death-1-dependent programmed cell death. Our results suggest that acute myocardial infarction and reperfusion accelerate immunosenescence in cytomegalovirus-seropositive patients.

Id3 Controls Cell Death of 2B4+ Virus-Specific CD8+ T Cells in Chronic Viral Infection.

Sustained Ag persistence in chronic infection results in a deregulated CD8(+) T cell response that is characterized by T cell exhaustion and cell death of Ag-specific CD8(+) T cells. Yet, the underlying transcriptional mechanisms regulating CD8(+) T cell exhaustion and cell death are poorly defined. Using the experimental mouse model of lymphocytic choriomeningitis virus infection, we demonstrate that the transcriptional regulator Id3 controls cell death of virus-specific CD8(+) T cells in chronic infection. By comparing acute and chronic infection, we showed that Id3 (-) virus-specific CD8(+) T cells were less abundant, whereas the absolute numbers of Id3 (+) virus-specific CD8(+) T cells were equal in chronic and acute infection. Phenotypically, Id3 (-) and Id3 (+) cells most prominently differed with regard to expression of the surface receptor 2B4; although Id3 (-) cells were 2B4(+), almost all Id3 (+) cells lacked expression of 2B4. Lineage-tracing experiments showed that cells initially expressing Id3 differentiated into Id3 (-)2B4(+) cells; in turn, these cells were terminally differentiated and highly susceptible to cell death under conditions of persisting Ag. Enforced Id3 expression specifically increased the persistence of 2B4(+) virus-specific CD8(+) T cells by decreasing susceptibility to Fas/Fas ligand-mediated cell death. Thus, our findings reveal that the transcriptional regulator Id3 promotes the survival of virus-specific CD8(+) T cells in chronic infection and suggest that targeting Id3 might be beneficial for preventing cell death of CD8(+) T cells in chronic infection or for promoting cell death of uncontrolled, hyperactive CD8(+) T cells to prevent immunopathology.

T cell expansion is the limiting factor of virus control in mice with attenuated TCR signaling: implications for human immunodeficiency.

Defining the minimal thresholds for effective antiviral T cell immunity is important for clinical decisions in immunodeficient patients. TCR signaling is critical for T cell development, activation, and effector functions. In this article, we analyzed which of these TCR-mediated processes is limiting for antiviral immunity in a mouse strain with reduced expression of SLP-76 (twp mice). Despite severe T cell activation defects in vitro, twp mice generated a normal proportion of antiviral effector T cells postinfection with lymphocytic choriomeningitis virus (LCMV). Twp CD8(+) T cells showed impaired polyfunctional cytokine production, whereas cytotoxicity as the crucial antiviral effector function for LCMV control was normal. The main limiting factor in the antiviral response of twp mice was impaired T cell proliferation and survival, leading to a 5- to 10-fold reduction of antiviral T cells at the peak of the immune response. This was still sufficient to control infection with the LCMV Armstrong strain, but the more rapidly replicating LCMV-WE induced T cell exhaustion and viral persistence. Thus, under conditions of impaired TCR signaling, reduced T cell expansion was the limiting factor in antiviral immunity. These findings have implications for understanding antiviral immunity in patients with T cell deficiencies.

TGF-ß downregulates KLRG1 expression in mouse and human CD8(+) T cells.

The inhibitory receptor killer cell lectin-like receptor G1 (KLRG1) and the integrin αE (CD103) are expressed by CD8(+) T cells and both are specific for E-cadherin. However, KLRG1 ligation by E-cadherin inhibits effector T-cell function, whereas binding of CD103 to E-cadherin enhances cell-cell interaction and promotes target cell lysis. Here, we demonstrate that KLRG1 and CD103 expression in CD8(+) T cells from untreated and virus-infected mice are mutually exclusive. Inverse correlation of KLRG1 and CD103 expression was also found in human CD8(+) T cells-infiltrating hepatocellular carcinomas. As TGF-ß is known to induce CD103 expression in CD8(+) T cells, we examined whether this cytokine also regulates KLRG1 expression. Indeed, our data further reveal that TGF-ß signaling in mouse as well as in human CD8(+) T cells downregulates KLRG1 expression. This finding provides a rationale for the reciprocal expression of KLRG1 and CD103 in different CD8(+) T-cell subsets. In addition, it points to the limitation of KLRG1 as a marker for terminally differentiated CD8(+) T cells if lymphocytes from tissues expressing high levels of TGF-ß are analyzed.

Infection history determines the differentiation state of human CD8+ T cells.

UNLABELLED: After the resolution of the acute phase of infection, otherwise quiescent antigen-experienced CD8(+) T cells confer rapid protection upon reinfection with viral pathogens or, in the case of persistent viruses, help to maintain control of the infection. Depending on the type of virus, antigen-specific CD8(+) T cells have distinct traits, ranging from typical memory cell properties in the case of rapidly cleared viruses to immediate effector functions for persistent viruses. We here show that both the differentiation stage, defined by the expression of cell surface markers, such as CD45RA, CCR7, CD28, and CD27, and distinct expression levels of T-bet and eomesodermin (Eomes) predict the functional profile of antigen-experienced CD8(+) T cells. Furthermore, virus-specific CD8(+) T cells targeting different respiratory syncytial virus-, influenza A virus-, Epstein-Barr virus (EBV)-, human cytomegalovirus (hCMV)-, and HIV-1-specific epitopes adopt distinct T-bet and Eomes expression patterns that appear to be installed early during the primary response. Importantly, the associations between surface phenotype, T-bet/Eomes expression levels, and the expression of markers that predict CD8(+) T-cell function change according to viral infection history, particularly against the background of HIV-1 and, to lesser extent, of human cytomegalovirus and/or Epstein-Barr virus infection. Thus, the functionality of human antigen-experienced CD8(+) T cells follows at least two dimensions, one outlined by the surface phenotype and another by the T-bet/Eomes expression levels, which are determined by previous or persistent viral challenges. IMPORTANCE: Functional human CD8(+) T-cell subsets have been defined using surface markers like CD45RA, CCR7, CD28, and CD27. However, the induction of function-defining traits, like granzyme B expression, is controlled by transcription factors like T-bet and Eomes. Here, we describe how T-bet and Eomes levels distinctly relate to the expression of molecules predictive for CD8(+) T-cell function in a surface phenotype-independent manner. Importantly, we found that central memory and effector memory CD8(+) T-cell subsets differentially express T-bet, Eomes, and molecules predictive for function according to viral infection history, particularly so in the context of HIV-1 infection and, to lesser extent, of latent EBV- and/or hCMV-infected, otherwise healthy adults. Finally, we show that the distinct phenotypes and T-bet/Eomes levels of different virus-specific CD8(+) T-cell populations are imprinted early during the acute phase of primary infection in vivo. These findings broaden our understanding of CD8(+) T-cell differentiation.

Clonal evolution of CD8+ T cell responses against latent viruses: relationship among phenotype, localization, and function.

UNLABELLED: Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, interleukin-7 receptor α (IL-7Rα)-expressing cells contain the precursors for long-lived antigen-experienced CD8(+) T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here, we studied whether IL-7Rα-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool. Using flow cytometry and functional assays, we found that the IL-7Rα(+) hCMV-specific T cell population comprises cells that have a memory phenotype and lack effector features. We used next-generation sequencing of the T cell receptor to compare the clonal repertoires of IL-7Rα(+) and IL-7Rα(-) subsets. We observed limited overlap of clones between these subsets during acute infection and after 1 year. When we compared the hCMV-specific repertoire between PB and paired LNs, we found many identical clones but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only rarely identical to the unique LN clones. Thus, although PB IL-7Rα-expressing and LN hCMV-specific CD8(+) T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8(+) T cell pool during latency or upon antigen recall. IL-7Rα(+) PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8(+) effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naive CD8(+) T cell pool. IMPORTANCE: Insight into the self-renewal properties of long-lived memory CD8(+) T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is, however, not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: circulating acute-phase IL-7Rα-expressing hCMV-specific CD8(+) T cells and lymph node (LN)-residing hCMV-specific (central) memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation seldom originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues or are recruited from the naive CD8(+) T cell pool.