Contribution of 4–1BBL on radioresistant cells in providing survival signals through 4–1BB expressed on CD8+ memory T cells in the bone marrow

The persistence of memory lymphocytes is a critical feature of adaptive immunity. The TNF family ligand 4–1BBL supports the antigen‐independent survival of CD8+ memory T cells. Here, we show that mice lacking 4–1BB only on αβ T cells show a similar defect in CD8+ T‐cell recall responses, as previously shown in 4–1BBL‐deficient mice. We show that 4–1BB is selectively expressed on BM CD8+ but not CD4 + memory T cells of unimmunized mice. Its ligand, 4–1BBL, is found on VCAM‐1+ stromal cells, CD11c+ cells, and a Gr1^lo myeloid population in unimmunized mice. Adoptive transfer of in vitro generated memory T cells into mice lacking 4–1BBL only on radioresistant cells recapitulates the defect in CD8 + T‐cell survival seen in the complete knockout mice, with smaller effects of 4–1BBL on hematopoietic cells. In BM, adoptively transferred DsRed CD8+ memory T cells are most often found in proximity to VCAM‐1+ cells or Gr1+ cells, followed by B220+ cells and to a much lesser extent near CD11c+ cells. Thus, a VCAM‐1+CD45^? stromal cell is a plausible candidate for the radioresistant cell that provides 4–1BBL to CD8+ memory T cells in the BM.     

Adoptive transfer of cytomegalovirus‐specific effector CD4+ T cells provides antiviral protection from murine CMV infection

Cytomegalovirus (CMV) infects a majority of the human population and establishes a life‐long persistence. CMV infection is usually asymptomatic but the virus carries pathogenic potential and causes severe disease in immunocompromised individuals. T‐cell‐mediated immunity plays an essential role in control of CMV infection and adoptive transfer of CMV‐specific CD8⁺ T cells restores viral immunity in immunosuppressed patients but a role for CD4⁺ T cells remains elusive. Here, we analyzed in adoptive transfer studies the features and antiviral functions of virus‐specific CD4⁺ T cells during primary murine CMV (MCMV) infection. MCMV‐specific CD4⁺ T cells expanded upon MCMV infection and displayed an effector phenotype and function. Adoptive transfer of in vivo activated MCMV‐specific CD4⁺ T cells to immune‐compromised mice was protective during pathogenic MCMV infection and IFN‐γ was a crucial mediator of this protective capacity. Moreover, co‐transfer of low doses of both MCMV‐specific CD4⁺ T cells and CD8⁺ T cells synergized in control of lytic viral replication in immune‐compromised mice. Our data reveal a pivotal antiviral role for virus‐specific CD4⁺ T cells in protection from pathogenic CMV infection and provide evidence for their antiviral therapeutic potential.     

Increasing inflationary T‐cell responses following transient depletion of MCMV‐specific memory T cells

Murine CMV (MCMV) infection induces effector CD8⁺ T cells that continue to increase in frequency after acute infection (“inflation”) and are stably maintained at a high frequency, with up to 20% of the CD8⁺ T‐cell compartment being specific for one epitope, although the flexibility and turnover of these populations is not fully defined. Here we report that effector/memory CD8⁺ T cells induced by MCMV can be paradoxically boosted following transient depletion of epitope specific CD8⁺ T cells. Treatment of MCMV‐infected mice with MHC‐Class I‐saporin tetramers led to partial (80–90%) depletion of epitope‐specific CD8⁺ T cells—rapidly followed by a rebound, leading to expansion and maintenance of up to 40% of total CD8⁺ T cells, with minimal changes in response to a control epitope (M45). These data indicate the tight balance between host and virus during persistent infection and the functional flexibility of the “inflated” CD8⁺ T cell responses during persistent infection.     

c‐IAP ubiquitin protein ligase activity is required for 4‐1BB signaling and CD8+ memory T‐cell survival

Cellular inhibitor of apoptosis proteins (c‐IAP) 1 and 2 are widely expressed ubiquitin protein ligases that regulate a variety of cellular functions, including the sensitivity of T cells to costimulation. 4‐1BB is a TNF receptor family member that signals via a complex that includes TRAF family members and the c‐IAPs to upregulate NF‐κB and ERK, and has been implicated in memory T‐cell survival. Here, we show that effector and memory T cells from mice expressing a dominant negative E3‐inactive c‐IAP2 (c‐IAP2^H570A) have impaired signaling downstream of 4‐1BB. When infected with lymphocytic choriomeningitis virus, unlike mice in which c‐IAPs were acutely downregulated by c‐IAP antagonists, the primary response of c‐IAP2^H570A mice was normal. However, the number of antigen‐specific CD8⁺ but not CD4⁺ T cells declined more rapidly and to a greater extent in c‐IAP2^H570A mice than in WT controls. Studies with T‐cell adoptive transfer demonstrated that the enhanced decay of memory cells was T‐cell intrinsic. Thus, c‐IAP E3 activity is required for 4‐1BB coreceptor signaling and maintenance of CD8⁺ T‐cell memory.     

Dispensable role for 4-1BB and 4-1BBL in development of vaccinia virus-specific CD8 T cells

CD8 T cells are strongly induced in response to certain strains of vaccinia virus (VACV) and the generation of this population is tightly regulated by two Tumor Necrosis Factor (TNF)/TNFR superfamily members, OX40 (CD134) and CD27. In this study, we examined the role of another member of the TNFR superfamily, 4-1BB (CD137, TNFRSF9), and its ligand (4-1BBL, CD137L, TNFSF9), that have been described to control the generation of memory CD8 T cell populations elicited by other viruses such as influenza. Expression of 4-1BB and 4-1BBL was observed in wild-type mice during the primary infection, but we found that both 4-1BB and 4-1BBL deficient mice generated normal numbers of VACV-specific effector CD8 T cells that produced IFN-γ and TNF. Additionally, CD8 T cells deficient in 4-1BB were able to expand and persist comparably to wild-type T cells in response to VACV infection. Furthermore, the knockout mice also showed no defect in development of VACV-specific CD8 memory T cell populations. Lastly, showing alternate control mechanisms were not active in the gene-deficient environments that masked any activity, blocking 4-1BB/4-1BBL interactions using neutralizing antibody also had no effect on the number of VACV-specific memory CD8 T cells induced. Thus, our data demonstrate that 4-1BB and 4-1BBL do not play a strong or dominant role in driving the generation of high frequencies of VACV-specific CD8 T cells.     

Salivary gland resident APCs are Flt3L‐ and CCR2‐independent macrophage‐like cells incapable of cross‐presentation

Cytomegaloviruses (CMVs) disseminate within the human population via mucosal excretions, for example, from the salivary glands (SGs), which represent a privileged site of viral immune evasion and persistence. The murine CMV (MCMV) model has served to identify factors that maintain a unique virus–host relationship in this organ. In contrast to all other organs, the SG is resistant to CD8⁺ T‐cell mediated control of MCMV replication due to virally induced MHC class I downregulation, which is exceptionally efficient in acinar glandular epithelial cells. Uniquely to the SG, IFN‐γ producing CD4⁺ T cells are required for virus control. While T‐cell responses have been extensively characterized in the SG, the ontogeny and function of APCs in this organ remain to be assessed. Here, we show that macrophage‐like cells constitute the population of SG‐resident APCs in steady state and during MCMV‐induced inflammation in mice. Inflammatory monocytes, monocyte‐derived DCs as well as conventional, Flt3L‐dependent DCs do not contribute to this population. Despite supporting contact formation to CD4⁺ and CD8⁺ T cells in principle, SG‐resident APCs fail to activate the latter due to their inability to cross‐present MCMV‐derived antigen.     

Cytomegalovirus‐seropositivity has a profound influence on the magnitude of major lymphoid subsets within healthy individuals

Cytomegalovirus (CMV) infects most individuals and elicits a strong CMV‐specific immune response. We have studied the influence of CMV‐seropositivity on the size of lymphoid subsets in healthy donors and demonstrate that the virus substantially modulates the peripheral lymphoid pool. CD8⁺ T cell numbers are increased in all CMV‐seropositive individuals because of a striking 60% increment in the CD8⁺ T cell memory pool. The CD45RA⁺ resting memory pool is doubled after CMV infection and increases further with age. The magnitude of the naïve CD8⁺ T cell pool is dramatically reduced in CMV‐seropositive individuals at all ages, and this accelerates the physiological decline by approximately 40 years. The number of CD4⁺ effector memory T cells is increased in CMV‐seropositive individuals and is differentially accommodated by a reduction in the number of naïve and central memory CD4⁺ T cells in young and elderly donors respectively. CMV‐seropositivity also increases the total number of B cells in older donors and suppresses the number of CD5⁺ B cells. These data reveal that CMV has a profound influence on the immune system of all healthy individuals and add to growing concern regarding the clinical and immunomodulatory significance of CMV infection in healthy donors.     

Strong and sustained effector function of memory‐ versus naïve‐derived T cells upon T‐cell receptor RNA transfer: Implications for cellular therapy

Current protocols used to select CMV‐specific T cells for adoptive immunotherapy focus on virus‐specific memory T cells from seropositive donors. However, this strategy is not feasible in patients undergoing allogeneic haematopoietic stem‐cell transplantation (HSCT) from CMV‐seronegative donors. Here, we redirected T cells of CMV‐seronegative donors with a human genetically engineered TCR recognizing an HLA‐A*0201‐binding peptide epitope of CMVpp65. To facilitate clinical translation of this approach, we used a non‐viral expression system based on in vitro transcribed RNA and electroporation. Although memory and naïve‐derived T‐cell subsets were both efficiently transfected by TCR‐RNA, memory‐derived T cells showed much stronger levels of HLA‐A*0201‐restricted cytolytic activity to CMV‐infected fibroblasts and maintained acquired function for 5–10 days. In addition to redirection of CD8⁺ cytotoxic T cells, TCR‐RNA transfection was capable of redirecting CD4⁺ T cells into potent Ag‐specific Th cells that efficiently triggered maturation of DCs. Our data suggest that memory rather than naïve‐derived T cells are the preferred subset for transient TCR expression by RNA electroporation, providing more efficient and sustained virus‐specific CD4⁺ and CD8⁺ T‐cell function. CMV TCR‐RNA may represent a suitable therapeutic ‘off‐the‐shelf’ reagent to be used in severe CMV infections of HSCT patients when endogenous CMV‐specific T‐cell immunity is insufficient.     

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 (T_EM), terminally differentiated T‐cell (T_EMRA) and CD57⁺ T_EMRA cell populations. Expression of NK‐cell receptors, LIR‐1 and KLRG1 on CD4⁺ and CD8⁺ CD57⁺ T_EM and T_EMRA 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.     

Effector T‐cell differentiation during viral and bacterial infections: Role of direct IL‐12 signals for cell fate decision of CD8+ T cells

To study the role of IL‐12 as a third signal for T‐cell activation and differentiation in vivo, direct IL‐12 signaling to CD8⁺ T cells was analyzed in bacterial and viral infections using the P14 T‐cell adoptive transfer model with CD8⁺ T cells that lack the IL‐12 receptor. Results indicate that CD8⁺ T cells deficient in IL‐12 signaling were impaired in clonal expansion after Listeria monocytogenes infection but not after infection with lymphocytic choriomeningitis virus, vaccinia virus or vesicular stomatitis virus. Although limited in clonal expansion after Listeria infection, CD8⁺ T cells deficient in IL‐12 signaling exhibited normal degranulation activity, cytolytic functions, and secretion of IFN‐γ and TNF‐α. However, CD8⁺ T cells lacking IL‐12 signaling failed to up‐regulate KLRG1 and to down‐regulate CD127 in the context of Listeria but not viral infections. Thus, direct IL‐12 signaling to CD8⁺ T cells determines the cell fate decision between short‐lived effector cells and memory precursor effector cells, which is dependent on pathogen‐induced local cytokine milieu.     

Mice deficient in stem cell antigen‐1 (Sca1, Ly‐6A/E) develop normal primary and memory CD4+ and CD8+ T‐cell responses to virus infection

Stem cell antigen‐1 (Sca1, Ly‐6A/E) is a well‐established marker of murine hematopoietic stem cells, and also is expressed on memory T cells. It has been suggested that the functional maintenance of T‐cell memory requires the expression of Sca1 on a specialized population of memory T cells termed “memory stem cells”. Here, we evaluate the requirement for Sca1 in the primary T‐cell response to virus infection, and in the establishment and maintenance of T‐cell memory. We find that Sca1 expression increases on almost all CD4⁺ and CD8⁺ T cells during virus infection, and remains high on virus‐specific memory cells. However, Sca1‐deficient (Sca1KO) mice generate normal primary T‐cell responses to infection; the kinetics, the immunodominance hierarchy, and the absolute numbers of CD4⁺ and CD8⁺ T cells are essentially indistinguishable from those observed in WT mice. Furthermore, by several criteria, primary and memory T cells in Sca1‐deficient mice are phenotypically and functionally normal. These data indicate that Sca1, although perhaps a useful marker of virus‐specific memory T cells, is not required for the regulation of T‐cell quantity or quality, or for the development of a competent pool of memory cells.     

Viral inoculum dose impacts memory T‐cell inflation

Memory T‐cell inflation develops during certain persistent viral infections and is characterized by the accumulation and maintenance of large numbers of effector‐memory T cells, albeit with varying degrees in size and phenotype among infected hosts. The underlying mechanisms that control memory T‐cell inflation are not yet fully understood. Here, we dissected CMV‐specific memory T‐cell formation and its connection to the initial infectious dose by varying the inoculum size. After low dose inoculum with mouse CMV, the accumulation of inflationary memory T cells was severely hampered and correlated with reduced reservoirs of latent virus in nonhematopoietic cells and diminished antigen‐driven T‐cell proliferation. Moreover, lowering of the initial viral dose turned the characteristic effector memory‐like inflationary T cells into more central memory‐like cells as evidenced by the cell‐surface phenotype of CD27^high, CD62L⁺, CD127⁺, and KLRG1^?, and by improved secondary expansion potential. These data show the impact of the viral inoculum on the degree of memory T‐cell inflation and provide a rationale for the observed variation of human CMV‐specific T‐cell responses in terms of magnitude and phenotype.     

Intrinsic 4-1BB signals are indispensable for the establishment of an influenza-specific tissue-resident memory CD8 T-cell population in the lung

The induction of long-lived heterotypic T-cell protection against influenza virus remains elusive, despite the conservation of T-cell epitopes. T-cell protection against influenza is critically dependent on lung-resident memory T cells (Trm). Here we show that intranasal administration of 4-1BBL along with influenza nucleoprotein in a replication-defective adenovirus vector to influenza pre-immune mice induces a remarkably stable circulating effector memory CD8 T-cell population characterized by higher IL-7Rα expression than control-boosted T cells, as well as a substantial lung parenchymal CD69⁺ CD8 Trm population, including both CD103⁺ and CD103⁻ cells. These T-cell responses persist to greater than 200 days post-boost and protect against lethal influenza challenge in aged (year old) mice. The expansion of the nucleoprotein-specific CD8 Trm population during boosting involves recruitment of circulating antigen-specific cells and is critically dependent on local rather than systemic administration of 4-1BBL as well as on 4-1BB on the CD8 T cells. Moreover, during primary influenza infection of mixed bone marrow chimeras, 4-1BB-deficient T cells fail to contribute to the lung-resident Trm population. These findings establish both endogenous and supraphysiological 4-1BBL as a critical regulator of lung-resident memory CD8 T cells during influenza infection.     

Human peripheral blood and bone marrow Epstein–Barr virus‐specific T‐cell repertoire in latent infection reveals distinct memory T‐cell subsets

EBV infection leads to life‐long viral persistence. Although EBV infection can result in chronic disease and malignant transformation, most carriers remain disease‐free as a result of effective control by T cells. EBV‐specific IFN‐γ‐producing T cells could be demonstrated in acute and chronic infection as well as during latency. Recent studies, however, provide evidence that assessing IFN‐γ alone is insufficient to assess the quantity and quality of a T‐cell response. Using overlapping peptide pools of latent EBV nuclear antigen 1 and lytic BZLF‐1 protein and multicolor flow cytometry, we demonstrate that the majority of ex vivo EBV‐reactive T cells in healthy virus carriers are indeed IL‐2‐ and/or TNF‐producing memory cells, the latter being significantly more frequent in BM. After in vitro expansion, a substantial number of EBV‐specific CD4⁺ and CD8⁺ T cells retained a CC‐chemokine receptor 7 (CCR7)‐positive memory phenotype. Based on their cytokine profiles, six different EBV‐specific T‐cell subsets could be distinguished with TNF‐single or TNF/IL‐2‐double producing cells expressing the highest CCR7 levels resembling early‐differentiated memory T cells. Our study delineates the memory T‐cell profile of a protective immune response and provides a basis for analyzing T‐cell responses in EBV‐associated diseases.     

TRAF2 regulates peripheral CD8+ T‐cell and NKT‐cell homeostasis by modulating sensitivity to IL‐15

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8⁺ T‐cell and NKT‐cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8⁺ T‐cell subsets. IL‐15‐dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8⁺CD44^hiCD122⁺ T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8⁺CD44^hi T cells exhibited impaired dose‐dependent proliferation to exogenous IL‐15. In contrast, TRAF2TKO CD8⁺ T cells proliferated normally to anti‐CD3 and TRAF2TKO CD8⁺CD44^hi T cells exhibited normal proliferation to exogenous IL‐2. TRAF2TKO CD8⁺ T cells expressed normal levels of IL‐15‐associated receptors and possessed functional IL‐15‐mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8⁺CD44^hiCD122⁺ and NKT cells was mechanistically linked to an inability to respond to IL‐15. The reduced CD8⁺CD44^hiCD122⁺ T‐cell and NKT‐cell populations in TRAF2TKO mice were rescued in the presence of high dose IL‐15 by IL‐15/IL‐15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8⁺ CD44^hiCD122⁺ T‐cell and NKT‐cell homeostasis by modulating sensitivity to T‐cell intrinsic growth factors such as IL‐15.     

Soluble TNF‐α but not transmembrane TNF‐α sensitizes T cells for enhanced activation‐induced cell death

In addition to its proinflammatory effects, TNF‐α exhibits immunosuppression. Here, we compared the capacities of transmembrane TNF‐α (tmTNF) and soluble TNF‐α (sTNF) in regulating expansion of activated T cells by apoptosis. Splenic CD4⁺ T cells from wtTNF, TNF‐α‐deficient (TNF^?/?) and TNF^?/? mice expressing a non‐cleavable mutant tmTNF showed comparable proliferation rates upon TCR‐mediated stimulation. Activation‐induced cell death (AICD), however, was significantly attenuated in tmTNF and TNF^?/?, compared with wtTNF CD4⁺ T cells. Addition of sTNF during initial priming was sufficient to enhance susceptibility to AICD in tmTNF and TNF^?/? CD4⁺ T cells to levels seen in wtTNF CD4⁺ T cells, whereas addition of sTNF only during restimulation failed to enhance AICD. sTNF‐induced, enhanced susceptibility to AICD was dependent on both TNF receptors. The reduced susceptibility of tmTNF CD4⁺ T cells for AICD was also evident in an in vivo model of adoptively transferred CD4⁺ T‐cell‐mediated colonic inflammation. Hence, the presence of sTNF during T‐cell priming may represent an important mechanism to sensitize activated T cells for apoptosis, thereby attenuating the extent and duration of T‐cell reactivities and subsequent T‐cell‐mediated, excessive inflammation.     

Age‐associated Epstein–Barr virus‐specific T cell responses in seropositive healthy adults

Epstein–Barr virus (EBV) is present in 95% of the world's adult population. The immune response participates in immune vigilance and persistent infection control, and this condition is maintained by both a good quality (functionality) and quantity of specific T cells throughout life. In the present study, we evaluated EBV‐specific CD4⁺ and CD8⁺ T lymphocyte responses in seropositive healthy individuals younger and older than 50 years of age. The assessment comprised the frequency, phenotype, functionality and clonotypic distribution of T lymphocytes. We found that in both age groups a similar EBV‐specific T cell response was found, with overlapping numbers of tumour necrosis factor (TNF)‐α⁺ T lymphocytes (CD4⁺ and CD8⁺) within the memory and effector cell compartments, in addition to monofunctional and multi‐functional T cells producing interleukin (IL)‐2 and/or interferon (IFN)‐γ. However, individuals aged more than 50 years showed significantly higher frequencies of IL‐2‐producing CD4⁺ T lymphocytes in association with greater production of soluble IFN‐γ, TNF‐α and IL‐6 than subjects younger than 50 years. A polyclonal T cell receptor (TCR)‐variable beta region (Vβ) repertoire exists in both age groups under basal conditions and in response to EBV; the major TCR families found in TNF‐α⁺/CD4⁺ T lymphocytes were Vβ1, Vβ2, Vβ17 and Vβ22 in both age groups, and the major TCR family in TNF‐α⁺/CD8⁺ T cells was Vβ13·1 for individuals younger than 50 years and Vβ9 for individuals aged more than 50 years. Our findings suggest that the EBV‐specific T cell response (using a polyclonal stimulation model) is distributed throughout several T cell differentiation compartments in an age‐independent manner and includes both monofunctional and multi‐functional T lymphocytes.     

Effect of standard tuberculosis treatment on naive,memory and regulatory T‐cell homeostasis in tuberculosis–diabetes co‐morbidity

Perturbations in CD4⁺ and CD8⁺ T‐cell phenotype and function are hallmarks of tuberculosis–diabetes co‐morbidity. However, their contribution to the pathogenesis of this co‐morbidity and the effect of anti‐tuberculosis treatment on the phenotype of the T‐cell subsets is poorly understood. In this study, we examined the frequency of different T‐cell subsets in individuals with pulmonary tuberculosis (PTB) with diabetes mellitus (DM) or without coincident diabetes mellitus (NDM) before, during and after completion of anti‐tuberculosis chemotherapy. PTB‐DM is characterized by heightened frequencies of central memory CD4⁺ and CD8⁺ T cells and diminished frequencies of naive, effector memory and/or effector CD4⁺ and CD8⁺ T cells at baseline and after 2 months of treatment but not following treatment completion in comparison with PTB‐NDM. Central memory CD4⁺ and CD8⁺ T‐cell frequencies exhibited a positive correlation with fasting blood glucose and glycated haemoglobin A1c levels, whereas the frequencies of naive and effector memory or effector CD4⁺ and CD8⁺ T cells exhibited a negative correlation. However, the frequencies of CD4⁺ and CD8⁺ T‐cell subsets in individuals with PTB exhibited no significant relationship with bacterial burdens. Finally, although minor alterations in the T‐cell subset compartment were observed at 2 months of treatment, significantly decreased frequencies of central memory and significantly enhanced frequencies of naive CD4⁺ and CD8⁺ T cells were observed at the completion of treatment. Our data reveal a profound effect of coexistent diabetes on the altered frequencies of central memory, effector memory and naive T cells and its normalization following therapy.     

Cytomegalovirus drives Vδ2neg γδ T cell inflation in many healthy virus carriers with increasing age

Cytomegalovirus (CMV) usually causes lifelong asymptomatic infection, but over time can distort immune profiles. Recent reports describe selective expansion of Vδ2^neg γδ T cells in healthy and immunocompromised CMV carriers. Having shown previously that virus‐specific CD8⁺ and CD4⁺ T cell responses are increased significantly in elderly CMV carriers, probably driven by chronic stimulation, we hypothesized that Vδ2^neg γδ T cells may also be expanded with age. Our results show that Vδ2^neg γδ T cells are increased significantly in CMV‐seropositive healthy individuals compared to CMV‐seronegative controls in all age groups. The differences were most significant in older age groups (P < 0·0001). Furthermore, while Vδ2^neg γδ T‐ cells comprise both naive and memory cells in CMV‐seronegative donors, highly differentiated effector memory cells are the dominant phenotype in CMV carriers, with naive cells reduced significantly in numbers in CMV‐seropositive elderly. Although phenotypically resembling conventional CMV‐specific T cells, Vδ2^neg γδ T cells do not correlate with changes in magnitude of CMV‐specific CD4⁺ or CD8⁺ T cell frequencies within those individuals, and do not possess ex‐vivo immediate effector function as shown by CMV‐specific CD4⁺ and CD8⁺ T cells. However, after short‐term culture, Vδ2^neg γδ T cells demonstrate effector T cell functions, suggesting additional requirements for activation. In summary, Vδ2^neg γδ T cells are expanded in many older CMV carriers, demonstrating a further level of lymphocyte subset skewing by CMV in healthy individuals. As others have reported shared reactivity of Vδ2^neg γδ T cells towards tumour cells, the composition of γδ T cell subsets may also have implications for risk of developing cancer in elderly people.